POLYNUCLEOTIDES AND POLYPEPTIDES OF PLANT AND BACTERIAL ORIGIN FOR PROTECTING PLANTS FROM PATHOGENIC FUNGI

- EVOGENE LTD.

The present invention relates to polynucleotides and polypeptides derived from plants and bacteria which are associated with increased resistance of plants to pathogenic fungi and/or Oomycetes. The fungicidal polypeptides of the invention are particularly effective in combating fungi inducing root rot and stalk rot in plants. The present invention further provides methods of using the polynucleotides and polypeptides of the invention for controlling plant fungal/Oomycetes pathogens and for producing transgenic plants having increased resistance to the pathogens.

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Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser. No. 17/046,110, filed Oct. 8, 2020; which is a national phase of PCT International Application No. PCT/IL2019/050399, filed on Apr. 10, 2019; which claims priority to U.S. Application No. 62/656,377, filed on Apr. 12, 2018, which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The Sequence Listing, submitted herewith through Patent Center, as an eXtensible Markup Language file (2023-04-05_Sequence_listing_169.0121-US01.xml, created on Apr. 5, 2023; 8,133 bytes) is incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to polynucleotides and polypeptides of plant and bacterial origin which are involved in enhancing resistance of plants towards pathogenic fungi and/or Oomycetes, particularly to fungi inducing root rot and stalk rot in plants, and use thereof for controlling plant diseases associated with the fungal pathogens and for producing genetically engineered plants having increased resistance to the pathogenic fungi.

BACKGROUND OF THE INVENTION

During all developmental stages, plants are exposed to an extremely wide range of biotic and abiotic stress conditions leading to plant diseases. In the production of crop plants, damages caused by biotic stresses, particularly by pathogenic agents, which may be further enhanced under conditions of abiotic stress, pose a major problem and significantly affect the crop yield and profitability.

Many plant diseases are caused by plant pathogenic fungi, and damages to both monocotyledonous and dicotyledonous crop plants are of billions of US$ loss in yield in the U.S. only. For example, stalk rot, caused by a complex of Fusarium spp. and other fungi is one of the most serious challenges in maize production. At present, the majority of both inbreed and hybrid maize lines are susceptible. Fusarium graminearum (Fg) and Fusarium verticillioides (Fv) are the two main causal agents of stalk rot caused by Fusarium spp. in maize, but more than 10 additional Fusarium spp. can cause stalk rot. Natural infection is initiated by a mixture of the local Fusarium spp., but typically during the progress of the disease one species predominates. Gibberella stalk rot (caused by Fusarium graminearum Schwabe) is more prevalent in maize grown in cool regions; while Fusarium stalk rot (caused by Fusarium verticillioides) is most common in dry, warm regions. Fusarium graminearum and other Fusarium species are also responsible for Fusarium head blight (FHB) of wheat, which is a major disease problem for wheat and barley production worldwide, and for various root rots in wide range of different hosts. Colletotrichum spp. infects many grain crops such as barley, wheat, sorghum and corn. C. graminicola is one of the other major pathogens causing mainly maize stalk rots and being capable of infecting many other parts of the corn plant. It has been suggested that C. graminicola behaves as a wilt fungus which efficiently colonizes and moves through the fiber cells that surround the vascular bundles and underlie the epidermal cells in the stalk rind. Movement through the mostly non-living fibers may allow the fungus to avoid host defenses, providing a base from which it can invade adjacent parenchyma cells.

Powdery mildews are fungal diseases caused by different species of fungi in the order Erysiphales that affect a wide range of plants including cereals, grasses, vegetables, ornamentals, weeds, shrubs, fruit trees, broad-leaved shade and forest trees.

Traditionally, plant diseases have been controlled by agronomic practices that include crop rotation, the use of agrochemicals, and conventional breeding techniques. The use of chemicals to control plant pathogens, while being effective, increases the production costs, and moreover, is opposed to by the public and government regulators due to the increased awareness to the harmful effects of such chemicals on the ecosystem and animal health.

Upon the plant recognition of an agent as a pathogen, an array of biochemical responses is activated by the plant. As of today, it is acknowledged that the initial plant response involves induction of several local responses in the cells immediately surrounding the infection site. In the hypersensitive response, cells contacted by the pathogen, and often neighboring cells, rapidly collapse and dry in a necrotic fleck. Other responses include the deposition of callose, the physical thickening of cell walls by lignification, and the synthesis of various antibiotic small molecules and proteins. Genetic factors in both the host and the pathogen determine the specificity of these local responses, which can be very effective in limiting spread of the infection.

Resistance to Fusarium, as an example, is a polygenic trait and can be seen as consisting of two major components: (1) resistance to initial penetration, and (2) resistance to the spreading of the pathogen in host tissue. Though there is no evidence of complete resistance to Fusarium stalk rot in maize, genetic variation for resistance exists within maize germplasm. Resistance to C. graminicola is also primarily quantitative, although a few sources of major gene resistance have been described.

The constantly growing volume of research regarding the mechanisms involved in plant resistance to pathogens and the genetic basis of such mechanism, together with advances in biotechnology have presented new opportunities for protecting plants against pathogen infection through genetic engineering.

Many genes have been identified to participate in the plant defense mechanisms. For example, Sanghyun S et al. (2008. J Exp Bot. 2008:59(9):2371-8) showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions. Zhu X et al. (2012. Funct Integr Genomics. 12(3):481-488) described that overexpression of wheat lipid transfer protein gene TaLTP5 increases resistances to Cochliobolus sativus and Fusarium graminearum in transgenic wheat. Perochon A et al. (2015. Plant Physiol 169(4):2895-2906) reported the functional characterization of an orphan gene (Triticum aestivum Fusarium Resistance Orphan Gene [TaFROG]) as a component of resistance to Fusarium head blight (FHB). Zuo D Y et al. (2016. Phytopatol. 106(6):614-623) showed that a deoxynivalenol-activated methionyl-tRNA synthetase gene from wheat encodes a nuclear localized protein and protects plants against Fusarium pathogens infection and mycotoxins. Dowd P F and Johnson E T (2016. J Plant Res. 129(1):13-20) showed that the maize peroxidase Px5, the pericarp expression of which has been shown to be associated with resistance to Aspergillus flavus growth and to insects in a set of inbred plant lines has a highly conserved sequence which enhances fungal and insect resistance.

Many defense response genes are induced in wheat and other plants during F. graminearum infection and may play a role in reducing FHB. These response genes were thus investigated in an attempt to produce resistant wheat lines. Mackintosh C A et al. (2007. Plant Cell Rep 26(4):479-488) examined overexpression of the defense response genes alpha-1-purothionin, thaumatin-like protein 1 (tlp-1), and beta-1,3-glucanase in wheat, and reported that all the genes reduced at least part of the disease symptoms. A beta-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, deoxynivalenol (DON) mycotoxin concentration, and percent of visually scabby kernels (VSK) compared to a control plant. Sasaki K et al. (2016. J Biotechnol 228:3-7) also showed that overexpression of TAD1 (Triticum aestivum defensin 1), a protein induced during cold acclimation in winter wheat and encoding a plant defensin with antimicrobial activity, increased resistance against Fusarium graminearum in the transformed wheat plants.

Various additional genetic manipulation of gene expression for improving resistance to pathogenic fungi has also been reported. For example, Brewer H C et al. (2014. BMC Plant Biol 14(1):317) showed that mutations in the Arabidopsis homoserine kinase gene DMR1 confer enhanced resistance to F. culmorum and F. graminearum. Tundo S et al. (2016. Mol Plant Microbe Interact 29(8):629-639) produced transgenic plants with a combination of genes encoding proteins involved in inhibiting the activity of cell wall-degrading enzymes (CWDEs) secreted by pathogens to breach the plant cell-wall barrier. They showed that pyramiding polygalacturonase (PG) inhibiting protein (PGIP2) and TAXI-III, a xylanase inhibitor that controls the activity of xylanases, enhanced resistance against Fusarium graminearum, while pyramiding PGIP2 and pectin methyl esterase inhibitor (PMEI) did not reach the same effect. Li X et al. (2015. Mol Plant Microbe Interact 28(11):1237-1246) demonstrated that transgenic wheat expressing a barley UDP-glucosyltransferase detoxifies deoxynivalenol and provides high levels of resistance to Fusarium graminearum.

Among others, International Application Publication Nos. WO/2006/085965 and WO 2006/091219 disclose methods for protecting plants from plant pathogenic fungi by enhancing fungal pathogen resistance in a plant using the nucleotide sequences disclosed therein. Further disclosed therein are methods comprising introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antifungal polypeptide as well as transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antifungal polypeptide or variant or fragment thereof.

U.S. Pat. No. 9,359,615 discloses plants which overexpress a p33kD or BURP protein, or an ortholog thereof, and exhibit an increased pre-formed resistance to pathogens, particularly fungal pathogens.

U.S. Pat. No. 9,732,354 discloses new gene that is able to provide plants with resistance against pathogens, particularly Verticillium, Ralstonia or Fusarium. The gene is typical for Brassicaceae, but may confer resistance to other plants. Further provided are host cells with a nucleotide construct encoding the protein and methods for enhancing the pathogen resistance of plants.

International (PCT) Patent Application Publication No. WO 2018/131037 to the Applicant of the present invention discloses polynucleotides and polypeptides associated with increased resistance of plant to pathogenic fungi, particularly to fungi inducing root rot and stalk rot in plants, and use thereof for controlling plant fungal pathogens and for producing transgenic plants having increased resistance to pathogenic fungi.

Intact microorganisms, particularly bacteria, which can survive on or be toxic to fungi, have been used as biocontrol agents. For example, U.S. Pat. No. 9,485,994 discloses methods and compositions for control of pathogenic fungal or Oomycetous infection. Particularly, the patent discloses an antifungal or an anti-Oomycetous composition comprising bacteria of the genus Collimonas and bacteria of the genus Bacillus, together exhibiting a synergistic antifungal or a synergistic anti-Oomycetous effect, and methods of use thereof. However, it is difficult both to apply biological organisms to large areas and to cause such living organisms to remain in the treated area or on the treated plant species for an extended time, and therefore use of intact bacteria as biocontrol agents is still limited.

Therefore, attempts were made to identify bacterial proteins and genes encoding same responsible for the biocontrol activity. Targets, mechanism of action and prospective applications of anti-fungal proteins of various organisms, including bacteria are reviewed, for example, by Theis and Stahl (Theis T and Stahl U. 2004 CMLS, Cell. Mol. Life Sci. 61:437-455). Proteomic approach taken to investigate Bacillus strains having anti-fungal activity revealed the presence of lytic enzymes, cellulases, proteases, 1,4-β-glucanases and hydrolases, all of which contribute to degradation of the pathogen cell wall. Further proteomic investigations showed that proteins involved in metabolism, protein folding, protein degradation, translation, recognition and signal transduction cascade play an important role in the control of Fusarium oxysporum (Baysal O. et al., 2013. Plose One 8(1):e53182). Li et al., (Li Jing et al., J Zhejiang Univ Sci B 10(4):264-272) describe the purification of a particular antifungal protein from Bacillus subtilis, designated B29I, which exhibited inhibitory activity on mycelial growth of Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia sclerotiorum. Yadav et al. (Yadav V et al., 2010. Intl J Microbiol, Article ID 196363, doi:10.1155/2010/196363) described the isolation of an antifungal protein from Escherichia coli BL21 (PPEBL21), predicted to be alcohol dehydrogenase (ADH). Additional example of enzymes having a significant role in the anti-fungal activity of bacteria includes chitinases (see, e.g. Hjort K et al., 2013. Appl Microbiol Biotechnol DOI 10.1007/s00253-013-5287-x).

Song et al. (Song C et al., 2015. BMC Genomics 16:1103) explored the genomic traits of the fungus-feeding bacterial genus Collimonas, and Haack et al. (Haack F S et al., 2016. Frontiers in Microbiology 7 Article 1668) investigated the molecular keys of the interactions of Janthinobacterium and Duganella species with the plant pathogenic fungi Fusarium graminearum.

However, as of today, there is a need for further developed methods and compositions for protecting plants from fungal pathogen.

SUMMARY OF THE INVENTION

The present invention provides isolated polynucleotides, constructs comprising same and isolated polypeptides useful in conferring and/or enhancing resistance of a plant towards pathogenic fungi and/or Oomycetes. The present invention further provides genetically altered plants and plant cells with enhanced resistance to pathogenic fungi and/or Oomycetes and methods for producing and selecting same as well as compositions comprising the polypeptides of the invention useful in protecting plants from the pathogenic fungi and/or Oomycetes.

According to one aspect, the present invention provides a method for enhancing the resistance of a plant or a part thereof to at least one pathogenic fungus and/or Oomycete, comprising modulating the expression and/or activity of at least one polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506 within at least one cell of the plant or part thereof, thereby enhancing the resistance of said plant or part thereof to the at least one pathogenic fungus and/or Oomycete compared to the resistance of a control plant. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the method comprises modulating the expression and/or activity of at least one polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the at least one polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, 81-301. Each possibility represents a separate embodiment of the present invention.

According to other embodiments, the at least one polypeptide is encoded by a polynucleotide comprising the nucleic acid sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-305. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, enhancing the resistance of the plant or part thereof to the pathogenic fungus and/or Oomycete comprises enhancing the expression and/or activity of at least one polypeptide compared to its expression and/or activity in the control plant.

According to certain exemplary embodiments, the polypeptide the expression and/or activity of which is to be enhanced comprises an amino acid sequence at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-506. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the polypeptide the expression and/or activity of which is to be enhanced comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-508. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the polypeptide the expression and/or activity of which is to be enhanced is an endogenous polypeptide of the at least one cell. According to other embodiments, the polypeptide the expression and/or activity of which is to be enhanced is a polypeptide heterologous to the at least one plant cell. According to certain embodiments, enhancing the expression and/or activity of the polypeptide comprises modulating the expression of an endogenous polynucleotide encoding said polypeptide within the at least one cell of the plant or part thereof.

Modulating, according to certain embodiments enhancing, the expression of the endogenous polynucleotide can be affected at the genomic and/or the transcript level using a variety of methods that induce the transcription and/or translation of the polypeptide.

According to certain embodiments, enhancing the expression and/or activity of the endogenous polypeptide comprises subjecting the at least one cell of the plant or part thereof to genome editing using artificially engineered nucleases as is known in the art.

According to certain embodiments, isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position as to enhance transcription of the endogenous polynucleotide. According to some embodiments, the regulatory element is selected from the group consisting of, but not limited to, a promoter and an enhancer.

According to certain embodiments, the polypeptide the expression of which is to be enhanced is encoded by a polynucleotide at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41, 43-61, 63-79, 81, 83-301. According to some embodiments, the polypeptide the expression of which is to be enhanced is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41, 43-61, 63-79, 81, 83-305.

According to other embodiments, at least one mutation may be inserted within the endogenous polynucleotide as long as the mutation results in enhanced expression of the encoded polypeptide. Any method for mutagenesis as is known in the art can be used according to the teachings of the present invention including chemical mutagenesis, radio-mutagenesis and site directed mutagenesis, for example using genome editing techniques.

According to certain embodiments, enhancing the resistance of the plant to the pathogenic fungus and/or Oomycete comprises reducing the expression of at least one polypeptide compared to its expression and/or activity in the control plant. According to certain exemplary embodiments, the polypeptide the expression and/or activity of which is to be reduced comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NO:328, SEQ ID NO:361, and SEQ ID NO:348. According to further exemplary embodiments, the polypeptide the expression and/or activity of which is to be reduced comprises the amino acid sequence set forth in any one of SEQ ID NO:328, SEQ ID NO:361 and SEQ ID NO:348.

According to certain embodiments, enhancing the resistance of the plant to the pathogenic fungus and/or Oomycete comprises reducing the expression and/or activity of at least one polynucleotide compared to its expression in the control plant. According to certain exemplary embodiments, the polynucleotide having reduced expression comprises a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in any one of SEQ ID NO:42, SEQ ID NO:82, and SEQ ID NO:62. According to certain exemplary embodiments, the polynucleotide having reduced expression comprises the nucleic acid sequence set forth in any one of SEQ ID NO:42, SEQ ID NO:82 and SEQ ID NO:62.

Any method as is known in the art for reducing the expression and/or activity of a plant endogenous protein and the polynucleotide encoding same can be used according to the teachings of the resent invention.

According to certain embodiments, reducing the expression and/or activity of the polypeptide comprises down-regulating the expression of the endogenous polynucleotide encoding said polypeptide within the at least one cell of the plant or part thereof.

According to certain embodiments, reducing the expression and/or activity of the polypeptide comprises modulating the endogenous polynucleotide as to encode a non-functional polypeptide.

According to certain embodiments, expression of the polynucleotide is affected at the genomic and/or the transcript level using a variety of molecules that interfere with transcription and/or translation (e.g., antisense, siRNA, Ribozyme, or DNAzyme) of the polynucleotide. Inserting a mutation into the polynucleotide, including deletions, insertions, site specific mutations, mutations mediated by artificially engineered nucleases (including zinc-finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR/Cas system) can be also used, as long as the mutations result in down-regulation of the gene expression or in the production of non-functional protein. Alternatively, expression can be inhibited at the protein level using, e.g., antagonists, enzymes that cleave the polypeptide, and the like.

According to additional aspect, the present invention provides a method for enhancing the resistance of a plant or a part thereof to at least one pathogenic fungus and/or Oomycete, comprising enhancing the expression and/or activity of at least one polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, an active fragment and/or a variant thereof within at least one cell of the plant or part thereof, thereby enhancing the resistance of said plant or part thereof to the at least one pathogenic fungus and/or Oomycete compared to the resistance of a control plant. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the method comprises enhancing the expression and/or activity of at least one polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, an active fragment and/or a variant thereof. Each possibility represents a separate embodiment of the present invention.

According to the certain exemplary embodiments, the fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:4661, 4647-4660, and 4662-4799.

According to certain embodiments, the at least one polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443, a fragment and/or a variant thereof. Each possibility represents a separate embodiment of the present invention.

According to other embodiments, the at least one polypeptide is encoded by a polynucleotide comprising the nucleic acid sequence set forth in any one of SEQ ID NOs:783, 592-782, 784-2443, a fragment and/or a variant thereof. Each possibility represents a separate embodiment of the present invention.

Enhancing the polypeptide expression can be affected at the genomic and/or the transcript and/or translation level using a variety of methods that induce the transcription and/or translation of the polypeptide.

According to certain embodiments, enhancing the expression and/or activity of the polypeptide comprises expressing an exogenous polynucleotide encoding said at least one polypeptide within the at least one cell of the plant or the part thereof.

According to certain embodiments, enhancing the expression and/or activity of the polypeptide comprises transforming at least one cell of the plant or part thereof with an exogenous polynucleotide encoding the polypeptide, thereby producing a transgenic plant over-expressing said polypeptide.

Any method as is known in the art for introducing an exogenous polynucleotide into a plant cell can be used according to the teachings of the present invention. According to some embodiments, the exogenous polynucleotide is transformed into the plant cell using a suitable vector.

According to certain embodiments, genome editing is employed to edit the genome of the at least one cell as to express a heterologous polypeptide of the invention.

According to certain exemplary embodiments, the polynucleotide the expression of which is to be enhanced comprises a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41, 43-61, 63-79, 81, and 83-301. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the polynucleotide the expression of which is to be enhanced comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41, 43-61, 63-79, 81, and 83-305.

According to further exemplary embodiments, the polynucleotide the expression of which is to be enhanced comprises a nucleic acid sequence at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443 and a fragment thereof. According to some embodiments, the polynucleotide the expression of which is to be enhanced comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443 and a fragment thereof.

Gene stacking is often desirable in the field of plant genetic engineering, leading to a desired phenotype and/or improvement of a desired phenotype. According to certain aspects, the present invention provides a method for enhancing the resistance of a plant or a part thereof to at least one pathogenic fungus and/or Oomycete, comprising transforming at least one cell of the plant or part thereof with at least one DNA construct comprising nucleic acid sequence encoding at least two polypeptides, wherein each polypeptide is capable of enhancing resistance to the at least one pathogenic fungus and/or Oomycete, thereby enhancing the resistance of said plant or part thereof to said at least one pathogenic fungus and/or Oomycete compared to the resistance of a control plant. According to certain embodiments, the present invention provides a method for enhancing the resistance of a plant or a part thereof to at least one pathogenic fungus and/or Oomycete, comprising transforming at least one cell of a plant or a part thereof with a DNA construct comprising a first polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:311 and a second polynucleotide encoding a polypeptide designated LFS57 having the amino acid sequence set forth in SEQ ID NO:617 of International Application Publication No. WO 2018/131037, thereby enhancing the resistance of said plant or part thereof to the at least one pathogenic fungus and/or Oomycete compared to the resistance of a control plant.

According to certain embodiments, the first polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:66 and the second polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NO:101 and SEQ ID NO:165 of WO 2018/131037.

According to certain embodiments, the present invention provides a method for enhancing the resistance of a plant or a part thereof to at least one pathogenic fungus and/or Oomycete, comprising transforming at least one cell of a plant or a part thereof with a DNA construct comprising a first polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:310 and a second polynucleotide encoding a polypeptide designated LFS46 having the amino acid sequence set forth in SEQ ID NO:608 of International Application publication No. WO 2018/131037, thereby enhancing the resistance of said plant or part thereof to the at least one pathogenic fungus and/or Oomycete compared to the resistance of a control plant.

According to certain embodiments, the first polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:65 and the second polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NO:92 and SEQ ID NO:157 of WO 2018/131037.

According to some embodiments, the control plant is a plant not manipulated to have modulated expression and/or activity of the polypeptide. According to some embodiments, the control plant is of the same species. According to some embodiments, the control plant comprises the same genetic background.

According to another aspect, the present invention provides a method for producing a population of plants each having an enhanced resistance to at least one pathogenic fungus and/or Oomycete, comprising the steps of:

    • (a) modulating the expression and/or activity of at least one polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, and 361-506 within at least one cell of each plant of a plant population as to produce a genetically engineered plant population;
    • (b) inoculating each plant of the genetically engineered plant population with the at least one pathogenic fungus and/or Oomycete; and
    • (c) selecting plants showing an enhanced resistance to said at least one pathogenic fungus and/or Oomycete compared to a control plant or to a pre-determined resistance score value;
    • thereby producing a population of genetically engineered plants having enhanced resistance to said at least one pathogenic fungus and/or Oomycete.

According to certain embodiments, the method comprises modulating the expression and/or activity of at least one polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508. According to certain embodiments, the method comprises modulating the expression and/or activity of at least one polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs:507-508. According to certain embodiments, the method comprises modulating the expression and/or activity of at least one polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs:507-509. According to other embodiments, the method comprises modulating the expression and/or activity of at least one polypeptide having the amino acid sequence set forth in SEQ ID NO:509. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the method comprises enhancing the expression and/or activity of the at least one polypeptide. According to these embodiments, the method comprises enhancing the expression and/or activity of at least one polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-506. According to some embodiments, the method comprises enhancing the expression and/or activity of at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-509. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the method comprises reducing the expression and/or activity of the at least one polypeptide. According to these embodiments, the method comprises reducing the expression and/or activity of at least one polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:328, SEQ ID NO:361 and SEQ ID NO:348. According to some embodiments, the method comprises reducing the expression and/or activity of at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:328, SEQ ID NO:361 and SEQ ID NO:348.

According to yet another aspect, the present invention provides a method for producing a population of plants each having an enhanced resistance to at least one pathogenic fungus and/or Oomycete, comprising the steps of:

    • (a) enhancing the expression and/or activity of at least one polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, an active fragment and/or variant thereof within at least one cell of each plant of a plant population as to produce a genetically engineered plant population;
    • (b) inoculating each plant of the genetically engineered plant population with the at least one pathogenic fungus and/or Oomycete; and
    • (c) selecting plants showing an enhanced resistance to said at least one pathogenic fungus and/or Oomycete compared to a control plant or to a pre-determined resistance score value;
    • thereby producing a population of genetically engineered plants having enhanced resistance to said at least one pathogenic fungus and/or Oomycete.

According to certain embodiments, the method comprises modulating the expression and/or activity of at least one polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs:2635, 2444-2634, 2636-4252, an active fragment and/or a variant thereof.

According to the certain exemplary embodiments, the polypeptide fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:4661, 4647-4660, and 4662-4799. The methods by which the expression and/or activity of the at least polypeptide can be enhanced or reduced are as is known in the art and as described hereinabove.

According to certain embodiments, the method comprises modulating the expression of at least one polynucleotide comprising a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, 81-301.

According to some embodiments, the method comprises modulating the expression of at least one polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, and 81-305. According to some embodiments, the method comprises modulating the expression of at least one polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:304-305. According to some embodiments, the method comprises modulating the expression of at least one polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:304-306. According to some embodiments, the method comprises modulating the expression of at least one polynucleotide comprising a nucleic acid sequence set forth in SEQ ID NO:306. Each possibility represents a separate embodiment of the present invention. According to some embodiments, modulating the expression of the at least one polynucleotide comprises enhancing its expression. According to these embodiments, the polynucleotide is at least 80% identical to a polynucleotide having the nucleic acid sequence selected from the group consisting of 40, 80, 22-39, 41, 43-61, 63-79, 81, and 83-301. According to some embodiments, the the polynucleotide comprises the nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41, 43-61, 63-79, 81, and 83-306. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, enhancing the expression comprises transforming the at least one cell of the plant or part thereof with a polynucleotide at least 80% identical to a polynucleotide having an amino acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, 81, and 83-301. According to certain embodiments, enhancing the expression comprises transforming the at least one cell of the plant or part thereof with a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, 81, and 83-306.

According to some embodiments, modulating the expression of the at least one polynucleotide comprises reducing its expression. According to these embodiments, the polynucleotide is at least 80% identical to a polynucleotide having the nucleic acid sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:82 and SEQ ID NO:62. According to some embodiments, the the polynucleotide comprises the nucleic acid sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:82 and SEQ ID NO:62.

According to certain embodiments, the method comprises enhancing the expression of at least one polynucleotide comprising a nucleic acid sequence at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443, a variant and/or a fragment thereof. According to some embodiments, the method comprises enhancing the expression of at least one polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443, a variant and/or a fragment thereof.

The methods by which the expression of the at least one polynucleotide can be enhanced or reduced are as is known in the art and as described hereinabove.

According to additional aspect, the present invention provides a method for selecting a plant having an enhanced resistance to at least one pathogenic fungus and/or Oomycete, comprising the steps of:

    • (a) providing a plurality of plants each comprising at least one cell with modulated expression and/or activity of a polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506.
    • (b) inoculating the plurality of plants with the at least one pathogenic fungus and/or Oomycete; and
    • (c) selecting plants showing an enhanced resistance to said at least one pathogenic fungus and/or Oomycete compared to a control plant or to a pre-determined resistance score value.

According to certain embodiments, the method comprises providing a plurality of plants each having a modulated expression and/or activity of at least one polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508. Each possibility represents a separate embodiment of the present invention. According to other embodiments, the method comprises providing a plurality of plants each having a modulated expression and/or activity of a polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs:507-509. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the method comprises providing a plurality of plants each having a modulated expression and/or activity of a polypeptide having the amino acid sequence set forth in SEQ ID NO:509.

According to certain embodiments, the method comprises providing a plurality of plants each having modulated expression of at least one polynucleotide comprising a nucleic acid sequence at least 80% identical to a polynucleotide having the nucleic acid sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-301. According to some embodiments, the at least one polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, 81-305. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the at least one polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO:306.

According to certain embodiments, the modulated expression and/or activity is selected from enhanced expression and/or activity and reduced expression and/or activity. Modulating the expression can be performed by any method as is known in the art and as described hereinabove. The polypeptides and polynucleotides the expression of which is to be enhanced or reduced are as described hereinabove. According to yet further aspect, the present invention provides a method for selecting a plant having an enhanced resistance to at least one pathogenic fungus and/or Oomycete, comprising the steps of:

    • (a) providing a plurality of plants each comprising at least one cell with enhanced expression and/or activity of a polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, an active fragment and/or a variant thereof;
    • (b) inoculating the plurality of plants with the at least one pathogenic fungus and/or Oomycete; and
    • (c) selecting plants showing an enhanced resistance to said at least one pathogenic fungus and/or Oomycete compared to a control plant or to a pre-determined resistance score value.

According to certain embodiments, the method comprises providing a plurality of plants each having an enhanced expression and/or activity of at least one polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs:2635, 2444-2634, 2636-4252, an active fragment and/or a variant thereof. Each possibility represents a separate embodiment of the present invention.

According to the certain exemplary embodiments, the fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:4661, 4647-4660, and 4662-4799. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the method comprises providing a plurality of plants each having enhanced expression of at least one polynucleotide comprising a nucleic acid sequence at least 70% identical to a polynucleotide having the nucleic acid sequence set forth in any one of SEQ ID NOs:783, 592-782, 784-2443. According to some embodiments, the at least one polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:4783, 592-782, 784-2443.

According to certain embodiments, the control plant is a plant not manipulated to have modulated expression and/or activity of the polypeptide. According to some embodiments, the control plant is of the same species. According to some embodiments, the control plant comprises the same genetic background.

According to certain embodiments, the pre-determined resistance score value is obtained by a method comprising the steps of inoculating a plurality of corresponding plants susceptible to the at least one pathogenic fungus or Oomycete; scoring the infection degree; and setting an average resistance score value.

Modulating (enhancing or reducing) the expression and/or activity of the polypeptide can be achieved as described hereinabove and by any other method as is known in the art.

According to certain embodiments, the plant part is selected from the group consisting of seeds, roots, shoots, leaves, flowers and the like. Each possibility represents separate embodiment of the present invention. According to certain exemplary embodiments, the plant part is a root. Tissue cultures comprising cells derived from the plant having a modulated expression and/or activity of a polypeptide of the invention are also encompassed within the scope of the present invention.

According to additional aspect, the present invention provides a genetically engineered plant having enhanced resistance to at least one fungus and/or Oomycete compared to a non-engineered control plant, the genetically engineered plant comprising at least one cell having modified expression and/or activity of at least one polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506 compared to the polypeptide expression and/or activity in the non-engineered control plant. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having enhanced expression and/or activity of at least one polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-506.

According to certain embodiments, the genetically engineered plant comprises at least one cell having reduced expression and/or activity of at least one polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:328, 361 and 348.

According to additional aspect, the present invention provides a genetically engineered plant having enhanced resistance to at least one fungus and/or Oomycete compared to a non-engineered control plant, the genetically engineered plant comprising at least one cell having enhanced expression and/or activity of at least one polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, an active fragment or a variant thereof compared to the polypeptide expression and/or activity in the non-engineered control plant. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having modified expression and/or activity of at least one polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508. Each possibility represents a separate embodiment of the present invention. According to certain embodiments, the genetically engineered plant comprises at least one cell having modified expression and/or activity of at least one polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NOs:507-508. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having enhanced expression and/or activity of at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-508. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having reduced expression and/or activity of at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:328, 361, and 348. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having enhanced expression and/or activity of at least one polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, an active fragment and/or a variant thereof. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having modified expression of at least one polynucleotide comprising a nucleic acid sequence at least 80% identical to a polynucleotide having the nucleic acid sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-301. According to some embodiments, the at least one polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, 81-305. According to some embodiments, the at least one polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:302-305. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having enhanced expression of at least one polynucleotide comprising a nucleic acid sequence at least 80% identical to a polynucleotide having the nucleic acid sequence set forth in any one of SEQ ID NOs: 40, 80, 22-39, 41, 43-61, 63-79, 81, and 83-301. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having reduced expression of at least one polynucleotide comprising a nucleic acid sequence at least 80% identical to a polynucleotide having the nucleic acid sequence set forth in any one of SEQ ID NOs:42, 82 and 62. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having enhanced expression of at least one polynucleotide comprising a nucleic acid sequence at least 70% identical to a polynucleotide having the nucleic acid sequence set forth in any one of SEQ ID NOs:783, 592-782, 784-2443. According to some embodiments, the at least one polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises a polynucleotide encoding a modified form of the at least one polypeptide, wherein the modified form has reduced or no activity compared to the unmodified form, thereby having an enhanced resistance to the at least one fungus.

According to additional aspect, the present invention provides an isolated or recombinant polynucleotide encoding a polypeptide comprising an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, and 362-506, wherein the polypeptide, when expressed in a plant, is capable of modulating the resistance of the plant to at least one pathogenic fungus and/or Oomycete. According to certain embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 362-506. According to certain embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs:507-508. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the polypeptide, when expressed in a plant, is capable of enhancing the resistance of the plant to at least one pathogenic fungus and/or Oomycete, said polypeptide is at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, and 362-506. According to certain embodiments, said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, and 362-508. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the polynucleotide encoding a polypeptide capable of modulating the resistance of the plant to at least one pathogenic fungus and/or Oomycete when expressed in said plant comprises a nucleic acid sequence at least 80% identical to a nucleic acids sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-301. According to other embodiments, the polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-301. According to yet further embodiments, the polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs:302-305.

According to certain embodiments, the polynucleotide encoding a polypeptide capable of enhancing the resistance of the plant to at least one pathogenic fungus and/or Oomycete when expressed in said plant comprises a nucleic acid sequence at least 80% identical to a nucleic acids sequence set forth in any one of SEQ ID NOs: 40, 80, 22-39, 41, 43-61, 63-79, 81, and 83-301. According to other embodiments, the polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 40, 80, 22-39, 41, 43-61, 63-79, 81, and 83-301. According to yet further embodiments, the polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs:302-305.

According to yet additional aspect, the present invention provides an isolated or recombinant polynucleotide encoding a polypeptide comprising an amino acid sequence at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, wherein the polypeptide, when expressed in a plant, is capable of enhancing the resistance of the plant to at least one pathogenic fungus and/or Oomycete. According to certain embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs:2635, 2444-2634, 2636-4252.

According to yet another aspect, the present invention provides an isolated or recombinant polynucleotide, a fragment or a variant thereof, the polynucleotide comprising a nucleic acid sequence at least 80% identical to a nucleic acids sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41, 43-61, 63-79, 81, and 83-301, wherein said polynucleotide, when expressed in a plant, is capable of enhancing the resistance of the plant to at least one pathogenic fungus and/or Oomycete. According to certain embodiments, the polynucleotide comprises the nucleic acid sequence set forth in any one if SEQ ID NOs:40, 80, 22-39, 41, 43-61, 63-79, 81, and 83-305.

According to yet further aspect, the present invention provides an isolated or recombinant polynucleotide, a fragment or a variant thereof, the polynucleotide comprising a nucleic acid sequence at least 70% identical to a nucleic acids sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443, wherein said polynucleotide, when expressed in a plant, is capable of enhancing the resistance of the plant to at least one pathogenic fungus and/or Oomycete. According to certain embodiments, the polynucleotide comprises the nucleic acid sequence set forth in any one if SEQ ID NOs:783, 592-782, 784-2443.

According to additional aspect, the present invention provides a recombinant polynucleotide optimized for polypeptide expression in a bacterial expression system, wherein the polypeptide is capable of inhibiting the growth of at least one pathogenic fungus and/or Oomycete.

According to certain exemplary embodiment the bacterium is Escherichia coli. According to these embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4468, 4450-4467, and 4469-4646. According to certain embodiments, the recombinant polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:4271, 4253-4270, and 4272-4449.

According to certain embodiments, the recombinant polynucleotide optimized for polypeptide expression in the bacterial expression system further comprises said bacterium signal peptide.

According to a further aspect, the present invention provides a fungicidal composition comprising an effective amount of at least one polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof, wherein the fungicidal composition is effective in inhibiting the growth and/or development of at least one plant pathogenic fungi and/or Oomycetes. According to certain embodiments, the fungicidal composition comprises at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 4468, 4661, 2444-2634, 2636-4252, 4450-4467, 4469-4660, and 4662-4799. According to some embodiments, the fungicidal composition comprises at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:4468, 4661, 4450-4467, 4469-4660, and 4662-4799. According to certain embodiments, the fungicidal composition further comprises at least one agriculturally compatible agent selected from the group consisting of a carrier, a stabilizer, a diluent, a surfactant, a mineral and an adjuvant.

According to yet additional aspect, the present invention provides a fungicidal composition comprising bacteria comprising at least one polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof, wherein the fungicidal composition is effective in inhibiting the growth and/or development of at least one plant pathogenic fungi and/or Oomycetes. According to certain embodiments, the bacteria comprise at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 4468, 4661, 2444-2634, 2636-4252, 4450-4467, 4469-4660, and 4662-4799. According to some embodiments, the bacteria comprise at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:4468, 4661, 4450-4467, 4469-4660, and 4662-4799. Each possibility represents a separate embodiment of the present invention. The bacteria can be of the same species or of different species, with the proviso that the bacteria have no adverse effect on a plant. The bacteria can be alive or dead. According to certain exemplary embodiments, the bacteria are selected from the group consisting of Bacillus subtilis Lactococcus lacti, Corynebacterium glutamicum; and Bacillus brevis.

According to certain embodiments, the fungicidal composition further comprises at least one agriculturally compatible agent selected from the group consisting of a carrier, a stabilizer, a diluent, a surfactant, a mineral and an adjuvant.

According to additional aspect, the present invention provides a nucleic acid construct comprising a polynucleotide according to some embodiments of the present invention, further comprising at least one regulatory element for directing the expression of the polynucleotide within a plant cell. According to certain embodiment, the regulatory element is a promoter. The promoter can be endogenous or heterologous to the plant comprising the nucleic acid construct.

The polypeptides and polynucleotides disclosed herein may be used to confer resistance to a wide variety of fungal and Oomycetous pathogens that cause commercial damage to crop and ornamental plants.

According to certain embodiments, the fungal or Oomycetous pathogens can be one or more fungi or Oomycetes from a class selected from the group consisting of Ascomycetes, Plasmodiophoromycetes, Chytridiomycetes, Zygomycetes, Basidiomycetes, Deuteromycetes, and Sordariomycetes. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the fungal pathogens can be one or more fungi from a genus selected from the group consisting of Fusarium, Colletotrichum, Geotrichum, Aspergillus, Alternaria, Athelia, Botryosphaeria, Botrytis, Cryphonectria, Choanephora, Cercospora, Magnaporthe Monilinia, Mycosphaerella, Melampsora, Puccinia, Phakopsora, Rhizoctonia, Septoria, Uromyces, Ustilago and Verticillium.

According to some embodiments, the Oomycetous pathogen can be from the class Oomycetes (synonym Peronosporomycetes). In some embodiments, said Oomycetous infection comprises infection by an Oomycete from a genus selected from the group consisting of Blumeria, Macrophomina, Oidium, Pythium, and Phytophthora. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the fungal or Oomycetous pathogen is selected from the group consisting of Botrytis cinerea, Mycosphaerella graminicola, Mycosphaerella fijiensis, Septoria lycopersici, Magnaporthe oryza, Rhizoctonia solani, Ustilago maydis, Sclerotium rolfsii, and Blumeria graminis.

According to certain exemplary embodiments, the fungus of the genus Fusarium is selected from the group consisting of Fusarium verticilloides and Fusarium graminearum. According to additional exemplary embodiments, the F. verticilloides is F. verticillioides strain A-00149-FGSC 7600. According to further exemplary embodiments, the F. graminearum is F. graminearum strain CBS 110260.

According to other exemplary embodiments, the fungus of the genus Colletotrichum is Colletotrichum graminicola.

The polynucleotides and polypeptides of the present invention can be used to confer resistance to any plant type. According to certain embodiments, the plant is a cereal plant. According to some embodiments, the cereal plant is selected from the group consisting of maize, wheat, barley, sorghum, rice, oat, and rye. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the cereal plant is maize (Zea mays). According to other embodiments, the plant is a field-crop plant. According to some embodiments, the field crop plant is selected from the group consisting of tomato, potato, sweet potato, cassava, beets, ginger, horseradish, radish, ginseng, turnip, any root or tuber crop, pepper, eggplant, ground cherry, tomatillo, okra, other fruiting vegetables, cucumber, cantaloupe, melon, muskmelon, squash, watermelon and other cucurbit plants.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration of the pQ6sVN plasmid. pQ6sVN used for expression of the isolated polynucleotide sequences of some embodiments of the invention in Brachypodium. “35S(V)”=35S promoter (SEQ ID NO:37); “NOS ter”=nopaline synthase terminator; “Bar_GA”=BAR open reading frame optimized for expression in Brachypodium (SEQ ID NO:39); “Hyg”=Hygromycin resistance gene. “Ubil promoter”=SEQ ID NO: 11; the isolated polynucleotide sequences of some embodiments of the invention were cloned into the Multiple cloning site of the vector (downstream of the “35S(V)” promoter) using one or more of the indicated restriction enzyme sites.

FIGS. 2A-H depict an exemplary design of Homology Directed Repair according to some embodiments of the invention. FIG. 2A depicts the sequence of the endogenous 5′-upstream flanking region of the genomic sequence GRMZM2G069095 (SEQ ID NO:45). FIG. 2B depicts the sequence of the endogenous 3′-downstream flanking region of the genomic sequence GRMZM2G069095 having the nucleic acid sequence set forth in SEQ ID NO:46. FIG. 2C depicts the sequence of the 5′-UTR gRNA (SEQ ID NO:43). FIG. 2D depicts the sequence of the 5′-UTR gRNA without NGG nucleotides (SEQ ID NO:47). FIG. 2E depicts the sequence of the 3′-UTR gRNA (SEQ ID NO:44). FIG. 2F depicts the sequence of the 3′-UTR gRNA after cut (SEQ ID NO:48). FIG. 2G depicts the coding sequence (from the “ATG” start codon to the “TGA” termination codon, marked by bold and underlined) of the desired LFS127_H4 sequence (SEQ ID NO:50) encoding the polypeptide set forth by SEQ ID NO:487. FIG. 2H depicts the exemplary repair template (SEQ ID NO:49) which includes (1) the upstream flanking region (1 kbp) sequence including part of the gRNA after cutting (SEQ ID NO:47; shown in bold and italics); (2) 5′ UTR of genomic DNA from Cas9 cutting site to ATG; (3) the coding sequence (CDS) of the desired LFS127_H4 sequence (SEQ ID NO:50) marked in lower case with the start (ATG) and the stop (TGA) codons marked in bold and underlined; (4) 3′ UTR of genomic DNA from the stop codon to Cas9 cutting site including the predicted part of the gRNA after cutting (SEQ ID NO:48, shown in bold and underlined and (5) the downstream flanking region (1 kbp) sequence.

FIG. 3A-E depicts an exemplary design of polynucleotide knockout (KO) using CRISPR/CAS system. FIG. 3A depicts the sequence of the KO gRNA (SEQ ID NO:51); FIG. 3B depicts the sequence of the KO gRNA after cut (SEQ ID NO:52); FIG. 3C depicts the coding sequence (from the “ATG” start codon to the “TAG” termination codon, marked by bold and underlined) of the desired LFS132 sequence (SEQ ID NO:53); FIG. 3D (targeted region in bold) and FIG. 3E depict the anticipated change in the coding sequence of the exemplified KO gene (SEQ ID NO:54).

 DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses means and methods for conferring and/or enhancing the resistance of a plant to pathogenic fungi and/or Oomycetes. Particularly, the present invention provides isolated polypeptides conferring or enhancing plant resistance to pathogenic fungi and/or Oomycetes, isolated polynucleotides encoding same, nucleic acid constructs comprising the polynucleotides and plant cells transformed with same. The present invention further provides methods for producing and selecting plants having increased resistance to at least one pathogenic fungus and/or Oomycete and plant with enhanced resistance to the at least one pathogenic fungus and/or Oomycete, in which the expression of the polynucleotides and/or polypeptides of the invention is modulated. In additional aspects, the present invention provides fungicidal compositions comprising isolated fungicidal polypeptides of the invention and/or bacteria comprising same.

The present invention is based in part on bioinformatics tools that have been used to identify polynucleotides associated with resistance or reduced sensitivity of plants to at least one pathogenic fungus or pathogenic Oomycete, as well as to identify polynucleotides of bacterial origin that encode for proteins with potential antifungal activity. Cereal plants, including maize (Zea), wheat (Triticum), millet (Sitaria italica) and the closely related Aegilops and Brachypodium species were used as representative genera to identify genes overexpressed or downregulated in plants showing increased resistance to fungal/Oomycetes infection. Genes comprising the nucleic acids sequence set forth in any one of SEQ ID NOs:22-62 and 302-303, encoding polypeptides having the amino acid sequence set forth in any one of SEQ ID NOs:308-348, 507, and 510 were identified. Homologous genes and encoded proteins were also identified in wider genera of plant, as described in details and presented in Table 7 hereinbelow. Bacteria isolated from soils known to contain Fusaria species were used for screening and identification of potential anti-fungal bacterial proteins. Genes comprising the nucleic acids sequence set forth in any one of SEQ ID NOs:592-789, encoding polypeptides having the amino acid sequence set forth in any one of SEQ ID NOs:2444-2641 were identified. Homologous genes and encoded proteins were also identified in wider genera of bacteria, as described in details and presented in Table 16 hereinbelow.

Polynucleotides of plant origin according to some embodiments of the present invention were cloned into binary vectors, and transformed into plants of the species Brachypodium distachyon (Example 6 hereinbelow) to validate the effect of the genes on the resistance of the transformed plants towards the fungi/Oomycetes (Example 7).

Polynucleotides of bacterial origin according to some embodiments of the present invention were cloned, transformed into E. coli, and the expressed proteins were isolated (Examples 14-15). The anti-fungal effect of the isolated proteins was validated by their ability to inhibit the growth of Fusarium verticilioides mycelia (Example 16, Table 21).

Definitions

The terms “comprise”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the agricultural, chemical, pharmacological, biological, biochemical and medical arts.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

As used herein, the phrase “exogenous polynucleotide” refers to a heterologous nucleic acid sequence which is not naturally expressed within the plant (e.g., a nucleic acid sequence from a different species) or to an endogenous nucleic acid of which overexpression in the plant is desired. The exogenous polynucleotide may be introduced into the plant in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule and/or a polypeptide molecule. The term “endogenous” as used herein refers to a polynucleotide or polypeptide which is naturally present and/or naturally expressed within a plant or a cell thereof.

The terms “modulating”, “modifying” and “altering” with reference to the expression or activity of a polynucleotide, gene, polypeptide or a protein within a cell or a plurality of cells, particularly plant cell(s), are used herein interchangeably and refer to changing their level of expression within the cell, particularly plant cell. The change can be an increase or a decrease; and it can be measured as compared to any one of the polynucleotide, gene, polypeptide and/or protein level within the same cell(s) before modulation and as compared to the level in a control plant or an average level from a plurality of control plants in which the expression was not modified by man.

According to certain embodiments, the control plant is a wild type plant not manipulated to have modulated expression and/or activity of the polynucleotide and/or polypeptide. According to some embodiments, the control plant is of the same species. According to some embodiments, the control plant comprises the same genetic background.

According to certain embodiments, the examined plant and the control plant are grown under the same growing conditions.

As used herein, the term “resistance” with regard to plants pathogenic fungus and/or Oomycete refers to a plant that is resistant to infection by a fungal or Oomycetous pathogen or resistant to the symptoms of fungal or Oomycetous pathogen infection. For example, a plant resistant to a fungal or Oomycetous pathogen can exhibit a lack of infection, or reduced symptoms of infection, when challenged with a pathogen. As another example, a plant resistant to a fungal or Oomycetous pathogen can be infected by the fungal or Oomycetous pathogen and yet exhibit a reduced number or degree of symptoms of said infection. As yet another example, a plant resistant to a fungal or Oomycetous pathogen can be infected by the pathogen and exhibit one or more symptoms of infection by the pathogen and yet exhibit a reduction in an effect of the infection or symptom thereof. For instance, a plant resistant to a fungal or Oomycetous pathogen can be infected by the pathogen, and exhibit one or more symptoms selected from the group consisting of leaf wilt, leaf or vascular discoloration (e.g., yellowing), spike bleaching etc., and yet not exhibit a reduction in yield loss in comparison to a plant that is not resistant (susceptible) to the fungal or Oomycetous pathogen.

Accordingly, “confer resistance to a pathogenic fungus and/or Oomycete” or “enhanced resistance to a pathogenic fungus and/or Oomycete” refer to a phenotype in which a plant has greater health, growth, multiplication, fertility, vigor, strength (e.g., stem strength and resistance), yield, or less severe symptoms associated with infection of the pathogenic fungus or Oomycete during or after a fungal or Oomycete infection than an organism that does not have enhanced resistance to the pathogen. Where a plant is tested for resistance, a control plant is used to assess the degree of the plant resistance. According to certain embodiments of the present invention, the control plant is a plant not manipulated to have modified expression of at least one polynucleotide and/or polypeptide of the present invention. The control plant is typically, but not necessarily of the same species as the examined plant. According to some embodiments the control plant is of the same specifies and has the same genetic background as the examined plant. The enhancement can be manifested as an increase of 0.1%, 0.2%, 0.3%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in health, growth, multiplication, fertility, vigor, strength, or yield, as compared to a control plant. The enhancement can be a decrease of 0.1%, 0.2%, 0.3%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in the symptoms associated with the pathogenic fungus and/or Oomycete as compared to the control plant. According to certain exemplary embodiments, the examined plant and the control plant are grown under the same conditions.

According to certain embodiments of the invention, enhancing the resistance of a plant to a pathogenic fungus comprises enhancing the expression and/or activity of a polypeptide of the invention within at least one cell of the plant. As used herein, the expression of a polynucleotide or polypeptide of the invention is “enhanced” or “up-regulated” if the level of the polynucleotide or polypeptide is enhanced by at least 50%, i.e. the polynucleotide or polypeptide level is at least 1.5-fold higher compared to its level in a control plant or compared to a predetermined threshold level. According to some embodiments, the level of the polynucleotide or polypeptide expression is enhanced by at least 60%, 70%, 80%, 90%, 100%, 200%, 300% and more.

According to certain embodiments, the pre-determined resistance score value is obtained by inoculating a population of corresponding plants susceptible to the at least one pathogenic fungus, scoring the infection degree and setting an average resistance score value.

According to certain embodiments of the invention, enhancing the resistance of a plant to a pathogenic fungus comprises reducing the expression and/or activity of a polynucleotide and/or polypeptide of the invention within at least one cell of the plant. As used herein, the expression of a polynucleotide or polypeptide of the invention is “reduced”, “inhibited”, “down regulated” or “knocked out” or “knocked down” if the level of the polynucleotide or polypeptide is reduced by at least 30% compared to its level in a control plant or compared to a predetermined threshold level. According to certain embodiments, the level of the polynucleotide or polypeptide is reduced by at least 40%, 50%, 60%, 70%, 80%, 90% and more. According to some embodiments, the term “reduced expression” refers to 100% inhibition or “knockout” of a polynucleotide function and/or expression.

As used herein the term “polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

As used herein the phrase “complementary polynucleotide sequence” refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.

The term “isolated” refers to at least partially separated from the natural environment e.g., from a plant cell.

As used herein the phrase “genomic polynucleotide sequence” refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.

It should be noted that the nucleic acid sequence of a polynucleotide encoding a polypeptide which is provided in the sequence listing as a single strand refers to the sense direction which is equivalent to the mRNA transcribed from the polynucleotide.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

As used herein, the phrase “exogenous polynucleotide” refers to a heterologous nucleic acid sequence which is not naturally expressed within the plant (e.g., a nucleic acid sequence from a different species) or to an endogenous nucleic acid of which overexpression in the plant is desired. The exogenous polynucleotide may be introduced into the plant in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule and/or a polypeptide molecule.

The term “endogenous” as used herein refers to a polynucleotide or polypeptide which is naturally present and/or naturally expressed within a plant or a cell thereof.

The term “heterologous” as used herein refers to polynucleotide or polypeptide which is not naturally present and/or naturally expressed within a plant or a cell thereof.

According to one aspect, the present invention provides a method for enhancing the resistance of a plant or a part thereof to at least one pathogenic fungus and/or Oomycete, comprising modulating the expression and/or activity of at least one polypeptide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506 and 2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof within at least one cell of the plant or part thereof, thereby enhancing the resistance of said plant or part thereof to the at least one pathogenic fungus and/or Oomycetes compared to the resistance of a control plant. Each possibility represents a separate embodiment of the present invention.

According to another aspect, the present invention provides a method for enhancing the resistance of a plant or a part thereof to at least one pathogenic fungus and/or Oomycete, comprising modulating the expression of at least one polynucleotide encoding a polypeptide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2380-4175; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:339-537 and 2380-4175, a fragment and/or a variant thereof within at least one cell of the plant or part thereof, thereby enhancing the resistance of said plant or part thereof to the at least one pathogenic fungus and/or Oomycete compared to the resistance of a corresponding control plant. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the polypeptide is 70%-79% homologous to any one of the polypeptides set forth in SEQ ID NOs:2635, 2444-2634, 2636-4252. According to other embodiments, the polypeptide is 75%-79% homologous to any one of the polypeptides set forth in SEQ ID NOs:2635, 2444-2634, 2636-4252.

According to certain embodiments, the polypeptide is 80%-99% homologous to any one of the polypeptides set forth in SEQ ID NOs:326, 360, 308-325, 327-359, 361-506, 2635, 2444-2634, and 2636-4252. According to other embodiments, the polypeptide is 85%-95% homologous to any one of the polypeptides set forth in SEQ ID NOs: 326, 360, 308-325, 327-359, 361-506, 2635, 2444-2634, and 2636-4252. According to other embodiments, the polypeptide is 90%-99% homologous to any one of the polypeptides set forth in SEQ ID NOs:326, 360, 308-325, 327-359, 361-506, 2635, 2444-2634, and 2636-4252. According to certain embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 326, 360, 308-325, 327-359, 361-508, 2635, 2444-2634, and 2636-4252. Each possibility represents a separate embodiment of the present invention. According to certain embodiments, the polypeptide consists of the amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508 and 2635, 2444-2634, 2636-4252. Each possibility represents a separate embodiment of the present invention.

According to yet additional aspect, the present invention provides a method for enhancing the resistance of a plant or a part thereof to at least one pathogenic fungus and/or Oomycete, comprising modulating the expression of at least one polynucleotide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to a polynucleotide having an nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, 81-301, 783, 592-782, 784-2443 within at least one cell of the plant or part thereof, thereby enhancing the resistance of said plant or part thereof to the at least one pathogenic fungus and/or Oomycetes compared to the resistance of a control plant. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the polynucleotide is 70%-79% homologous to any one of the polynucleotides set forth in SEQ ID NOs:783, 592-782, 784-2443. According to other embodiments, the polynucleotide is 75%-79% homologous to any one of the polynucleotides set forth in SEQ ID NOs:783, 592-782, 784-2443. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the polynucleotide is 80%-99% homologous to any one of the polynucleotides set forth in SEQ ID NOs:40, 80, 22-39, 41-79, 81-301, 783, 592-782, and 784-2443. According to other embodiments, the polynucleotide is 85%-95% homologous to any one of the polynucleotides set forth in SEQ ID NOs:40, 80, 22-39, 41-79, 81-301, 783, 592-782, and 784-2443. According to other embodiments, the polynucleotide is 90%-99% homologous to any one of the polynucleotides set forth in SEQ ID NOs:40, 80, 22-39, 41-79, 81-301, 783, 592-782, and 784-2443. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the polynucleotide comprises a nucleic acids sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-305, 783, 592-782, 784-2443. According to other embodiments, the exogenous polynucleotide consists of a nucleic acids sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-305, 783, 592-782, and 784-2443. Each possibility represents a separate embodiment of the present invention.

According to another aspect, the present invention provides a method for producing a population of plants each having an enhanced resistance to at least one pathogenic fungus or Oomycete, comprising the steps of:

    • (a) modulating the expression and/or activity of at least one polypeptide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506, 2635, 2444-2634, and 2636-4252, a fragment and/or a variant thereof within at least one cell of each plant of the plant population as to produce a genetically engineered plant population;
    • (b) inoculating each plant of the genetically engineered plant population with the at least one pathogenic fungus; and
    • (c) selecting plants showing an enhanced resistance to said at least one pathogenic fungus compared to a control or to a pre-determined resistance score value;
    • thereby producing a population of genetically engineered plants having enhanced resistance to said at least one pathogenic fungus.

According to certain embodiments, the method comprises modulating the expression and/or activity of a polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508, 2635, 2444-2634, and 2636-4252. According to other embodiments, the method comprises enhancing the expression and/or activity of a polypeptide having the amino acid sequence set forth in SEQ ID NO:509. According to yet additional embodiments, the method comprises enhancing the expression and/or activity of a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-509, 2635, 2444-2634, and 2636-4252.

According to additional aspect, the present invention provides a method for selecting a plant having an enhanced resistance to at least one pathogenic fungus and/or Oomycete, comprising the steps of:

    • (a) providing a plurality of plants each comprising at least one cell with modulated expression and/or activity of a polypeptide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506, 2635, 2444-2634, and 2636-4252, a fragment and/or a variant thereof;
    • (b) inoculating the plurality of plants with the at least one pathogenic fungus or Oomycete; and
    • (c) selecting plants showing an enhanced resistance to said at least one pathogenic fungus or Oomycete compared to a control plant or to a pre-determined resistance score value.

According to certain embodiments, the method comprises providing a plurality of plants each comprising at least one cell with modulated expression and/or activity of at least one polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508, 2635, 2444-2634, and 2636-4252. Each possibility represents a separate embodiment of the present invention. According to other embodiments, the method comprises providing a plurality of plants each having modulated expression and/or activity of a polypeptide having the amino acid sequence set forth SEQ ID NO:509. According to other embodiments, the method comprises providing a plurality of plants each having modulated expression and/or activity of at least one polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 326, 360, 308-325, 327-359, 361-509, 2635, 2444-2634, and 2636-4252.

The plurality of plants having modulated expression and/or activity of the polypeptide may include plants having at least one cell with enhanced expression and/or activity of a polypeptide, plants having at least one cell with reduced expression and/or activity of a polypeptide or a combination thereof. Enhancing or reducing the expression and/or activity of the polypeptide can be performed as is known in the Art and as described hereinbelow.

According to certain embodiments, the polypeptide the expression and/or activity of which is to be enhanced comprises an amino acid sequence at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2635, 2444-2634, 2636-4252 or at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-506. According to some embodiments, the polypeptide the expression and/or activity of which is to be enhanced comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-508, 2635, 2444-2634, and 2636-4252. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, enhancing the resistance of the plant to the pathogenic fungus and/or Oomycete comprises reducing the expression and/or activity of at least one polypeptide compared to its expression and/or activity in the control plant. According to certain exemplary embodiments, the polypeptide the expression and/or activity of which is to be reduced comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NO:328, SEQ ID NO:361, and SEQ ID NO:348. According to further exemplary embodiments, the polypeptide the expression and/or activity of which is to be reduced comprises the amino acid sequence set forth in any one of SEQ ID NO:328, SEQ ID NO:361 and SEQ ID NO:348.

According to another aspect, the present invention provides a method for conferring and/or enhancing the resistance of a grafted plant to at least one pathogenic fungus and/or Oomycete, the method comprising providing a scion and a rootstock, wherein the rootstock exhibits enhanced resistance to the at least one pathogenic fungus and/or Oomycete, said rootstock comprises at least one cell with a modulated expression of a polynucleotide encoding a polypeptide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous, or identical to the amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506, 2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof compared to the scion and grafting said scion onto said rootstock, thereby producing a grafted plant having an enhanced resistance to said at least one pathogenic fungus and/or Oomycete.

According to another aspect, the present invention provides a method for conferring and/or enhancing the resistance of a grafted plant to at least one pathogenic fungus and/or Oomycete, the method comprising providing a scion and a rootstock, the scion having an enhances resistance to the at least one pathogenic fungus and/or Oomycete, said scion comprises at least one cell with modulated expression of at least one polynucleotide encoding a polypeptide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous, or identical to the amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506, 2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof compared to the rootstock, and grafting said scion onto said rootstock, thereby producing a grafted plant having an enhanced resistance to the at least one pathogenic fungus and/or Oomycete.

According to certain embodiments, the polypeptide expressed in the scion or in the rootstock comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508, 2635, 2444-2634, and 2636-4252.

According to certain embodiments, the scion or rootstock having enhanced resistance to the at least one fungus and/or Oomycete comprises at least one cell with enhanced expression and/or activity of at least one polypeptide or the nucleotide encoding same. According to other embodiments, the scion or rootstock having enhanced resistance to the at least one fungus and/or Oomycete comprises at least one cell with reduced expression and/or activity of at least one polypeptide or the nucleotide encoding same. The polypeptides the expression of which is enhanced or reduced are as described hereinabove.

According to some embodiments, the at least one polynucleotide is constitutively expressed in the transgenic rootstock. According to some embodiments, the at least one polynucleotide is expressed in the transgenic rootstock in a tissue specific or inducible manner. According to some embodiments, the expression of the at least one polynucleotide is induced by biotic stress, particularly by fungi infection.

According to additional aspect, the present invention provides a method of growing a crop plant having enhanced resistance to at least one pathogenic fungus and/or Oomycete comprising the steps of:

    • (a) selecting a parent plant having a modulated expression of at least one polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous, or identical to the amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506, 2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof for enhanced resistance to at least one pathogenic fungus and/or Oomycete; and
    • (b) growing a progeny crop plant of the parent plant, wherein the progeny crop plant having modulated expression of the polynucleotide has an enhanced resistance to the at least one pathogenic fungus and/or Oomycete.

According to certain embodiments, the encoded polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508, 2635, 2444-2634, and 2636-4252. According to certain embodiments, the encoded polypeptide comprises the amino acid sequence set forth in SEQ ID NOs:5509. According to some embodiments, the encoded polypeptide consists of the amino acid sequence set forth in any one of SEQ ID NOs: 326, 360, 308-325, 327-359, 361-509, 2635, 2444-2634, 2636-4252. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the polynucleotide comprises a nucleic acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to a nucleic acid sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-301, 783, 592-782, and 784-2443. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-305, 783, 592-782, and 784-2443. According to certain embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:306. According to some embodiments, the polynucleotide consists of the nucleic acid sequence set forth in any one of SEQ ID NOs: 40, 80, 22-39, 41-79, 81-306, 783, 592-782, 784-2443. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the modulated expression comprises up-regulation of at least one polynucleotide expression. According to certain embodiments, the modulated expression comprises down-regulation of at least one polynucleotide expression.

According to certain exemplary embodiments, the gene to be upregulated comprises a nucleic acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to a nucleic acid sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41, 43-61, 63-79, 81, 83-301. According to some embodiments, the gene to be upregulated comprises a polynucleotide selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41, 43-61, 63-79, 81, 83-306.

According to certain exemplary embodiments, the gene the expression of which is to be downregulated comprises a nucleic acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to a nucleic acid sequence set forth in any one of SEQ ID NOs:42, 62 and 82. According to certain embodiments, the gene the expression of which is to be downregulated comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:42, 62 and 82.

According to certain embodiments, the encoded polypeptide the expression and/or activity of which is to be downregulated is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to an amino acid sequence set forth in any one of SEQ ID NOs:328, 248 and 361. According to certain embodiments, the polypeptide the expression and/or activity of which is to be downregulated comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:328, 248 and 361. Each possibility represents a separate embodiment of the present invention.

According to yet additional aspect, the present invention provides a method of producing seeds of a crop comprising the steps of:

    • (a) selecting a parent plant having a modulated expression of at least one polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous, or identical to the amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506, 2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof for enhanced resistance to at least one pathogenic fungus and/or Oomycete;
    • (b) growing the selected parent plant of step (a) to produce seeds;
    • (c) harvesting the produced seeds.

According to certain embodiments, the encoded polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508 and 2635, 2444-2634, 2636-4252. According to certain embodiments, the encoded polypeptide comprises the amino acid sequence set forth in SEQ ID NO:509. According to some embodiments, the encoded polypeptide consists of the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508 and 2635, 2444-2634, 2636-4252. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the modulated expression of the least one polynucleotide comprises up-regulation of a polynucleotide expression as described hereinabove. According to certain embodiments, the modulated expression of the least one polynucleotide comprises down-regulation of a polynucleotide expression as described hereinabove.

According to certain embodiments, the seeds produced by the methods of the invention comprise at least one cell having modulated expression of the polynucleotide. According to some embodiments, plants grown from the produced seeds have enhanced resistance to at least one pathogenic fungus and/or Oomycete.

The present invention encompasses polynucleotides identified to be associated with resistance to at least one pathogenic fungus and/or Oomycete polypeptides encoded by same and homologs thereto.

According to certain embodiments, the exogenous isolated or recombinant polynucleotides employed in the methods of the present invention encode a polypeptide comprising an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to an amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-509 and 2635, 2444-2634, 2636-4252.

According to certain embodiments, the exogenous isolated or recombinant polynucleotides employed in the methods of the present invention comprise a nucleic acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous to, or identical to a nucleic acid sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-306 and 783, 592-782, 784-2443.

Homologous sequences include both orthologous and paralogous sequences. The term “paralogous” relates to gene-duplications within the genome of a species leading to paralogous genes. The term “orthologous” relates to homologous genes in different organisms due to ancestral relationship. Thus, orthologs are evolutionary counterparts derived from a single ancestral gene in the last common ancestor of given two species (Koonin E V and Galperin M Y 2003. Sequence—Evolution—Function: Computational Approaches in Comparative Genomics. Boston: Kluwer Academic; Chapter 2, Evolutionary Concept in Genetics and Genomics) and therefore have great likelihood of having the same function.

One option to identify orthologues in monocot or in dicot plant species is by performing a reciprocal blast search. This may be done by a first blast involving blasting the sequence-of-interest against any sequence database, such as the publicly available NCBI database which may be found at: ncbi.nlm.nih.gov. If orthologues in rice were sought, the sequence-of-interest would be blasted against, for example, the 28,469 full-length cDNA clones from Oryza sativa Nipponbare available at NCBI. The blast results may be filtered. The full-length sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequences of the organism from which the sequence-of-interest is derived. The results of the first and second blasts are then compared. An ortholog is identified when the sequence resulting in the highest score (best hit) in the first blast identifies in the second blast the query sequence (the original sequence-of-interest) as the best hit. Using the same rational a paralog (homolog to a gene in the same organism) is found. In case of large sequence families, the ClustalW program may be used (ebi.ac.uk/Tools/clustalw2/index.html), followed by a neighbor-joining tree (Wikipedia.org/wiki/Neighbor-joining) which helps visualizing the clustering.

Homology (e.g., percent homology, sequence identity+sequence similarity) can be determined using any homology comparison software computing a pairwise sequence alignment.

As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff J G. (Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915-9).

Identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

According to some embodiments of the invention, the identity is a global identity, i.e., an identity over the entire amino acid or nucleic acid sequences of the invention and not over portions thereof.

According to some embodiments of the invention, the term “homology” or “homologous” refers to identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences; or the identity of an amino acid sequence to one or more nucleic acid sequence.

According to some embodiments of the invention, the homology is a global homology, i.e., a homology over the entire amino acid or nucleic acid sequences of the invention and not over portions thereof.

According to some embodiments of the invention, the homology is a partial homology, i.e. a homology over part of the amino acid or nucleic acid sequences of the invention. According to certain exemplary embodiments, the partial sequence is of polynucleotides and polypeptides of embodiments of the present invention lacking a signal or transit peptide.

The degree of homology or identity between two or more sequences can be determined using various known sequence comparison tools. Following is a non-limiting description of such tools which can be used along with some embodiments of the invention.

Pairwise global alignment was defined by S. B. Needleman and C. D. Wunsch, “A general method applicable to the search of similarities in the amino acid sequence of two proteins” Journal of Molecular Biology, 1970, pages 443-53, volume 48).

For example, when starting from a polypeptide sequence and comparing to other polypeptide sequences, the EMBOSS-6.0.1 Needleman-Wunsch algorithm (available from emboss(dot)sourceforge(dot)net/apps/cvs/emboss/apps/needle(dot)html) can be used to find the optimum alignment (including gaps) of two sequences along their entire length—a “Global alignment”. Default parameters for Needleman-Wunsch algorithm (EMBOSS-6.0.1) include: gapopen=10; gapextend=0.5; datafile=EBLOSUM62; brief=YES.

According to some embodiments of the invention, the parameters used with the EMBOSS-6.0.1 tool (for protein-protein comparison) include: gapopen=8; gapextend=2; datafile=EBLOSUM62; brief=YES.

According to some embodiments of the invention, the threshold used to determine homology using the EMBOSS-6.0.1 Needleman-Wunsch algorithm is 80%, 81%, 82%, 83%, 84%, 8%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

When starting from a polypeptide sequence and comparing to polynucleotide sequences, the OneModel FramePlus algorithm [Halperin, E., Faigler, S. and Gill-More, R. (1999)—FramePlus: aligning DNA to protein sequences. Bioinformatics, 15, 867-873) (available from biocceleration(dot)com/Products(dot)html] can be used with following default parameters: model=frame+_p2n.model mode=local.

According to some embodiments of the invention, the parameters used with the OneModel FramePlus algorithm are model=frame+_p2n.model, mode=qglobal.

According to some embodiments of the invention, the threshold used to determine homology using the OneModel FramePlus algorithm is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

When starting with a polynucleotide sequence and comparing to other polynucleotide sequences the EMBOSS-6.0.1 Needleman-Wunsch algorithm (available from emboss(dot)sourceforge(dot)net/apps/cvs/emboss/apps/needle(dot)html) can be used with the following default parameters: (EMBOSS-6.0.1) gapopen=10; gapextend=0.5; datafile=EDNAFULL; brief=YES.

According to some embodiments of the invention, the parameters used with the EMBOSS-6.0.1 Needleman-Wunsch algorithm are gapopen=10; gapextend=0.2; datafile=EDNAFULL; brief=YES.

According to some embodiments of the invention, the threshold used to determine homology using the EMBOSS-6.0.1 Needleman-Wunsch algorithm for comparison of polynucleotides with polynucleotides is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 8%, 88%, 89%, 90%, 91%, 9%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

According to some embodiment, determination of the degree of homology further requires employing the Smith-Waterman algorithm (for protein-protein comparison or nucleotide-nucleotide comparison).

Default parameters for GenCore 6.0 Smith-Waterman algorithm include: model=sw.model.

According to some embodiments of the invention, the threshold used to determine homology using the Smith-Waterman algorithm is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 8%, 88%, 89%, 90%, 91%, 9%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

According to some embodiments of the invention, the global homology is performed on sequences which are pre-selected by local homology to the polypeptide or polynucleotide of interest (e.g., 60% identity over 60% of the sequence length), prior to performing the global homology to the polypeptide or polynucleotide of interest (e.g., 80% global homology on the entire sequence). For example, homologous sequences are selected using the BLAST software with the Blastp and tBlastn algorithms as filters for the first stage, and the needle (EMBOSS package) or Frame+algorithm alignment for the second stage. Local identity (Blast alignments) is defined with a very permissive cutoff−60% Identity on a span of 60% of the sequences lengths because it is used only as a filter for the global alignment stage. In this specific embodiment (when the local identity is used), the default filtering of the Blast package is not utilized (by setting the parameter “-F F”).

In the second stage, homologs are defined based on a global identity of at least 80% to the core gene polypeptide sequence.

According to some embodiments of the invention, two distinct forms for finding the optimal global alignment for protein or nucleotide sequences are used:

1. Between Two Proteins (Following the Blastp Filter):

EMBOSS-6.0.1 Needleman-Wunsch algorithm with the following modified parameters: gapopen=8 gapextend=2. The rest of the parameters are unchanged from the default options listed here:

Standard (Mandatory) Qualifiers:

    • [-aswquence] sequence filename and optional format, or reference (input USA)
    • [-bsequence] sequall Sequence(s) filename and optional format, or reference (input USA)
    • -gapopen float [10.0 for any sequence]. The gap open penalty is the score taken away when a gap is created. The best value depends on the choice of comparison matrix. The default value assumes you are using the EBLOSUM62 matrix for protein sequences, and the EDNAFULL matrix for nucleotide sequences. (Floating point number from 1.0 to 100.0)
    • -gapextend float [0.5 for any sequence]. The gap extension, penalty is added to the standard gap penalty for each base or residue in the gap. This is how long gaps are penalized. Usually you will expect a few long gaps rather than many short gaps, so the gap extension penalty should be lower than the gap penalty. An exception is where one or both sequences are single reads with possible sequencing errors in which case you would expect many single base gaps. You can get this result by setting the gap open penalty to zero (or very low) and using the gap extension penalty to control gap scoring. (Floating point number from 0.0 to 10.0)
    • [-outfile] align [*needle] Output alignment file name

Additional (Optional) qualifiers:

    • -datafile matrixf [EBLOSUM62 for protein, EDNAFULL for DNA]. This is the scoring matrix file used when comparing sequences. By default, it is the file ‘EBLOSUM62’ (for proteins) or the file ‘EDNAFULL’ (for nucleic sequences). These files are found in the ‘data’ directory of the EMBOSS installation.

Advanced (Unprompted) qualifiers:

    • -[no]brief boolean [Y] Brief identity and similiarity

Associated Qualifiers:

“-asequence” associated qualifiers -sbegin1 integer Start of the sequence to be used -send1 integer End of the sequence to be used -sreverse1 boolean Reverse (if DNA) -sask1 boolean Ask for begin/end/reverse -snucleotide1 boolean Sequence is nucleotide -sprotein1 boolean Sequence is protein -slower1 boolean Make lower case -supper1 boolean Make upper case -sformat1 string Input sequence format -sdbname1 string Database name -sid1 string Entryname -ufo1 string UFO features -fformat1 string Features format -fopenfile1 string Features file name “-bsequence” associated qualifiers -sbegin2 integer Start of each sequence to be used -send2 integer End of each sequence to be used -sreverse2 boolean Reverse (if DNA) -sask2 boolean Ask for begin/end/reverse -snucleotide2 boolean Sequence is nucleotide -sprotein2 boolean Sequence is protein -slower2 boolean Make lower case -supper2 boolean Make upper case -sformat2 string Input sequence format -sdbname2 string Database name -sid2 string Entryname -ufo2 string UFO features -fformat2 string Features format -fopenfile2 string Features file name “-outfile” associated qualifiers -aformat3 string Alignment format -aextension3 string File name extension -adirectory3 string Output directory -aname3 string Base file name -awidth3 integer Alignment width -aaccshow3 boolean Show accession number in the header -adesshow3 boolean Show description in the header -ausashow3 boolean Show the full USA in the alignment -aglobal3 boolean Show the full sequence in alignment

 -auto  boolean Turn off prompts  -stdout  boolean Write first file to standard output  -filter  boolean Read first file from standard input, write  first file to standard output  -options  boolean Prompt for standard and additional values  -debug  boolean Write debug output to program.dbg  -verbose  boolean Report some/full command line options  -help  boolean Report command line options.           More information on associated and general qualifiers can be found with -help -verbose  -warning  boolean Report warnings  -error  boolean Report errors  -fatal  boolean Report fatal errors  -die  boolean Report dying program messages

2. Between a Protein Sequence and a Nucleotide Sequence (Following the Tblastn Filter):

GenCore 6.0 OneModel application utilizing the Frame+algorithm with the following parameters: model=frame+_p2n.model mode=qglobal -q=protein.sequence -db=nucleotide. sequence. The rest of the parameters are unchanged from the default options:

Usage:

    • om -model=<model_fname>[-q=]query [-db=]database [options]
    • -model=<model_fname> Specifies the model that you want to run. All models supplied by Compugen are located in the directory $CGNROOT/models/.

Valid command line parameters:

    • -dev=<dev_name> Selects the device to be used by the application.

Valid Devices are:

    • bic—Bioccelerator (valid for SW, XSW, FRAME_N2P, and FRAME_P2N models).
    • xlg—BioXL/G (valid for all models except XSW).
    • xlp—BioXL/P (valid for SW, FRAME+_N2P, and
    • FRAME_P2N models).
    • xlh—BioXL/H (valid for SW, FRAME+_N2P, and
    • FRAME_P2N models).
    • soft—Software device (for all models).
    • -q=<query> Defines the query set. The query can be a sequence file or a database reference. You can specify a query by its name or by accession number. The format is detected automatically. However, you may specify a format using the -qfmt parameter. If you do not specify a query, the program prompts for one. If the query set is a database reference, an output file is produced for each sequence in the query.
    • -db=<database name> Chooses the database set. The database set can be a sequence file or a database reference. The database format is detected automatically. However, you may specify a format using -dfmt parameter.
    • -qacc Add this parameter to the command line if you specify query using accession numbers.
    • -dacc Add this parameter to the command line if you specify a database using accession numbers.
    • -dfmt/-qfmt=<format_type> Chooses the database/query format type. Possible formats are:
    • fasta—fasta with seq type auto-detected.
    • fastap—fasta protein seq.
    • fastan—fasta nucleic seq.
    • gcg—gcg format, type is auto-detected.
    • gcg9seq—gcg9 format, type is auto-detected.
    • gcg9seqp—gcg9 format protein seq.
    • gcg9seqn—gcg9 format nucleic seq.
    • nbrf—nbrf seq, type is auto-detected.
    • nbrfp—nbrf protein seq.
    • nbrfn—nbrf nucleic seq.
    • embl—embl and swissprot format.
    • genbank—genbank format (nucleic).
    • blast—blast format.
    • nbrf_gcg—nbrf-gcg seq, type is auto-detected.
    • nbrf_gcgp—nbrf-gcg protein seq.
    • nbrf_gcgn—nbrf-gcg nucleic seq.
    • raw—raw ascii sequence, type is auto-detected.
    • rawp—raw ascii protein sequence.
    • rawn—raw ascii nucleic sequence.
    • pir—pir codata format, type is auto-detected.
    • profile—gcg profile (valid only for -qfmt
    • in SW, XSW, FRAME_P2N, and FRAME+_P2N).
    • -out=<out_fname> The name of the output file.
    • -suffix=<name> The output file name suffix.
    • -gapop=<n> Gap open penalty. This parameter is not valid for FRAME+. For FrameSearch the default is 12.0. For other searches the default is 10.0.
    • -gapext=<n> Gap extend penalty. This parameter is not valid for FRAME+. For FrameSearch the default is 4.0. For other models: the default for protein searches is 0.05, and the default for nucleic searches is 1.0.
    • -qgapop=<n> The penalty for opening a gap in the query sequence. The default is 10.0. Valid for XSW.
    • -qgapext=<n> The penalty for extending a gap in the query sequence. The default is 0.05. Valid for XSW.
    • -start=<n> The position in the query sequence to begin the search.
    • -end=<n> The position in the query sequence to stop the search.
    • -qtrans Performs a translated search, relevant for a nucleic query against a protein database. The nucleic query is translated to six reading frames and a result is given for each frame.

Valid for SW and XSW.

    • -dtrans Performs a translated search, relevant for a protein query against a DNA database. Each database entry is translated to six reading frames and a result is given for each frame.

Valid for SW and XSW.

Note: “-qtrans” and “-dtrans” options are mutually exclusive.

    • -matrix=<matrix_file> Specifies the comparison matrix to be used in the search. The matrix must be in the BLAST format. If the matrix file is not located in $CGNROOT/tables/matrix, specify the full path as the value of the -matrix parameter.
    • -trans=<transtab_name> Translation table. The default location for the table is $CGNROOT/tables/trans.
    • -onestrand Restricts the search to just the top strand of the query/database nucleic sequence.
    • -list=<n> The maximum size of the output hit list. The default is 50.
    • -docalign=<n> The number of documentation lines preceding each alignment. The default is 10.
    • -thr_score=<score_name> The score that places limits on the display of results. Scores that are smaller than -thr_min value or larger than -thr_max value are not shown. Valid options are: quality.
    • zscore.
    • escore.
    • -thr_max=<n> The score upper threshold. Results that are larger than -thr_max value are not shown.
    • -thr_min=<n> The score lower threshold. Results that are lower than -thr_min value are not shown.
    • -align=<n> The number of alignments reported in the output file.
    • -noalign Do not display alignment.

Note: “-align” and “-noalign” parameters are mutually exclusive.

    • -outfmt=<format_name> Specifies the output format type. The default format is PFS. Possible values are:
    • PFS—PFS text format
    • FASTA—FASTA text format
    • BLAST—BLAST text format
    • -nonorm Do not perform score normalization.
    • -norm=<norm_name> Specifies the normalization method. Valid options are:
    • log—logarithm normalization.
    • std—standard normalization.
    • stat—Pearson statistical method.

Note: “-nonorm” and “-norm” parameters cannot be used together.

Note: Parameters -xgapop, -xgapext, -fgapop, -fgapext, -ygapop, -ygapext, -delop, and -delext apply only to FRAME+.

    • -xgapop=<n> The penalty for opening a gap when inserting a codon (triplet). The default is 12.0.
    • -xgapext=<n> The penalty for extending a gap when inserting a codon (triplet). The default is 4.0.
    • -ygapop=<n> The penalty for opening a gap when deleting an amino acid. The default is 12.0.
    • -ygapext=<n> The penalty for extending a gap when deleting an amino acid. The default is 4.0.
    • -fgapop=<n> The penalty for opening a gap when inserting a DNA base. The default is 6.0.
    • -fgapext=<n> The penalty for extending a gap when inserting a DNA base. The default is 7.0.
    • -delop=<n> The penalty for opening a gap when deleting a DNA base. The default is 6.0.
    • -delext=<n> The penalty for extending a gap when deleting a DNA base. The default is 7.0.
    • -silent No screen output is produced.
    • -host=<host_name> The name of the host on which the server runs. By default, the application uses the host specified in the file $CGNROOT/cgnhosts.
    • -wait Do not go to the background when the device is busy. This option is not relevant for the Parseq or Soft pseudo device.
    • -batch Run the job in the background. When this option is specified, the file “$CGNROOT/defaults/batch.defaults” is used for choosing the batch command. If this file does not exist, the command “at now” is used to run the job.

Note: “-batch” and “-wait” parameters are mutually exclusive.

    • -version Prints the software version number.
    • -help Displays this help message. To get more specific help type:
      • “om -model=<model_fname>-help”.

According to some embodiments the homology is a local homology or a local identity.

Local alignments tools include, but are not limited to the BlastP, BlastN, BlastX or TBLASTN software of the National Center of Biotechnology Information (NCBI), FASTA, and the Smith-Waterman algorithm.

A tblastn search allows the comparison between a protein sequence to the six-frame translations of a nucleotide database. It can be a very productive way of finding homologous protein coding regions in unannotated nucleotide sequences such as expressed sequence tags (ESTs) and draft genome records (HTG), located in the BLAST databases est and htgs, respectively.

Default parameters for blastp include: Max target sequences: 100; Expected threshold: e-5; Word size: 3; Max matches in a query range: 0; Scoring parameters: Matrix—BLOSUM62; filters and masking: Filter—low complexity regions.

Local alignments tools, which can be used include, but are not limited to, the tBLASTX algorithm, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. Default parameters include: Max target sequences: 100; Expected threshold: 10; Word size: 3; Max matches in a query range: 0; Scoring parameters: Matrix—BLOSUM62; filters and masking: Filter—low complexity regions.

Modulating the expression and/or activity of the polypeptides of the present invention within a plant cell as to enhance the resistance of the plant to the pathogenic fungi and/or Oomycetes may include enhancing the expression and/or activity of polypeptides identified to positively contribute to the plant defense mechanism against the pathogenic fungi, or reducing the expression and/or activity of those polypeptides found to be associated with susceptibility to the fungus or Oomycete infection.

According to certain embodiments, enhancing the resistance of the plant or part thereof to the pathogenic fungus and/or Oomycete comprises enhancing the expression and/or activity of the at least one polypeptide compared to its expression and/or activity in the control plant.

According to certain embodiments, enhancing the expression and/or activity of the polypeptide comprises introducing into at least one cell of the plant or part thereof an exogenous polynucleotide encoding said polypeptide, thereby producing a transgenic plant over-expressing said polypeptide compared to the control plant.

According to certain embodiments, the exogenous polynucleotide encodes a polypeptide endogenous to the at least one cell. According to other embodiments, the exogenous polynucleotide encodes a polypeptide heterologous to the at least one plant cell.

Nucleic acid sequences encoding the polypeptides of the present invention may be optimized for expression. Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization.

The phrase “codon optimization” refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment thereof that approaches codon usage within the plant of interest, and/or to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Therefore, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant. The nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure. For example (see U.S. Pat. No. 7,214,862), the standard deviation of codon usage (SDCU), a measure of codon usage bias, may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation. The formula used is:

∑ n = 1 N [ ( X n - Y n ) / Y n ] ⁢ 2 / N

    • wherein Xn refers to the frequency of usage of codon n in highly expressed plant genes, where Yn to the frequency of usage of codon n in the gene of interest and N refers to the total number of codons in the gene of interest. A Table of codon usage from highly expressed genes of dicotyledonous plants is compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).

Alternative method of optimizing the nucleic acid sequence in accordance with the preferred codon usage for a particular plant cell type is based on the direct use, without performing any extra statistical calculations, of codon optimization Tables such as those provided on-line at the Codon Usage Database through the NIAS (National Institute of Agrobiological Sciences) DNA bank in Japan (www.kazusa.or.jp/codon/). The Codon Usage Database contains codon usage tables for a number of different species, with each codon usage Table having been statistically determined based on the data present in Genbank.

By using the tables described above to determine the most preferred or most favored codons for each amino acid in a particular species (for example, rice), a naturally-occurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular plant species. This is affected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored. However, one or more less-favored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5′ and 3′ ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively affect mRNA stability or expression.

The naturally-occurring encoding nucleotide sequence may already, in advance of any modification, contain a number of codons that correspond to a statistically-favored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically-favored with regards to a particular plant, and modifying these codons in accordance with a codon usage table of the particular plant to produce a codon optimized derivative. A modified nucleotide sequence may be fully or partially optimized for plant codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application Publication No. WO 93/07278.

Bacterial genes quite often possess multiple methionine initiation codons in proximity to the start of the open reading frame. Translation initiation at one or more of these start codons often leads to generation of a functional protein, and it is not always predetermined which of these codons are used naturally in the bacterium. These start codons can include ATG codons, but additional codons, such GTG, may be used, for example by Bacillus sp. as a start codon, and proteins that initiate translation at GTG codons contain a methionine at the first amino acid. Thus, it is understood that use of one of the alternate methionine codons may also lead to generation of proteins capable of conferring resistance to plants against pathogenic filamentous fungi and/or Oomycetes. These proteins are encompassed within the scope of the present invention. It will be understood that, when expressed in plants, it will be necessary to alter the alternate start codon to ATG for proper translation. In addition, the translation initiator methionine of a polypeptide of the disclosure may be cleaved off post translationally. One skilled in the art understands that the N-terminal translation initiator methionine can be removed by methionine aminopeptidase in many cellular expression systems.

As is known to the skilled Artisan, the polynucleotide coding sequence can be modified to add a codon at the position following the methionine start codon to create a restriction enzyme site for recombinant cloning purposes and/or for expression purposes.

A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels, J. J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). A signal peptide may form part of the polypeptides of the invention or may be added as described hereinabove. In plants, the signal peptide may preferably direct the protein to the apoplast, the primary site of fungal colonization. When the antifungal proteins of the invention are of bacterial origin, the native fungal signal peptide should be replaced by a plant signal peptide, for example by the maize β-expansin 1 signal peptide (Fursova O et al., Ann Bot. 2012 110(1):47-56; Wu Y et al., Plant Physiol. 2001 126(1) 222-232). Bacterial-originated anti-fungal protein may also include lipid-anchor sequences, which should be modified and/or replaced for effective expression in plant cells.

According to certain embodiments of the present invention, a signal peptide required for expression in specific bacterium or plant species needs to be added or replace the native signal peptide. It is to be explicitly understood that polynucleotides and polypeptides optimized for expression in plant or bacterial cells by modification of their native N-terminus are encompassed within the scope of the present invention, although the global identity of the modified polypeptide to its parent peptide may be less than 70%. A polypeptide that was modified by removal of a native signal peptide thereof is considered herein as a “fragment polypeptide” or a “derived polypeptide”, which includes the amino acid sequence of the mature polypeptide, without the native signal peptide of either a curated or an isolated natural polypeptide. As used herein, the term “optimized polypeptide” refers to a polypeptide encoded by a polynucleotide modified for optimized expression in a desired organism.

According to additional aspect, the present invention provides an isolated or recombinant polynucleotide encoding a polypeptide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous, or identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506 and 2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof. According to certain embodiments, the present invention provides an isolated polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508 and 2635, 2444-2634, 2636-4252. According to additional embodiments, the present invention provides an isolated polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508 and 2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof. According to certain embodiments, the present invention provides an isolated or recombinant polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs:507-598. According to certain embodiments, the present invention provides an isolated or recombinant polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs:507-508. Each possibility represents a separate embodiment of the present invention.

The invention also encompasses fragments of the above described polynucleotides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or man induced, either randomly or in a targeted fashion.

Expression in plants of polypeptides isolated from bacteria according to some embodiments of the present invention may require modifying the polypeptide sequences for optimized expression. For example, signal peptides forming part of bacterial polypeptides should be typically removed, and optionally replaced with adequate signal peptides which will direct the localization of the polypeptide to a desired plant compartment.

According to certain embodiments, the bacterial polypeptide fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:4647-4799.

According to additional aspect, the present invention provides an isolated polynucleotide, a fragment or a mutant thereof, the polynucleotide comprising a nucleic acids sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous, or identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, 81-301 and 783, 592-782, 784-2443. According to certain embodiments, the present invention provides an isolated polynucleotide, a fragment or a mutant thereof, the polynucleotide comprising the nucleic acid sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-305 and 783, 592-782, 784-2443. According to additional embodiments, the present invention provides an isolated polynucleotide consisting of the nucleic acid sequence set forth in any one of SEQ ID NOs:40, 80, 22-39, 41-79, 81-305 and 783, 592-782, 784-2443.

According to certain aspects, the present invention provides a recombinant polynucleotide optimized for polypeptide expression in a bacterial expression system, wherein the polypeptide is capable of inhibiting the growth of at least one pathogenic fungus and/or Oomycete.

According to certain exemplary embodiment the bacterium is Escherichia coli. According to these embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:4468, 4450-4467, and 4469-4646. According to certain embodiments, the recombinant polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:4271, 4253-4270, and 4272-4449.

According to certain exemplary embodiments, the recombinant polynucleotide optimized for polypeptide expression in the bacterial expression system further comprises said bacterium signal peptide.

The isolated polynucleotides and polypeptides of the present invention and the fragment thereof are associated with conferring and/or increasing the resistance of a plant to at least one pathogenic fungus and/or Oomycete.

According to some embodiments of the invention, the exogenous polynucleotide is a non-coding RNA.

As used herein the phrase “non-coding RNA” refers to an RNA molecule which does not encode an amino acid sequence (a polypeptide). Examples of such non-coding RNA molecules include, but are not limited to, an antisense RNA, a pre-miRNA (precursor of a microRNA), or a precursor of a Piwi-interacting RNA (piRNA).

Thus, the invention encompasses nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.

According to additional aspect, the present invention provides a nucleic acid construct comprising the isolated polynucleotide of the invention, further comprising at least one regulatory element for directing transcription of the nucleic acid sequence in a host plant cell.

According to some embodiments of the invention, expressing the exogenous polynucleotide of the invention within a plant is affected by transforming one or more cells of the plant with the exogenous polynucleotide, followed by generating a mature plant from the transformed cells and cultivating the mature plant under conditions suitable for expressing the exogenous polynucleotide within the mature plant.

According to some embodiments of the invention, the transformation is affected by introducing to the plant cell a nucleic acid construct which includes the exogenous polynucleotide of some embodiments of the invention and at least one promoter for directing transcription of the exogenous polynucleotide in a host cell (a plant cell). Further details of suitable transformation approaches are provided hereinbelow.

As mentioned, the nucleic acid construct according to some embodiments of the invention comprises a promoter sequence and the isolated polynucleotide of some embodiments of the invention.

According to some embodiments of the invention, the isolated polynucleotide is operably linked to the promoter sequence.

A coding nucleic acid sequence is “operably linked” to a regulatory sequence (e.g., promoter) if the regulatory sequence is capable of exerting a regulatory effect on the coding sequence linked thereto.

As used herein, the term “promoter” refers to a region of DNA which lies upstream of the transcriptional initiation site of a gene to which RNA polymerase binds to initiate transcription of RNA. The promoter controls where (e.g., which portion of a plant) and/or when (e.g., at which stage or condition in the lifetime of an organism) the gene is expressed.

According to some embodiments of the invention, the promoter is heterologous to the isolated polynucleotide and/or to the host cell.

As used herein the phrase “heterologous promoter” refers to a promoter from a different species or from the same species but from a different gene locus as of the isolated polynucleotide sequence.

According to some embodiments of the invention, the isolated polynucleotide is heterologous to the plant cell (e.g., the polynucleotide is derived from a different plant species when compared to the plant cell, thus the isolated polynucleotide and the plant cell are not from the same plant species).

Any suitable promoter sequence can be used by the nucleic acid construct of the present invention. Preferably the promoter is selected from the group consisting of a constitutive promoter, a tissue-specific, or biotic-stress specific promoter, particularly promoters inducible by fungi infection.

According to some embodiments of the invention, the promoter is a plant promoter, which is suitable for expression of the exogenous polynucleotide in a plant cell.

Suitable promoters for expression in planta include, but are not limited to, Wheat SPA promoter (SEQ ID NO:1; Albanietal, 1997. Plant Cell, 9:171-184); wheat LMW [SEQ ID NO:2 (longer LMW promoter) and SEQ ID NO:3 (LMW promoter)]; HMW glutenin-1 [SEQ ID NO:4; (Wheat HMW glutenin-1 longer promoter); and SEQ ID NO:5 (Wheat HMW glutenin-1 Promoter); Thomas and Flavell, 1990. The Plant Cell 2:1171-1180; Furtado et al., 2009. Plant Biotechnology Journal 7:240-253]; wheat alpha, beta and gamma gliadins [e.g., SEQ ID NO:6 (wheat alpha gliadin, B genome, promoter); SEQ ID NO:7 (wheat gamma gliadin promoter); Rafalski J A et al. 1984. EMBO 3:1409-1415], wheat TdPR60 [SEQ ID NO:8 (wheat TdPR60 longer promoter) or SEQ ID NO:9 (wheat TdPR60 promoter); Kovalchuk et al., 2009. Plant Mol Biol 71:81-98], maize Ub1 Promoter [cultivar Nongda 105 (SEQ ID NO:10); GenBank: DQ141598.1; Taylor et al., 1993. Plant Cell Rep 12: 491-495; and cultivar B73 (SEQ ID NO: 11; Christensen, A H et al. 1992. Plant Mol. Biol. 18(4):675-689); rice actin 1 (SEQ ID NO:12; Mc Elroy et al. 1990, The Plant Cell (2):163-171 rice GOS2 [SEQ ID NO:13 (rice GOS2 longer promoter) and SEQ ID NO:14 (rice GOS2 Promoter); De Pater et al. 1992. Plant J. 2: 837-44], arabidopsis Pho1 [SEQ ID NO:15 (arabidopsis Pho1 Promoter); Hamburger et al., Plant Cell. 2002; 14: 889-902,], ExpansinB promoters, e.g., rice ExpB5 [SEQ ID NO:16 (rice ExpB5 longer promoter) and SEQ ID NO: 17 (rice ExpB5 promoter)] and Barley ExpB1 [SEQ ID NO: 18 (barley ExpB1 Promoter); Won et al. Mol Cells. 2010. 30:369-76], barley SS2 (sucrose synthase 2; SEQ ID NO: 19; Guerin and Carbonero, 1997. Plant Physiology 114(1):55-62), and rice PG5a (SEQ ID NO:20; U.S. Pat. No. 7,700,835; Nakase et al., 1996. Plant Mol Biol. 32:621-30).

Suitable constitutive promoters include, for example, CaMV 35S promoter [SEQ ID NO:21 (CaMV 35S (pQXNc) Promoter); SEQ ID NO:22 (PJJ 35S from Brachypodium); SEQ ID NO:23 (CaMV 35S (OLD) Promoter; Odell et al., Nature 313:810-812, 1985)], Arabidopsis At6669 promoter [SEQ ID NO:24 (Arabidopsis At6669 (OLD) Promoter; see PCT Publication No. WO04081173 or the new At6669 promoter (SEQ ID NO:25 (Arabidopsis At6669 (NEW) Promoter)]; maize Ubl Promoter [cultivar Nongda 105 (SEQ ID NO: 10); and cultivar B73 (SEQ ID NO:11)]; rice actin 1 (SEQ ID NO:12); pEMU (Last et al., 1991. Theor. Appl. Genet. 81:581-588); CaMV 19S (Nilsson et al., 1997. Physiol. Plant 100:456-462); rice GOS2 [SEQ ID NO:13 (rice GOS2 longer Promoter) and SEQ ID NO: 14 (rice GOS2 Promoter); RBCS promoter (SEQ ID NO:26); Rice cyclophilin (Bucholz et al., 1994 Plant Mol Biol. 25(5):837-43); Maize H3 histone (Lepetit et al., 1992 Mol. Gen. Genet. 231: 276-285); Actin 2 (An et al., 1996. Plant J. 10(1); 107-121) and Synthetic Super MAS (Ni et al., 1995. The Plant Journal 7: 661-676). Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

Suitable tissue-specific promoters include, but are not limited to, leaf-specific promoters [e.g., AT5G06690 (Thioredoxin), high expression, SEQ ID NO:27); AT5G61520 (AtSTP3, low expression, SEQ ID NO:28, described in Buttner et al., 2000. Plant, Cell and Environment 23:175-184); or the promoters described in Yamamoto et al., 1997. Plant J. 12:255-265; Kwon et al., 1994. Plant Physiol. 105:357-67; Yamamoto et al., 1994. Plant Cell Physiol. 35:773-778; Gotor et al., 1993. Plant J. 3:509-18; Orozco et al., Plant Mol. Biol. 1993. 23:1129-1138; and Matsuoka et al., 1993. Proc. Natl. Acad. Sci. USA 90:9586-9590; as well as Arabidopsis STP3 (AT5G61520) promoter (Buttner et al., 2000. Plant, Cell and Environment 23:175-184]; seed-preferred promoters [e.g., Napin (originated from Brassica napus which is characterized by a seed specific promoter activity; Stuitje A. R. et. al. 2003. Plant Biotechnology Journal 1(4):301-309; SEQ ID NO:29 (Brassica napus NAPIN Promoter) from seed specific genes (Simon, et al., 1985. Plant Mol. Biol. 5:191; Scofield, et al., 1987. J. Biol. Chem. 262:12202; Baszczynski, et al., 1990. Plant Mol. Biol. 14:633), rice PG5a (SEQ ID NO:20; U.S. Pat. No. 7,700,835), early seed development Arabidopsis BAN (AT1G61720) (SEQ ID NO:30, US 2009/0031450), late seed development Arabidopsis ABI3 (AT3G24650) (SEQ ID NO:31 (Arabidopsis ABI3 (AT3G24650) longer Promoter) or SEQ ID NO:32 (Arabidopsis ABI3 (AT3G24650) Promoter)) (Ng et al., 2004. Plant Molecular Biology 54: 25-38), Brazil Nut albumin (Pearson' et al., 1992. Plant Mol. Biol. 18: 235-245), legumin (Ellis, et al. 1988. Plant Mol. Biol. 10: 203-214), Glutelin (rice) (Takaiwa et al., 1986. Mol. Gen. Genet. 208:15-22; Takaiwa et al., 1987. FEBS Letts. 221: 43-47), Zein (Matzke et al., 1990. Plant Mol Biol, (143):323-332), napA (Stalberg et al., 1996. Planta 199:515-519); Wheat SPA (SEQ ID NO:1); sunflower oleosin (Cummins et al., 1992. Plant Mol. Biol. 19: 873-876); endosperm specific promoters [e.g., wheat LMW (SEQ ID NO:2; Wheat LMW Longer Promoter), and SEQ ID NO:3 (Wheat LMW Promoter)] and HMW glutenin-1 [(SEQ ID NO:4 (Wheat HMW glutenin-1 longer Promoter); and SEQ ID NO:5 (Wheat HMW glutenin-1 Promoter); Colot et al., Mol Gen Genet 216:81-90, 1989; Olin et al., NAR 17:461-2, 1989), wheat alpha, beta and gamma gliadins (SEQ ID NO:6 (wheat alpha gliadin (B genome) promoter); SEQ ID NO:7 (wheat gamma gliadin promoter);, Barley ltrl promoter, barley B1, C, D hordein (Cho et al., Theor Appl Gen 98:1253-62, 1999; Muller et al., Plant J 4:343-55, 1993; Sorenson et al., Mol Gen Genet 250:750-60, 1996), Barley DOF (Mena et al., 1998. The Plant Journal 116(1):53-62), Biz2 (EP99106056.7), Barley SS2 (SEQ ID NO:19), wheat Tarp60 (Kovalchuk et al., 2009. Plant Mol Biol 71:81-98), barley D-hordein (D-Hor) and B-hordein (B-Hor) (Agnelo F et al., 2009. Plant Biotech J 793):240-253)], Synthetic promoter (Vicente-Carbajosa et al., 1998. Plant J. 13: 629-640), rice prolamin NRP33, rice -globulin Glb-1 (Wu et al., 1998. Plant Cell Physiology 39(8) 885-889), rice alpha-globulin REB/OHP-1 (Nakase et al. 1997. Plant Mol. Biol. 33: 513-S22), rice ADP-glucose PP (Russell et al., Trans Res 6:157-68, 1997), maize ESR gene family (Opsahl-Ferstad et al., Plant J 12:235-46, 1997), sorgum gamma-kafirin (DeRose et al., PMB 32:1029-35, 1996)], embryo specific promoters [e.g., rice OSH1 (Sato et al., Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996), KNOX (Postma-Haarsma et al., 1999. Plant Mol. Biol. 39:257-71), rice oleosin (Wu et al., 1998. J. Biochem., 123:386], and flower-specific promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer et al., 1990. Plant Mol. Biol. 15, 95-109), LAT52 (Twell et al., 1989. Mol. Gen Genet 217:240-245), Arabidopsis apetala-3 (Tilly et al., 1998. Development 125:1647-57), Arabidopsis APETALA 1 (AT1G69120, API) (SEQ ID NO:33 (Arabidopsis (AT1G69120) APETALA 1)) (Hempel et al., 1997. Development 124:3845-3853)], and root promoters [e.g., the ROOTP promoter [SEQ ID NO:34]; rice ExpB5 [SEQ ID NO:17 (rice ExpB5 Promoter); or SEQ ID NO:16 (rice ExpB5 longer Promoter)] and barley ExpB1 promoters (SEQ ID NO:18) (Won et al. 2010. Mol. Cells 30: 369-376); Arabidopsis ATTPS-CIN (AT3G25820) promoter (SEQ ID NO:35; Chen et al., 2004. Plant Phys 135:1956-66); Arabidopsis Pho1 promoter (SEQ ID NO: 15), which is also slightly induced by stress].

The nucleic acid construct of some embodiments of the invention can further include an appropriate selectable marker and/or an origin of replication. According to some embodiments of the invention, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible with propagation in cells. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.

The nucleic acid construct of some embodiments of the invention can be utilized to stably or transiently transform plant cells. In stable transformation, the exogenous polynucleotide is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.

There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev. Plant. Physiol. & Plant. Mol. Biol. 1991. 42:205-225; Shimamoto et al., 1989. Nature 338:274-276).

The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:

    • (i) Agrobacterium-mediated gene transfer: Klee et al., (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Amtzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.
    • (ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217; glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. See, e.g., Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.

There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.

Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. The new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced from the seedlings to meet production goals. During stage three, the tissue samples grown in stage two are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.

According to some embodiments of the invention, the transgenic plant is generated by transient transformation of leaf cells, meristematic cells or the whole plant.

Transient transformation can be performed by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.

Viruses that have been shown to be useful for the transformation of plant hosts include, for example, Cauliflower mosaic virus (CaMV), Tobacco mosaic virus (TMV), brome mosaic virus (BMV) and Bean Common Mosaic Virus (BV or BCMV). Methods for transformation of plants using plant viruses are well known in the art; see, e.g. U.S. Pat. No. 4,855,237; Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988); and Mortimer C et al., 2015. Current Opinion in Biotechnology 32:85-92). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants are described in WO 87/06261.

According to some embodiments of the invention, the virus used for transient transformations is avirulent and thus is incapable of causing severe symptoms such as reduced growth rate, mosaic, ring spots, leaf roll, yellowing, streaking, pox formation, tumor formation and pitting. A suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus. Virus attenuation may be performed by using methods well known in the art including, but not limited to, sub-lethal heating, chemical treatment or by directed mutagenesis techniques such as described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4:259-269, 2003), Gal-On et al., J Gener Viriol 73: 2183-87 (1992), Atreya et al. Viriology 191:106-11 (1992) and Huet et al. Viriology 75: 1407-14 (1994).

Suitable virus strains can be obtained from available sources such as, for example, the American Type culture Collection (ATCC) or by isolation from infected plants. Isolation of viruses from infected plant tissues can be effected by techniques well known in the art such as described, for example by Foster and Taylor, Eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology Vol 81 Humana Press, 1998). Briefly, tissues of an infected plant believed to contain a high concentration of a suitable virus, preferably young leaves and flower petals, are ground in a buffer solution (e.g., phosphate buffer solution) to produce a virus infected sap which can be used in subsequent inoculations.

Construction of plant RNA viruses for the introduction and expression of non-viral exogenous polynucleotide sequences in plants is demonstrated by the above references as well as by Dawson, W. O. et al., 1989. Virology 172:285-292; Takamatsu et al. 1987. EMBO J 6:307-311; French et al. 1986. Science 231:1294-1297; Takamatsu et al. 1990. FEBS Letters 269:73-76; and U.S. Pat. No. 5,316,931.

When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.

In one embodiment, a plant viral polynucleotide is provided in which the native coat protein coding sequence has been deleted from a viral polynucleotide, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral polynucleotide, and ensuring a systemic infection of the host by the recombinant plant viral polynucleotide, has been inserted. Alternatively, the coat protein gene may be inactivated by insertion of the non-native polynucleotide sequence within it, such that a protein is produced. The recombinant plant viral polynucleotide may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Non-native (foreign) polynucleotide sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one polynucleotide sequence is included. The non-native polynucleotide sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral polynucleotide is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non-native coat protein coding sequence.

In a third embodiment, a recombinant plant viral polynucleotide is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral polynucleotide. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Non-native polynucleotide sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.

In a fourth embodiment, a recombinant plant viral polynucleotide is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.

The viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral polynucleotide to produce a recombinant plant virus. The recombinant plant viral polynucleotide or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral polynucleotide is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (exogenous polynucleotide) in the host to produce the desired protein.

Techniques for inoculation of viruses to plants may be found, for example, in Foster and Taylor, Eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998; Maramorosh and Koprowski, Eds. “Methods in Virology” 7 vols, Academic Press, New York 1967-1984; Hill, S. A. “Methods in Plant Virology”, Blackwell, Oxford, 1984; Walkey, D. G. A. “Applied Plant Virology”, Wiley, New York, 1985; and Kado and Agrawa, eds. “Principles and Techniques in Plant Virology”, Van Nostrand-Reinhold, New York.

In addition to the above, the polynucleotide of the present invention can also be introduced into a chloroplast genome thereby enabling chloroplast expression.

A technique for introducing exogenous polynucleotide sequences to the genome of the chloroplasts is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous polynucleotide is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous polynucleotide molecule into the chloroplasts. The exogenous polynucleotides selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily performed by enzymes inherent to the chloroplast. To this end, the exogenous polynucleotide includes, in addition to a gene of interest, at least one polynucleotide stretch which is derived from the chloroplast's genome. In addition, the exogenous polynucleotide includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous polynucleotide. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference. A polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.

According to some embodiments of the invention, the transformed plant is homozygote to the transgene (i.e., the exogenous polynucleotide of some embodiments of the invention), and accordingly all seeds generated thereby include the transgene.

Expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing multiple nucleic acid constructs, each including a different exogenous polynucleotide, into a single plant cell. The transformed cell can then be regenerated into a mature plant using the methods described hereinabove.

Alternatively, expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing into a single plant-cell a single nucleic-acid construct including a plurality of different exogenous polynucleotides. Such a construct can be designed with a single promoter sequence which can transcribe a polycistronic messenger RNA including all the different exogenous polynucleotide sequences. To enable co-translation of the different polypeptides encoded by the polycistronic messenger RNA, the polynucleotide sequences can be inter-linked via an internal ribosome entry site (IRES) sequence which facilitates translation of polynucleotide sequences positioned downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule encoding the different polypeptides described above will be translated from both the capped 5′ end and the two internal IRES sequences of the polycistronic RNA molecule to thereby produce in the cell all different polypeptides. Alternatively, the construct can include several promoter sequences each linked to a different exogenous polynucleotide sequence.

The plant cell transformed with the construct including a plurality of different exogenous polynucleotides can be regenerated into a mature plant, using the methods described hereinabove.

Alternatively, expressing a plurality of exogenous polynucleotides in a single host plant can be effected by introducing different nucleic acid constructs, including different exogenous polynucleotides, into a plurality of plants. The regenerated transformed plants can then be cross-bred and resultant progeny selected for superior abiotic stress tolerance, water use efficiency, fertilizer use efficiency, early flowering, grain filling period, harvest index, plant height, growth, biomass, yield and/or vigor traits, using conventional plant breeding techniques.

According to certain embodiments, enhancing the resistance of the plant or part thereof to the pathogenic fungus comprises reducing the expression and/or activity of the at least one polypeptide compared to its expression and/or activity in the control plant.

Any method as is known in the art for reducing the expression and/or activity of a plant endogenous protein and the polynucleotide encoding same can be used according to the teachings of the resent invention.

According to certain embodiment of the invention, reducing the expression and/or activity of a polypeptide of the invention within a plant cell comprising transforming the plant cell with a polynucleotide that inhibits the expression of said polypeptide. The polynucleotide may inhibit the transcription or translation of a polynucleotide encoding said polypeptide or can encode for an inhibitory polypeptide interfering with the translation or activity of said polypeptide.

Polynucleotide-Based Methods

According to some embodiments of the present disclosure, a plant is transformed with a polynucleotide that inhibits the expression of a polypeptide of the invention. According to certain exemplary embodiments, the plant cell is transformed within a construct capable of expressing the inhibitory polynucleotide. The term “expression” as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. For example, for the purposes of the present invention, a construct capable of expressing the inhibitory polynucleotide is capable of producing an RNA molecule that inhibits the transcription and/or translation of a polypeptide of the invention. The “expression” or “production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide, while the “expression” or “production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide. Examples of polynucleotides that inhibit the expression of a CCT polypeptide are given below.

Sense Suppression/Co-suppression

According to certain embodiments, inhibition of the expression of a polypeptide of the invention may be obtained by sense suppression or co-suppression. For co-suppression, a construct is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding the polypeptide in the “sense” orientation. Over-expression of the RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the co-suppression constructs are screened to identify those that show the greatest inhibition of the polypeptide expression.

The polynucleotide used for co-suppression may correspond to all or part of the sequence encoding the polypeptide of the invention, all or part of the 5′ and/or 3′ untranslated region of said polypeptide transcript or all or part of both the coding sequence and the untranslated regions of a transcript encoding said polypeptide. In some embodiments where the polynucleotide comprises all or part of the coding region for said polypeptide, the construct is designed to eliminate the start codon of the polynucleotide so that no protein product will be translated.

Co-suppression may be used to inhibit the expression of plant genes to produce plants having undetectable protein levels for the proteins encoded by these genes. See, for example, Broin, et al., (2002) Plant Cell 14:1417-1432. Co-suppression may also be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Methods for using co-suppression to inhibit the expression of endogenous genes in plants are described, for example, in Yu, et al., Phytochemistry (2003) 63:753-763 and U.S. Pat. Nos. 5,034,323, 5,283,184 and 5,942,657. The efficiency of co-suppression may be increased by including a poly-dT region in the expression cassette at a position 3′ to the sense sequence and 5′ of the polyadenylation signal. See for example, US Patent Application Publication Number 2002/0048814. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, optimally greater than about 65% sequence identity, more optimally greater than about 85% sequence identity, most optimally greater than about 95% sequence identity (U.S. Pat. Nos. 5,283,184 and 5,034,323).

Antisense Suppression

According to some embodiments, inhibition of the expression of a polypeptide of the invention may be obtained by antisense suppression. For antisense suppression, the construct is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the polypeptide. Over-expression of the antisense RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the antisense RNA are screened to identify those that show the greatest inhibition of said polypeptide expression.

The polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the polypeptide of the invention, all or part of the complement of the 5′ and/or 3′ untranslated region of its transcript or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding said polypeptide. In addition, the antisense polynucleotide may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 400, 450, 500, 550 or greater may be used. Methods for using antisense suppression to inhibit the expression of endogenous genes in plants are described, for example, in Liu, et al., (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657. Efficiency of antisense suppression may be increased by including a poly-dT region in the expression cassette at a position 3′ to the antisense sequence and 5′ of the polyadenylation signal (see, e.g. US Patent Application Publication Number 2002/0048814).

Double-Stranded RNA Interference

According to some embodiments, inhibition of the expression of a polypeptide of the invention may be obtained by double-stranded RNA (dsRNA) interference. For dsRNA interference, a sense RNA molecule like that described above for co-suppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished by designing the DNA construct to comprise both a sense sequence and an antisense sequence. Alternatively, separate constructs may be used for the sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference construct(s) are then screened to identify plant lines that show the greatest inhibition of the expression of the polypeptide. Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964, Liu, et al., (2002) Plant Physiol. 129:1732-1743 and WO 1999/49029, WO 1999/53050, WO 1999/61631 and WO 2000/49035.

Hairpin RNA Interference and Intron-Containing Hairpin RNA Interference

According to some embodiments, inhibition of the expression of a polypeptide of the invention may be obtained by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference. These methods are highly efficient at inhibiting the expression of endogenous genes. See, Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38 and the references cited therein.

For hpRNA interference, the construct is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem. The base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited and an antisense sequence that is fully or partially complementary to the sense sequence. Thus, the base-paired stem region of the molecule generally determines the specificity of the RNA interference. hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731 and Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38. Methods for using hpRNA interference to inhibit or silence the expression of genes are described, for example, in US Patent Application Publication Number 2003/0175965. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga, et al., (2003) Mol. Biol. Rep. 30:135-140.

For Intron-Containing Hairpin RNA (ihpRNA) interference, the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron that is capable of being spliced in the cell in which the ihpRNA is expressed. The use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith, et al., (2000) Nature 407:319-320. In fact, Smith, et al., shows 100% suppression of endogenous gene expression using ihpRNA-mediated interference. Methods for using ihpRNA interference to inhibit the expression of endogenous plant genes are described, for example, in US Patent Application Publication Number 2003/0180945.

The expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. Thus, it is the loop region that determines the specificity of the RNA interference. See, for example, WO 2002/00904.

Amplicon-Mediated Interference

Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus. The viral sequences present in the transcription product of the construct allow the transcription product to direct its own replication. The transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e., the messenger RNA for the polypeptide of the invention). Methods of using amplicons to inhibit the expression of endogenous plant genes are described, for example, in Angell and Baulcombe, (1997) EMBO J. 16:3675-3684, Angell and Baulcombe, (1999) Plant J. 20:357-362.

Ribozymes

According to some embodiments, the polynucleotide expressed by the construct of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of a polypeptide of the invention. Thus, the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of said polypeptide. This method is described, for example, in U.S. Pat. No. 4,987,071.

Small Interfering RNA or Micro RNA

According to certain embodiments of the invention, inhibition of the expression of a polypeptide of the invention may be obtained by RNA interference by expression of a gene encoding a micro RNA (miRNA). miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNAs are highly efficient at inhibiting the expression of endogenous genes. See, for example, Palatnikl J F et al., (2003) Nature 425:257-263.

For miRNA interference, the construct is designed to express an RNA molecule that is modeled on an endogenous miRNA gene. The miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence). For suppressing the expression of a polypeptide of the invention, the 22-nucleotide sequence is selected from the polypeptide transcript sequence and contains 22 nucleotides of said transcript sequence in sense orientation and 21 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence. miRNA molecules are highly efficient at inhibiting the expression of endogenous genes and the RNA interference they induce is inherited by subsequent generations of plants.

Polypeptide-Based Inhibition of Gene Expression

According to certain additional or alternative embodiments, the inhibitory polynucleotide encodes a zinc finger protein that binds to a gene encoding a polypeptide of the invention, resulting in reduced expression of the gene. In particular embodiments, the zinc finger protein binds to a regulatory region of a polypeptide encoding gene. In other embodiments, the zinc finger protein binds to a messenger RNA encoding said polypeptide and prevents its translation. Methods of selecting sites for targeting by zinc finger proteins have been described, for example, in U.S. Pat. No. 6,453,242 and methods for using zinc finger proteins to inhibit the expression of genes in plants are described, for example, in U.S. Pat. No. 7,151,201.

Polypeptide-Based Inhibition of Protein Activity

According to certain additional or alternative embodiments, the polynucleotide encodes an antibody that binds to a polypeptide of the invention and reduces the activity of the polypeptide. In another embodiment, the binding of the antibody results in increased turnover of the antibody-polypeptide complex by cellular quality control mechanisms. The expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald, (2003) Nature Biotech. 21:35-36.

According to some embodiments of the invention, up-regulation or down regulation of the expression and/or activity of the polypeptide of the invention is achieved by means of genome editing.

Genome editing is a reverse genetics method which uses artificially engineered nucleases to cut and create specific double-stranded breaks at a desired location(s) in the genome, which are then repaired by cellular endogenous processes such as, homology directed repair (HDR) and non-homologous end-joining (NHEJ). NHEJ directly joins the DNA ends in a double-stranded break, while HDR utilizes a homologous sequence as a template for regenerating the missing DNA sequence at the break point. In order to introduce specific nucleotide modifications to the genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. Genome editing cannot be performed using traditional restriction endonucleases since most restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location. To overcome this challenge and create site-specific single- or double-stranded breaks, several distinct classes of nucleases have been discovered and bioengineered to date. These include the meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR/Cas system.

Genome editing is a powerful tool to impact target traits by modifications of the target plant genome sequence. Such modifications can result in new or modified alleles or regulatory elements.

In addition, the traces of genome-edited techniques can be used for marker assisted selection (MAS) as is further described hereinunder. Target plants for the mutagenesis/genome editing methods according to the invention are any plants of interest including monocot or dicot plants.

Over-expression of a polypeptide by genome editing can be achieved by: (i) replacing an endogenous sequence encoding the polypeptide of interest or a regulatory sequence under which it is placed, and/or (ii) inserting a new gene encoding the polypeptide of interest in a targeted region of the genome, and/or (iii) introducing point mutations which result in up-regulation of the gene encoding the polypeptide of interest (e.g., by altering the regulatory sequences such as promoter, enhancers, 5′-UTR and/or 3′-UTR, or mutations in the coding sequence).

Down regulation of the expression of a polypeptide by gnome editing can be achieved by (i) replacing an endogenous sequence encoding a polypeptide negatively affecting a desired plant trait, according to some embodiments of the invention enhancing susceptibility of the plant to pathogenic fungi and/or Oomycete or replacing a regulatory sequence under which the endogenous sequence encoding the polypeptide is placed, and/or (ii) introducing point mutations which result in down-regulation of the gene encoding the polypeptide of interest (e.g., by altering the regulatory sequences such as promoter, enhancers, 5′-UTR and/or 3′-UTR, or mutations in the coding sequence).

Genome Editing Systems Overview

Several systems have been reported to enable genome editing implementation. Examples detailed herein below:

Meganucleases—Meganucleases are commonly grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLIDADG family are characterized by having either one or two copies of the conserved LAGLIDADG motif. The four families of meganucleases are widely separated from one another with respect to conserved structural elements and, consequently, DNA recognition sequence specificity and catalytic activity. Meganucleases are found commonly in microbial species and have the unique property of having very long recognition sequences (>14 bp) thus making them naturally very specific for cutting at a desired location. This can be exploited to make site-specific double-stranded breaks directing modifications in regulatory elements or coding regions upon introduction of the desired sequence. One of skill in the art can use these naturally occurring meganucleases, however the number of such naturally occurring meganucleases is limited. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to create hybrid enzymes that recognize a new sequence. Alternatively, DNA interacting amino acids of the meganuclease can be altered to design sequence specific meganucleases (see e.g., U.S. Pat. No. 8,021,867). Meganucleases can be designed using the methods described in e.g., Certo, M T et al., 2012. Nature Methods 9:073-975; U.S. Pat. Nos. 8,304,222; 8,021,867; 8,119,381; 8,124,369; 8,129,134; 8,133,697; 8,143,015; 8,143,016; 8,148,098; or 8,163,514. Alternatively, meganucleases with site specific cutting characteristics can be obtained using commercially available technologies e.g., Precision Biosciences' Directed Nuclease Editor™ genome editing technology.

ZFNs and TALENs—Two distinct classes of engineered nucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have both proven to be effective at producing targeted double-stranded breaks (Christian et al., 2010. Genetics 186:757-761; Kim et al., 1996. Proc. Natl. Acad. Sci. 93:1156-1160; Li et al., 2011. Nucleic Acids Res 39:359-372; Mahfouz et al., 2011. Proc. Natl. Acad. Sci; 108:2623-2628; Miller et al., 2010. Nat Biotechnol. 29:143-148).

Basically, ZFNs and TALENs restriction endonuclease technology utilizes a non-specific DNA cutting enzyme which is linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively). Typically, a restriction enzyme whose DNA recognition site and cleaving site are separate from each other is selected. The cleaving portion is separated and then linked to a DNA binding domain, thereby yielding an endonuclease with very high specificity for a desired sequence. An exemplary restriction enzyme with such properties is Fokl. Additionally, Fokl has the advantage of requiring dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner recognizes a unique DNA sequence. To enhance this effect, Fokl nucleases have been engineered that can only function as heterodimers and have increased catalytic activity. The heterodimer functioning nucleases avoid the possibility of unwanted homodimer activity and thus increase specificity of the double-stranded break.

Thus, for example to target a specific site, ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind adjacent sequences at the targeted site. Upon transient expression in cells, the nucleases bind to their target sites and the FokI domains heterodimerize to create a double-stranded break. Repair of these double-stranded breaks through the nonhomologous end-joining (NHEJ) pathway most often results in small deletions or small sequence insertions. Since each repair made by NHEJ is unique, the use of a single nuclease pair can produce an allelic series with a range of different deletions at the target site. The deletions typically range anywhere from a few base pairs to a few hundred base pairs in length, but larger deletions have successfully been generated in cell culture by using two pairs of nucleases simultaneously (Carlson et al., 2012. Proc. Natl. Acad. Sci 109:17382-17387; Lee et al., 2010. Genome Res 20:81-89). In addition, when a fragment of DNA with homology to the targeted region is introduced in conjunction with the nuclease pair, the double-stranded break can be repaired via homology directed repair to generate specific modifications (Li et al., 2011, ibid; Miller et al., 2010, ibid; Urnov et al., 2005. Nature, 435:646-651).

Although the nuclease portions of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases is in their DNA recognition peptide. ZFNs rely on Cys2-His2 zinc fingers and TALENs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers typically found in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because both zinc fingers and TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities. Approaches for making site-specific zinc finger endonucleases include, e.g., modular assembly (where Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), OPEN (low-stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries, among others. ZFNs can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, Calif.).

Method for designing and obtaining TALENs are described in e.g. Reyon et al., 2012. Nature Biotechnology 30(5):460-5; Miller et al., 2011. Nat Biotechnol. 29:143-148; Cermak et al., 2011. Nucleic Acids Research 39 (12):e82 and Zhang et al., 2011 Nature Biotechnology 29 (2):149-53. A recently developed web-based program named Mojo Hand was introduced by Mayo Clinic for designing TAL and TALEN constructs for genome editing applications (can be accessed through www(dot)talendesign(dot)org). TALEN can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, Calif.).

The ZFN/TALEN system capability for precise targeting can be utilized for directing modifications in regulatory elements and/or coding regions upon introduction of the sequence of interest for trait improvement.

CRISPR/Cas9—The CRISPR/Cas system for genome editing contains two distinct components: a gRNA (guide RNA) and an endonuclease e.g. Cas9.

The gRNA is typically a 20-nucleotide sequence encoding a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. The binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break. Just as with ZFNs and TALENs, the double-stranded brakes produced by CRISPR/Cas can undergo homologous recombination or NHEJ.

The Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks in the genomic DNA.

A significant advantage of CRISPR/Cas is that the high efficiency of this system coupled with the ability to easily create synthetic gRNAs enables multiple genes to be targeted simultaneously. In addition, the majority of cells carrying the mutation present biallelic mutations in the targeted genes.

However, apparent flexibility in the base-pairing interactions between the gRNA sequence and the genomic DNA target sequence allows imperfect matches to the target sequence to be cut by Cas9.

Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called ‘nickases’. With only one active nuclease domain, the Cas9 nickase cuts only one strand of the target DNA, creating a single-strand break or ‘nick’. A single-strand break, or nick, is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a double-strand break, in what is often referred to as a ‘double nick’ CRISPR system. A double-nick can be repaired by either NHEJ or HDR depending on the desired effect on the gene target. Thus, if specificity and reduced off-target effects are crucial, using the Cas9 nickase to create a double-nick by designing two gRNAs with target sequences in close proximity and on opposite strands of the genomic DNA would decrease off-target effect as either gRNA alone will result in nicks that will not change the genomic DNA.

Modified versions of the Cas9 enzyme containing two inactive catalytic domains (dead Cas9, or dCas9) have no nuclease activity while still able to bind to DNA based on gRNA specificity. The dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains. For example, the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.

There is a number of publically available tools available to help choose and/or design target sequences as well as lists of bioinformatically determined unique gRNAs for different genes in different species such as the Feng Zhang lab's Target Finder, the Michael Boutros lab's Target Finder (E-CRISP), the RGEN Tools: Cas-OFFinder, the CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes and the CRISPR Optimal Target Finder.

In order to use the CRISPR system, both gRNA and Cas9 should be expressed in a target cell. The insertion vector can contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids. CRISPR plasmids are commercially available such as the px330 plasmid from Addgene.

Recombinant adeno-associated virus (rAAV) platform—this genome-editing platform is based on rAAV vectors which enable insertion, deletion or substitution of DNA sequences in the genomes of live mammalian cells. The rAAV genome is a single-stranded deoxyribonucleic acid (ssDNA) molecule, either positive- or negative-sensed, which is about 4.7 kb long. These single-stranded DNA viral vectors have high transduction rates and have a unique property of stimulating endogenous homologous recombination in the absence of double-strand DNA breaks in the genome. One of skill in the art can design a rAAV vector to target a desired genomic locus and perform both gross and/or subtle endogenous gene alterations in a cell. rAAV genome editing has the advantage in that it targets a single allele and does not result in any off-target genomic alterations. rAAV genome editing technology is commercially available, for example, the rAAV GENESIS™ system from Horizon™ (Cambridge, UK).

Methods for qualifying efficacy and detecting sequence alteration are well known in the art and include, but not limited to, DNA sequencing, electrophoresis, an enzyme-based mismatch detection assay and a hybridization assay such as PCR, RT-PCR, RNase protection, in-situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis.

Sequence alterations in a specific gene can also be determined at the protein level using e.g. chromatography, electrophoretic methods, immunodetection assays such as ELISA and Western blot analysis and immunohistochemistry.

In addition, one ordinarily skilled in the art can readily design a knock-in/knock-out construct including positive and/or negative selection markers for efficiently selecting transformed cells that underwent a homologous recombination event with the construct. Positive selection provides a means to enrich the population of clones that have taken up foreign DNA. Non-limiting examples of such positive markers include glutamine synthetase, dihydrofolate reductase (DHFR), markers that confer antibiotic resistance, such as neomycin, hygromycin, puromycin, and blasticidin S resistance cassettes. Negative selection markers are necessary to select against random integrations and/or elimination of a marker sequence (e.g. positive marker). Non-limiting examples of such negative markers include the herpes simplex-thymidine kinase (HSV-TK) which converts ganciclovir (GCV) into a cytotoxic nucleoside analog, hypoxanthine phosphoribosyltransferase (HPRT) and adenine phosphoribosytransferase (ARPT).

Recombination Procedures—Common to Different Genome Editing Systems

“Hit and run” or “in-out”—involves a two-step recombination procedure. In the first step, an insertion-type vector containing a dual positive/negative selectable marker cassette is used to introduce the desired sequence alteration. The insertion vector contains a single continuous region of homology to the targeted locus and is modified to carry the mutation of interest. This targeting construct is linearized with a restriction enzyme at a one site within the region of homology, electroporated into the cells, and positive selection is performed to isolate homologous recombinants. These homologous recombinants contain a local duplication that is separated by intervening vector sequence, including the selection cassette. In the second step, targeted clones are subjected to negative selection to identify cells that have lost the selection cassette via intrachromosomal recombination between the duplicated sequences. The local recombination event removes the duplication and, depending on the site of recombination, the allele either retains the introduced mutation or reverts to wild type. The end result is the introduction of the desired modification without the retention of any exogenous sequences.

The “double-replacement” or “tag and exchange” strategy—involves a two-step selection procedure similar to the hit and run approach, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3′ and 5′ homology arms is used to insert a dual positive/negative selectable cassette near the location where the mutation is to be introduced. After electroporation and positive selection, homologous targeted clones are identified. Next, a second targeting vector that contains a region of homology with the desired mutation is electroporated into targeted clones, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation while eliminating unwanted exogenous sequences.

Site-Specific Recombinases—The Cre recombinase derived from the P1 bacteriophage and Flp recombinase derived from the yeast Saccharomyces cerevisiae are site-specific DNA recombinases each recognizing a unique 34 base pair DNA sequence (termed “Lox” and “FRT”, respectively) and sequences that are flanked with either Lox sites or FRT sites can be readily removed via site-specific recombination upon expression of Cre or Flp recombinase, respectively. For example, the Lox sequence is composed of an asymmetric eight base pair spacer region flanked by 13 base pair inverted repeats. Cre recombines the 34 base pair lox DNA sequence by binding to the 13 base pair inverted repeats and catalyzing strand cleavage and religation within the spacer region. The staggered DNA cuts made by Cre in the spacer region are separated by 6 base pairs to give an overlap region that acts as a homology sensor to ensure that only recombination sites having the same overlap region recombine. Basically, the site-specific recombinase system offers means for the removal of selection cassettes after homologous recombination. This system also allows for the generation of conditional altered alleles that can be inactivated or activated in a temporal or tissue-specific manner. Of note, the Cre and Flp recombinases leave behind a Lox or FRT “scar” of 34 base pairs. The Lox or FRT sites that remain are typically left behind in an intron or 3′ UTR of the modified locus, and current evidence suggests that these sites usually do not interfere significantly with gene function. Thus, Cre/Lox and Flp/FRT recombination involves introduction of a targeting vector with 3′ and 5′ homology arms containing the mutation of interest, two Lox or FRT sequences and typically a selectable cassette placed between the two Lox or FRT sequences. Positive selection is applied and homologous recombinants that contain targeted mutation are identified. Transient expression of Cre or Flp in conjunction with negative selection results in the excision of the selection cassette and selects for cells where the cassette has been lost. The final targeted allele contains the Lox or FRT scar of exogenous sequences.

Transposases—As used herein, the term “transposase” refers to an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome.

As used herein the term “transposon” refers to a mobile genetic element comprising a nucleotide sequence which can move around to different positions within the genome of a single cell. In the process the transposon can cause mutations and/or change the amount of a DNA in the genome of the cell. A number of transposon systems that are able to also transpose in cells e.g. vertebrates have been isolated or designed, such as Sleeping Beauty [Izsvik and Ivics Molecular Therapy (2004) 9: 147-156], piggyBac [Wilson et al. Molecular Therapy (2007) 15: 139-145], Tol2 [Kawakami et al. PNAS (2000) 97 (21): 11403-11408] or Frog Prince [Miskey et al. Nucleic Acids Res. December 1, (2003) 31(23): 6873-6881]. Generally, DNA transposons translocate from one DNA site to another in a simple, cut-and-paste manner. Each of these elements has their own advantages, for example, Sleeping Beauty is particularly useful in region-specific mutagenesis, whereas Tol2 has the highest tendency to integrate into expressed genes. Hyperactive systems are available for Sleeping Beauty and piggyBac. Most importantly, these transposons have distinct target site preferences, and can therefore introduce sequence alterations in overlapping, but distinct sets of genes. Therefore, to achieve the best possible coverage of genes, the use of more than one element is particularly preferred. The basic mechanism is shared between the different transposases, therefore the piggyBac (PB) is described as an example. PB is a 2.5 kb insect transposon originally isolated from the cabbage looper moth, Trichoplusia ni. The PB transposon consists of asymmetric terminal repeat sequences that flank a transposase, PBase. PBase recognizes the terminal repeats and induces transposition via a “cut-and-paste” based mechanism, and preferentially transposes into the host genome at the tetranucleotide sequence TTAA. Upon insertion, the TTAA target site is duplicated such that the PB transposon is flanked by this tetranucleotide sequence. When mobilized, PB typically excises itself precisely to reestablish a single TTAA site, thereby restoring the host sequence to its pretransposon state. After excision, PB can transpose into a new location or be permanently lost from the genome. Typically, the transposase system offers an alternative means for the removal of selection cassettes after homologous recombination quite similar to the use Cre/Lox or Flp/FRT. Thus, for example, the PB transposase system involves introduction of a targeting vector with 3′ and 5′ homology arms containing the mutation of interest, two PB terminal repeat sequences at the site of an endogenous TTAA sequence and a selection cassette placed between PB terminal repeat sequences. Positive selection is applied and homologous recombinants that contain targeted mutation are identified. Transient expression of PBase removes in conjunction with negative selection results in the excision of the selection cassette and selects for cells where the cassette has been lost. The final targeted allele contains the introduced mutation with no exogenous sequences.

For PB to be useful for the introduction of sequence alterations, there must be a native TTAA site in relatively close proximity to the location where a particular mutation is to be inserted.

Homology Directed Repair (HDR) Homology Directed Repair (HDR) can be used to generate specific nucleotide changes (also known as gene “edits”) ranging from a single nucleotide change to large insertions. In order to utilize HDR for gene editing, a DNA “repair template” containing the desired sequence must be delivered into the cell type of interest with e.g. the guide RNA [gRNA(s)] and Cas9 or Cas9 nickase or other genome editing method (examples herein below). The repair template must contain the desired edit as well as additional homologous sequence immediately upstream and downstream of the target (termed left and right homology arms). The length and binding position of each homology arm is dependent on the size of the change being introduced. The repair template can be a single stranded oligonucleotide, double-stranded oligonucleotide, or double-stranded DNA plasmid depending on the specific application.

The HDR method was successfully used for targeting a specific modification in a coding sequence of a gene in plants [Budhagatapalli Nagaveni et al. (2015) “Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley”. G3 (Bethesda). 5(9): 1857-1863). Thus, the gfp-specific transcription activator-like effector nucleases were used along with a repair template that, via HDR, facilitates conversion of gfp into yfp, which is associated with a single amino acid exchange in the gene product. The resulting yellow-fluorescent protein accumulation along with sequencing confirmed the success of the genomic editing.

Similarly, Zhao Yongping et al. 2016 (An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design. Scientific Reports 6, Article number: 23890 (2016)) describe co-transformation of Arabidopsis plants with a combinatory dual-sgRNA/Cas9 vector that successfully deleted miRNA gene regions (MIR169a and MIR827a) and second construct that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct to provide both targeted deletion and donor repair for targeted gene replacement by HDR.

Specific considerations for Homology Directed Repair (HDR) utilizing CRISPR/Cas9 system are described herein: It should be noted that the repair template should not include a sequence that exhibits more than 90% identity to the gRNA designed to the genomic DNA or to the reverse complement sequence of the gRNA which is designed to the genomic sequence, otherwise the repair template becomes a suitable target for Cas9 cleavage. Additionally or alternatively, when using a short repair template (e.g., about 40-200 base pairs) the repair template should preferably lack the Protospacer Adjacent Motif (PAM) sequence. For example, the PAM could be mutated such that it is no longer present, but the coding region of the gene is not affected (i.e. a silent mutation).

Introduction of large double stranded DNA as repair template can be performed using plasmids, yet, the plasmid should be linearized before transfection.

Activation of Target Genes Using CRISPR/Cas9 System

Many bacteria and archaea contain endogenous RNA-based adaptive immune systems that can degrade nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeat (CRISPR) genes that produce RNA components and CRISPR associated (Cas) genes that encode protein components. The CRISPR RNAs (crRNAs) contain short stretches of homology to specific viruses and plasmids and act as guides to direct Cas nucleases to degrade the complementary nucleic acids of the corresponding pathogen. Studies of the type II CRISPR/Cas system of Streptococcus pyogenes have shown that three components form an RNA/protein complex and together are sufficient for sequence-specific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs homologous to the target sequence, and a trans-activating crRNA (tracrRNA) (Jinek et al. Science (2012) 337: 816-821). It was further demonstrated that a synthetic chimeric guide RNA (gRNA) composed of a fusion between crRNA and tracrRNA could direct Cas9 to cleave DNA targets that are complementary to the crRNA in vitro. It was also demonstrated that transient expression of CRISPR-associated endonuclease (Cas9) in conjunction with synthetic gRNAs can be used to produce targeted double-stranded brakes in a variety of different species.

The CRISPR/Cas9 system is a remarkably flexible tool for genome manipulation. A unique feature of Cas9 is its ability to bind target DNA independently of its ability to cleave target DNA. Specifically, both RuvC- and HNH-nuclease domains can be rendered inactive by point mutations (D10A and H840A in SpCas9), resulting in a nuclease dead Cas9 (dCas9) molecule that cannot cleave target DNA. The dCas9 molecule retains the ability to bind to target DNA based on the gRNA targeting sequence. The dCas9 can be tagged with transcriptional activators, and targeting these dCas9 fusion proteins to the promoter region results in robust transcription activation of downstream target genes. The simplest dCas9-based activators consist of dCas9 fused directly to a single transcriptional activator. Importantly, unlike the genome modifications induced by Cas9 or Cas9 nickase, dCas9-mediated gene activation is reversible, since it does not permanently modify the genomic DNA.

Indeed, genome editing was successfully used to over-express a protein of interest in a plant by, for example, mutating a regulatory sequence, such as a promoter to overexpress the endogenous polynucleotide operably linked to the regulatory sequence. For example, U.S. Patent Application Publication No. 20160102316 to Rubio Munoz, Vicente et al., describes plants with increased expression of an endogenous DDA1 plant nucleic acid sequence wherein the endogenous DDA1 promoter carries a mutation introduced by mutagenesis or genome editing which results in increased expression of the DDA1 gene, using for example, CRISPR. The method involves targeting of Cas9 to the specific genomic locus, in this case DDA1, via a 20-nucleotide guide sequence of the single-guide RNA. An online CRISPR Design Tool can identify suitable target sites (tools.genome-engineering.org; Ran et al. (2013) Nature Protocols, 8911:2281-2308).

The CRISPR-Cas system was used for altering (increasing or decreasing) gene expression in plants as described in U.S. Patent Application publication No. 20150067922 to Yang; Yinong et al. The engineered, non-naturally occurring gene editing system comprises two regulatory elements, wherein the first regulatory element (a) operable in a plant cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA (gRNA) that hybridizes with the target sequence in the plant, and a second regulatory element (b) operable in a plant cell operably linked to a nucleotide sequence encoding a Type-II CRISPR-associated nuclease, wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets the target sequence and the CRISPR-associated nuclease cleaves the DNA molecule, thus altering the expression of a gene product in a plant. It should be noted that the CRISPR-associated nuclease and the guide RNA do not naturally occur together.

In addition, as described above, point mutations which activate a gene-of-interest and/or which result in over-expression of a polypeptide-of-interest can be also introduced into plants by means of genome editing. Such mutation can be for example, deletions of repressor sequences which result in activation of the gene-of-interest; and/or mutations which insert nucleotides and result in activation of regulatory sequences such as promoters and/or enhancers.

According to additional aspect, the present invention provides a genetically engineered plant having enhanced resistance to at least one fungus and/or Oomycete compared to a non-engineered control plant, the genetically engineered plant comprises at least one cell having modified expression and/or activity of at least one polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252 or at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506 and 2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof compared to the polypeptide expression and/or activity in the non-engineered control plant. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having modified expression and/or activity of at least one polypeptide having the amino acid sequences selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508 and 2635, 2444-2634, 2636-4252. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant having enhanced resistance to the at least one fungus and/or Oomycete comprises at least one cell with enhanced expression and/or activity of at least one polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2635, 2444-2634, 2636-4252 or at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-506.

According to certain embodiments, the genetically engineered plant comprises at least one cell transformed with an exogenous polynucleotide encoding the at least one polypeptide, the polynucleotide is at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443 or at least 80% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41, 43-61, 63-79, 81, and 83-301, thereby having an enhanced resistance to the at least one fungus. The exogenous polynucleotide can be endogenous to the plant cell or heterologous to the plant cell.

According to certain embodiments, the genetically engineered plant comprises at least one cell edited to express an exogenous polynucleotide encoding the at least one polypeptide, thereby having an enhanced resistance to the at least one fungus.

According to certain embodiments, the genetically engineered plant comprises at least one cell edited to over-express an endogenous polynucleotide encoding the at least one polypeptide, thereby having an enhanced resistance to the at least one fungus.

According to certain embodiments, the genetically engineered plant having enhanced resistance to the at least one fungus comprises at least one cell with enhanced expression of at least one polynucleotide encoding a polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252 or at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-506. According to certain exemplary embodiments, the polynucleotide expression in the genetically engineered plant is enhanced in comparison to the polynucleotide expression in a control plant.

According to certain embodiments, the genetically engineered plant having enhanced resistance to the at least one fungus comprises at least one cell with reduced expression and/or activity of the at least one polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:328, 348 and 361. According to certain exemplary embodiments, the polypeptide expression and/or activity in the genetically engineered plant is reduced in comparison to the polypeptide expression and/or activity in a control plant.

According to certain embodiments, the genetically engineered plant having reduced expression and/or activity of the at least one polypeptide comprises at least one cell having reduced expression of a polynucleotide encoding said at least one polypeptide, thereby having an enhanced resistance to the at least one fungus.

According to certain embodiments, the genetically engineered plant comprises a polynucleotide encoding a modified form of the at least one polypeptide, wherein the modified form has reduced or no activity compared to the unmodified form, thereby having an enhanced resistance to the at least one fungus.

According to additional aspect, the present invention provides a genetically engineered plant having enhanced resistance to at least one fungus and/or Oomycete compared to a control plant, the genetically engineered plant comprises at least one cell having modified expression and/or activity of at least one polypeptide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous, or identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-506 and 2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof compared to the polypeptide expression and/or activity in a control plant. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having modified expression and/or activity of at least one polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327-359, 361-508 and 2635, 2444-2634, 2636-4252. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having modified expression of a polynucleotide encoding the at least one polypeptide.

According to additional aspect, the present invention provides a genetically engineered plant having enhanced resistance to at least one fungus and/or Oomycete compared to a control plant, the genetically engineered plant comprises at least one cell having modified expression of at least one polynucleotide at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79% or more homologous to a polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs:783, 592-782, 784-2443; or at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more homologous, or identical to a polynucleotide having an nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, 81-301 and 783, 592-782, 784-2443 compared to the polynucleotide expression and/or activity in a control plant. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the genetically engineered plant comprises at least one cell having modified expression of at least one polynucleotide having the nucleic acid sequence selected from the group consisting of SEQ ID NOs:40, 80, 22-39, 41-79, 81-305 and 783, 592-782, 784-2443. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, modified expression/and or activity of a polypeptide or polynucleotide encoding same comprises enhanced expression and/or activity. According to certain embodiments, modified expression/and or activity of a polypeptide or polynucleotide encoding same comprises reduced expression and/or activity.

According to certain embodiments, the genetically engineered plant having enhanced resistance to the at least one fungus and/or Oomycete comprises at least one cell with enhanced expression and/or activity of at least one polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:326, 360, 308-325, 327, 329-347, 349-359, 362-506 and 2635, 2444-2634, 2636-4252. According to certain exemplary embodiments, the polypeptide expression and/or activity in the genetically engineered plant is enhanced in comparison to the polypeptide expression and/or activity in a control plant.

According to certain embodiments, the genetically engineered plant having enhanced resistance to the at least one fungus and/or Oomycete comprises at least one cell with reduced expression and/or activity of at least one polypeptide at least 80% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:328, 361 and 348. According to certain exemplary embodiments, the polypeptide expression and/or activity in the genetically engineered plant is reduced in comparison to the polypeptide expression and/or activity in a control plant.

According to certain embodiments, the genetically engineered plant having reduced expression and/or activity of the at least one polypeptide comprises at least one cell having reduced expression of a polynucleotide encoding said at least one polypeptide, thereby having an enhanced resistance to the at least one fungus.

According to certain embodiments, the genetically engineered plant comprises a polynucleotide encoding a modified form of the at least one polypeptide, wherein the modified form has reduced or no activity compared to the unmodified form, thereby having an enhanced resistance to the at least one fungus.

Once expressed within the plant cell or the entire plant, the level of the polypeptide encoded by the exogenous polynucleotide can be determined by methods well known in the art such as, activity assays, Western blots using antibodies capable of specifically binding the polypeptide, Enzyme-Linked Immuno Sorbent Assay (ELISA), radio-immuno-assays (RIA), immunohistochemistry, immunocytochemistry, immunofluorescence and the like.

Methods of determining the level in the plant of the RNA transcribed from the exogenous polynucleotide are well known in the art and include, for example, Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR) analysis (including quantitative, semi-quantitative or real-time RT-PCR) and RNA-in situ hybridization.

The sequence information and annotations uncovered by the present teachings can be harnessed in favor of classical breeding. Thus, sub-sequence data of those polynucleotides described above, can be used as markers for marker assisted selection (MAS), in which a marker is used for indirect selection of a genetic determinant or determinants of a trait of interest (e.g., biomass, growth rate, oil content, yield, abiotic stress tolerance, water use efficiency, nitrogen use efficiency and/or fertilizer use efficiency). Nucleic acid data of the present teachings (DNA or RNA sequence) may contain or be linked to polymorphic sites or genetic markers on the genome such as restriction fragment length polymorphism (RFLP), microsatellites and single nucleotide polymorphism (SNP), DNA fingerprinting (DFP), amplified fragment length polymorphism (AFLP), expression level polymorphism, polymorphism of the encoded polypeptide and any other polymorphism at the DNA or RNA sequence.

Examples of marker assisted selections include, but are not limited to, selection for a morphological trait (e.g., a gene that affects form, coloration, male sterility or resistance such as the presence or absence of awn, leaf sheath coloration, height, grain color, aroma of rice); selection for a biochemical trait (e.g., a gene that encodes a protein that can be extracted and observed; for example, isozymes and storage proteins); selection for a biological trait (e.g., pathogen races or insect biotypes based on host pathogen or host parasite interaction can be used as a marker since the genetic constitution of an organism can affect its susceptibility to pathogens or parasites).

The polynucleotides and polypeptides described hereinabove can be used in a safe and cost-effective manner in a wide range of economical plants, exemplary species of which are described hereinabove.

It will be appreciated that some genes involved in a plant defense mechanism conferring resistance to a particular fungus species may also be involved in resistance to other species, regulated by the same or homologous genes. Of course, the overall defense mechanism is related, not identical, and therefore not all genes involved in resistance to one pathogen will confer resistance to other pathogens. Nonetheless, if a gene confers or enhances resistance to one of the pathogen species, it would be apparent to one skilled in the art to test for resistance to other pathogens, specifically to pathogen of the same genus or that cause similar symptoms.

According to certain embodiments, the fungus and/or Oomycete is selected from, but not limited to, Fusarium verticillioides; Fusarium graminearum; Collotetrichum graminicola; Fusarium avenaceum; Fusarium culmorum; Fusarium oxysporum; Fusarium roseum; Fusarium semitectum; Fusarium solani; Fusarium verticillioides; Fusarium verticillioides var. subglutinans; Acremonium strictum; Albugo candida; Albugo tragopogonis; Alternaria alternate; Alternaria brassicae; Alternaria helianthi; Alternaria zinnia; Aphanomyces euteiches; Ascochyta sorghina; Ascochyta tritici; Aspergillus flavus; Bipolaris maydis O; Bipolaris sorghicola; Bipolaris sorokiniana; Botrytis cinerea; Cephalosporium acremonium; Cephalosporium gramineum; Cephalosporium maydis; Cercospora kikuchii; Cercospora medicaginis; Cercospora sojina; Cercospora sorghi; Cladosporium herbarum; Clavibacter michiganense subsp. Nebraskense; Clavibacter michiganese subsp. Insidiosum; Claviceps purpurea; Claviceps sorghi; Cochliobolus heterostrophus; Colletotrichum dematium (Colletotichum truncatum); Colletotrichum trifolii; Colletotrichum sublineolum; Corn stunt spiroplasma; Corynespora cassiicola; Curvularia inaequalis; Curvularia lunata; Curvularia pallescens; Diaporthe phaseolorum var. caulivora; Diaporthe phaseolorum var. sojae (Phomopsis sojae); Diplodia macrospora; Erwinia carotovora; Erwinia carotovorum pv. Carotovora; Erwinia chrysanthemi pv. Zea; Erwinia stewartii; Erysiphe cichoracearum; Erysiphe graminis fsp. tritici; Exserohilum turcicum I, II & III; Gaeumannomyces graminis var. tritici; Gibberella zeae (Fusarium graminearum); Gloeocercospora sorghi; Glomerella glycines; Helminthosporium carbonum I, II & III (Cochliobolus carbonum); Helminthosporium pedicellatum; Helminthosporium sorghicola; Kabatiella maydis; Leptosphaeria maculans; Leptosphaerulina briosiana; Leptotrichila medicaginis; Macrophomina phaseolina; Microsphaera diffusa; Mycosphaerella brassicicola; Nigrospora oryzae; Penicillium oxalicum; Perconia circinata; Peronosclerospora maydis; Peronosclerospora philippinensis; Peronosclerospora sacchari; Peronosclerospora sorghi; Peronospora manshurica; Peronospora parasitica; Peronospora trifoliorum; Phakopsora pachyrhizi; Phialophora gregata; Phoma insidiosa; Phoma macdonaldii; Phoma medicaginis var. medicaginis; Phomopsis helianthi; Phyllachara sacchari; Phyllosticta maydis; Phyllosticta sojicola; Physoderma maydis; Physopella zeae; Phytophthora cryptogea; Phytophthora megasperma; Phytophthora megasperma fsp. Glycinea; Plasmopora halstedii; Pseudocercosporella herpotrichoides; Pseudomonas andropogonis; Pseudomonas avenae; Pseudomonas avenae (Pseudomonas alboprecipitans); Pseudomonas syringae p.v. atrofaciens; Pseudomonas syringae p.v. glycinea; Pseudomonas syringae p.v. syringae; Pseudopeziza medicaginis; Puccinia graminis fsp. tritici; Puccinia helianthi; Puccinia polysora; Puccinia purpurea; Puccinia recondita fsp. tritici; Puccinia sorghi; Puccinia striiformis; Pyrenophora tritici-repentis; Pythium aphanidermatum; Pythium arrhenomanes; Pythium debaryanum; Pythium gramicola; Pythium graminicola; Pythium irregular; Pythium splendens; Pythium ultimum; Ramulispora sorghi; Ramulispora sorghicola; Rhizoctonia cerealis; Rhizoctonia solani; Rhizopus arrhizus; Rhizopus oryzae; Rhizopus stolonifera; Sclerophthona macrospora; Sclerospora graminicola; Sclerotinia sclerotiorum; Sclerotinia trifoliorum; Sclerotium rolfsii; Septoria avenae; Septoria glycines; Septoria helianthi; Septoria nodorum; Septoria tritici; Exserohilum turcicum; Sphacelotheca cruenta; Sporisorium reilianum (Sphacelotheca reiliana); Sporisorium sorghi; Stagonospora meliloti; Stemphylium alfalfa; Stemphylium botryosum; Stemphylium herbarum; Stenocarpella maydi (Diplodia maydis); Tilletia indica; Tilletia laevis; Tilletia tritici; Trichoderma viride; Urocystis agropyri; Uromyces striatus; Ustilago maydis; Ustilago tritici; Verticillium albo-atrum; Verticillium dahlia; Xanthomonas campestris p.v. alfalfa; Xanthomonas campestris p.v. holcicola; Xanthomonas campestris p.v. phaseoli; and Xanthomonas campestris p.v. translucens. Each possibility represents a separate embodiment of the present invention.

Specific pathogenic fungi or Oomycetes are known to cause dramatic crop lose due to disease symptoms which negatively affect the quality of the crop. For example, Fusarium verticilloides and Fusarium graminearum cause rot in maize (specifically stalk rot), wheat, sweet paper, eggplants and head blight is wheat. Fusarium oxysporum causes sudden death syndrome (SDS) in soybeans, yellow spots in sugar beet, Panama disease in Banana, and wilt in tomato, sweet pepper, eggplants, potatoes and various plant of the Cucurbitaceae family. Colletotrichum spp. cause stalk rot in maize, anthracnose in sugar beet, tomato and sweet pepper. Botrytis cinerea causes gray mold in tomato, sweet pepper, eggplants and potato. Rust is caused by Puccinia spp. in maize, wheat and sunflower, by Uromyces spp. in sunflower and by Phakopsora in soybean. Phytophthora causes root rot in soybean, late blight in tomato and potato, blight in eggplant and blight fruit rot in sweet pepper. Mycosphaerella graminicola causes leaf blotch in wheat. Mycosphaerella fijiensis causes black leaf streak disease (BLSD; aka black Sigatoka leaf spot) in banana. Septoria lycopersici causes leaf spots in tomato. Verticillium spp. cause wilt disease in canola, sugar beet, tomato, sweet pepper, eggplant and potato. Magnaporthe oryza causes rice blast. Pythium spp. cause damping off disease in maize, soybean, tomato, sweet pepper, eggplant and potato and black vessels in sugar beet. Sclerotinia causes stem rot in soybean and white mold in tomato, sweet pepper, eggplant and potato. Rhizoctonia solani causes root crown rot in sugar beet, sheath blight in rice, and damping off disease in tomato, sweet pepper, eggplant and potato. Maize smut is caused by Ustilago maydis. Alternaria spp. cause leaf spots in sugar beet and sweet pepper, early blight in tomato and potato, and fruit rot in sweet pepper and eggplants. Cercospora causes leaf blight in soybean and leaf spots in sugar beet, sweet pepper, eggplants and potato. Macrophomina causes charcoal rot in maize, wheat, soybean, tomato and potato. Sclerotium rolfsii causes Southern blight in sweet pepper and eggplants. Oidium spp. cause powdery mildew in tomato, sweet pepper, eggplants and potato. Powdery mildew is also caused by Blumeria graminis.

Methods for identifying symptoms caused by various fungi and Oomycetes upon infection of specific plant species, and for measuring the degree of the plant susceptibility/resistance to the infection are well known to those skilled in the art.

The term “plant” as used herein encompasses a whole plant, a grafted plant, ancestor(s) and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), rootstock, scion, and plant cells, tissues and organs. The plant or part thereof may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia villosa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypefjhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonafjhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize, wheat, barley, rye, oat, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Alternatively, algae and other non-Viridiplantae can be used for the methods of the present invention.

According to some embodiments, the plant used according to the teachings of the present invention is a crop plant such as maize, rice, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, Leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, poplar and cotton. According to certain exemplary embodiments, the crop plant is maize (Zea mays).

According to some embodiment, the plant used according to the teachings of the present invention is a field crop plant selected from the group consisting of tomato, potato, sweet potato, cassava, beets, ginger, horseradish, radish, ginseng, turnip, any root or tuber crop, pepper, eggplant, ground cherry, tomatillo, okra, other fruiting vegetables, cucumber cantaloupe, melon, muskmelon, squash, watermelon and other cucurbit plants.

According to some embodiments of the invention the plant is a dicotyledonous plant.

According to some embodiments of the invention the plant is a monocotyledonous plant.

According to some embodiments the present invention provides a plant cell expressing the exogenous polynucleotide of some embodiments of the invention, the nucleic acid construct comprising the exogenous polynucleotide of some embodiments of the invention and/or the polypeptide of some embodiments of the invention.

According to a further aspect, the present invention provides a fungicidal composition comprising an effective amount of at least one polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof, wherein the fungicidal composition is effective in inhibiting the growth and/or development of at least one plant pathogenic fungi and/or oomycetes. According to certain embodiments, the fungicidal composition comprises at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 4468, 4661, 2444-2634, 2636-4252, 4450-4467, 4469-4660, and 4662-4799. According to some embodiments, the fungicidal composition comprises at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:4468, 4661, 4450-4467, 4469-4660, and 4662-4799.

According to yet additional aspect, the present invention provides a fungicidal composition comprising bacteria comprising at least one polypeptide at least 70% identical to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 2444-2634, 2636-4252, a fragment and/or a variant thereof, wherein the fungicidal composition is effective in inhibiting the growth and/or development of at least one plant pathogenic fungi and/or oomycetes. According to certain embodiments, the bacteria comprise at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2635, 4468, 4661, 2444-2634, 2636-4252, 4450-4467, 4469-4660, and 4662-4799. According to some embodiments, the bacteria comprise at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:4468, 4661, 4450-4467, 4469-4660, and 4662-4799. Each possibility represents a separate embodiment of the present invention. The bacteria can be of the same species or of different species, with the proviso that the bacteria have no adverse effect on the plant. The bacteria can be alive or dead. Any bacteria suitable for expression of exogenous genes and not having negative effects on plant can be used (see, e.g. Billman-Jacobe H. “Expression in bacteria other than E. coli 1996. Curr. Opiniom in Biotech 7:500). According to certain exemplary embodiments, the bacteria are selected from the group consisting of Bacillus subtilis, Lactococcus lacti, Corynebacterium glutamicum; and Bacillus brevis. Particular example is Bacillus Expression systems Lonza XS™ ToolBox.

According to certain embodiments, the fungicidal composition further comprises at least one agriculturally compatible carrier or diluent.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods and Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

Section I: Plant Genes Associated with Fungal Resistance

Example 1: Identification of Plant Genes Associated with Fungal Resistance

The inventors of the present invention have identified polynucleotides related to resistant to fungal infection, particularly to infection by Fusarium verticillioides, Fusarium graminearum or Colletotrichum graminicola. Modulating the expression of the polynucleotide in plants can increase the plant resistance to fungal infection.

The nucleotide sequence datasets used for the polynucleotide identification were originated from publicly available databases as well as from Applicant proprietary sequencing data obtained using the Solexa technology (form e.g. wheat, maize and sorghum). Sequence data from 200 different plant species was introduced into a single, comprehensive database. The information used to build the datasets included gene expression levels, protein annotation, enzymatic activity and involvement in biosynthetic pathways.

Major databases used included:

Genomic Databases

Arabidopsis genome [TAIR genome version 6 (arabidopsis.org/)];

Rice genome [IRGSP build 4.0 (rgp.dna.affrc.go.jp/IRGSP/)];

Poplar [Populus trichocarpa release 1.1 from JGI (assembly release v1.0) (genome.jgi-psf.org/)];

Brachypodium [JGI 4x assembly, brachpodium.org)];

Soybean [DOE-JGI SCP, version Glyma0 (phytozome.net/)];

Grape [French-Italian Public Consortium for Grapevine Genome Characterization grapevine genome (genoscope.cns.fr/)];

Castorbean [TIGR/J Craig Venter Institute 4x assembly [msc.jcvi.org/r communis];

Sorghum [DOE-JGI SCP, version Sbil [phytozome.net/)]; and

Maize “B73” [DOE-JGI SCP, version AGPv2 [phytozome.net/)];

Databases of Expressed Sequence Tag (EST) and mRNA Sequences:

GenBank ncbi.nlm.nih.gov/dbEST;

RefSeq (ncbi.nlm.nih.gov/RefSeq/);

TAIR (arabidopsis.org/);

Protein and Pathway Databases:

Uniprot [uniprot.org/];

AraCyc [arabidopsis.org/biocyc/index.jsp];

ENZYME [expasy.org/enzyme/];

Microarray datasets were downloaded from: GEO (ncbi.nlm.nih.gov/geo/); TAIR (arabidopsis.org/); and Applicant proprietary microarray data as described in PCT Patent Application Publication No. WO 2008/122980.

QTL (quantitative trait loci) and SNPs information was retrieved from Gramene [gramene.org/qtl/]; and Panzea [panzea.org/index.html].

Database Assembly

Database assembly was performed to build a wide, rich, reliable annotated and easy to analyze database. The assembly comprised data retrieved from publicly available genomic sequences, mRNA sequences, ESTs DNA sequences and QTL data, as well as information regarding gene expression, protein annotation, and involvement in biosynthesis pathway, all in various plant types. The assembly further comprised data retrieved from Applicant proprietary databases produced from various plant types including genomic sequences, mRNA sequences, expression, proteomic and metabolomic data, QTL and GWAS (genome-wide association studies) data.

Database assembly is comprised of a toolbox of gene refining, structuring and annotation as well as analysis tools enabling to construct a tailored database for each gene-discovery project. Gene refining and structuring tools enable to reliably detect splice variants and antisense transcripts, and understand various potential phenotypic outcomes of a single gene. The capabilities of the “LEADS” platform of Compugen LTD. for analyzing human genome have been confirmed and accepted by the scientific community [see e.g., “Widespread Antisense Transcription”, Yelin, et al. (2003) Nature Biotechnology 21, 379-85; “Splicing of Alu Sequences”, Lev-Maor, et al. (2003) Science 300 (5623), 1288-91; “Computational analysis of alternative splicing using EST tissue information”, Xie H et al. Genomics 2002], and have been proven most efficient in plant genomics as well.

EST Clustering and Gene Assembly

For gene clustering and assembly of organisms with available genome sequence data (Arabidopsis, rice, castorbean, grape, Brachypodium, poplar, soybean, sorghum, maize) the genomic LEADS version (GANG) was employed. This tool allows most accurate clustering of ESTs and mRNA sequences on genome, and predicts gene structure as well as alternative splicing events and antisense transcription.

For organisms with no available full genome sequence data, “expressed LEADS” clustering software was applied.

Gene Annotation

Predicted genes and proteins were annotated as follows: BLAS™ search [blast.ncbi.nlm.nih.gov/Blast.cgi] against all plant UniProt [uniprot.org/] sequences was performed. Open reading frames (ORFs) of each putative transcript were analyzed and longest ORF with highest number of homologues was selected as a predicted protein of the transcript. The predicted proteins were analyzed by InterPro [ebi.ac.uk/interpro/].

BLAS™ against proteins from AraCyc and ENZYME databases was used to map the predicted transcripts to AraCyc pathways.

Predicted proteins from different species were compared using BLAS™ algorithm (ncbi.nlm.nih.gov/Blast.cgi) to validate the accuracy of the predicted protein sequence, and for efficient detection of orthologs.

Gene Expression Profiling

Several data sources were exploited for gene expression profiling, namely microarray data and digital expression profile (see below). Genes were analyzed for expression patterns in different plant species and varieties. The analysis was based on differential expression under uninfected and infected conditions, wherein the infection was induced by different pathogens and the gene expression was measured in different plant organs and at different time points along the disease development. Publicly available microarray datasets were downloaded from TAIR and NCBI GEO sites, renormalized, and integrated into the database. Expression profiling is one of the most important resource data for identifying genes related to disease resistance.

A digital expression profile summary was compiled for each gene cluster according to all keywords included in the sequence records comprising the cluster. Digital expression, also known as electronic Northern Blot, is a tool that displays virtual expression profile based on the expressed sequence tag (EST) sequences forming the gene cluster. The tool provides the expression profile of a cluster in terms of plant anatomy (e.g., the tissue/organ in which the gene is expressed), developmental stage (the developmental stages at which a gene can be found) and profile of treatment (provides the physiological conditions under which a gene is expressed such as drought, cold, pathogen infection, etc.). Given a random distribution of ESTs in the different clusters, the digital expression provides a probability value that describes the probability of a cluster having a total of N ESTs to contain X ESTs from a certain collection of libraries. For the probability calculations, the following is taken into consideration: a) the number of ESTs in the cluster, b) the number of ESTs of the implicated and related libraries, c) the overall number of ESTs available representing the species. Thereby clusters with low probability values are highly enriched with ESTs from the group of libraries of interest indicating a specialized expression.

Recently, the accuracy of this system was demonstrated by Portnoy et al., 2009 (Analysis of The Melon Fruit Transcriptome Based on 454 Pyrosequencing, in: Plant & Animal Genomes XVII Conference, San Diego, Calif.). Transcriptomic analysis, based on relative EST abundance in data was performed by 454 pyrosequencing of cDNA representing mRNA of the melon fruit. Fourteen double strand cDNA samples obtained from two genotypes, two fruit tissues (flesh and rind) and four developmental stages were sequenced. GS FLX pyrosequencing (Roche/454 Life Sciences) of non-normalized and purified cDNA samples yielded 1,150,657 expressed sequence tags that assembled into 67,477 unigenes (32,357 singletons and 35,120 contigs). Analysis of the data obtained against the Cucurbit Genomics Database [icugi.org/] confirmed the accuracy of the sequencing and assembly. Expression patterns of selected genes fitted well their qRT-PCR data.

The produced datasets were used for further comparative analyses of gene expression in various plant species and lines in response to fungal infection.

Example 2: Production of Plant Transcriptomes for Discovery of Genes Correlating with Resistance to the Fungal Infection Experimental Procedures

The association of gene expression with fungal infection was examined in Brachypodium distachyon, Aegilops spp., Setaria italica, Triticum aestivum and Zea mays.

The following pathogenic fungi were used: Fusarium verticillioides (hereinafter F. verticillioides or Fv). A GFP transformant of F. verticillioides strain A-00149-FGSC 7600 (Oren et al., 2003, Appl. Environ. Microbiol. 69:1695) was used throughout the data generation and validation experiments; and Fusarium graminearum (hereinafter F. graminearum or Fg).

Fungi Infection

Fungal infection of plant roots: this method was used for inoculating plant root with F. verticillioides (Fv) and Fusarium graminearum (Fg). Sterilized seeds from tolerant and sensitive lines of the examined plant were germinated on water agar supplemented with Ampicillin (100 μg/ml) and inoculated after 6 days with an agar plug (1 cm diameter) covered with one-week old fungal mycelium applied to the root site (1 cm below the seed) for 2 hours four days after germination. Control plants were mock inoculated with an empty plug.

RNA extraction was performed using TRIzol Reagent from Invitrogen (invitrogen.com/content.cfm?pageid=469). Approximately 30-50 mg of sample tissue was taken for the analysis. The weighed tissues were ground using pestle and mortar in liquid nitrogen and resuspended in 500 μl of TRIzol Reagent. To the homogenized lysate, 100 μl of chloroform were added followed by precipitation using isopropanol and two washes with 75% ethanol. The RNA was eluted in 30 μl of RNase-free water. RNA samples were cleaned up using Qiagen's RNeasy minikit clean-up protocol as per the manufacturer's protocol (QIAGEN Inc, CA USA).

RNA was extracted from tissues of the infected and mock plants obtained from each treatment as follows:

Direct root infection—Root and basal stem tissues from plants growing under normal or fungal infection conditions were sampled at 6, 24 and 72 hours post infection (hpi) and RNA was extracted as described hereinabove.

Identification and Validation of Gene Associated with Fungal Infection

In order to discover gene expressed in the examined plant species and plant lines with fungal infection, the present inventors utilized available micro-arrays as described in details hereinbelow for each plant species examined. To define correlations between the levels of RNA expression and fungal resistance, parameters related to plant response to fungal infection were analyzed under normal and infected conditions. From plant identified as encompassing variance in the resistance or susceptibility spectrum, hybrids were selected for further association analysis between fungal infection and gene expression after the plants were challenged with F. verticillioides or F. graminearum as described hereinabove.

Fungal infection was phenotyped as follows:

Plants infected via the direct root infection were phenotyped (24 and 72 hpi) for fungal recovery from root and stem tissues by culturing sterilized explants on PDA for 4-5 days and validating the presence of the Fv-GFP strain. For plants infected with Fg, development of necrosis on the roots was monitored along 7 days.

Example 2.1: Production of B. distachyon Plant Transcriptomes

The association of gene expression in Brachypodium lines with fungal infection was investigated utilizing a Brachypodium oligonucleotide micro-array, produced by Agilent Technologies [chem.agilent.com/Scripts/PDS.asp?lPage=50879]. The array oligonucleotide represents about 27,500 Brachypodium genes and transcripts. To define correlations between the levels of RNA expression and fungal resistance, parameters related to responses to fungal infection were analyzed in 30 different Brachypodium lines under normal and infected conditions as described hereinabove. Among them, 5 lines encompassing susceptibility to F. graminearum (designated as ““sensitive”, Table 1) were selected for RNA expression analysis after challenge with F. verticillioides or F. graminearum as described hereinabove.

TABLE 1 Brachypodium varieties used for production of transcriptomic data and their phenotypic response to F. graminearum infection Response to Variety F. graminearum Bd20-1 Sensitive Bd25-1 Sensitive PI254868 Sensitive Bd9-1 Sensitive Bd26-1 Sensitive

Example 2.2: Production of Setaria italica Transcriptome for Discovery of Gene Correlating with Resistance to the Fungal Infection

The association of gene expression in Setaria italica lines with fungal infection was investigated utilizing a Setaria italica oligonucleotide micro-array, produced by Agilent Technologies [chem.agilent.com/Scripts/PDS.asp?lPage=50879]. The array oligonucleotide represents about 27,500 Setaria italica genes and transcripts designed based on data from Public databases (Example 1). To define correlations between the levels of RNA expression and fungal resistance related parameters in responses to fungal infection various plant characteristics of 30 different Setaria italica hybrids were analyzed under normal and infected conditions as described hereinabove. Among them, 5 hybrids encompassing medium resistance to F. graminearum (designated as “medium resistant”, Table 2) were selected for RNA expression analysis after challenge with F. verticillioides or F. graminearum

TABLE 2 Setaria italica varieties used for production of transcriptomic data and their phenotypic response to F. graminearum infection Response to Variety F. graminearum PI231736 Medium resistant PI473596 Medium resistant PI253494 Medium resistant PI269971 Medium resistant Ames27267 Medium resistant

Example 2.3: Production of Aegilops spp. Transcriptome

In order to define correlations between the levels of RNA expression with fungal resistance related parameters, responses to fungal infection of 30 different Aegilops varieties were analyzed under normal and infected conditions as described hereinabove. Among them, 5 hybrids encompassing resistance to F. graminearum (designated resistant”, Table 3) were selected for RNAseq expression analysis (Illumina) after challenge with F. verticillioides or F. graminearum.

TABLE 3 Aegilops varieties used for production of transcriptomic data and their phenotypic response to F. garminearum infection Response to Species Variety F. graminearum Aegilops geniculata Coll3-III Resistant Aegilops geniculata Coll3-VI Resistant Aegilops tauschii L84 TQ20 Resistant Aegilops tauschii TQ106 Resistant Aegilops sharonensis 6118VI Resistant

Example 2.4: Production of Maize Transcriptome

The association of gene expression in Maize lines with fungal infection was investigated utilizing a Maize oligonucleotide micro-array, produced by Agilent Technologies [chem.agilent.com/Scripts/PDS.asp?lPage=50879]. The array oligonucleotide represents about 60K Maize genes and transcripts designed based on data from Public databases (Example 1). To define correlations between the levels of RNA expression and fungal resistance related parameters responses to fungal infection of various plant characteristics of 30 different Maize hybrids were analyzed under normal and infected conditions as described hereinabove. Among them, 6 hybrids encompassing variance in the resistance spectrum to F. verticillioides or C. graminicola (designated as “tolerant” and “sensitive”, Table 4) were selected for RNA expression analysis after challenge with F. verticillioides or F. graminearum

TABLE 4 Maize varieties used for production of transcriptomic data and their phenotypic response to F. verticillioides infection Response to Variety F. verticillioides 32W86 Tolerant Klips Sensitive W182E Tolerant B84 Sensitive NC350 Tolerant Ky WS4 Sensitive

Example 2.5: Production of Wheat Transcriptome

The association of gene expression in wheat lines to fungal infection was investigated utilizing a wheat oligonucleotide micro-array, produced by Agilent Technologies [chem.agilent.com/Scripts/PDS.asp?lPage=50879]. The array oligonucleotide represents about 50,000 wheat genes and transcripts.

In order to define correlations between the levels of RNA expression with fungal resistance related parameters, responses to fungal infection of 30 different wheat varieties were analyzed under normal and infected conditions as described hereinabove. Among them, 6 hybrids encompassing variance in the resistance spectrum to F. verticillioides (designated as “tolerant” and “sensitive”, Table 5) were selected for RNA expression analysis after challenge with F. verticillioides or F. graminearum.

TABLE 5 Wheat varieties used for production of transcriptomic data and their phenotypic response to F. verticillioides infection Response to Variety F. verticillioides Aurora Sensitive Precoce Tolerant Barani Sensitive N46 Tolerant Bobwhite Sensitive Thacher Tolerant

Differential Expression Analysis

The analysis was preformed via proprietary differential expression algorithm.

The default query parameters used were: >2 fold change, p value<0.01, FDR <0.5 (FDR=false discovery rate). Stringency varied due to specific experimental context. The following queries were performed across species (aggregated through the use of proprietary ortholog determination), germplasm, organs, types of pathogens treated, and time post infection:

    • 1. Up regulation upon infection: the gene's expression level is higher in infected samples than in mock controls (both resistant and susceptible lines are queried).
    • 2. Stronger expression induction in resistant lines: the gene's expression induction is higher in resistant than in susceptible lines upon infection.
    • 3. Higher basal expression in resistant lines: the gene's expression is higher in resistant than in susceptible lines in uninfected samples.

No type of query is necessary nor sufficient but overall enrichment of positive indications is considered to identify genes significantly qualifying the above criteria

Results

The genes identified using the above differential expression analyses and the indications found per gene are described hereinbelow:

LFS90

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hours post infection—hpi).

Wheat—The wheat gene ortholog was upregulated in the roots and stem in response to Fv at early infection stages (6 and 72 hpi, respectively), and in the inflorescence in response to Fusarium graminearum (Fg) at early infection stages (30 and 50 hpi).

LFS91

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi).

Barley—The barley gene ortholog was upregulated at both early and late stages following inflorescence infection with Fusarium graminearum (Fg) or challenging assays with the mycotoxin Deoxynivalenol (DON) 1, 2, 3, 4- and 6-days post infection (dpi).

Brachypodium—The Brachypodium ortholog was upregulated in the roots of several genotypes in response to Fg at early infection stages (48 hpi).

Maize—The maize gene ortholog was upregulated in response to Colletotrichum graminicola (Cg) at early infection stage (120 hpi).

Sorghum—The sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (6, 24 and 72 hpi).

Wheat—The wheat ortholog was upregulated in the inflorescence in response to Fg at early infection stages (50 hpi). QTL—The rice ortholog mapped to a QTL for blast disease resistance.

LFS92

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi).

Wheat—The wheat ortholog was upregulated in the inflorescence in response to Fusarium graminearum (Fg) at early infection stages (50 hpi).

Maize—The maize ortholog was upregulated in the inflorescence in response to Fv at early infection stages (72 and 96 hpi). In addition, the ortholog was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early infection stages (96 and 120 hpi).

QTL—The soybean ortholog mapped to a QTL for fungal disease resistance.

LFS93

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi).

QTL—The maize ortholog mapped to QTLs for Fusarium ear rot resistance and for resistance to contamination with the mycotoxin fumonisin.

LFS94

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi).

Wheat—The wheat ortholog was upregulated in the roots of resistant genotypes in response to Fv at early infection stages (1.5 and 2.5 hpi) and in the inflorescence in response to Fusarium graminearum (Fg) at early infection stages (30 and 50 hpi)

LFS95

Brachypodium—The Brachypodium ortholog gene was upregulated following spikes inoculation with Fusarium graminearum (Fg) (96 hpi).

Wheat—The wheat ortholog was upregulated in the roots of resistant genotypes in response to Fv at early infection stages (1.5 and 2.5 hpi) and in the inflorescence in response to Fusarium graminearum (Fg) at early infection stages (30 and 50 hpi).

Maize—The maize ortholog was upregulated in the inflorescence in response to Fv at early infection stages (96 hpi). In addition, the ortholog was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early infection stages (96 and 120 hpi).

LFS96

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv), and in several genotypes also to Fusarium graminearum (Fg) at early infection stage (6 and 24 hpi).

Maize—The maize ortholog was upregulated in the inflorescence in response to Fv at early infection stages (72 and 96 hpi). In addition, the ortholog was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early infection stages (72, 96 and 120 hpi).

QTL—The rice ortholog mapped to a QTL for blast disease resistance.

LFS97

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi). In addition, an upregulation in several genotypes was observed in the roots in response to Fusarium graminearum (Fg) at early infection stages (6 and 24 hpi).

Barley—The barley ortholog was upregulated at early and late stages following inflorescence infection with Fg (72, 96 and 144 hpi) or challenging assays with the mycotoxin Deoxynivalenol (DON) (12 and 24 hpi).

Wheat—The wheat ortholog was upregulated in the inflorescence in response to Fg at early infection stages (30 and 50 hpi).

Maize—The maize ortholog was upregulated in the inflorescence in response to Fv at early infection stage (72 hpi). In addition, the gene was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early infection stage (120 hpi) and was upregulated in the inflorescence following Cg infection (72 hpi).

Sorghum—The sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (2.5 and 6 hpi).

QTL—The maize ortholog mapped to a QTL for resistance to the mycotoxin fumonisin contamination. The Sorghum ortholog mapped to a QTL for rust resistance.

LFS98

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hours post infection—hpi). In addition, an upregulation in several genotypes was observed in the roots, in response to Fusarium graminearum (Fg) at early infection stages (24 hpi).

Maize—The maize ortholog was upregulated in the roots in response to Fv and Fg at early infection stages (6 hpi and 72 hpi, respectively). In addition, the ortholog was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early infection stage (96 and 120 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (24 hpi).

Wheat—The wheat ortholog was upregulated in the roots in response to Fv at late infection stages (10 days post infection—dpi), and in the inflorescence in response to Fg at early infection stages (50 hpi).

QTL—The maize ortholog mapped to a QTL for Fusarium ear rot resistance. The rice ortholog mapped to a QTL for sheath blight disease resistance.

LFS99

Barley—The barley ortholog was upregulated at both early and late stages following inflorescence infection with Fusarium graminearum (Fg) or challenging assays with the mycotoxin Deoxynivalenol (DON) (1, 2, 3, 4 and 6 dpi).

Wheat—The wheat ortholog was upregulated in the roots in response to Fv at early infection stages (6 and 24 hpi). In addition, the ortholog was upregulated in the inflorescence in response to Fg at early infection stages (30 and 50 hpi).

LFS100

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hours post infection—hpi). In addition, an upregulation in several genotypes was observed in the roots, in response to Fusarium graminearum (Fg) at early infection stages (6 and 24 hpi).

Wheat—The wheat ortholog was upregulated in the roots of resistant genotypes in response to Fv at early infection stages (6 and 24 hpi) and in the inflorescence in response to Fg at early infection stages (30 and 50 hpi).

Brachypodium—The Brachypodium ortholog was upregulated in the roots at early stages of both Fv and Fg infections (6 and 24 hpi).

Barley—The barley ortholog was upregulated at both early and late stages following inflorescence infection with Fg or challenging assays with the mycotoxin Deoxynivalenol (DON) (1, 2, 3, 4 and 6 dpi).

QTL—The rice ortholog mapped to a QTL for blast disease resistance

LFS102

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hours post infection—hpi).

Wheat—The wheat ortholog was upregulated in the inflorescence in response to Fg at early infection stages (30 and 50 hpi).

Barley—The barley ortholog was upregulated at late stages following inflorescence infection with Fg (3, 4- and 6-days post infection—dpi) or at early stage following challenging assays with the mycotoxin Deoxynivalenol (DON) (2 and 4 dpi).

Maize—The maize ortholog was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early and late infection stages (120 hpi) and was upregulated in the inflorescence following Cg infection (4 and 6 dpi). QTL—The maize ortholog mapped to a QTL for fumonisin contamination resistance.

LFS104

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi). In addition, an upregulation in several genotypes was observed in the roots, in response to Fusarium graminearum (Fg) at early infection stages (24 hpi).

Wheat—The wheat ortholog was upregulated in the roots in response to Fv at early infection stages (6 and 24 hpi) and in the inflorescence in response to Fg at early infection stages (30 and 50 hpi).

Brachypodium—The Brachypodium ortholog was moderately upregulated following spikes inoculation with Fg (96 hpi).

Barley—The barley ortholog was upregulated at both early and late stages following inflorescence infection with Fg or challenging assays with the mycotoxin Deoxynivalenol (DON) (1, 2, 3, 4- and 6-days post infection—dpi).

Maize—The maize ortholog was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early and late infection stages (120 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (6 hpi).

QTL—The maize ortholog mapped to QTLs for fumonisin contamination resistance. The rice ortholog mapped to a QTL for blast disease resistance.

LFS105

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi).

Maize—The maize ortholog was upregulated in the inflorescence in response to Fv at early infection stages (72 hpi). In addition, the ortholog was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early infection stage (120 hpi).

Wheat—The wheat ortholog was upregulated in the roots in response to Fv at early infection stages (6 and 24 hpi) and in the inflorescence in response to Fusarium graminearum (Fg) at early infection stages (30 and 50 hpi).

QTL—The rice ortholog mapped to a QTL for blast disease resistance

LFS106

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi).

Brachypodium—The Brachypodium ortholog was upregulated in the roots of several genotypes in response to Fv at early infection stage (48 hpi). In addition, the gene was upregulated following spikes inoculation with Fusarium graminearum (Fg) (96 hpi).

Wheat—The wheat ortholog was upregulated in the inflorescence in response to Fg at early infection stages (50 hpi).

Maize—The maize ortholog was upregulated in the inflorescence in response to Fv at early infection stage (72 hpi). In addition, the gene was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early infection stage (120 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (6 and 24 hpi)

LFS107

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi). In addition, in several genotypes upregulation was observed in the roots in response to Fusarium graminearum (Fg) at early infection stages (24 hpi).

Foxtail millet (Setaria italica)—The foxtail millet ortholog was moderately upregulated in the roots in response to Fg at early infection stages (24 and 48 hpi).

Wheat—The wheat ortholog was upregulated in the roots in response to Fv at early infection stages (6 hpi) and in the inflorescence in response to Fg at early infection stages (30 and 50 hpi).

QTL—The rice ortholog mapped to a QTL for blast disease resistance.

LFS109

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi) and upregulated moderately in the roots in response to Fusarium graminearum (Fg) at early infection stage (6 and 24 hpi).

Brachypodium—The Brachypodium ortholog was upregulated in the roots of several genotypes in response to Fg at early infection stages (48 hpi).

Maize—The maize ortholog was upregulated in the inflorescence in response to Fv at early infection stages (72 hpi). In addition, the ortholog was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early infection stage (48, 96 and 120 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (6 and 24 hpi).

LFS110

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi). In addition, an upregulation in several genotypes was observed in the roots, in response to Fusarium graminearum (Fg) at early infection stages (24 hpi).

Barley—The barley ortholog was upregulated at early infection stages following inflorescence infection with Fg (96 hpi) or challenging assays with the mycotoxin Deoxynivalenol (DON) (1 and 12 hpi).

Maize—The maize ortholog was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early infection stages (120 hpi).

QTL—The maize ortholog mapped to QTLs for Fusarium ear rot resistance. The rice ortholog mapped to a QTL for blast disease resistance. The soybean ortholog mapped to a QTL for fungal disease resistance.

LFS111

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi).

Foxtail millet (Setaria italica)—The foxtail millet ortholog was moderately upregulated in the roots of several genotypes in response to Fusarium graminearum (Fg) at early infection stages (48 hpi).

Brachypodium—The Brachypodium ortholog was moderately upregulated in the roots of several genotypes in response to Fg at early infection stages (48 hpi).

QTL—The maize ortholog mapped to QTLs for resistance to fumonisin contamination. The rice ortholog mapped to a QTL for blast disease resistance.

LFS112

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and hpi).

Foxtail millet (Setaria italica)—The foxtail millet ortholog was upregulated in the roots of several genotypes at early infection stages of Fv infection (6 and 24 hpi).

Maize—The maize ortholog was upregulated in the inflorescence in response to Fv at early infection stage (72 hpi). In addition, the gene was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early and late infection stages (120 hpi) and was upregulated in the inflorescence following Cg infection (4 and 6 dpi).

QTL—The rice ortholog mapped to a QTL for sheath blast disease resistance. The soybean ortholog is mapped to a QTL for a fungal disease resistance

LFS113

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi).

Barley—The barley ortholog was upregulated at both early and late stages following inflorescence infection with Fusarium graminearum (Fg) or challenging assays with the mycotoxin Deoxynivalenol (DON) (1, 2, 3, 4 and 6 dpi).

Foxtail millet (Setaria italica)—The foxtail millet ortholog was upregulated in the roots of several genotypes in response to Fg at early infection stages (24 and 48 hpi).

Brachypodium—The Brachypodium ortholog was upregulated in the roots of several genotypes in response to Fg at early infection stages (48 hpi).

Wheat—The wheat ortholog was upregulated in the inflorescence in response to Fg at early infection stages (30 and 50 hpi).

QTL—The Sorghum ortholog mapped to a QTL for anthracnose resistance. The rice ortholog mapped to a QTL for blast disease resistance.

LFS114

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) at early infection stage (6 and 24 hpi).

Brachypodium—The gene was upregulated in the roots in response to Fg at early infection stage (24 and 48 hpi).

Barley—The barley ortholog was upregulated at early stages following inflorescence challenging with the mycotoxin Deoxynivalenol (DON) (1 and 12 hpi).

Wheat—The wheat ortholog was upregulated in inflorescence in response to Fg at early infection stages (30 and 50 hpi).

Maize—The maize ortholog was upregulated in the roots in response to Fv at early (6 hpi) and late (6 and 14 dpi) infection stages. In addition, the ortholog was upregulated in the inflorescence in response to Fv at early infection stage (72 hpi). The ortholog was also upregulated in the roots in response to Fg at early infection stages (24 and 72 hpi). An upregulation was also observed in the leaves in response to Colletotrichum graminicola (Cg) infection (96 and 120 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (6 and 24 hpi)

LFS115

Aegilops—The gene was upregulated in the roots in response to Fusarium graminearum (Fg) at early infection stage (6 and 24 hpi).

LFS116

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi).

Brachypodium—The Brachypodium ortholog was upregulated following spikes inoculation with Fusarium graminearum (Fg) (96 hpi).

Wheat—The wheat ortholog was upregulated in the inflorescence in response to Fg at early infection stages (30 and 50 hpi).

QTL—The rice ortholog mapped to a QTL for blast disease resistance.

LFS117

Aegilops—The gene was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hours post infection—hpi).

Brachypodium—The Brachypodium ortholog was upregulated following spikes inoculation with Fusarium graminearum (Fg) (96 hpi).

Maize—The maize ortholog was upregulated in the roots in response to Fg at early infection stages (72 hpi).

QTL—The maize ortholog mapped to a QTL for Fusarium ear rot resistance

LFS121

Foxtail millet (Setaria italica)—The gene was upregulated in the roots at early stages of Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) infection (6 and 24 hpi).

Aegilops—The Aegilops ortholog was upregulated in the roots at early infection stages of Fg and Fv infection (6 and 24 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (6 and 24 hpi).

Wheat—The wheat ortholog was upregulated in the roots of resistant genotypes in response to Fv at early infection stages (6 hpi) and in the inflorescence in response to Fg at early infection stages (30 and 50 hpi).

Maize—The maize ortholog was upregulated in the leaves in response to Colletotrichum graminicola (Cg) at early infection stages (120 hpi).

QTL—The maize ortholog mapped to a QTL for Fusarium ear rot resistance.

LFS122

Foxtail millet (Setaria italica)—The gene was upregulated in the roots of several genotypes in response to Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) at early infection stages (6 and 24 hpi).

Aegilops—The Aegilops ortholog was upregulated in the roots in response to Fv at early infection stage (6 and 24 hpi)

LFS123

Foxtail millet (Setaria italica)—The gene was upregulated in the roots at early stages of Fusarium graminearum (Fg) infection (24 and 48 hpi).

LFS124

Foxtail millet (Setaria italica)—The gene was upregulated in the roots at early infection stages of both Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) infections (6 and 24 hpi).

Aegilops—The Aegilops ortholog was upregulated in the roots in response to Fv at early infection stage (6 and 24 hpi). In addition, an upregulation in several genotypes was observed in the roots, in response to Fg at early infection stages (24 hpi).

Wheat—The wheat ortholog was upregulated in the roots in response to Fv at early infection stages (24 and 72 hpi) and in the inflorescence in response to Fg at early infection stages (50 hpi).

Barley—The barley ortholog was upregulated at both early and late stages following inflorescence infection with Fg or challenging assays with the mycotoxin Deoxynivalenol (DON) (1, 2, 3, 4 and 6 dpi).

Maize—The maize ortholog was upregulated in the inflorescence in response to Fv at early infection stage (72 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (6 and 24 hpi).

QTL—The rice ortholog mapped to a QTL for blast disease resistance. The Sorghum ortholog mapped to a QTL for rust resistance.

LFS125

Foxtail millet (Setaria italica)—The foxtail millet gene was upregulated in the roots in response to Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) at early infection stages (24 hpi).

Aegilops—The Aegilops ortholog was upregulated in the roots in response to Fv at early infection stage (6 and 24 hpi).

Maize—The maize ortholog was upregulated in the inflorescence of resistant genotype in response to Fv at early infection stages (96 hpi). In addition, the ortholog was upregulated in the leaves of resistant genotype in response to Colletotrichum graminicola (Cg) at early infection stage (48 and 120 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (6 hpi).

QTL—The maize ortholog mapped to a QTL for fumonisin contamination resistance.

LFS126

Foxtail millet (Setaria italica)—The gene was upregulated in the roots at early stages of Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) infection (24 and 48 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stages (72 hpi).

QTL—The Sorghum ortholog mapped to a QTL for anthracnose resistance.

LFS127

Foxtail millet (Setaria italica)—The gene was upregulated in the roots at early stages of Fusarium graminearum (Fg) infection (24 and 48—hpi).

Brachypodium—The Brachypodium ortholog was upregulated in the roots at early stages of Fg and Fusarium verticillioides (Fv) infection (48 hpi).

Aegilops—The Aegilops ortholog was upregulated in the roots of several genotypes at early stages of Fg and Fv infection (24 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fv at late infection stages (5 dpi).

Maize—The maize ortholog was upregulated in the leaves of a resistant genotype in response to Colletotrichum graminicola (Cg) at early infection stage (120 hpi).

Wheat—The wheat ortholog was upregulated in the roots in response to Fv at early and late infection stages (6, 24 and hpi, 5 and 10 dpi). QTL—The rice ortholog mapped to a QTL for blast disease resistance. The sorghum ortholog mapped to QTLs for anthracnose resistance and for rust resistance.

LFS128

Foxtail millet (Setaria italica)—The gene was upregulated in the roots at early infection stages of Fusarium graminearum (Fg) infection (24 and 48 hpi).

Aegilops—The Aegilops ortholog was moderately upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 hpi).

Sorghum—The Sorghum ortholog was upregulated in the roots in response to Fv at early infection stages (2.5 hpi)

LFS129

Aegilops—The Aegilops ortholog was upregulated in the roots in response to Fusarium verticillioides (Fv) at early infection stage (6 and 24 hpi).

Wheat—The wheat ortholog was upregulated in the inflorescence in response to Fg at early infection stages (30 and 50 hpi).

Barley—The barley ortholog was upregulated at both early and late stages following inflorescence infection with Fg (72, 96 and 144 hpi).

Brachypodium—The Brachypodium ortholog was upregulated in the roots of several genotypes in response to Fg at early infection stage (48 hpi).

QTL—The rice ortholog mapped to a QTL for sheath blight disease resistance.

LFS130

Aegilops—Computational evidences indicate up-regulation of the gene mainly at early infection stages of Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) in roots.

LFS131 Foxtail mille (Setaria italica)—The gene was upregulated in the roots and stem in response to Fusarium verticillioides (Fv) at early infection stages.

LFS132

Maize—Computational evidences indicate upregulation of the gene in roots of sensitive lines at early infection stages of Fusarium verticillioides (Fv).

LFS82

Maize—Computational evidences indicate upregulation of the gene mainly at early infection stages of Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) in different organs Wheat—The wheat ortholog was upregulated in the roots and stem in response to Fv at early infection stages.

LFS83

Wheat—Computational evidences indicate upregulation of the gene mainly at early infection stages of Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) in different organs. Aegilops and Maize—The gene orthologs were upregulated in the roots and stem in response to Fusarium verticillioides (Fv) at early infection stages.

LFS84

Wheat—Computational evidences indicate upregulation of the gene and its Aegilops ortholog mainly at early infection stages of Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) in different organs

Maize—The gene was up regulated as a result of Colletotrichum graminicola (Cg) infection in stem tissue.

LFS86

Maize—Computational evidences indicate upregulation of the gene and its Sorghum ortholog mainly at early infection stages of Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) in different organs.

LFS87

Maize—Computational evidences indicate upregulation of the gene and its wheat ortholog mainly at early infection stages of Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) in different organs

LFS89

Wheat—Computational evidences indicate upregulation of the gene at late infection stages of Fusarium graminearum (Fg) in heads and its Aegilops ortholog mainly at early infection stages of Fusarium verticillioides (Fv) and Fusarium graminearum (Fg) roots.

Maize—Computational evidences indicate a positive correlation of the gene expression to the base diameter, stalk width.

The plant genes listed in Table 6 below were identified as candidates to have a major impact on plant resistance to at least one of Fusarium verticilloides, Fusarium graminearum and Colletotrichum graminicola when expression thereof is modulated in plants. The identified gene name, the plant from which it is derived, and the amino acid and nucleic acid sequences of each gene are summarized in Table 6, hereinbelow.

TABLE 6 Genes associated with plant resistance to fungal infection Polyn. Polyp. SEQ ID SEQ ID Gene Name Organism NO: NO: LFS82 Zea mays 22 308 LFS83 Triticum aestivum 23 309 LFS84 Triticum aestivum 24 310 LFS86 Zea mays 25 311 LFS87 Zea mays 26 312 LFS89 Triticum aestivum 27 313 LFS90 Aegilops tauschii 28 314 LFS91 Aegilops tauschii 29 315 LFS92 Aegilops tauschii 30 316 LFS93 Aegilops tauschii 31 317 LFS94 Aegilops tauschii 32 318 LFS95 Aegilops tauschii 33 319 LFS96 Aegilops tauschii 34 320 LFS97 Aegilops tauschii 35 321 LFS98 Aegilops tauschii 36 322 LFS99 Aegilops tauschii 37 323 LFS100 Aegilops tauschii 38 324 LFS102 Aegilops tauschii 39 325 LFS104 Aegilops tauschii 40 326 LFS105 Aegilops tauschii 302 510 LFS106 Aegilops tauschii 41 327 LFS107 Aegilops tauschii 42 328 LFS109 Aegilops tauschii 43 329 LFS110 Aegilops tauschii 44 330 LFS111 Aegilops tauschii 45 331 LFS112 Aegilops tauschii 303 507 LFS113 Aegilops tauschii 46 332 LFS114 Aegilops tauschii 47 333 LFS115 Aegilops tauschii 48 334 LFS116 Aegilops tauschii 49 335 LFS117 Aegilops tauschii 50 336 LFS121 Setaria italica 51 337 LFS122 Setaria italica 52 338 LFS123 Setaria italica 53 339 LFS124 Setaria italica 54 340 LFS125 Setaria italica 55 341 LFS126 Setaria italica 56 342 LFS127 Setaria italica 57 343 LFS128 Setaria italica 58 344 LFS129 Setaria italica 59 345 LFS130 Aegilops tauschii 60 346 LFS131 Setaria italica 61 347 LFS132 Zea mays 62 348 “polyn.” = polynucleotide; “polyp.” = polypeptide.

Example 3: Identification of Homologous Sequences Associated with Fungal Resistance

The concepts of orthology and paralogy have recently been applied to functional characterizations and classifications on the scale of whole-genome comparisons. Orthologs and paralogs constitute two major types of homologs: The first evolved from a common ancestor by specialization, and the latter are related by duplication events. It is assumed that paralogs arising from ancient duplication events are likely to have diverged in function while true orthologs are more likely to retain identical function over evolutionary time.

To further investigate and identify putative orthologs of the genes identified to affect plant resistance to Fusarium verticilloides, Fusarium graminearum or Colletotrichum graminicola, all sequences were aligned using the BLAST™ (Basic Local Alignment Search Tool). Sequences sufficiently similar were tentatively grouped. These putative orthologs were further organized under a Phylogram—a branching diagram (tree) assumed to be a representation of the evolutionary relationships among the biological taxa. Putative ortholog groups were analyzed as to their agreement with the phylogram and in cases of disagreements these ortholog groups were broken accordingly.

Expression data were analyzed and the EST libraries were classified using a fixed vocabulary of custom terms such as developmental stages (e.g., genes showing similar expression profile through development with up-regulation at specific stage, such as at seed germination, time points during seedling growth and at maturity) and/or plant organ (e.g., genes showing similar expression profile across their organs with up-regulation at specific organs such as roots and stems). The annotations from all the ESTs clustered to a gene were analyzed statistically by comparing their frequency in the cluster versus their abundance in the database, allowing the construction of a numeric and graphic expression profile of that gene, which is termed “digital expression”. The rationale of using these two complementary methods with methods of phenotypic association studies of QTLs, SNPs and phenotype expression correlation is based on the assumption that true orthologs are likely to retain identical function over evolutionary time. These methods provide different sets of indications on function similarities between two homologous genes, including similarities in the sequence level—identical amino acids in the protein domains and similarity in expression profiles.

The search and identification of homologous genes involves the screening of sequence information available, for example, in public databases such as the DNA Database of Japan (DDBJ), GenBank, and the European Molecular Biology Laboratory Nucleic Acid Sequence Database (EMBL) or versions thereof or the MIPS database. A number of different search algorithms have been developed, including but not limited to the suite of programs referred to as BLAS™ programs. There are five implementations of BLAST™, three designed for nucleotide sequence queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Altschul, S. F et al., (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410). Such methods involve alignment and comparison of sequences. The BLAS™ algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAS™ analysis is publicly available through the National Centre for Biotechnology Information. Other such software or algorithms are GAP, BESTFIT, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.

The homologous genes may belong to the same gene family. The analysis of a gene family may be carried out using sequence similarity analysis. To perform this analysis, one may use standard programs for multiple alignments e.g. Clustal W. A neighbor-joining tree of the proteins homologous to the proteins encoded by the genes identified in this invention may be used to provide an overview of structural and ancestral relationships. Sequence identity may be calculated using an alignment program as described above. It is expected that plants other that those examined in the present invention will carry a similar functional gene (ortholog) or a family of similar genes and those genes will provide the same preferred phenotype as the genes presented here. Advantageously, these family members may be useful in the methods of the invention. Example of other plants are included here but not limited to, barley (Hordeum vulgare), Arabidopsis (Arabidopsis thaliana), maize (Zea mays), cotton (Gossypium), Oilseed rape (Brassica napus), Rice (Oryza sativa), Sugar cane (Saccharum officinarum), Sorghum (Sorghum bicolor), Soybean (Glycine max), Sunflower (Helianthus annuus), Tomato (Lycopersicon esculentum), and Wheat (Triticum aestivum).

The above-mentioned analyses for sequence homology can be carried out on a full-length sequence, but may also be based on a comparison of certain regions such as conserved domains. The identification of such domains would also be well within the realm of the person skilled in the art and would involve, for example, a computer readable format of the nucleic acids of the present invention, the use of alignment software programs and the use of publicly available information on protein domains, conserved motifs and boxes. This information is available in the PRODOM (biochem.ucl.ac.uk/bsm/dbbrowser/protocol/prodomqry.html), PIR (pir.Georgetown.edu/) or Pfam (sanger.ac.uk/Software/Pfam/) databases. Sequence analysis programs designed for motif searching may be used for identification of fragments, regions and conserved domains as mentioned above. Preferred computer programs include, but are not limited to, MEME, SIGNALSCAN, and GENESCAN.

A person skilled in the art may use the homologous sequences provided herein to find similar sequences in other species and other organisms. Homologues of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. To produce such homologues, amino acids of the protein may be replaced by other amino acids having similar properties (conservative changes, such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheet structures). Conservative substitution Tables are well known in the art (see for example Creighton T E (1984) Proteins. W.H. Freeman and Company). Homologues of a nucleic acid encompass nucleic acids having nucleotide substitutions, deletions and/or insertions relative to the unmodified nucleic acid in question and having similar biological and functional activity as the unmodified nucleic acid from which they are derived.

Polynucleotides and polypeptides with significant homology to the identified genes described in Table 6 (Example 2) were identified from the databases using BLAS™ software with the Blastp and tBlastn algorithms as filters for the first stage, and the needle (EMBOSS package) or Frame+algorithm alignment for the second stage. Local identity (BLAS™ alignments) was defined with a very permissive cutoff—60% Identity on a span of 60% of the sequences lengths because it is used only as a filter for the global alignment stage. The default filtering of the BLAS™ package was not utilized (by setting the parameter “-F F”).

In the second stage, homologs were defined based on a global identity of at least 80% to the core gene polypeptide sequence. Two distinct forms for finding the optimal global alignment for protein or nucleotide sequences were used in this application:

1. Between two proteins (following the BLASTP filter):

EMBOSS-6.0.1 Needleman-Wunsch algorithm with the following modified parameters: gapopen=8 gapextend=2. The rest of the parameters were unchanged from the default options described hereinabove.

2. Between a protein sequence and a nucleotide sequence (following the TBLASTN filter):

GenCore 6.0 OneModel application utilizing the Frame+algorithm with the following parameters: model=frame+_p2n.model mode=qglobal -q=protein.sequence -db=nucleotide.sequence. The rest of the parameters are unchanged from the default options described hereinabove.

The query polypeptide sequences were the sequences listed in Table 6 (Example 2). The subject sequences are protein sequences identified in the database based on greater than 80% global identity to the predicted translated sequences of the query nucleotide sequences or to the polypeptide sequences. Homology was calculated as % of identity over the aligned sequences. The identified orthologous and homologous sequences having at least 80% global sequence identity to said sequences are provided in Table 7, below. These homologous genes are expected to increase plant resistance to fungal infection caused by the mentioned pathogens

TABLE 7 Homologues (e.g., orthologues) of genes associated with plant resistance to fungal infection Polyn. Hom. to Polyp. Hom. to % SEQ ID Gene SEQ ID SEQ ID glob. NO: Name Organism NO: NO: Iden. Algor. 100 LFS122 Panicum virgatum — 338 90.76 glotblastn 101 LFS122 Panicum virgatum 369 338 84.3 globlastp 102 LFS109 Hordeum vulgare 370 329 81.2 globlastp 103 LFS86 Sorghum bicolor 371 311 80.3 globlastp 104 LFS132 Sorghum bicolor — 348 80.33 glotblastn 105 LFS107 Triticum aestivum 372 328 90.6 globlastp 106 LFS116 Triticum aestivum 373 335 99.5 globlastp 107 LFS116 Hordeum vulgare 374 335 90.9 globlastp 108 LFS121 Panicum virgatum 375 337 85 globlastp 109 LFS121 Panicum virgatum 376 337 84.3 globlastp 110 LFS121 Zea mays 377 337 80.9 globlastp 111 LFS92 Triticum aestivum 378 316 94.6 globlastp 112 LFS92 Secale cereale 379 316 87.5 globlastp 113 LFS95 Aegilops tauschii 380 354 88.7 globlastp 114 LFS95 Triticum aestivum 381 354 88.3 globlastp 115 LFS95 Secale cereale 382 354 86 globlastp 116 LFS95 Secale cereale 383 354 84.8 globlastp 117 LFS95 Secale cereale 384 354 84.3 globlastp 118 LFS95 Secale cereale — 354 83.88 glotblastn 119 LFS95 Secale cereale — 354 83.47 glotblastn 120 LFS95 Triticum aestivum 385 354 83.1 globlastp 121 LFS95 Triticum aestivum 386 354 80.2 globlastp 122 LFS106 Secale cereale 387 327 89.2 globlastp 123 LFS106 Triticum aestivum 388 327 85.5 globlastp 124 LFS106 Hordeum vulgare 389 327 83.1 globlastp 125 LFS111 Triticum aestivum — 331 96.71 glotblastn 126 LFS111 Hordeum vulgare 390 331 88.6 globlastp 127 LFS97 Triticum aestivum 391 321 99.8 globlastp 128 LFS97 Triticum aestivum 392 321 99 globlastp 129 LFS97 Triticum aestivum 393 321 99 globlastp 130 LFS97 Hordeum vulgare 394 321 98.1 globlastp 131 LFS97 Secale cereale 395 321 97.7 globlastp 132 LFS97 Brachypodium distachyon 396 321 94.7 globlastp 133 LFS97 Avena sativa 397 321 94.7 globlastp 134 LFS97 Brachypodium distachyon 398 321 93 globlastp 135 LFS97 Oryza sativa 399 321 89.1 globlastp 136 LFS97 Panicum virgatum 400 321 84.4 globlastp 137 LFS97 Setaria italica 401 321 83.6 globlastp 138 LFS97 Aegilops tauschii — 321 82.56 glotblastn 139 LFS97 Sorghum bicolor 402 321 81.2 globlastp 140 LFS98 Triticum aestivum 403 357 97.2 globlastp 141 LFS113 Triticum aestivum 404 332 98.8 globlastp 142 LFS113 Hordeum vulgare 405 332 97.5 globlastp 143 LFS113 Brachypodium distachyon 406 332 84.6 globlastp 144 LFS113 Oryza sativa 407 332 82.7 globlastp 145 LFS113 Oryza sativa — 332 81.53 glotblastn 146 LFS113 Secale cereale — 363 98.25 glotblastn 147 LFS113 Secale cereale 408 363 97.9 globlastp 148 LFS113 Setaria italica 409 363 84 globlastp 149 LFS113 Sorghum bicolor 410 363 83.1 globlastp 150 LFS113 Zea mays 411 363 82.2 globlastp 151 LFS117 Triticum aestivum — 365 96.87 glotblastn 152 LFS100 Triticum aestivum — 359 92.49 glotblastn 153 LFS100 Aegilops tauschii — 359 89.67 glotblastn 154 LFS100 Secale cereale — 359 80.47 glotblastn 155 LFS94 Secale cereale — 318 94.7 glotblastn 156 LFS94 Secale cereale — 318 93.32 glotblastn 157 LFS94 Triticum aestivum — 318 93.09 glotblastn 158 LFS94 Hordeum vulgare 412 318 92.2 globlastp 159 LFS94 Secale cereale 413 318 91.2 globlastp 160 LFS94 Aegilops tauschii 414 318 90.1 globlastp 161 LFS94 Brachypodium distachyon 415 318 86.3 globlastp 162 LFS94 Brachypodium distachyon 416 318 85.8 globlastp 163 LFS94 Oryza sativa 417 318 83.3 globlastp 164 LFS94 Secale cereale 418 318 83.1 globlastp 165 LFS94 Zea mays 419 318 80 globlastp 166 LFS99 Triticum aestivum — 358 80 glotblastn 167 LFS95 Hordeum vulgare 420 319 89 globlastp 168 LFS95 Aegilops tauschii 421 319 87.9 globlastp 169 LFS96 Triticum aestivum 422 320 98.1 globlastp 170 LFS96 Aegilops tauschii — 320 86.79 glotblastn 171 LFS130 Triticum aestivum 423 346 98.5 globlastp 172 LFS130 Hordeum vulgare 424 346 94.8 globlastp 173 LFS130 Brachypodium distachyon 425 346 83.1 globlastp 174 LFS82 Panicum virgatum 426 349 94.7 globlastp 175 LFS82 Panicum virgatum 427 349 93.9 globlastp 176 LFS82 Oryza sativa 428 349 88.6 globlastp 177 LFS82 Brachypodium distachyon 429 349 87.1 globlastp 178 LFS82 Triticum aestivum 430 349 86.4 globlastp 179 LFS82 Secale cereale 431 349 86.2 globlastp 180 LFS82 Hordeum vulgare 432 349 86.1 globlastp 306 LFS112 Triticum aestivum 509 507 99.2 globlastp 305 LFS112 Hordeum vulgare 508 507 95.6 globlastp 181 LFS129 Panicum virgatum 433 345 92 globlastp 182 LFS129 Panicum virgatum 434 345 80.5 globlastp 183 LFS94 Triticum aestivum 435 353 99.8 globlastp 184 LFS94 Secale cereale 436 353 96.8 globlastp 185 LFS94 Secale cereale 437 353 90.8 globlastp 186 LFS94 Setaria italica 438 353 80.7 globlastp 187 LFS89 Aegilops tauschii 313 313 100 globlastp 188 LFS84 Hordeum vulgare 439 310 94.3 globlastp 189 LFS84 Secale cereale 440 310 90 globlastp 190 LFS84 Aegilops tauschii 441 310 89.4 globlastp 191 LFS84 Brachypodium distachyon 442 310 87.4 globlastp 192 LFS84 Panicum virgatum 443 310 85.8 globlastp 193 LFS84 Sorghum bicolor 444 310 85.3 globlastp 194 LFS84 Setaria italica 445 310 84.9 globlastp 195 LFS84 Zea mays — 310 83.65 glotblastn 196 LFS84 Zea mays 446 310 83.2 globlastp 197 LFS84 Oryza sativa — 310 80.27 glotblastn 198 LFS125 Zea mays 447 341 83.8 globlastp 199 LFS115 Secale cereale 448 334 99.8 globlastp 200 LFS115 Triticum aestivum 449 334 92.7 globlastp 201 LFS114 Hordeum vulgare 450 364 92.3 globlastp 202 LFS114 Secale cereale 451 364 91.4 globlastp 203 LFS114 Triticum aestivum 452 364 80.8 globlastp 204 LFS93 Hordeum vulgare 453 317 92.8 globlastp 205 LFS93 Aegilops tauschii — 317 89.24 glotblastn 206 LFS93 Triticum aestivum — 317 84.39 glotblastn 207 LFS93 Brachypodium distachyon — 317 80.38 glotblastn 208 LFS126 Panicum virgaium 454 342 91.2 globlastp 209 LFS126 Panicum virgatum 455 342 91.2 globlastp 210 LFS126 Zea mays 456 342 88 globlastp 211 LFS126 Sorghum bicolor 457 342 88 globlastp 212 LFS126 Oryza sativa 458 342 80.5 globlastp 213 LFS100 Triticum aestivum 459 324 81.9 globlastp 214 LFS83 Triticum aestivum 460 350 99.7 globlastp 215 LFS83 Secale cereale 461 350 95.4 globlastp 216 LFS83 Lolium multiflorum 462 350 87.5 globlastp 217 LFS83 Lolium multiflorum 463 350 87.5 globlastp 218 LFS83 Brachypodium distachyon 464 350 83.7 globlastp 219 LFS83 Setaria italica 465 350 80.5 globlastp 220 LFS131 Setaria italica 466 347 83.2 globlastp 221 LFS131 Setaria italica 467 347 81.2 globlastp 222 LFS131 Sorghum bicolor 468 347 81.2 globlastp 223 LFS91 Triticum aestivum 315 315 100 globlastp 224 LFS91 Triticum aestivum 469 315 93.1 globlastp 225 LFS91 Secale cereale — 315 91.16 glotblastn 226 LFS91 Secale cereale — 315 89.23 glotblastn 227 LFS91 Secale cereale 470 315 81.4 globlastp 228 LFS91 Brachypodium distachyon 471 315 80.7 globlastp 229 LFS83 Secale cereale 472 309 95.6 globlastp 230 LFS83 Secale cereale — 309 82.07 glotblastn 231 LFS83 Aegilops tauschii 473 309 82 globlastp 232 LFS83 Sorghum bicolor 474 309 80.8 globlastp 233 LFS83 Triticum aestivum 475 309 80.5 globlastp 234 LFS102 Triticum aestivum 476 325 96.9 globlastp 235 LFS102 Hordeum vulgare 477 325 90.2 globlastp 236 LFS102 Aegilops tauschii — 325 87.5 glotblastn 237 LFS104 Hordeum vulgare 478 326 97.8 globlastp 238 LFS104 Secale cereale 479 326 83.1 globlastp 239 LFS104 Zea mays — 326 81.37 glotblastn 240 LFS99 Avena sativa — 323 93 glotblastn 241 LFS99 Triticum aestivum — 323 87 glotblastn 242 LFS99 Triticum aestivum 480 323 86 globlastp 243 LFS99 Triticum aestivum — 323 82 glotblastn 244 LFS90 Triticum aestivum 481 314 99.8 globlastp 245 LFS90 Triticum aestivum 482 314 97.2 globlastp 246 LFS90 Triticum aestivum 483 314 95.5 globlastp 247 LFS90 Triticum aestivum 484 314 93.9 globlastp 248 LFS90 Triticum aestivum 485 314 93.7 globlastp 249 LFS82 Sorghum bicolor — 308 97.24 glotblastn 250 LFS87 Zea mays 486 312 96.9 globlastp 251 LFS87 Zea mays 486 312 96.9 globlastp 252 LFS87 Zea mays — 312 95.92 glotblastn 253 LFS87 Sorghum bicolor — 312 95.92 glotblastn 254 LFS87 Saccharum officinarum — 312 95.92 glotblastn 255 LFS87 Cenchrus ciliaris — 312 94.9 glotblastn 256 LFS87 Setaria italica — 312 94.9 glotblastn 257 LFS87 Zea mays — 312 94.9 glotblastn 258 LFS87 Panicum virgatum — 312 94.9 glotblastn 259 LFS87 Panicum virgatum — 312 94.9 glotblastn 260 LFS87 Zea mays — 312 93.88 glotblastn 261 LFS87 Pennisetum glaucum — 312 93.88 glotblastn 262 LFS87 Echinochloa colona — 312 91.84 glotblastn 263 LFS87 Zea mays — 312 89.8 glotblastn 264 LFS87 Oryza sativa — 312 88.78 glotblastn 265 LFS87 Oryza sativa — 312 87.76 glotblastn 266 LFS87 Hordeum vulgare — 312 86.73 glotblastn 267 LFS87 Aegilops tauschii — 312 85.71 glotblastn 268 LFS87 Secale cereale — 312 85.71 glotblastn 269 LFS87 Secale cereale — 312 85.71 glotblastn 270 LFS87 Triticum aestivum — 312 85.71 glotblastn 271 LFS87 Triticum aestivum — 312 85.71 glotblastn 272 LFS87 Brachypodium distachyon — 312 84.69 glotblastn 273 LFS87 Cocos nucifera — 312 82.65 glotblastn 274 LFS87 Phalaenopsis aphrodite — 312 82.65 glotblastn 275 LFS87 Cocos nucifera — 312 81.63 glotblastn 276 LFS87 Cynodon dactylon — 312 81.63 glotblastn 277 LFS87 Elaeis guineensis — 312 81.63 glotblastn 278 LFS87 Lolium multiflorum — 312 80.61 glotblastn 279 LFS87 Avena sativa — 312 80.61 glotblastn 280 LFS87 Elaeis guineensis — 312 80.61 glotblastn 281 LFS104 Brachypodium distachyon 487 360 92.6 globlastp 282 LFS104 Setaria italica 488 360 86.7 globlastp 283 LFS104 Sorghum bicolor 489 360 85.6 globlastp 284 LFS104 Zea mays 490 360 83.8 globlastp 285 LFS104 Oryza sativa 491 360 80.9 globlastp 286 LFS127 Panicum virgatum 492 343 86.2 globlastp 287 LFS127 Sorghum bicolor 493 343 85.2 globlastp 288 LFS127 Panicum virgatum 494 343 82.1 globlastp 289 LFS127 Zea mays 495 343 81.5 globlastp 290 LFS125 Panicum virgatum 496 367 92.3 globlastp 291 LFS125 Panicum virgatum 497 367 91.6 globlastp 292 LFS125 Zea mays 498 367 85.5 globlastp 293 LFS125 Sorghum bicolor 499 367 84.7 globlastp 294 LFS125 Oryza sativa 500 367 82.1 globlastp 295 LFS117 Hordeum vulgare 501 336 94.3 globlastp 296 LFS117 Secale cereale — 336 80.97 glotblastn 297 LFS124 Setaria italica 502 340 90.1 globlastp 298 LFS124 Sorghum bicolor 503 340 85.7 globlastp 299 LFS124 Zea mays 504 340 83.3 globlastp 300 LFS124 Panicum virgatum 505 340 82.3 globlastp 301 LFS124 Panicum virgatum 506 340 81.8 globlastp “Polyn.” = polynucleotide; “Polyp.” = polypeptide; “Algor.” = algorithm (used for sequence alignment and determination of percent homology); “Hom.”-homology; “iden.”-identity; “glob.”-global.

The output of the functional genomics approach described herein is a set of genes highly predicted to improve resistance of a plant to fungal infection by Fusarium verticilloides, Fusarium graminearum or Colletotrichum graminicola when their expression is modulated (enhanced or down-regulated) in said plant. Although each gene is predicted to have its own impact, modifying the mode of expression of more than one gene is expected to provide an additive or synergistic effect on the plant resistance Altering the expression of each gene described herein alone or of a set of genes together increases the overall plant resistance, hence expects to increase agricultural productivity.

Example 4: Identification of Domains Comprised within Identified Genes

A polypeptide domain refers to a set of conserved amino acids located at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved, and particularly amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.

The Integrated Resource of Protein Families, Domains and Sites (InterPro) database is an integrated interface for the commonly used signature databases for text- and sequence-based searches. The InterPro database combines these databases, which use different methodologies and varying degrees of biological information about well-characterized proteins to derive protein signatures. Collaborating databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted at the Sanger Institute server in the United Kingdom.

Interpro is hosted at the European Bioinformatics Institute in the United Kingdom. InterProScan is the software package that allows sequences (protein and nucleic) to be scanned against InterPro's signatures. Signatures are predictive models, provided by several different databases that make up the InterPro consortium.

InterProScan 5.32-71.0 was used to analyze the polypeptides of the present invention (core and homologues/orthologs) for common domains (Jones P et al., 2014. Bioinformatics, January 2014 (doi:10.1093/bioinformatics/btu031). Briefly, InterProScan is based on scanning methods native to the InterPro member databases. It is distributed with pre-configured method cut-offs recommended by the member database experts and which are believed to report relevant matches. All cut-offs are defined in configuration files of the InterProScan programs. Matches obtained with the fixed cut-off are subject to the following filtering:

Pfam filtering: Each Pfam family is represented by two hidden Markov models (HMMs)—ls and fs (full-length and fragment). An HMM model has bit score cut-offs (for each domain match and the total model match) and these are defined in the Gathering threshold (GA) lines of the Pfam database. Initial results are obtained with quite a high common cut-off and then the matches of the signature with a lower score than the family specific cut-offs are dropped.

If both the fs and ls model for a particular Pfam hits the same region of a sequence, the AM field in the Pfam database is used to determine which model should be chosen—globalfirst (LS); localfirst (FS) or byscore (whichever has the highest e-value).

Another type of filtering has been implemented since release of version 4.1. It is based on Clan filtering and nested domains. Further information on Clan filtering can be found in the Pfam website (www.sanger.ac.uk/Pfam) for more information on Clan filtering.

TIGRFAMs filtering: Each TIGRFAM HMM model has its own cut-off scores for each domain match and the total model match. These bit score cut-offs are defined in the “trusted cut-offs” (TC) lines of the database. Initial results are obtained with quite a high common cut-off and then the matches (of the signature or some of its domains) with a lower score compared to the family specific cut-offs are dropped.

PRINTS filtering: All matches with p-value more than a pre-set minimum value for the signature, are dropped.

SMART filtering: The publicly distributed version of InterProScan has a common e-value cut-off corresponding to the reference database size. A more sophisticated scoring model is used on the SMART web server and in the production of pre-calculated InterPro Match data.

Exact scoring thresholds for domain assignments are proprietary data. The InterProMatches data production procedure uses these additional smart.thresholds data. It is to be noted that the given cut-offs are e-values (i.e. the number of expected random hits) and therefore are valid only in the context of reference database size and smart.desc data files to filter out results obtained with higher cut-off.

It implements the following logic: If the whole sequence E-value of a found match is worse than the ‘cut_low’, the match is dropped. If the domain E-value of a found match is worse than the ‘repeat’ cut-off (where defined) the match is dropped. If a signature is a repeat, the number of significant matches of that signature to a sequence must be greater than the value of ‘repeats’ in order for all matches to be accepted as true (T).

If the signature is part of a family (‘family_cut’ is defined), if the domain E-value is worse than the domain cut off (‘cutoff’), the match is dropped. If the signature has “siblings” (because it has a family_cut defined), and they overlap, the preferred sibling is chosen as the true match according to information in the overlaps file.

PROSITE patterns CONFIRMation: ScanRegExp is able to verify PROSITE matches using corresponding statistically-significant CONFIRM patterns. The default status of the PROSITE matches is unknown (?) and the true positive (T) status is assigned if the corresponding CONFIRM patterns match as well. The CONFIRM patterns were generated based on the true positive SWISS-PROT PROSITE matches using eMOTIF software with a stringency of 10e-9 P-value.

PANTHER filtering: Panther has pre- and post-processing steps. The pre-processing step is intended to speed up the HMM-based searching of the sequence and involves blasting the HMM sequences with the query protein sequence in order to find the most similar models above a given e-value. The resulting HMM hits are then used in the HMM-based search.

Panther consists of families and sub-families. When a sequence is found to match a family in the blast run, the sub-families are also scored using HMMER tool (that is, unless there is only 1 sub-family, in which case, the family alone is scored against).

Any matches that score below the e-value cut-off are discarded. Any remaining matches are searched to find the HMM with the best score and e-value and the best hit is then reported (including any sub-family hit).

GENE3D filtering: Gene3D also employs post-processing of results by using a program called DomainFinder. This program takes the output from searching the Gene3D HMMs against the query sequence and extracts all hits that are more than 10 residues long and have an e-value better than 0.001. If hits overlap at all, the match with the better e-value is chosen.

The polypeptides of the invention associated with conferring and/or enhancing the resistance of a plant to at least one fungal pathogen can be characterized by specific amino acid domains. According to certain embodiments, particular domains are conserved within a family of polypeptides as described in Table 8 hereinbelow. Without wishing to be bound by any specific theory or mechanism of action, the conserved domain(s) may indicate common functionally of the polypeptides comprising same. The domains are presented by an identifier (number). Table 9 provides the details of each domain.

TABLE 8 Core and homologous polypeptides comprising the same domains Core Poly- peptide Gene Homologous Polypeptides (SEQ Name Characteristic Domains Comprising the Domains ID NO) (Core) (Domain identifier) (SEQ ID NOs) 308 LFS82 1; 2; 3; 4; 5; 6; 7; 8; 9; 10 in core, 426; 427; 428; 429; 430; 431; 432 cloned and homologs 309 LFS83 11; 12; 13; 14; 15; 16 in core, cloned 460; 461; 462; 463; 464; 465; 472; 473; and homologs 474; 475 310 LFS84 2; 17; 18; 5 in core, cloned and 439; 440; 441; 442; 443; 444; 445; 446 homologs 311 LFS86 19 in core, cloned and homologs 371 312 LFS87 20; 21 in core and homologs 486 313 LFS89 22; 23; 24; 25; 26; 27 in core, cloned 313 and homologs 314 LFS90 28; 29; 30 in core, cloned and 481; 482; 483; 484; 485 homologs 315 LFS91 31; 32 in core, cloned and 315; 469; 470; 471 homologs 316 LFS92 23; 24; 33 in core, cloned and 378; 379 homologs 317 LFS93 34 in core, cloned and homologs 453 318 LFS94 35; 36 in core, cloned and 412; 413; 414; 415; 416; 417; 418; 419; homologs 435; 436; 437; 438 319 LFS95 37 in core, cloned and homologs 380; 381; 382; 383; 384; 385; 386; 420;421 320 LFS96 38; 39 in core, cloned and 422 homologs 321 LFS97 40; 41; 42; 43 in core, cloned and 391; 392; 393; 394; 395; 396; 397; 398; homologs 399; 400; 401; 402 322 LFS98 44; 45; 32; 46 in core, cloned and 403 homologs 323 LFS99 no domains in core 324 LFS100 47; 48 in core, cloned and 459 homologs 325 LFS102 49; 50 in core, cloned and 476; 477 homologs 326 LFS104 51; 52; 53; 54; 55; 56; 57 in core, 478; 479; 487; 488; 489; 490; 491 cloned and homologs 327 LFS106 no domains in core 328 LFS107 35; 36 in core, cloned and 372 homologs 329 LFS109 58; 59; 60; 21; 61; 20; 62 in core, 370 cloned and homologs 330 LFS110 63 in core and cloned 331 LFS111 34 in core, cloned and homologs 390 332 LFS113 65; 66; 67 in core, cloned and 404; 405; 406; 407; 408; 409; 410; 411 homologs 333 LFS114 68; 69 in core, cloned and 450; 451; 452 homologs 334 LFS115 1; 2; 3; 4; 5; 70; 9; 71; 10 in core, 448; 449 cloned and homologs 335 LFS116 20; 72; 21 in core, cloned and 373; 374 homologs 336 LFS117 73; 74; 75 in core, cloned and 501 homologs 337 LFS121 76; 77; 78 in core, cloned and 375; 376; 377 homologs 338 LFS122 79 in core, cloned and homologs 369 339 LFS123 80; 81; 82; 83; 84 in core and cloned 340 LFS124 85; 86; 87; 88 in core, cloned and 502; 503; 504; 505; 506 homologs 341 LFS125 89 in core, cloned and homologs 447; 496; 497; 498; 499; 500 342 LFS126 62; 59; 60 in core, cloned and 454; 455; 456; 457; 458 homologs 343 LFS127 90; 91; 92; 93; 94 in core, cloned and 492; 493; 494; 495 homologs 344 LFS128 95; 96; 97; 98 in core and cloned 345 LFS129 99; 100; 101; 48; 102; 103; 104; 105; 433; 434 106; 107 in core, cloned and homologs 346 LFS130 108 in core and homologs 423; 424; 425 347 LFS131 109; 110; 111; 112 in core and 466; 467; 468 homologs 348 LFS132 35; 36 in core 507 LFS112 64 in core, cloned and homologs 508; 509 510 LFS105 no domains in core

TABLE 9 Details of identified domains Domain InterPro Identifier No. Accession No. Description 1 IPR023298 SSF81665 P-type ATPase, transmembrane domain superfamily 2 IPR036412 SSF56784 HAD-like superfamily 3 IPR023299 G3DSA:3.40.1110.10 P-type ATPase, cytoplasmic domain N 4 IPR008250 SSF81653 P-type ATPase, A domain superfamily 5 IPR023214 G3DSA:3.40.50.1000 HAD superfamily 6 IPR032631 PF16209 Phospholipid-translocating ATPase N-terminal P-type ATPase, N-terminal 7 IPR032630 PF16212 Phospholipid-translocating P-type ATPase C- terminal P-type ATPase, C-terminal 8 IPR006539 TIGR01652 ATPase-Plipid: phospholipid-translocating P-type ATPase, flippase P-type ATPase, subfamily IV 9 IPR001757 TIGR01494 ATPase_P-type: HAD ATPase, P-type, family IC P-type ATPase 10 IPR018303 PS00154 E1-E2 ATPases phosphorylation site. P-type ATPase, phosphorylation site 11 IPR000909 PF00388 Phosphatidylinositol-specific phospholipase C, X domain Phosphatidylinositol-specific phospholipase C, X domain 12 IPR017946 G3DSA:3.20.20.190 PLC-like phosphodiesterase, TIM beta/alpha- barrel domain superfamily 13 IPR001192 PR00390 Phospholipase C signature Phosphoinositide phospholipase C family 14 IPR035892 G3DSA:2.60.40.150 C2 domain superfamily 15 IPR000008 SM00239 C2 domain 16 IPR001711 PF00387 Phosphatidylinositol-specific phospholipase C, Y domain Phospholipase C, phosphatidylinositol-specific, Y domain 17 IPR006379 TIGR01484 HAD-SF-IIB: HAD hydrolase, family IIB HAD- superfamily hydrolase, subfamily IIB 18 IPR003337 PF02358 Trehalose-phosphatase 19 IPR008889 PF05678 VQ motif 20 IPR000719 PS50011 Protein kinase domain profile. Protein kinase domain 21 IPR011009 SSF56112 Protein kinase-like domain superfamily 22 IPR012967 PF08100 Dimerisation domain Plant methyltransferase 23 IPR036390 SSF46785 Winged helix DNA-binding domain superfamily 24 IPR036388 G3DSA:1.10.10.10 Winged helix-like DNA-binding domain superfamily 25 IPR029063 SSF53335 S-adenosyl-L-methionine-dependent methyltransferase 26 IPR001077 PF00891 O-methyltransferase domain 27 IPR016461 PIRSF005739 O-methyltransferase COMT-type 28 IPR008998 PF07468 Agglutinin domain 29 IPR036242 SSF50382 Agglutinin domain superfamily 30 IPR005830 PF01117 Aerolysin toxin Aerolysin 31 IPR001509 PF01370 NAD dependent epimerase/dehydratase family 32 IPR036291 SSF51735 NAD(P)-binding domain superfamily 33 IPR000232 PS00434 HSF-type DNA-binding domain signature. Heat shock factor (HSF)-type, DNA-binding 34 IPR005202 PS50985 GRAS family profile. Transcription factor GRAS 35 IPR003480 PF02458 Transferase family Transferase 36 IPR023213 G3DSA:3.30.559.10 Chloramphenicol acetyltransferase-like domain superfamily 37 IPR007770 PF05078 Protein of unknown function (DUF679) Protein DMP 38 IPR035995 SSF57247 Bowman-Birk type proteinase inhibitor 39 IPR000877 cd00023 BBI Proteinase inhibitor I12, Bowman-Birk 40 IPR003593 SM00382 AAA+ ATPase domain 41 IPR003959 PF00004 ATPase family associated with various cellular activities (AAA) ATPase, AAA-type, core 42 IPR025753 PF14363 Domain associated at C-terminal with AAA AAA-type ATPase, N-terminal domain 43 IPR027417 SSF52540 P-loop containing nucleoside triphosphate hydrolase 44 IPR011032 SSF50129 GroES-like superfamily 45 IPR020843 SM00829 Polyketide synthase, enoylreductase domain 46 IPR013149 PF00107 Zinc-binding dehydrogenase Alcohol dehydrogenase, C-terminal 47 IPR003245 PS51485 Phytocyanin domain profile. Phytocyanin domain 48 IPR008972 SSF49503 Cupredoxin 49 IPR035897 SSF52200 Toll/interleukin-1 receptor homology (TIR) domain superfamily 50 IPR000157 PF01582 TIR domain Toll/interleukin-1 receptor homology (TIR) domain 51 IPR020833 PS00711 Lipoxygenases iron-binding region signature 1. Lipoxygenase, iron binding site 52 IPR036392 SSF49723 PLAT/LH2 domain superfamily 53 IPR036226 SSF48484 Lipoxigenase, C-terminal domain superfamily 54 IPR013819 PR00087 Lipoxygenase signature Lipoxygenase, C-terminal 55 IPR027433 G3DSA:4.10.372.10 Lipoxygenase, domain 3 56 IPR020834 PS00081 Lipoxygenases iron-binding region signature 2. Lipoxygenase, conserved site 57 IPR001246 PR00468 Plant lipoxygenase signature Lipoxygenase, plant 58 IPR017441 PS00107 Protein kinases ATP-binding region signature. Protein kinase, ATP binding site 59 IPR001611 PS51450 Leucine-rich repeat profile. Leucine-rich repeat 60 IPR032675 G3DSA:3.80.10.10 Leucine-rich repeat domain superfamily 61 IPR008271 PS00108 Serine/Threonine protein kinases active-site signature. 62 IPR003591 SM00369 Leucine-rich repeat, typical subtype 63 IPR015425 PS51444 Formin homology-2 (FH2) domain profile. Formin, FH2 domain 64 IPR020864 SM00457 Membrane attack complex component/perforin (MACPF) domain 65 IPR036259 SSF103473 MFS transporter superfamily 66 IPR020846 cd06174 MFS Major facilitator superfamily domain 67 IPR000109 PF00854 POT family Proton-dependent oligopeptide transporter family 68 IPR029058 SSF53474 Alpha/Beta hydrolase fold 69 IPR013094 PF07859 alpha/beta hydrolase fold Alpha/beta hydrolase fold-3 70 IPR004014 PF00690 Cation transporter/ATPase, N-terminus Cation- transporting P-type ATPase, N-terminal 71 IPR006534 TIGR01647 ATPase-IIIA_H: plasma-membrane proton-efflux P-type ATPase P-type ATPase, subfamily IIIA 72 IPR001245 PF07714 Protein tyrosine kinase Serine-threonine/tyrosine- protein kinase, catalytic domain 73 IPR036955 G3DSA:3.30.730.10 AP2/ERF domain superfamily 74 IPR016177 SSF54171 DNA-binding domain superfamily 75 IPR001471 PR00367 Ethylene responsive element binding protein signature AP2/ERF domain 76 IPR001005 cd00167 SANT SANT/Myb domain 77 IPR009057 SSF46689 Homeobox-like domain superfamily 78 IPR017930 PS51294 Myb-type HTH DNA-binding domain profile. Myb domain 79 IPR025886 PF14299 Phloem protein 2 Phloem protein 2-like 80 IPR005630 PF03936 Terpene synthase family, metal binding domain Terpene synthase, metal-binding domain 81 IPR036965 G3DSA:1.50.10.130 Terpene synthase, N-terminal domain superfamily 82 IPR008949 G3DSA:1.10.600.10 Isoprenoid synthase domain superfamily 83 IPR008930 SSF48239 Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid 84 IPR001906 PF01397 Terpene synthase, N-terminal domain 85 IPR016039 SSF53901 Thiolase-like 86 IPR001099 PF00195 Chalcone and stilbene synthases, N-terminal domain Chalcone/stilbene synthase, N-terminal 87 IPR011141 PIRSF000451 Polyketide synthase, type III 88 IPR012328 PF02797 Chalcone and stilbene synthases, C-terminal domain Chalcone/stilbene synthase, C-terminal 89 IPR002123 SM00563 Phospholipid/glycerol acyltransferase 90 IPR011051 SSF51182 RmlC-like cupin domain superfamily 91 IPR006045 SM00835 Cupin 1 92 IPR014710 G3DSA:2.60.120.10 RmlC-like jelly roll fold 93 IPR001929 PR00325 Germin signature Germin 94 IPR019780 PS00725 Germin family signature. Germin, manganese binding site 95 IPR026961 PF13962 Domain of unknown function PGG domain 96 IPR036770 G3DSA:1.25.40.20 Ankyrin repeat-containing domain superfamily 97 IPR020683 cd00204 ANK Ankyrin repeat-containing domain 98 IPR002110 PS50088 Ankyrin repeat profile. Ankyrin repeat 99 IPR034289 cd13897 CuRO_3_LCC_plant Laccase, third cupredoxin domain 100 IPR011706 PF07731 Multicopper oxidase Multicopper oxidase, type 2 101 IPR011707 PF07732 Multicopper oxidase Multicopper oxidase, type 3 102 IPR033138 PS00079 Multicopper oxidases signature 1. Multicopper oxidases, conserved site 103 IPR002355 PS00080 Multicopper oxidases signature 2. Multicopper oxidase, copper-binding site 104 IPR001117 PF00394 Multicopper oxidase Multicopper oxidase, type 1 105 IPR017761 TIGR03389 laccase: laccase 106 IPR034285 cd13875 CuRO_2_LCC_plant Laccase, second cupredoxin domain 107 IPR034288 cd13849 CuRO_1_LCC_plant Laccase, first cupredoxin domain 108 IPR004326 PF03094 Mlo family Mlo-related protein 109 IPR036396 G3DSA:1.10.630.10 Cytochrome P450 superfamily 110 IPR001128 PF00067 Cytochrome P450 Cytochrome P450 111 IPR017972 PS00086 Cytochrome P450 cysteine heme-iron ligand signature. Cytochrome P450, conserved site 112 IPR002401 PR00463 E-class P450 group I signature Cytochrome P450, E-class, group I

Example 5: Gene Cloning and Generation of Binary Vectors for Expression in Plants

To validate the role of genes identified hereinabove in increasing resistance to fungal infection, selected genes were over-expressed in plants, as follows.

Cloning Strategy

Selected genes from those presented in Examples 1-4 hereinabove were cloned into binary vectors for the generation of transgenic plants. In addition, to examine the effect of gene stacking, two pairs of genes were also cloned. For cloning, the full-length open reading frames (ORFs) were identified. EST clusters and in some cases mRNA sequences were analyzed to identify the entire open reading frame by comparing the results of several translation algorithms to known proteins from other plant species.

In order to clone the full-length cDNAs, reverse transcription (RT) followed by polymerase chain reaction (PCR; RT-PCR) was performed on total RNA extracted from roots or shoots challenged by the relevant pathogen. Total RNA extraction, production of cDNA and PCR amplification was performed using standard protocols described elsewhere (Sambrook J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, New York) which are well known to those skilled in the art. PCR products were purified using PCR purification kit (Qiagen).

Typically, 2 sets of primers were prepared for the amplification of each gene, via nested PCR (if required). Both sets of primers were used for amplification on a cDNA. In case no product was obtained, a nested PCR reaction was performed. Nested PCR was performed by amplification of the gene using external primers and then using the produced PCR product as a template for a second PCR reaction, where the internal set of primers was used. Alternatively, one or two of the internal primers were used for gene amplification, both in the first and the second PCR reactions (meaning only 2-3 primers are designed for a gene). To facilitate further cloning of the cDNAs, an 8-12 base pairs (bp) extension was added to the 5′ of each internal primer. The primer extension includes an endonuclease restriction site. The restriction sites were selected using two parameters: (a) the restriction site does not exist in the cDNA sequence; and (b) the restriction sites in the forward and reverse primers were designed such that the digested cDNA was inserted in the sense direction into the binary vector utilized for transformation.

PCR products were digested with the restriction endonucleases (New England BioLabs Inc.) according to the sites designed in the primers. Each digested/undigested PCR product was inserted into a high copy vector pUC19 (New England BioLabs Inc.), or into plasmids originating from this vector. In some cases, the undigested PCR product was inserted into pCR-Blunt II-TOPO (Invitrogen) or into pJET1.2 (CloneJET PCR Cloning Kit, Thermo Scientific) or directly into the binary vector. The digested/undigested products and the linearized plasmid vector were ligated using T4 DNA ligase enzyme (Roche, Switzerland or other manufacturers). In cases where pCR-Blunt II-TOPO is used no T4 ligase was needed.

Sequencing of the inserted genes was performed using the ABI 377 sequencer (Applied Biosystems). In some cases, after confirming the sequences of the cloned genes, the cloned cDNA was introduced into pQ6sVN plasmid.

Several DNA sequences of the selected genes were synthesized by GeneArt™ (Life Technologies, Grand Island, N.Y., USA). Synthetic DNA was designed in silico. Suitable restriction enzyme sites were added to the cloned sequences at the 5′ end and at the 3′ end to enable later cloning into the desired binary vector.

Binary vectors—The pPI plasmid vector was constructed by inserting a synthetic poly-(A) signal sequence, originating from pGL3 basic plasmid vector (Promega, GenBank Accession No. U47295; nucleotides 4658-4811) into the HindIII restriction site of the binary vector pBI101.3 (Clontech, GenBank Accession No. U12640). pGI is similar to pPI, but the original gene in the backbone is GUS-Intron and not GUS.

The modified pGI vector (e.g., pQFN, pQFNc, pQFNd, pQYN_6669, pQNa_RP, pQFYN, pQXNc or pQ6sVN (FIG. 1) is a modified version of the pGI vector in which the cassette is inverted between the left and right borders so the gene and its corresponding promoter are close to the right border and the hygromycin-resistance conferring gene is close to the left border.

In case of Brachypodium transformation, after confirming the sequences of the cloned genes, the cloned cDNAs were introduced into pQ6sVN (FIG. 1) containing 35S promoter (SEQ ID NO:37) and the NOS terminator (SEQ ID NO:36) via digestion with appropriate restriction endonucleases. The genes were cloned downstream to the 35S promoter and upstream to the NOS terminator. In the pQ6sVN vector the Hygromycin resistance gene cassette and the Bar_GA resistance gene cassette replaced the NPTII resistance gene cassette. pQ6sVN contains the 35S promoter (SEQ ID NO:37). Bar_GA resistance gene (SEQ ID NO: 39) is an optimized sequence of the BAR gene for expression in Brachypodium plants (ordered from GeneArt™).

BAR gene coding sequence is provided in GenBank Accession No. JQ293091.1 (SEQ ID NO:38); further description is provided in Akama K, et al. “Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker”, Plant Cell Rep. 1995, 14(7):450-4; Christiansen P, et al. “A rapid and efficient transformation protocol for the grass Brachypodium distachyon”, Plant Cell Rep. 2005 March; 23(10-11):751-8. Epub 2004 Oct. 19; and Pacurar D I, et al. “A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L”, Transgenic Res. 2008 17(5):965-75; each of which is fully incorporated herein by reference in its entirety.

The pQ6sVN vector, in addition to the multiple cloning sites (MCS) between the promoter and terminator, contains additional restriction sites (PmeI, I-SceI, and BsaI) downstream of the expression cassette to allow cloning of a second expression cassette into the vector. Stacking two genes (two expression cassettes) into pQ6sVN was performed as follows: the first gene was cloned into pQ6sVN via the MCS. The second gene was cloned into the vector pUCsVN. Genes cloned into this high copy vector via its MCS are flanked by a 35S promoter and a NOS terminator—the same elements found in the pQ6sVN vector. The expression cassette of pUCsVN is flanked by restriction sites for PmeI, I-SceI and BsaI. The cassette containing the second gene was excised from pUCsVN by digestion with one of the aforementioned enzymes, and ligated into the pQ6sVN plasmid already carrying the first gene, linearized with the same restriction enzyme. This resulted in a single vector harboring two “stacked” cassettes. The sequences and orientations of both cassettes were verified by Sanger sequencing and restriction digests.

In case genomic DNA was cloned, the genes were amplified by direct PCR on genomic DNA extracted from leaf tissue using the DNAeasy kit (Qiagen Cat. No. 69104).

Table 10 hereinbelow provides a list of the gene cloned, including gene name, the organism from which the gene is derived, the SEQ ID NO. of the primer used and the SEQ ID NO. of the gene polynucleotide and encoded polypeptide.

TABLE 10 Cloned genes Polyn. Polyp. Gene Primers used SEQ ID SEQ ID Name Organism SEQ ID NOs NO NO LFS82 Zea mays 535, 561, 574, 588 63 349 LFS83 Triticum aestivum 536, 562, 575, 589 64 350 LFS84 Triticum aestivum — 65 310 LFS86 Zea mays — 66 311 LFS89 Triticum aestivum — 67 313 LFS90 Aegilops tauschii 511, 537, 563, 576 68 351 LFS91 Aegilops tauschii 512, 538 69 315 LFS92 Aegilops tauschii 513, 539 70 352 LFS93 Aegilops tauschii — 71 317 LFS94 Aegilops tauschii 514, 540, 564 72 353 LFS95 Aegilops tauschii 515, 541, 565, 577 73 354 LFS96 Aegilops tauschii 516, 542 74 355 LFS97 Aegilops tauschii 517, 543, 566, 578 75 356 LFS98 Aegilops tauschii 518, 544, 567, 579 76 357 LFS99 Aegilops tauschii 519, 545, 568, 580 77 358 LFS100 Aegilops tauschii 520, 546, 569, 581 78 359 LFS102 Aegilops tauschii 521, 547, 582 79 325 LFS104 Aegilops tauschii 547, 582, 522, 548 80 360 LFS105 Aegilops tauschii 590, 591 307 510 LFS106 Aegilops tauschii 523, 549 81 327 LFS107 Aegilops tauschii 524, 550 82 361 LFS109 Aegilops tauschii 525, 551 83 362 LFS110 Aegilops tauschii — 84 330 LFS111 Aegilops tauschii — 85 331 LFS112 Aegilops tauschii — 304 507 LFS113 Aegilops tauschii 526, 552, 570, 583 86 363 LFS114 Aegilops tauschii 527, 553, 571, 584 87 364 LFS115 Aegilops tauschii — 88 334 LFS116 Aegilops tauschii 528, 554, 572, 585 89 335 LFS117 Aegilops tauschii 529, 555 90 365 LFS121 Setaria italica — 91 337 LFS122 Setaria italica 530, 556, 573, 586 92 338 LFS123 Setaria italica 531, 557, 587 93 366 LFS124 Setaria italica — 94 340 LFS125 Setaria italica 532, 558 95 367 LFS126 Setaria italica — 96 342 LFS127 Setaria italica — 97 343 LFS128 Setaria italica 533, 559 98 368 LFS129 Setaria italica 534, 560 99 345 “polyn.” = polynucleotide; “polyp.” = polypeptide.

Example 6: Transformation of Brachypodium distachyon Plants with the Polynucleotides of the Invention

Similar to the Arabidopsis model plant, Brachypodium distachyon has several features that recommend it as a model plant for functional genomics studies, especially in the grasses. Traits that make it an ideal model include its small genome (˜160 Mbp for a diploid genome and 355 Mbp for a polyploidy genome), small physical stature, a short lifecycle, and few growth requirements. Brachypodium is related to the major cereal grain species but is understood to be more closely related to the Triticeae (wheat, barley) than to the other cereals. Brachypodium, with its polyploidy accessions, can serve as an ideal model for these grains (whose genomic size and complexity is a major barrier to biotechnological improvement).

Brachypodium distachyon embryogenic calli were transformed using the procedure described by Vogel and Hill (2008. High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3. Plant Cell Rep 27:471-478); Vain et al (2008. Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis. Plant Biotechnology J 6: 236-245), and Vogel J, et al. (2006. Agrobacterium mediated transformation and inbred line development in the model grass Brachypodium distachyon. Plant Cell Tiss Org. Cult. 85:199-211), each of which is fully incorporated herein by reference, with some minor modifications, which are briefly summarized hereinbelow.

Callus initiation—Immature spikes (about 2 months after seeding) were harvested at the very beginning of seeds filling. Spikes were then husked and surface sterilized with 3% NaClO containing 0.10% Tween 20, shaken on a gyratory shaker at low speed for 20 minutes. Following three rinses with sterile distilled water, embryos were excised under a dissecting microscope in a laminar flow hood using fine forceps.

Excised embryos (size ˜0.3 mm, bell shaped) were placed on callus induction medium (CIM) [LS salts (Linsmaier, E. M. & Skoog, F. 1965. Physiol. Plantarum 18, 100) and vitamins plus 3% sucrose, 6 mg/L CuSO4, 2.5 mg/l 2,4-Dichlorophenoxyacetic Acid, pH 5.8 and 0.25% phytagel (Sigma)] with the scutellum side down, 50 or 100 embryos on a plate, and incubated at 28° C. in the dark. One week later, the embryonic calli were cleaned from emerging shoots and somatic calli, and subcultured onto fresh CIM medium. During culture, yellowish embryogenic calli (EC) appear and were further selected (e.g., picked and transferred) for further incubation in the same conditions for additional 2 weeks. Twenty-five pieces of sub-cultured calli were then separately placed on 90×15 mm petri plates, and incubated as before for three additional weeks.

Transformation—As described in Vogel and Hill (2008, Supra), Agrobacterium is scraped off 2-day-old MGL plates (plates with the MGL medium which contains: Tryptone 5 gr/L, Yeast Extract 2.5 gr/L, NaCl 5 gr/L, D-Mannitol 5 g/l, MgSO4*7H2O 0.204 gr/L, K2HPO4 0.25 gr/L, Glutamic Acid 1.2 gr/L, Plant Agar 7.5 gr/L) and resuspended in liquid MS medium supplemented with 200 μM acetosyringone to an optic density (OD) at 600 nm (OD600) of 0.6 to 1.0. Once the desired OD was attained, 1 ml of 10% Synperonic PE/F68 (Sigma) per 100 ml of inoculation medium is added.

To begin inoculation, 300 callus pieces were placed in approximately 12 plates (25 callus pieces in each plate) and covered with the Agrobacterium suspension (8-10 ml). The callus was incubated in the Agrobacterium suspension for 5 to 20 minutes. After incubation, the Agrobacterium suspension was aspirated off and the calli were then transferred into co-cultivation plates, prepared by placing a sterile 7-cm diameter filter paper in an empty 90×15 mm petri plate. The calli pieces are then gently distributed on the filter paper. One co-cultivation plate is used for two starting callus plates (50 initial calli pieces). The co-cultivation plates were then sealed with Parafilm M® or a plastic wrap [e.g., Saran™ wrap (Dow Chemical Company)] and incubated at 24° C. in the dark for 3 days.

The callus pieces were then individually transferred into CIM medium as described above, which was further supplemented with 200 mg/L Ticarcillin (to kill the Agrobacterium) and Bialaphos (5 mg/L) or Hygromycin B (40 mg/L) (for selection of the transformed resistant embryogenic calli sections), and incubated at 28° C. in the dark for 14 days. The calli pieces were then transferred to shoot induction media (SIM; LS salts and vitamins plus 3% Maltose monohydrate) supplemented with 400 mg/L Ticarcillin, Bialaphos (5 mg/L) or Hygromycin B (40 mg/L), Indol-3-acetic acid (IAA) (0.25 mg/L), and 6-Benzylaminopurine (BAP) (1 mg/L), and were cultivated in conditions as described below. After 10-15 days calli were sub-cultured on the same fresh media for additional 10-15 days (total of 20-30 days). At each sub-culture all the pieces from a single callus were kept together to maintain their independence and were incubated under the following conditions: light to a level of 60 IE m−2 s−1, a 16-hours light, 8-hours dark photoperiod and a constant 24° C. temperature. During the period of 20 to 30 days from the beginning of cultivation of calli on shoot induction media (SIM) plantlets start to emerge from the transformed calli.

When plantlets were large enough to handle without damage, they were transferred to plates containing the above-mentioned shoot induction media (SIM) with Bialaphos or Hygromycin B. Each plantlet is considered as a different event. After two weeks of growth, the plantlets were transferred to 2-cm height Petri plates (De Groot, Catalog No. 60-664160) containing MSnoH media (MS salts 4.4 gr/L, sucrose 30 gr/L, supplemented with Hygromycin B (40 mg/L) and Ticarcillin (400 mg/L). Roots usually appear within 2 weeks. Rooted and non-rooted plants were transferred to a fresh MSnoH media supplemented with Hygromycin B and Ticarcillin as described above. In case roots do not appear in the non-rooted plants after two weeks on the MSnoH media (which is supplemented with Hygromycin B and Ticarcillin), then the non-rooted plants are further transferred to the rooting induction medium [RIM; MS salts and vitamins 4.4 gr/L, sucrose 30 gr/L with Ticarcillin 400 mg/L, Indol-3-acetic acid (IAA) (1 mg/L), and α-Naphthalene acetic acid (NAA) (2 mg/L)]. After additional two weeks of incubation at 24° C., the plantlets are transferred to 0.5 modified RIM medium [MS modified salts 4.4 gr/L, MS vitamins 103 mg/L, sucrose 30 gr/L with α-Tocopherol (2 mg/L), Indol-3-acetic acid (IAA) (1 mg/L), and α-Naphthalene acetic acid (NAA) (2 mg/L)] and are incubated at 28° C. for additional 15-20 days, till the roots appear.

If needed, in the tillering stage the plantlets can grow axillary tillers and eventually become bushy on the above-mentioned media (SIM) without Bialaphos or Hygromycin B. Each bush from the same plant (event ID) is then divided to tissue culture boxes (“Humus”) containing “rooting medium” [MS basal salts, 3% sucrose, 3 gr/L phytagel, 2 mg/L α-Naphthalene Acetic Acid (NAA) and 1 mg/L IAA and Ticarcillin 400 mg/L, PH 5.8]. All plants in a “Humus box” are individual plants of the same transformation event.

When plantlets establish roots, they are transplanted to the soil and grown in the greenhouse. Before transfer to greenhouse, 20 randomly selected events are tested every month for expression of the BAR_GA gene (SEQ ID NO:39, BAR gene) which is responsible for resistance to Bialaphos, using AgraStrip® LL strip test seed check (Romer labs). Briefly, the expression of the BAR gene is determined as follows: Leaves (about 0.5 cm long leave) are grounded using a pellet pestle in an Eppendorf tube containing 150 μl of water until the water turns green in color. A strip test is then added to the Eppendorf tube and the results are read within 30-60 seconds. Appearance of two pink bands means that the plant is transgenic. On the other hand, appearance of one pink band means that the plant is not transgenic or not expressing BAR gene.

To verify the transgenic status of plants containing the gene of interest, T1 plants are subjected to PCR as previously described by Vogel et al. 2006 [Agrobacterium mediated transformation and inbred line development in the model grass Brachypodium distachyon. Plant Cell Tiss Org. Cult. 85:199-211].

Example 7: Validation Assays

The transgenic Brachypodium plants obtained as described hereinabove were used to validate the effect of the transformed gene(s) on fungal penetration and spreading within the plant by evaluation of fungal penetration and spreading within inoculated seedlings grown under controlled conditions.

Each validation assay evaluates the gene performance by quantitative and/or qualitative measure of specific traits as described in Table 11 below.

TABLE 11 Allocation of fungal parameters to specific traits # Parameters Traits 1 Fungal biomass in root Fungal presence in tissue

The validation assay was performed with inoculated transgenic plants grown under controlled conditions till seedling stage (1-2 tillers).

Transgenic Brachypodium seeds were sown in trays contained sterilized vermiculite soaked with 0.1% BASTA solution for selection of transgenic plants. The trays were placed in the refrigerator at 4° C. for cold treatment for 3 days (stratification step) following by trays placement in controlled growth chamber for germination up to 5 days (26° C.±1, humidity of 50%).

The viable transgenic seedlings were inoculated with Fusarium verticillioides (Fv 149 strain, GFP-tagged and bar-resistant) spore solution (106/ml) and then transplanted to 180 cm3 pots filled with sterilized vermiculite. The plants were grown for 7 days in the growth chamber.

For fungal biomass recovery, roots externally sterilized and then grinded. Serial dilutions of the extracts from each one of the biological repeats (1 biological repeat is a pool of 6 roots) were plated in Petri dishes and incubated for 5 days at 25° C. Developing colonies were counted to provide Colony Forming Units (CFU) using a florescent binocular and data were transformed to logarithmic scale for further analysis.

Fungal Biomass was computed as Log CFU of fungal root extraction 7 days after inoculation

Statistical analyses—To identify genes conferring significantly improved tolerance to fungal penetration and fungal spreading, the results obtained from the transgenic plants were compared to those obtained from control plants. To identify outperforming genes and constructs, results from the independent transformation events tested were analyzed separately. Analysis was conducted on the log CFU data using Student's t-test. The JMP statistics software package was used (Version 5.2.1, SAS Institute Inc., Cary, N.C., USA). Table 12 hereinbelow shows the reduction in CFU in the tested infected plants and its significance compared to control plants.

TABLE 12 Validation results (single genes): reduction of fungi burden in Brachypodium plant infected with Fusarium verticilloides Gene % change of logCFU relative to control % change of CFU Name Origin (p value) relative to control LFS90 Aegilops first assay: first assay: Event 13869.1: 11.45% (significant, p_value = 0.00) Event 13869.1: 179.54% Event 13870.1: 6.93% (significant, p_value = 0.02) Event 13870.1: 86.38% Event 13865.1: 9.40% (significant, p_value = 0.00) Event 13865.1: 132.57% Event 13866.1: 18.38% (significant, p_value = 0.00) Event 13866.1: 420.96% Event 13868.1: 10.57% (significant, p_value = 0.00) Event 13868.1: 158.22% Event 13874.1: 9.78% (significant, p_value = 0.00) Event 13874.1: 140.59% second assay: second assay: Event 13869.1: −1.72% (not significant, p_value = 0.25) Event 13869.1: -14.02% Event 13865.1: −2.28% (not significant, p_value = 0.18) Event 13865.1: −18.22% Event 13866.1: 3.23% (not significant, p_value = 0.11) Event 13866.1: 32.90% Event 13868.1: −3.61% (significant, p_value = 0.08) Event 13868.1: −27.22% Event 13874.1: 0.44% (not significant, p_value = 0.44) Event 13874.1: 3.95% LFS91 Aegilops first assay: first assay: Event 13884.1: 8.87% (significant, p_value = 0.00) Event 13884.1: 134.98% Event 13883.1: −6.99% (significant, p_value = 0.01) Event 13883.1: −49.02% Event 13885.1: −3.76% (not significant, p_value = 0.10) Event 13885.1: −30.38% Event 13891.1: 0.26% (not significant, p_value = 0.46) Event 13891.1: 2.55% Event 13890.1: 9.90% (significant, p_value = 0.00) Event 13890.1: 159.62% Event 13894.1: 1.62% (not significant, p_value = 0.29) Event 13894.1: 16.93% second assay: second assay: Event 13883.1: −9.31% (significant, p_value = 0.00) Event 13883.1: −61.15% LFS92 Aegilops first assay: first assay: Event 14188.1: −0.38% (not significant, p_value = 0.44) Event 14188.1: −3.18% Event 14202.1: −4.87% (significant, p_value = 0.08) Event 14202.1: −33.67% Event 14152.1: −5.65% (significant, p_value = 0.04) Event 14152.1: −37.92% Event 14155.1: −1.66% (not significant, p_value = 0.31) Event 14155.1: −13.09% Event 14187.1: −1.17% (not significant, p_value = 0.37) Event 14187.1: −9.42% Event 14201.1: −0.06% (not significant, p_value = 0.50) Event 14201.1: −0.52% second assay: second assay: Event 14202.1: −1.85% (not significant, p_value = 0.29) Event 14202.1: −16.59% Event 14152.1: −11.40% (significant, p_value = 0.00) Event 14152.1: −67.31% LFS93 Aegilops first assay: first assay: Event 14149.1: −2.25% (not significant, p_value = 0.28) Event 14149.1: −18.91% Event 14150.1: 10.52% (significant, p_value = 0.00) Event 14150.1: 166.28% Event 14108.1: −1.06% (not significant, p_value = 0.38) Event 14108.1: −9.38% Event 14147.1: −5.21% (significant, p_value = 0.07) Event 14147.1: −38.44% Event 14156.1: 8.77% (significant, p_value = 0.01) Event 14156.1: 126.22% Event 14157.1: 7.58% (significant, p_value = 0.01) Event 14157.1: 102.48% second assay: second assay: Event 14147.1: −3.07% (not significant, p_value = 0.14) Event 14147.1: −25.88% LFS94 Aegilops first assay: first assay: Event 14165.1: 4.96% (significant, p_value = 0.07) Event 14165.1: 49.56% Event 14164.1: 2.87% (not significant, p_value = 0.19) Event 14164.1: 26.25% Event 14162.1: 0.03% (not significant, p_value = 0.50) Event 14162.1: 0.23% Event 14171.1: 8.46% (significant, p_value = 0.01) Event 14171.1: 98.73% Event 14172.1: 8.30% (significant, p_value = 0.01) Event 14172.1: 96.09% Event 14168.1: 3.84% (not significant, p_value = 0.12) Event 14168.1: 36.52% LFS95 Aegilops first assay: first assay: Event 14213.1: 9.19% (significant, p_value = 0.01) Event 14213.1: 135.70% Event 14212.1: 4.42% (not significant, p_value = 0.12) Event 14212.1: 51.10% Event 14208.1: 5.86% (significant, p_value = 0.05) Event 14208.1: 72.71% Event 14205.1: 0.68% (not significant, p_value = 0.43) Event 14205.1: 6.50% Event 14210.1: 6.97% (significant, p_value = 0.03) Event 14210.1: 91.54% Event 14214.1: −5.60% (significant, p_value = 0.06) Event 14214.1: −40.68% second assay: second assay: Event 14214.1: 6.12% (significant, p_value = 0.04) Event 14214.1: 82.23% LFS96 Aegilops first assay: first assay: Event 14017.34: −10.34% (significant, p_value = 0.00) Event 14017.34: −63.12% Event 14018.29: −7.66% (significant, p_value = 0.01) Event 14018.29: −52.23% Event 14014.38: −18.53% (significant, p_value = 0.00) Event 14014.38: −83.26% Event 14015.40: −2.21% (not significant, p_value = 0.25) Event 14015.40: −19.17% Event 14019.34: −8.21% (significant, p_value = 0.01) Event 14019.34: −54.70% Event 14020.36: −5.72% (significant, p_value = 0.04) Event 14020.36: −42.40% second assay: second assay: Event 14017.34: −1.34% (not significant, p_value = 0.28) Event 14017.34: −13.44% Event 14018.29: 2.48% (not significant, p_value = 0.15) Event 14018.29: 30.61% Event 14014.38: −2.69% (not significant, p_value = 0.13) Event 14014.38: −25.17% Event 14019.34: −2.40% (not significant, p_value = 0.15) Event 14019.34: −22.82% Event 14020.36: −0.85% (not significant, p_value = 0.36) Event 14020.36: −8.74% LFS97 Aegilops first assay: first assay: Event 13932.1: −1.43% (not significant, p_value = 0.28) Event 13932.1: −12.81% Event 13938.1: −2.60% (not significant, p_value = 0.15) Event 13938.1: −22.09% Event 13936.1: −1.96% (not significant, p_value = 0.22) Event 13936.1: −17.12% Event 13937.1: −5.62% (significant, p_value = 0.01) Event 13937.1: −41.63% Event 13939.1: 1.19% (not significant, p_value = 0.32) Event 13939.1: 12.11% Event 13933.1: −0.78% (not significant, p_value = 0.38) Event 13933.1: −7.17% second assay: second assay: Event 13937.1: 3.78% (not significant, p_value = 0.13) Event 13937.1: 41.87% Event 13937.1: −6.16% (significant, p_value = 0.00) Event 13937.1: −48.55% LFS98 Aegilops first assay: first assay: Event 13911.1: −0.83% (not significant, p_value = 0.42) Event 13911.1: −7.43% Event 13917.1: −2.20% (not significant, p_value = 0.22) Event 13917.1: −18.51% Event 13910.1: −5.18% (significant, p_value = 0.03) Event 13910.1: −38.17% Event 13909.1: −0.66% (not significant, p_value = 0.43) Event 13909.1: −5.97% Event 13915.1: 8.77% (significant, p_value = 0.00) Event 13915.1: 125.80% Event 13919.1: 6.10% (significant, p_value = 0.01) Event 13919.1: 76.17% second assay: second assay: Event 13910.1: −4.21% (not significant, p_value = 0.12) Event 13910.1: −34.81% LFS99 Aegilops first assay: first assay: Event 14138.1: 12.65% (significant, p_value = 0.00) Event 14138.1: 158.53% Event 14137.1: 6.25% (significant, p_value = 0.02) Event 14137.1: 59.92% Event 14140.1: 12.90% (significant, p_value = 0.00) Event 14140.1: 163.35% Event 14134.1: 8.96% (significant, p_value = 0.00) Event 14134.1: 95.88% Event 14135.1: −4.95% (significant, p_value = 0.05) Event 14135.1: −31.05% Event 14142.1: 24.44% (significant, p_value = 0.00) Event 14142.1: 526.43% second assay: second assay: Event 14138.1: −2.38% (not significant, p_value = 0.23) Event 14138.1: −20.09% Event 14137.1: 13.83% (significant, p_value = 0.00) Event 14137.1: 268.06% Event 14140.1: 9.65% (significant, p_value = 0.00) Event 14140.1: 148.18% Event 14135.1: 0.57% (not significant, p_value = 0.46) Event 14135.1: 5.54% Event 14141.1: −0.95% (not significant, p_value = 0.35) Event 14141.1: −8.60% Event 14142.1: 5.81% (significant, p_value = 0.06) Event 14142.1: 72.84% LFS100 Aegilops first assay: first assay: Event 13984.27: 8.05% (significant, p_value = 0.01) Event 13984.27: 125.39% Event 14070.35: −2.27% (not significant, p_value = 0.27) Event 14070.35: −20.44% Event 14052.30: −3.67% (not significant, p_value = 0.14) Event 14052.30: −30.97% Event 14051.31: −2.02% (not significant, p_value = 0.30) Event 14051.31: −18.41% Event 14077.38: −4.53% (significant, p_value = 0.10) Event 14077.38: −36.71% Event 13987.36: −4.48% (not significant, p_value = 0.10) Event 13987.36: −36.40% second assay: second assay: Event 14077.38: −1.98% (not significant, p_value = 0.27) Event 14077.38: −18.25% LFS104 Aegilops first assay: first assay: Event 13943.39: −0.15% (not significant, p_value = 0.47) Event 13943.39: −1.54% Event 14068.24: −2.02% (not significant, p_value = 0.29) Event 14068.24: −18.43% Event 14069.22: 2.54% (not significant, p_value = 0.27) Event 14069.22: 29.23% Event 14022.32: −11.15% (significant, p_value = 0.00) Event 14022.32: −67.60% Event 14023.24: −9.31% (significant, p_value = 0.02) Event 14023.24: −60.99% Event 14067.17: 4.44% (not significant, p_value = 0.14) Event 14067.17: 56.62% second assay: second assay: Event 14022.32: −19.32% (significant, p_value = 0.00) Event 14022.32: −86.85% Event 14023.24: −20.99% (significant, p_value = 0.00) Event 14023.24: −88.96% LFS105 Aegilops first assay: first assay: Event 14026.1: −8.60% (significant, p_value = 0.00) Event 14026.1: −56.52% Event 14035.1: −1.32% (not significant, p_value = 0.29) Event 14035.1: −12.04% Event 14031.1: −3.52% (significant, p_value = 0.09) Event 14031.1: −28.86% Event 14025.1: −2.91% (not significant, p_value = 0.12) Event 14025.1: −24.57% Event 14034.1: −2.54% (not significant, p_value = 0.16) Event 14034.1: −21.80% Event 14029.1: −2.34% (not significant, p_value = 0.17) Event 14029.1: −20.30% second assay: second assay: Event 14026.1: −4.00% (significant, p_value = 0.08) Event 14026.1: −32.37% Event 14031.1: 2.04% (not significant, p_value = 0.23) Event 14031.1: 22.08% LFS106 Aegilops first assay: first assay: Event 13993.33: 6.18% (significant, p_value = 0.02) Event 13993.33: 80.50% Event 13996.23: 8.88% (significant, p_value = 0.00) Event 13996.23: 133.43% Event 14000.41: 7.34% (significant, p_value = 0.01) Event 14000.41: 101.56% Event 14003.37: 2.66% (not significant, p_value = 0.21) Event 14003.37: 28.96% Event 13999.42: 2.15% (not significant, p_value = 0.24) Event 13999.42: 22.80% Event 14001.37: 11.19% (significant, p_value = 0.00) Event 14001.37: 191.28% second assay: second assay: Event 13993.33: −7.16% (significant, p_value = 0.05) Event 13993.33: −52.83% Event 13996.23: −5.75% (significant, p_value = 0.09) Event 13996.23: −45.32% Event 14000.41: −2.56% (not significant, p_value = 0.27) Event 14000.41: −23.57% Event 14001.37: −4.58% (not significant, p_value = 0.14) Event 14001.37: −38.15% LFS107 Aegilops first assay: first assay: Event 13951.32: 10.34% (significant, p_value = 0.00) Event 13951.32: 153.93% Event 13948.31: 11.64% (significant, p_value = 0.00) Event 13948.31: 185.33% Event 13946.32: 8.99% (significant, p_value = 0.00) Event 13946.32: 124.83% Event 13956.45: 11.85% (significant, p_value = 0.00) Event 13956.45: 190.80% Event 13960.33: 4.67% (significant, p_value = 0.06) Event 13960.33: 52.37% second assay: second assay: Event 13951.32: 1.24% (not significant, p_value = 0.34) Event 13951.32: 11.52% Event 13948.31: 3.36% (significant, p_value = 0.09) Event 13948.31: 34.45% Event 13946.32: 1.90% (not significant, p_value = 0.21) Event 13946.32: 18.24% Event 13956.45: 4.76% (significant, p_value = 0.02) Event 13956.45: 52.17% LFS109 Aegilops first assay: first assay: Event 14193.1: −5.39% (significant, p_value = 0.00) Event 14193.1: −39.83% Event 14194.1: 1.98% (not significant, p_value = 0.14) Event 14194.1: 20.52% Event 14197.1: 0.05% (not significant, p_value = 0.48) Event 14197.1: 0.51% Event 14192.1: 0.49% (not significant, p_value = 0.38) Event 14192.1: 4.67% Event 14198.1: −5.18% (significant, p_value = 0.00) Event 14198.1: −38.60% Event 14191.1: −1.98% (not significant, p_value = 0.15) Event 14191.1: −16.99% second assay: second assay: Event 14193.1: 2.69% (not significant, p_value = 0.16) Event 14193.1: 30.08% LFS110 Aegilops first assay: first assay: Event 14254.1: −3.90% (significant, p_value = 0.07) Event 14254.1: −33.07% Event 14283.1: 0.61% (not significant, p_value = 0.40) Event 14283.1: 6.45% Event 14255.1: 4.83% (significant, p_value = 0.03) Event 14255.1: 64.37% Event 14284.1: −6.43% (significant, p_value = 0.01) Event 14284.1: −48.43% Event 14250.1: 0.60% (not significant, p_value = 0.41) Event 14250.1: 6.40% Event 14252.1: 0.17% (not significant, p_value = 0.47) Event 14252.1: 1.81% second assay: second assay: Event 14254.1: 0.22% (not significant, p_value = 0.44) Event 14254.1: 2.24% Event 14284.1: −0.97% (not significant, p_value = 0.32) Event 14284.1: −9.28% LFS111 Aegilops first assay: first assay: Event 14064.1: 17.84% (significant, p_value = 0.00) Event 14064.1: 433.55% Event 14058.1: −9.20% (significant, p_value = 0.00) Event 14058.1: −57.84% Event 14060.1: −8.68% (significant, p_value = 0.00) Event 14060.1: −55.73% Event 14073.1: 10.65% (significant, p_value = 0.00) Event 14073.1: 171.77% Event 14057.1: −5.24% (significant, p_value = 0.03) Event 14057.1: −38.82% Event 14059.1: 13.43% (significant, p_value = 0.00) Event 14059.1: 252.78% second assay: second assay: Event 14058.1: −2.16% (not significant, p_value = 0.20) Event 14058.1: −17.33% Event 14057.1: 3.96% (not significant, p_value = 0.17) Event 14057.1: 42.13% Event 14060.1: −0.35% (not significant, p_value = 0.45) Event 14060.1: −3.33% LFS112 Aegilops first assay: first assay: Event 14307.1: −5.30% (significant, p_value = 0.01) Event 14307.1: −40.36% Event 14309.1: −1.49% (not significant, p_value = 0.25) Event 14309.1: −13.54% Event 14298.1: 0.65% (not significant, p_value = 0.40) Event 14298.1: 6.53% Event 14301.1: −4.38% (significant, p_value = 0.03) Event 14301.1: −34.78% Event 14302.1: −9.07% (significant, p_value = 0.00) Event 14302.1: −58.75% Event 14305.1: −1.56% (not significant, p_value = 0.24) Event 14305.1: −14.12% second assay: second assay: Event 14307.1: 0.90% (not significant, p_value = 0.33) Event 14307.1: 9.44% Event 14301.1: −3.62% (significant, p_value = 0.04) Event 14301.1: −30.53% Event 14302.1: −4.12% (significant, p_value = 0.02) Event 14302.1: −33.92% LFS113 Aegilops first assay: first assay: Event 13974.29: 4.15% (significant, p_value = 0.05) Event 13974.29: 44.75% Event 13979.31: 4.39% (significant, p_value = 0.04) Event 13979.31: 47.89% LFS114 Aegilops first assay: first assay: Event 14179.1: −14.48% (significant, p_value = 0.00) Event 14179.1: −75.12% Event 14181.1: −4.51% (significant, p_value = 0.03) Event 14181.1: −35.14% Event 14175.1: −8.02% (significant, p_value = 0.00) Event 14175.1: −53.70% Event 14184.1: −7.70% (significant, p_value = 0.00) Event 14184.1: −52.26% Event 14174.1: −20.43% (significant, p_value = 0.00) Event 14174.1: −85.95% Event 14177.1: −9.03% (significant, p_value = 0.00) Event 14177.1: −58.00% second assay: second assay: Event 14179.1: −1.72% (not significant, p_value = 0.28) Event 14179.1: −15.43% Event 14181.1: −1.90% (not significant, p_value = 0.26) Event 14181.1: −16.90% Event 14175.1: 0.26% (not significant, p_value = 0.46) Event 14175.1: 2.59% Event 14184.1: −1.44% (not significant, p_value = 0.31) Event 14184.1: −13.09% Event 14174.1: −3.09% (not significant, p_value = 0.14) Event 14174.1: −25.97% Event 14177.1: −2.13% (not significant, p_value = 0.23) Event 14177.1: −18.75% LFS115 Aegilops first assay: first assay: Event 14260.1: 1.19% (not significant, p_value = 0.34) Event 14260.1: 12.64% Event 14261.1: −0.84% (not significant, p_value = 0.39) Event 14261.1: −8.09% Event 14268.1: 4.73% (significant, p_value = 0.05) Event 14268.1: 60.69% Event 14259.1: 0.10% (not significant, p_value = 0.49) Event 14259.1: 0.97% Event 14263.1: −8.76% (significant, p_value = 0.00) Event 14263.1: −58.48% Event 14262.1: 4.26% (significant, p_value = 0.08) Event 14262.1: 53.37% second assay: second assay: Event 14263.1: −4.66% (significant, p_value = 0.01) Event 14263.1: −37.40% LFS116 Aegilops first assay: first assay: Event 13962.41: −2.38% (not significant, p_value = 0.30) Event 13962.41: −20.21% Event 13898.1: −2.67% (not significant, p_value = 0.27) Event 13898.1: −22.39% Event 13904.1: −7.55% (significant, p_value = 0.04) Event 13904.1: −51.20% Event 13961.30: −4.26% (not significant, p_value = 0.17) Event 13961.30: −33.27% Event 13902.1: −3.32% (not significant, p_value = 0.23) Event 13902.1: −27.05% Event 13903.1: −7.12% (significant, p_value = 0.06) Event 13903.1: −49.13% second assay: second assay: Event 13904.1: −1.77% (not significant, p_value = 0.29) Event 13904.1: −16.48% Event 13903.1: −0.56% (not significant, p_value = 0.44) Event 13903.1: −5.52% LFS117 Aegilops first assay: first assay: Event 14246.1: −5.66% (significant, p_value = 0.05) Event 14246.1: −42.61% Event 14203.1: 3.03% (not significant, p_value = 0.19) Event 14203.1: 34.55% Event 14245.1: −9.05% (significant, p_value = 0.00) Event 14245.1: −58.81% second assay: second assay: Event 14246.1: 3.99% (not significant, p_value = 0.14) Event 14246.1: 44.59% Event 14245.1: −1.48% (not significant, p_value = 0.34) Event 14245.1: −12.76% LFS122 Foxtail first assay: first assay: millet Event 13926.1: 10.35% (significant, p_value = 0.00) Event 13926.1: 160.27% Event 13880.1: 14.50% (significant, p_value = 0.00) Event 13880.1: 281.82% Event 13879.1: 12.68% (significant, p_value = 0.00) Event 13879.1: 222.64% Event 13908.1: 6.91% (significant, p_value = 0.01) Event 13908.1: 89.30% Event 13906.1: 0.21% (not significant, p_value = 0.47) Event 13906.1: 1.99% Event 13907.1: −3.63% (significant, p_value = 0.09) Event 13907.1: −28.52% second assay: second assay: Event 13907.1: −9.24% (significant, p_value = 0.00) Event 13907.1: −60.88% LFS123 Foxtail first assay: first assay: millet Event 14127.1: 3.23% (not significant, p_value = 0.11) Event 14127.1: 37.61% Event 14130.1: −0.64% (not significant, p_value = 0.40) Event 14130.1: −6.18% Event 14125.1: −13.66% (significant, p_value = 0.00) Event 14125.1: −74.11% Event 14121.1: −5.44% (significant, p_value = 0.02) Event 14121.1: −41.63% Event 14122.1: 7.25% (significant, p_value = 0.00) Event 14122.1: 104.81% Event 14131.1: 1.34% (not significant, p_value = 0.30) Event 14131.1: 14.13% second assay: second assay: Event 14125.1: 0.49% (not significant, p_value = 0.47) Event 14125.1: 4.42% Event 14121.1: −0.47% (not significant, p_value = 0.39) Event 14121.1: −4.06% LFS125 Foxtail first assay: first assay: millet Event 14043.1: 0.01% (not significant, p_value = 0.49) Event 14043.1: 0.05% Event 14056.1: −6.59% (significant, p_value = 0.02) Event 14056.1: −46.26% Event 14044.1: −6.57% (significant, p_value = 0.02) Event 14044.1: −46.15% Event 14048.1: 1.23% (not significant, p_value = 0.34) Event 14048.1: 12.26% Event 14042.1: 1.20% (not significant, p_value = 0.34) Event 14042.1: 11.95% Event 14053.1: −4.57% (significant, p_value = 0.08) Event 14053.1: −34.98% second assay: second assay: Event 14056.1: −6.84% (significant, p_value = 0.02) Event 14056.1: −50.22% Event 14044.1: −6.73% (significant, p_value = 0.02) Event 14044.1: −49.65% Event 14053.1: −3.49% (not significant, p_value = 0.15) Event 14053.1: −29.92% LFS126 Foxtail first assay: first assay: millet Event 14081.1: −4.75% (significant, p_value = 0.02) Event 14081.1: −37.78% Event 14089.1: −9.88% (significant, p_value = 0.00) Event 14089.1: −62.77% Event 14085.1: 6.71% (significant, p_value = 0.00) Event 14085.1: 95.51% Event 14082.1: −0.40% (not significant, p_value = 0.43) Event 14082.1: −3.88% Event 14086.1: −10.60% (significant, p_value = 0.00) Event 14086.1: −65.34% Event 14087.1: 3.21% (significant, p_value = 0.08) Event 14087.1: 37.82% second assay: second assay: Event 14081.1: −5.25% (significant, p_value = 0.06) Event 14081.1: −41.44% Event 14089.1: 2.31% (not significant, p_value = 0.23) Event 14089.1: 26.61% Event 14086.1: −4.17% (significant, p_value = 0.10) Event 14086.1: −34.67% LFS127 Foxtail first assay: first assay: millet Event 14102.1: −1.42% (not significant, p_value = 0.32) Event 14102.1: −14.65% Event 14104.1: −6.45% (significant, p_value = 0.01) Event 14104.1: −51.20% Event 14101.1: −3.76% (significant, p_value = 0.08) Event 14101.1: −34.14% Event 14096.1: −4.07% (significant, p_value = 0.07) Event 14096.1: −36.39% Event 14098.1: −7.82% (significant, p_value = 0.00) Event 14098.1: −58.07% Event 14100.1: −9.83% (significant, p_value = 0.00) Event 14100.1: −66.48% second assay: second assay: Event 14104.1: 2.31% (not significant, p_value = 0.29) Event 14104.1: 24.66% Event 14101.1: −0.33% (not significant, p_value = 0.47) Event 14101.1: −3.06% Event 14096.1: 7.82% (significant, p_value = 0.03) Event 14096.1: 110.92% Event 14098.1: −1.41% (not significant, p_value = 0.37) Event 14098.1: −12.56% Event 14100.1: 2.72% (not significant, p_value = 0.26) Event 14100.1: 29.66% LFS128 Foxtail first assay: first assay: millet Event 14222.1: −0.79% (not significant, p_value = 0.40) Event 14222.1: −7.05% Event 14223.1: −2.67% (not significant, p_value = 0.19) Event 14223.1: −21.93% Event 14225.1: −1.43% (not significant, p_value = 0.32) Event 14225.1: −12.39% Event 14226.1: −2.56% (not significant, p_value = 0.17) Event 14226.1: −21.10% Event 14229.1: −0.93% (not significant, p_value = 0.38) Event 14229.1: −8.23% Event 14220.1: 2.35% (not significant, p_value = 0.22) Event 14220.1: 24.38% “CFU” = Colony Forming Unit

An increase in the resistance of plants to fungal diseases may be achieved by two genes stack expression, which are addressing the same or different mode of action.

Two different combinations of gene stacking were expressed in Brachypodium plants. The combination of LFS57 (disclosed in WO 2018/131037), a gene linked to programmed cell death, and LFSS86 disclosed in the present invention, a gene related to plant defense signaling activation, addresses the effect of stacking two different mode of actions directed to increase resistance.

The combination of LFS46 (disclosed in WO 2018/131037) and LFS84 disclosed in the present invention addresses the effect of stacking two genes coding for enzymes processing the same metabolite involved in defense response signaling therefore increasing the efficacy of the reaction.

Table 13 hereinbelow summarizes the reduction of fungi burden in the transgenic Brachypodium plants comprising the stacked genes infected with Fusarium verticilloides.

TABLE 13 Validation results (stacked genes): reduction of fungi burden in Brachypodium plant infected with Fusarium verticilloides Construct Origin Origin % change of logCFU relative to control % change of CFU Name gene 1 gene 2 (p-value) relative to control Stack: LFS57 & Sorghum Maize first assay: first assay: LFS86 Event 13342.1: −11.07% Event 13342.1: −66.14% (significant, p_value = 0.00) Event 13345.1: −41.40% Event 13345.1: −5.46% Event 13339.1: −74.12% (significant, p_value = 0.04) Event 13344.1: 93.66% Event 13339.1: −13.82% Event 13331.1: 3.85% (significant, p_value = 0.00) Event 13340.1: −56.20% Event 13344.1: 6.76% (significant, p_value = 0.01) Event 13331.1: 0.39% (not significant, p_value = 0.45) Event 13340.1: −8.44% (significant, p_value = 0.00) second assay: second assay: Event 13342.1: −2.88% Event 13342.1: −23.99% (not significant, p_value = 0.13) Event 13345.1: −22.59% Event 13345.1: −2.69% Event 13339.1: −30.57% (not significant, p_value = 0.15) Event 13339.1: −3.83% (significant, p_value = 0.07) Stack: LFS46 & Sorghum Wheat first assay: first assay: LFS84 Event 13859.1: −7.47% Event 13859.1: −52.31% (significant, p_value = 0.01) Event 13852.1: −0.79% Event 13852.1: −0.08% Event 13848.1: −26 99% (not significant, p_value = 0.49) Event 13848.1: −3.18% (not significant, p_value = 0.17) Event 13850.1: −72.23% Event 13850.1: −12.93% Event 13851.1: 53.34% (significant, p_value = 0.00) Event 13851.1: 4.31% (not significant, p_value = 0.11) second assay: second assay: Event 13859.1: −4.10% Event 13859.1: −31.75% (not significant, p_value = 0.13) Event 13850.1: −74.82% Event 13850.1: −14.82% (significant, p_value = 0.00) “CFU” = Colony Forming Unit

Example 8: Overexpression of a Polypeptide by Genome Editing

Over-expression of a polypeptide according to certain embodiments of the present invention can be achieved using methods of gene editing. One example of such approach includes editing a selected genomic region as to express the polypeptide of interest. In the current example, the target genomic region is the maize locus GRMZM2G069095 (based on genome version Zea mays AGPv3) and the polypeptide to be over-expressed is the maize LFS127_H4 comprising the amino acid sequence set forth in SEQ ID NO:495 encoded by the polynucleotide having the nucleic acid sequence set forth in SEQ ID NO:289. It is to be explicitly understood that other genome loci can be used as targets for genome editing for over-expressing other polypeptides of the invention based on the same principles.

FIG. 2A depicts the sequence of the endogenous 5′ upstream flanking region of the genomic sequence GRMZM2G069095 (SEQ ID NO:12) and FIG. 2B depicts the sequence of the endogenous 3′-downstream flanking region of this genomic locus (SEQ ID NO: 13). FIG. 2C depicts the sequence of the 5′-UTR gRNA (SEQ ID NO:10) and FIG. 2D depicts the sequence of the 5′-UTR gRNA without NGG nucleotides following the 3 nucleotides after the Cas9 cutting (SEQ ID NO:14). FIG. 2E depicts the sequence of the 3′-UTR gRNA (SEQ ID NO: 11) and FIG. 2F depicts the sequence of the 3′-UTR gRNA after cut (SEQ ID NO:15). FIG. 2G depicts the coding sequence (from the “ATG” start codon to the “TAG” termination codon, marked by bold and underlined) of the desired LFS127_H4 sequence (SEQ ID NO:289) encoding the polypeptide set forth by SEQ ID NO:495.

The complete exemplary repair template (SEQ ID NO:16) is depicted in FIG. 2H. The repair template includes: (1) the upstream flanking region (1 kbp) sequence including part of the gRNA after cutting (SEQ ID NO: 14; shown in bold and italics); (2) 5′ UTR of genomic DNA from Cas9 cutting site to ATG; (3) the coding sequence (CDS) of the desired LFS127_H4 sequence (SEQ ID NO:17) marked in lower case with the start (ATG) and the stop (TGA) codons marked in bold and underlined; (4) 3′ UTR of genomic DNA from the stop codon to Cas9 cutting site including the predicted part of the gRNA after cutting (SEQ ID NO:15), shown in bold and underlined and (5) the downstream flanking region (1 kbp) sequence.

The repair template is delivered into the cell type of interest along with the 5′ and 3′guide RNA sequences (SEQ ID NO: 10 and SEQ ID NO:11, respectively).

Example 9: Knock Out of a Polypeptide by Genome Editing

Knock-out of a polypeptide according to certain embodiments of the present invention can be achieved using methods of gene editing.

In the current example, the target genomic region is the maize (Zea mays) LFS132 comprising the amino acid sequence set forth in SEQ ID NO:348 encoded by the polynucleotide having the nucleic acid sequence set forth in SEQ ID NO:62. It is to be explicitly understood that other genome loci can be used as targets for genome editing for over-expressing other polypeptides of the invention based on the same principles.

The Crisper/CAS9 or similar systems can generate double stranded breaks (DSBs) at any genomic locus under the guidance of an engineered single-guide RNA when delivered into the cell type of interest. Non-homologous end-joining (NHEJ) in the absence of DNA template accompanied with the modification of target genomic repair the DSBs but tends to be prone to insertion and/or deletion (indel) mutations at the junctional site therefore causing framshifts mutations that disrupt the targeted gene. (FIG. 3E)

FIG. 3A depicts the sequence of the KO gRNA (SEQ ID NO:51); FIG. 3C depicts the coding sequence (from the “ATG” start codon to the “TGA” termination codon, marked by bold and underlined) of the desired LFS132 sequence (SEQ ID NO:53); FIG. 3B depicts the sequence of the KO gRNA after cut (SEQ ID NO:52); FIG. 3D (targeted region in bold) and FIG. 3E depict the anticipated change in the coding sequence of the exemplified KO gene (SEQ ID NO:54).

Section II: Bacterial Genes Associated with Fungal Resistance

Example 10: Isolating Soil Bacteria with Potential Antifungal Activity

Bacteria were isolated from soil in five different locations in Israel with agriculture history for Fusaria spp. related diseases, by a two-layer functional assay against Fusarium verticillioides (Fv). In brief, fungal spores were plated on PDA agar medium with a thin R2A medium (Reasoner D J & Geldreich A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol 1985 49:1-7) overlay containing different dilutions of soil washes. Active bacteria were isolated from lytic zones and further characterized for the antifungal activity in dual-culture confrontation assays against Fv on R2A agar plates. Table 14 presents a list of the bacteria isolated. In parenthesis is the percentage of similarity between the isolate and the known genus or species.

TABLE 14 Bacterial isolates having antifungal activity Isolate Taxa Isolate No. (Percentage of similarity) 1 Arthrobacter globiformis (31.4) 2 Bacillus (95.0) 3 Pseudomonas (66.2) 4 Erwinia (17.8) 5 Arthrobacter (33.1) 6 Bacillus (75.5) 7 Bacillus (94.8) 8 Bacillus (94.8) 9 Pseudomonas (43.6) | Massilia (45.6) 10 Pseudomonas (43.6) |Massilia (45.6) 11 Pseudomonas (44.0) 12 Pseudomonas (61.4) 13 Pseudomonas (61.4) 14 Pseudomonas (61.4) 15 Pseudomonas (52.7) |Bacillus subtilis (80.1) 16 Bacillus (78.4) 17 Bacillus (95.2) 18 Bacillus (95.1) 19 Bacillus (93.5) 20 Pseudomonas (50.6) 21 Erwinia (17.9) 22 Lysobacter_capsici (85.2) | Erwinia (18.0) 23 Arthrobacter (36.1) 24 Arthrobacter_globiformis (29.2) 25 Pseudomonas (63.8) | Lysobacter (22.5) 26 Pseudomonas (63.8) | Lysobacter (22.5) 27 Pseudomonas (59.2) 28 Pseudomonas (59.2) 29 Pseudomonas (59.2) 30 Chryseobacterium_daeguense (28.1) 31 Chryseobacterium_daeguense (28.1) 32 Pseudomonas (44.0) 33 Pseudomonas (44.0) 34 Pseudomonas (61.4) 35 Pseudomonas (60.9) 36 Pseudomonas (60.9) 37 Pseudomonas (58.9) | Bacillus (77.2) 38 Bacillus (74.0) 39 Arthrobacter (19.8) 40 Pantoea_agglomerans (35.7) 41 Curtobaderium_UNCCL17 (82.3) | Pantoea_agglomerans (91.6) 42 Pantoea (83.6) 43 Pantoea (83.6) 44 Pseudomonas alcaligenes (38.6) 45 Pseudomonas (50.2) 46 Pseudomonas (41.2) 47 Pseudomonas (85.4) 48 Leifsonia (11.9) | Clavibacter michiganensis (9.3) 49 Microbacterium (19.8) 50 Microbacterium (19.8) 51 Pseudomonas (60.1) 52 Bacillus (94.5) 53 Pseudomonas s (50.1) 54 Pseudomonas (40.5) 55 Pseudomonas (40.5) 56 Pseudomonas (59.1) 57 Pseudomonas s (59.1) 58 Bacillus (92.4) 59 Pseudomonas (44.0) 60 Bacillus (92.4) 61 Pseudomonas (44.0) 62 Pseudomonas (56.0) 63 Bacillus (95.0) 64 Bacillus (74.0) 65 Pseudomonas (41.1) 66 Pseudomonas (41.1) 67 Pseudomonas (41.1) 68 Pseudomonas (60.1) 69 Pseudomonas (60.1) 70 Pseudomonas (60.1) 71 Pantoea (100); 72 Bacillus (100); 73 Arthrobacter (100); unclassified; 74 Paenibacillus (100); unclassified; 75 Paenibacillus (100); unclassified; 76 Pseudomonas (100); Pseudomonas frederiksbergensis (98) 77 Erwinia (91); unclassified; 78 Bacillus (100) 79 Pseudomonas chloraphis 80 Alcaligenaceae (100); unclassified 81 Bacillus_axarquiensis 82 Pseudomonas spp. 83 Pantoea agglomerans 84 Chryseobacterium_ginsenosidimutans 85 Bacillus_altitudinis

Example 11: Identifying Fungicidal Genes Eligible for Disease Control

The present inventors have identified 199 polynucleotides of bacterial origin that encode for proteins with potential antifungal activity. The antifungal activity of successfully purified soluble proteins was tested in an in vitro spore assay against Fusarium verticilloides. The antifungal activity is further examined by expressing the bacterial genes in planta, and evaluating fungal resistance conferred by the exogenous polynucleotides.

The polynucleotides and polypeptides of some embodiments of the invention having the antifungal activity originate from bacterial isolate proprietary genomes as listed in Table 14 and public bacterial genomes that were incorporated into a unified database, also containing gene expression data produced ad hoc, as well as gene phylogeny, protein annotation, enzymatic function and pathway.

Genomic and Transcriptomic Profiling of Bacterial Isolates for Gene Discovery Genomic Profiling

Genome assembly—genomes of all isolates were assembled using a proprietary pipeline.

Gene prediction—gene prediction was performed using Prokaryotic Dynamic Programming Genefinding Algorithm [Prodigal—BMC Bioinformatics. 2010 Mar. 8; 11(1):119].

Gene annotation—Predicted genes and proteins were annotated using BLAST™ search [blast.ncbi.nlm.nih.gov/Blast.cgi] against NCBI nr (non-redundant protein sequence database) and by further analysis by InterPro [ebi.ac.uk/interpro/].

Transcriptome Profiling

Eleven (11) bacterial strains (Serial Number 71, 72, 73, 74, 75, 77, 78, 79, 80, 83 of Table 14) were striped on a R2A medium in Petri dishes on the two opposite sides of a Fusarium verticillioides strip culture. Bacterial samples were collected after 3 days of co-culture after a clear inhibition zone was defined and the collected bacteria were used for transcriptomics analysis. Total RNA was extracted and submitted to Omega Bioservices contract lab for RNA sequencing. Transcriptomics data were evaluated for identification of highly expressed potential antifungal genes.

Differential Expression Data Analysis

RNA-seq uses next-generation sequencing (NGS) methods to sequence cDNA that has been derived from an RNA sample, and hence produces millions of short reads. These reads were then mapped onto a reference genome and the number of reads mapping within a genomic feature of interest (such as a gene) was used as a measure of the abundance of the feature in the analyzed sample, resulting in gene expression profile. According to the gene expression profile, a differential analysis was performed to identify genes with higher expression during fungal co-culture compared to pure bacteria cultures that are further associated with antifungal phenotypes.

Identification of Antifungal Genes from Bacterial Isolates

The 199 genes listed in Table 15 below were identified as potential antifungal and may confer fungal-resistance traits in planta. The Table shows the identified genes, their curated polynucleotide and polypeptide sequences.

TABLE 15 Identified antifungal genes from bacterial isolates Polyn. Polyp. Gene SEQ ID SEQ ID Name Gene Description Isolate Name NO NO BLFS1 antifungal protein Streptomyces clavuligerus ATCC 27064 592 2444 BLFS2 Hypothetical protein 15627EM_META_Alkanindiges1| 593 2445 BLFS3 Hypothetical protein 15627EM_META_ 594 2446 Enterococcus10|16v1|scaffold580442 G95 BLFS4 Hypothetical protein 15627EM_META_Klebsiella4 595 2447 BLFS5 Hypothetical protein Actinosporangium_NRRL_B_3428 596 2448 BLFS6 Hypothetical protein Actinosynnema mirum 597 2449 BLFS7 peptidase C11 Ammonifex_degensii_KC4 598 2450 clostripain BLFS8 Hypothetical protein Amycolatopsis azurea 599 2451 BLFS9 oxidoreductase Amycolatopsis azurea 600 2452 BLFS10 oxidoreductase Amycolatopsis azurea DSM 43854 601 2453 BLFS11 Hypothetical protein Arthrobacter spp. 602 2454 BLFS12 Hypothetical protein Arthrobacter spp. 603 2455 BLFS13 Hypothetical protein Arthrobacter spp. 604 2456 BLFS14 alpha integrin Arthrobacter spp. 605 2457 BLFS15 alpha integrin Arthrobacter_161MFSha2_1 606 2458 BLFS16 lysozyme Arthrobacter_161MFSha2_1 607 2459 BLFS17 D-alanyl-D-alanine Arthrobacter_161MFSha2_1 608 2460 carboxypeptidase BLFS18 Hypothetical protein Bacillus spp. 609 2461 BLFS19 peptidase p60 Bacillus spp. 610 2462 BLFS20 Cell division Bacillus spp. 611 2463 suppressor protein BLFS21 spoivb peptidase Bacillus spp. 612 2464 BLFS22 Hypothetical protein Bacillus spp. 613 2465 BLFS23 glycosyl hydrolase Bacillus spp. 614 2466 family 18 BLFS24 Hypothetical protein Bacillus spp. 615 2467 BLFS25 chitooligosaccharide Bacillus spp. 616 2468 deacetylase BLFS26 Hypothetical protein Bacillus spp. 617 2469 BLFS27 large protein of pyocin Bacillus spp. 618 2470 ap41 BLFS29 Hypothetical protein Bacillus_marmarensis_DSM_21297 619 2471 BLFS30 epsilon-toxin type b Bacillus_thuringiensis_serovar_indiana 620 2472 BLFS31 Hypothetical protein Bacillus velezensis 621 2473 BLFS32 Protease HtpX [htpX]. Bacillus velezensis 622 2474 BLFS33 zinc metalloprotease Bacillus velezensis 623 2475 BLFS34 Cell division Bacillus velezensis 624 2476 suppressor protein BLFS35 Hypothetical protein Bradyrhizobium japonicum 625 2477 BLFS36 Hypothetical protein Bradyrhizobium yuanmingense 626 2478 BLFS37 beta/gamma crystallin Brevibacillus laterosporus 627 2479 family protein BLFS38 jacalin-like lectin Brevibacillus laterosporus 628 2480 domain protein BLFS39 toxin ETX Brevibacillus laterosporus 629 2481 BLFS41 chitobiase Burkholderia gladioli 630 2482 BLFS42 chitin-binding protein Burkholderia gladioli 631 2483 BLFS43 carboxypeptidase Burkholderia gladioli 632 2484 regulatory-like domain protein BLFS44 ricin-type beta-trefoil Burkholderia gladioli 633 2485 lectin domain protein BLFS45 peptidase Burkholderia gladioli 634 2486 BLFS46 lysm domain protein Burkholderia gladioli 635 2487 BLFS47 Hypothetical protein Cellulosimicrobium spp. 636 2488 BLFS48 peptidase m48 Collimonas arenae 637 2489 BLFS49 Hypothetical protein Collimonas arenae 638 2490 BLFS50 chitin-binding protein Collimonas_arenae 639 2491 BLFS51 endoglucanase Collimonas_arenae 640 2492 BLFS52 Ricin B lectin Dactylosporangium_aurantiacum 641 2493 BLFS53 Etx/MtX1 toxin Dickeya_dadantii 642 2494 BLFS55 beta/gamma crystallin Duganella_HH101 643 2495 family protein BLFS56 aminopeptidase Duganella_HH101 644 2496 BLFS57 glycoside hydrolase Duganella_HH101 645 2497 family 12 BLFS58 17 kda surface antigen Duganella_HH101 646 2498 BLFS59 Hypothetical protein Duganella violaceinigra 647 2499 BLFS60 Hypothetical protein Erwinia spp. 648 2500 BLFS61 type i secretion target Erwinia spp. 649 2501 BLFS62 murein hydrolase Erwinia spp. 650 2502 activator nlpd BLFS63 n-acetylmuramoyl-1- Erwinia spp. 651 2503 alanine amidase amib BLFS64 NLP/P60 protein Geobacillus_Y4_1MC1 652 2504 BLFS65 extracellular repeat Janthinobacterium_HH010 653 2505 containing protein HAF family BLFS67 Beta/gamma crystallin Janthinobacterium_HH103 654 2506 BLFS68 putative exported Janthinobacterium MP5059B 655 2507 protein BLFS69 heme adhesion protein Janthinobacterium_Marseille 656 2508 BLFS70 Hypothetical protein Janthinobacterium_agaricidamnosum 657 2509 BLFS72 Carboxypeptidase Lysobacter spp. 658 2510 BLFS73 Hypothetical protein Paenibacillus spp. 659 2511 BLFS74 m1-852 Paenibacillus spp. 660 2512 BLFS75 Serine hydrolase Paenibacillus spp. 661 2513 BLFS76 Hypothetical protein Paenibacillus spp. 662 2514 BLFS77 glycoside hydrolase Paenibacillus spp. 663 2515 family 5 BLFS79 Hypothetical protein Paenibacillus spp. 664 2516 BLFS80 Hypothetical protein Paenibacillus spp. 665 2517 BLFS82 sspe Paenibacillus spp. 666 2518 BLFS83 exoglucanase a Paenibacillus spp. 667 2519 BLFS84 alpha/beta hydrolase Paenibacillus spp. 668 2520 BLFS85 alpha-amylase Paenibacillus spp. 669 2521 BLFS86 hypothetical protein Paenibacillus spp. 670 2522 BLFS87 endoglucanase b Paenibacillus spp. 671 2523 BLFS88 Hypothetical protein Paenibacillus spp. 672 2524 BLFS89 endoglucanase Paenibacillus spp. 673 2525 BLFS90 wgr domain- Paenibacillus spp. 674 2526 containing protein BLFS91 Hypothetical protein Paenibacillus spp. 675 2527 BLFS92 spoivb peptidase Paenibacillus spp. 676 2528 BLFS93 arabinogalactan endo- Paenibacillus spp. 677 2529 1,4-beta-galactosidase BLFS94 rhamnogalacturonan Paenibacillus spp. 678 2530 endolyase BLFS95 Hypothetical protein Paenibacillus spp. 679 2531 BLFS96 Hypothetical protein Paenibacillus curdlanolyticus 680 2532 BLFS97 Hypothetical protein Paenibacillus mucilaginosus 681 2533 BLFS98 Hypothetical protein Pantoea spp. 682 2534 BLFS99 uncharacterized Pantoea spp. 683 2535 protein yhcn BLFS100 uncharacterized Pantoea spp. 684 2536 protein yhcn BLFS101 metalloprotease loiP Pantoea spp. 685 2537 BLFS102 Hypothetical protein Pantoea spp. 686 2538 BLFS103 toxin tccc3 Pantoea spp. 687 2539 BLFS104 Hypothetical protein Pantoea spp. 688 2540 BLFS105 protein of unknown Pantoea spp. 689 2541 function (DUF1311} BLFS106 uncharacterized Pantoea spp. 690 2542 protein yqjd BLFS107 polyketide synthase Pantoea spp. 691 2543 BLFS108 toxin relE Pantoea spp. 692 2544 BLFS109 hemolysin Pantoea spp. 693 2545 BLFS110 hypothetical protein Pedobacter V48 694 2546 BLFS111 hypothetical protein Pseudogulbenkiania_ferrooxidans 695 2547 EGD HP2 BLFS112 Hypothetical protein Pseudomonas spp. 696 2548 BLFS113 transpeptidase Pseudomonas spp 697 2549 BLFS114 ATP-dependent zinc Pseudomonas spp 698 2550 protease BLFS115 probable periplasmic Pseudomonas spp 699 2551 serine endoprotease DegP-like BLFS116 lysophospholipase Pseudomonas spp 700 2552 BLFS118 peptidoglycan-binding Pseudomonas spp 701 2553 protein BLFS119 Hypothetical protein Pseudomonas spp 702 2554 BLFS120 supernuclease toxin 2 Pseudomonas spp. 703 2555 family protein BLFS121 glutamate Pseudomonas spp 704 2556 carboxypeptidase BLFS122 hpr Pseudomonas spp 705 2557 BLFS123 peptidase c1 Pseudomonas spp 706 2558 BLFS124 tigr02448 family Pseudomonas spp 707 2559 protein BLFS125 peptidase Pseudomonas spp 708 2560 BLFS126 Hypothetical protein Pseudomonas spp 709 2561 BLFS127 Hypothetical protein Pseudomonas spp 710 2562 BLFS128 peptidoglycan Pseudomonas spp 711 2563 hydrolase flgj BLFS129 alginate lyase Pseudomonas spp 712 2564 BLFS130 leucine-rich repeat- Pseudomonas spp.. 713 2565 containing protein BLFS131 uncharacterized Pseudomonas chlororaphis 714 2566 protein ycfj BLFS132 Hypothetical protein Pseudomonas chlororaphis 715 2567 BLFS133 Hypothetical protein Pseudomonas chlororaphis 716 2568 BLFS134 Protein of unknown Pseudomonas chlororaphis 717 2569 function DUF2782 BLFS135 multidrug transporter Pseudomonas chlororaphis 718 2570 BLFS136 Hypothetical protein Pseudoxanthomonas J31 719 2571 BLFS137 membrane protein Rhodococcus KB6 720 2572 BLFS138 uncharacterized Salinibacillus spp. 721 2573 peptidase BLFS139 peptidoglycan Salinibacillus spp. 722 2574 endopeptidase BLFS140 uncharacterized Salinibacillus spp. 723 2575 lipoprotein ybbd BLFS141 mannan endo-1,4-beta- Salinibacillus spp. 724 2576 mannosidase BLFS142 uncharacterized Salinibacillus spp. 725 2577 protein ykvt BLFS143 putative signal peptide Salinibacillus spp. 726 2578 peptidase sppa BLFS144 thermolysin Salinibacillus spp. 727 2579 BLFS145 d-alanyl-d-alanine Salinibacillus spp. 728 2580 carboxypeptidase dacb BLFS146 1-ala--d-glu Salinibacillus spp. 729 2581 endopeptidase BLFS147 beta-n- Salinibacillus spp. 730 2582 acetylglucosaminidase BLFS148 metalloprotease loip Serratia_fonticola_AU_P3 731 2583 BLFS149 chitobiase Serratia_fonticola_LMG_7882 732 2584 BLFS150 lytic enzyme Stenotrophomonas_maltophilia_ATCC_19867 733 2585 BLFS151 peptidase S33 family Stenotrophomonas_maltophilia_S028 734 2586 protein BLFS152 lipase Streptomyces_NRRL_F_4335 735 2587 BLFS153 peptidase Streptomyces albus 736 2588 BLFS154 peptidase s1 Streptomyces_clavuligerus_ATCC_27064 737 2589 BLFS155 mannose-binding Streptomyces griseorubens 738 2590 protein BLFS156 hypothetical protein Streptomyces_mobaraensis_NBRC_13819 739 2591 BLFS157 hypothetical protein Thalassobacter_arenae_DSM_19593 740 2592 BLFS158 bicupin, oxalate Virgibacillus_Vm_5 741 2593 decarboxylase family BLFS159 large Ala/Gln-rich Xanthomonas_hortorum_pv_carotae_M081 742 2594 protein PUB6 Hypothetical protein Bacillus_weihenstephanensis 743 2595 PUB23 Hypothetical protein Bacillus cereus 744 2596 PUB31 Hypothetical protein Bacillus_thuringiensis_serovar_andalousiensis 745 2597 PUB65 putative extracellular Variovorax_paradoxus B4 746 2598 carbohydrate- recognition protein ZLFS1 Arthropod defensin Actinomyces_oral_taxon_171 747 2599 ZLFS2 peptidase m14 Arthrobacter spp. 748 2600 ZLFS3 probable Bacillus_amyloliquefaciens_plantarum 749 2601 peptidoglycan endopeptidase lyte ZLFS5 Hypothetical protein Burkholderia_A1 750 2602 ZLFS6 type vi secretion Collimonas_fungivorans_Ter331 751 2603 protein ZLFS7 glycosyl hydrolase Collimonas_fungivorans_Ter331 752 2604 ZLFS8 chitin-binding protein Collimonas_fungivorans_Ter331 753 2605 ZLFS9 polyketide synthase- Collimonas_fungivorans_Ter331 754 2606 like protein ZLFS10 Hypothetical protein Collimonas_fungivorans_Ter331 755 2607 ZLFS11 patatin Collimonas_fungivorans_Ter331 756 2608 ZLFS12 pseudomonalisin Collimonas_fungivorans_Ter331 757 2609 ZLFS13 Hypothetical protein Collimonas_fungivorans_Ter331 758 2610 ZLFS14 Hypothetical protein Collimonas_fungivorans_Ter331 759 2611 ZLFS15 Beta-barrel assembly- Collimonas_fungivorans_Ter331 760 2612 enhancing protease [bepA]. ZLFS16 d-alanyl-d-alanine Collimonas_fungivorans_Ter331 761 2613 carboxypeptidase dacd ZLFS17 Antimicrobial protein Herbidospora cretacea 762 2614 MiAMP1 ZLFS18 peptidoglycan-binding Lysobacter enzymogenes 763 2615 protein ZLFS19 lipoprotein nlpd/lppb Lysobacter gummosus 764 2616 homolog ZLFS21 chitosanase Paenibacillus spp. 765 2617 ZLFS22 Hypothetical protein Paenibacillus spp. 766 2618 ZLFS23 beta-glucanase Pantoea spp. 767 2619 ZLFS24 lipase 1 Pantoea spp. 768 2620 ZLFS25 SGNH hydrolase-type Pantoea spp. 769 2621 esterase domain ZLFS27 lytic transglycosylase Pseudomonas_In5 770 2622 ZLFS28 hydrolase Pseudomonas_In5 771 2623 ZLFS29 Beta-barrel assembly- Pseudomonas_In5 772 2624 enhancing protease [bepA]. ZLFS30 dipeptidase Pseudomonas_In5 773 2625 ZLFS31 lipase 1 Serratia_plymuthica_S13 774 2626 ZLFS32 endoglucanase Serratia_plymuthica_S13 775 2627 ZLFS33 chitin-binding protein Serratia_plymuthica_S13 776 2628 ZLFS34 peptidyl-dipeptidase Serratia_plymuthica_S13 777 2629 dcp ZLFS35 Hypothetical protein Serratia_plymuthica_S13 778 2630 ZLFS36 Calcium-mediated Serratia_plymuthica_S13 779 2631 lectin ZLFS37 upf0339 protein yegp Serratia_plymuthica_S13 780 2632 ZLFS38 bacterioferritin Serratia_plymuthica_S13 781 2633 ZLFS39 Glycogen debranching Serratia_plymuthica_S13 782 2634 enzyme [glgX]. ZLFS42 killer toxin like Streptomyces_HPH0547 783 2635 ZLFS44 glycoside hydrolase Streptomyces_M10 784 2636 ZLFS45 Oxidoreductase Streptomyces_M10 785 2637 ZLFS47 Antimicrobial protein Streptomyces_scopuliridis_RB72 786 2638 MiAMP1 ZLFS48 Antimicrobial protein Streptosporangium_amethystogenes 787 2639 MiAMP1 ZLFS49 Hypothetical protein Curtobacterium spp. 788 2640 ZLFS51 Glycosyl hydrolase Bacillus_subtilis_subtilis_OH_131_1 789 2641 “polyn.” = polynucleotide; “polyp.” = polypeptide.

Example 12: Identification of Orthologous Sequences of Antifungal Proteins

Orthologues and paralogues constitute two major types of homologues: The first evolved from a common ancestor by specialization, and the latter are related by duplication events. It is assumed that paralogues arising from ancient duplication events are likely to have diverged in function while true orthologues are more likely to retain identical function over evolutionary time. Orthologues of the discovered antifungal genes are not only likely to be antifungal by themselves but also may hold improved potency or target different fungi spectra.

The search and identification of homologous genes involves the screening of sequence information available, for example, public databases such as the GenBank, and the European Molecular Biology Laboratory Nucleic Acid Sequence Database (EMBL).

Polynucleotides and polypeptides with significant homology to the identified genes described in Table 15 (Example 11) were identified from the databases using BLAST™ software with the Blastp and tBlastn algorithms as filters for the first stage, and the needle (EMBOSS package) or Frame+algorithm alignment for the second stage. Local identity (BLAST™ alignments) was defined with a very permissive cutoff−40% Identity on a span of 40% of the sequences lengths because it is used only as a filter for the global alignment stage. The default filtering of the BLAST™ package was not utilized (by setting the parameter “-F F”).

In the second stage, homologs were defined based on a global identity of at least 70% to the core gene polypeptide sequence. Two distinct forms for finding the optimal global alignment for protein or nucleotide sequences were used in this application:

1. Between two proteins (following the BLASTP filter):

EMBOSS-6.0.1 Needleman-Wunsch algorithm with the following modified parameters: gapopen=8 gapextend=2. The rest of the parameters were unchanged from the default options described hereinabove.

2. Between a protein sequence and a nucleotide sequence (following the TBLASTN filter): GenCore 6.0 OneModel application utilizing the Frame+algorithm with the following parameters: model=frame+p2n.model mode=global -q=protein. sequence -db=nucleotide.sequence. The rest of the parameters are unchanged from the default options described hereinabove.

Homology was calculated as % of identity over the aligned sequences. The query sequences were the polypeptide sequences depicted in Table 15 (Example 11). The subject sequences are protein sequences identified in the database based on greater than 80% global identity to the predicted translated sequences of the query nucleotide sequences or to the polypeptide sequences. The identified orthologous and homologous sequences having at least 700 global sequence identity to the sequences are provided in Table 16, below.

TABLE 16 Homologues (e.g., orthologues) of the identified antifungal genes/polypeptides Polyn. Hom. to Polyp. Hom. to % SEQ ID Gene SEQ ID SEQ ID glob. NO Name Organism NO NO Iden. Algor. 1187 BLFS87 Paenibacilhis sp. 3024 2523 96.2 globlastp 1188 BLFS87 Paenibacilhis sp. 3025 2523 95.5 globlastp 1189 BLFS87 Paenibacilhis sp. 3026 2523 94.8 globlastp 1190 BLFS87 Paenibacillus sp. 3027 2523 94.1 globlastp 1191 BLFS87 Paenibacillus sp. 3028 2523 93.5 globlastp 1192 BLFS87 Paenibacillus sp. 3029 2523 92 globlastp 1193 BLFS87 Paenibacillus sp. 3030 2523 91.1 globlastp 1194 BLFS87 Paenibacillus sp. 3031 2523 90.2 globlastp 1195 BLFS87 Paenibacillus sp. 3032 2523 89.4 globlastp 1196 BLFS87 Paenibacillus sp. 3033 2523 88.3 globlastp 1197 BLFS87 Paenibacillus sp. 3034 2523 71 globlastp 2052 ZLFS3 Bacillus sp. 3866 2601 98.5 globlastp 2053 ZLFS3 Bacillus sp. 3867 2601 97.8 globlastp 2054 ZLFS3 Bacillus sp. 3868 2601 97.4 globlastp 2055 ZLFS3 Bacillus sp. 3869 2601 94.9 globlastp 2056 ZLFS3 Bacillus sp. 3870 2601 94.6 globlastp 2057 ZLFS3 Bacillus sp. 3871 2601 94.2 globlastp 2058 ZLFS3 Bacillus sp. 3872 2601 93.8 globlastp 2059 ZLFS3 Bacillus sp. 3873 2601 84.1 globlastp 2060 ZLFS3 Bacillus sp. 3874 2601 83.8 globlastp 2061 ZLFS3 Bacillus sp. 3875 2601 76.1 globlastp 2062 ZLFS3 Bacillus sp. 3876 2601 75.7 globlastp 2063 ZLFS3 Bacillus sp. 3877 2601 75.5 globlastp 2064 ZLFS3 Bacillus sp. 3878 2601 75.4 globlastp 2065 ZLFS3 Bacillus sp. 3879 2601 75.2 globlastp 2066 ZLFS3 Bacillus sp. 3880 2601 74.9 globlastp 2067 ZLFS3 Bacillus sp. 3881 2601 74.5 globlastp 2068 ZLFS3 Bacillus sp. 3882 2601 74.1 globlastp 2069 ZLFS3 Bacillus sp. 3883 2601 73 globlastp 949 BLFS34 Bacillus sp. 2799 2476 99 globlastp 950 BLFS34 Bacillus sp. 2800 2476 98.1 globlastp 951 BLFS34 Bacillus sp. 2801 2476 95.1 globlastp 1013 BLFS56 Janthinobacterium sp. 2859 2496 98.7 globlastp 1014 BLFS56 Oxalobacteraceae sp. 2860 2496 93.2 globlastp 1015 BLFS56 Janthinobacterium sp. 2861 2496 72.5 globlastp 1016 BLFS56 Janthinobacterium sp. 2862 2496 72.2 globlastp 1017 BLFS56 Janthinobacterium sp. 2863 2496 71.6 globlastp 1018 BLFS56 Massilia sp. 2864 2496 71.4 globlastp 1019 BLFS56 Massilia sp. — 2496 71.02 glotblastn 1020 BLFS56 Massilia sp. — 2496 70.7 glotblastn 1021 BLFS56 Duganella sp. — 2496 70.04 glotblastn 1207 BLFS89 Paenibacillus sp. 3044 2525 99.2 globlastp 1208 BLFS89 Paenibacillus sp. 3045 2525 98.7 globlastp 1209 BLFS89 Paenibacillus sp. 3046 2525 97 globlastp 1210 BLFS89 Paenibacillus sp. 3047 2525 96.7 globlastp 1211 BLFS89 Paenibacillus sp. 3048 2525 95 globlastp 1212 BLFS89 Paenibacillus sp. 3049 2525 94.2 globlastp 1213 BLFS89 Paenibacillus sp. 3050 2525 93.4 globlastp 1214 BLFS89 Paenibacillus sp. 3051 2525 92.2 globlastp 1215 BLFS89 Paenibacillus sp. 3052 2525 91.9 globlastp 1216 BLFS89 Paenibacillus sp. — 2525 71.86 glotblastn 1217 BLFS89 Paenibacillus sp. — 2525 70.07 glotblastn 884 BLFS23 Bacillus sp. 2734 2466 96.4 globlastp 885 BLFS23 Bacillus sp. 2735 2466 95.2 globlastp 886 BLFS23 Bacillus sp. 2736 2466 94 globlastp 887 BLFS23 Bacillus sp. 2737 2466 93.1 globlastp 888 BLFS23 Bacillus sp. 2738 2466 93 globlastp 889 BLFS23 Bacillus sp. 2739 2466 92.4 globlastp 890 BLFS23 Bacillus sp. 2740 2466 92 globlastp 891 BLFS23 Bacillus sp. 2741 2466 91.8 globlastp 892 BLFS23 Bacillus sp. 2742 2466 91.2 globlastp 1062 BLFS64 Geobacillus sp. 2904 2504 98.8 globlastp 2194 ZLFS28 Pseudomonas sp. 4008 2623 94.1 globlastp 2195 ZLFS28 Pseudomonas sp. 4009 2623 93.8 globlastp 2196 ZLFS28 Pseudomonas sp. 4010 2623 92.2 globlastp 2197 ZLFS28 Pseudomonas sp. 4011 2623 91.4 globlastp 2198 ZLFS28 Pseudomonas sp. 4012 2623 89.5 globlastp 2199 ZLFS28 Pseudomonas sp. 4013 2623 88.3 globlastp 2200 ZLFS28 Pseudomonas sp. 4014 2623 87.9 globlastp 2201 ZLFS28 Pseudomonas sp. 4015 2623 87.5 globlastp 2202 ZLFS28 Pseudomonas sp. 4016 2623 86.7 globlastp 2203 ZLFS28 Pseudomonas sp. 4017 2623 86.3 globlastp 2204 ZLFS28 Pseudomonas sp. 4018 2623 85.9 globlastp 2205 ZLFS28 Pseudomonas sp. 4019 2623 85.5 globlastp 2206 ZLFS28 Pseudomonas sp. 4020 2623 82.7 globlastp 2207 ZLFS28 Pseudomonas sp. 4021 2623 80.1 globlastp 2208 ZLFS28 Collimonas sp. 4022 2623 72.1 globlastp 908 BLFS29 Bacillus sp. 2758 2471 75.3 globlastp 2104 ZLFS19 Environmental sample 3918 2616 84.6 globlastp 2105 ZLFS19 Lysobacter sp. 3919 2616 84.3 globlastp 2106 ZLFS19 Lysobacter sp. 3920 2616 83.9 globlastp 2107 ZLFS19 Lysobacter sp. 3921 2616 83.4 globlastp 2108 ZLFS19 Lysobacter sp. 3922 2616 82.4 globlastp 2109 ZLFS19 Environmental sample 3923 2616 81.9 globlastp 2110 ZLFS19 Environmental sample 3924 2616 81.5 globlastp 2111 ZLFS19 Environmental sample 3925 2616 80.6 globlastp 2112 ZLFS19 Lysobacter sp. 3926 2616 79.7 globlastp 2113 ZLFS19 Lysobacter sp. 3927 2616 76.9 globlastp 845 BLFS18 Bacillus sp. 2697 2461 95.5 globlastp 846 BLFS18 Bacillus sp. 2698 2461 84.6 globlastp 847 BLFS18 Bacillus sp. 2699 2461 82.1 globlastp 848 BLFS18 Bacillus sp. 2700 2461 81.9 globlastp 849 BLFS18 Bacillus sp. 2701 2461 80.1 globlastp 850 BLFS18 Bacillus sp. 2702 2461 78.1 globlastp 851 BLFS18 Bacillus sp. 2703 2461 77 globlastp 852 BLFS18 Bacillus sp. 2704 2461 76.3 globlastp 853 BLFS18 Bacillus sp. 2705 2461 75 globlastp 854 BLFS18 Bacillus sp. 2706 2461 74.2 globlastp 855 BLFS18 Bacillus sp. 2707 2461 73 globlastp 856 BLFS18 Kurthia sp. 2708 2461 72 globlastp 857 BLFS18 Bacillus sp. 2709 2461 71 globlastp 858 BLFS18 Anoxybacillus sp. 2710 2461 70.3 globlastp 1997 BLFS155 Streptomyces sp. 3811 2590 88.1 globlastp 1998 BLFS155 Streptomyces sp. 3812 2590 70.2 globlastp 592 BLFS156 Streptomyces sp. — 2591 71.07 glotblastn 1696 BLFS130 Pseudomonas sp. 3520 2565 98.8 globlastp 1697 BLFS130 Pseudomonas sp. 3521 2565 93.9 globlastp 1698 BLFS130 Pseudomonas sp. 3522 2565 81.2 globlastp 1699 BLFS130 Pseudomonas sp. 3523 2565 72.9 globlastp 1700 BLFS130 Pseudomonas sp. 3524 2565 70 globlastp 2426 ZLFS51 Bacillus sp. 4235 2641 99.1 globlastp 2427 ZLFS51 Bacillus sp. 4236 2641 98.1 globlastp 2428 ZLFS51 Bacillus sp. 4237 2641 97.2 globlastp 2429 ZLFS51 Bacillus sp. 4238 2641 96 globlastp 2430 ZLFS51 Salinibacillus sp. 4239 2641 95.3 globlastp 2431 ZLFS51 Bacillus sp. 4240 2641 94.6 globlastp 2432 ZLFS51 Bacillus sp. 4241 2641 93.9 globlastp 2433 ZLFS51 Bacillus sp. 4242 2641 87.6 globlastp 2434 ZLFS51 Bacillus sp. 4243 2641 86.2 globlastp 2435 ZLFS51 Bacillus sp. 4244 2641 85.7 globlastp 2436 ZLFS51 Bacillus sp. 4245 2641 78 globlastp 2437 ZLFS51 Bacillus sp. 4246 2641 77.1 globlastp 2438 ZLFS51 Bacillus sp. 4247 2641 76 globlastp 2439 ZLFS51 Bacillus sp. 4248 2641 75.2 globlastp 2440 ZLFS51 Bacillus sp. 4249 2641 74 globlastp 2441 ZLFS51 Bacillus sp. 4250 2641 73.8 globlastp 2442 ZLFS51 Bacillus sp. 4251 2641 71.1 globlastp 2443 ZLFS51 Bacillus sp. 4252 2641 70 globlastp 2389 ZLFS42 Streptomyces sp. 4202 2635 80.3 globlastp 2390 ZLFS42 Streptomyces sp. 4203 2635 78.6 globlastp 2391 ZLFS42 Streptomyces sp. 4204 2635 78.6 globlastp 2392 ZLFS42 Streptomyces sp. 4205 2635 77.8 globlastp 785 ZLFS42 Streptomyces sp. 2637 2635 77.8 globlastp 2393 ZLFS42 Streptomyces sp. 4206 2635 76.9 globlastp 2394 ZLFS42 Streptomyces sp. 4207 2635 76.1 globlastp 2395 ZLFS42 Streptomyces sp. 4207 2635 76.1 globlastp 2396 ZLFS42 Streptomyces sp. 4208 2635 75.2 globlastp 2397 ZLFS42 Streptomyces sp. 4209 2635 73.5 globlastp 2398 ZLFS42 Streptomyces sp. 4210 2635 73.5 globlastp 808 ZLFS42 Streptomyces sp. 2660 2635 70.1 globlastp 1792 BLFS136 Pseudoxanthomonas sp. 3616 2571 81 globlastp 1163 BLFS84 Paenibacillus sp. 3000 2520 98.3 globlastp 1164 BLFS84 Paenibacillus sp. 3001 2520 95.9 globlastp 1165 BLFS84 Paenibacillus sp. 3002 2520 95.1 globlastp 1166 BLFS84 Paenibacillus sp. 3003 2520 92.4 globlastp 1167 BLFS84 Paenibacillus sp. 3004 2520 89.5 globlastp 1168 BLFS84 Paenibacillus sp. 3005 2520 88.1 globlastp 1169 BLFS84 Paenibacillus sp. 3006 2520 87.5 globlastp 1129 BLFS79 Paenibacillus sp. 2966 2516 99.2 globlastp 1130 BLFS79 Paenibacillus sp. 2967 2516 98.5 globlastp 1131 BLFS79 Paenibacillus sp. 2968 2516 97 globlastp 1132 BLFS79 Paenibacillus sp. 2969 2516 96.6 globlastp 1133 BLFS79 Paenibacillus sp. 2970 2516 95.1 globlastp 1134 BLFS79 Paenibacillus sp. 2971 2516 94 globlastp 1135 BLFS79 Paenibacillus sp. 2972 2516 93.6 globlastp 1136 BLFS79 Paenibacillus sp. 2973 2516 93.2 globlastp 1137 BLFS79 Paenibacillus sp. 2974 2516 92.5 globlastp 1138 BLFS79 Paenibacillus sp. 2975 2516 91.7 globlastp 2235 ZLFS30 Pseudomonas sp. 4049 2625 94.6 globlastp 2236 ZLFS30 Pseudomonas sp. 4050 2625 90.5 globlastp 2237 ZLFS30 Pseudomonas sp. 4051 2625 89.1 globlastp 2238 ZLFS30 Pseudomonas sp. 4052 2625 88.3 globlastp 2239 ZLFS30 Pseudomonas sp. 4053 2625 87.2 globlastp 2240 ZLFS30 Pseudomonas sp. 4054 2625 86 globlastp 2241 ZLFS30 Pseudomonas sp. 4055 2625 85 globlastp 2242 ZLFS30 Erwinia sp. 4056 2625 84.1 globlastp 2243 ZLFS30 Pseudomonas sp. 4057 2625 83.1 globlastp 2244 ZLFS30 Pseudomonas sp. 4058 2625 82 globlastp 2245 ZLFS30 Pseudomonas sp. 4059 2625 81 globlastp 2246 ZLFS30 Pseudomonas sp. 4060 2625 80.1 globlastp 2247 ZLFS30 Pseudomonas sp. 4061 2625 79.1 globlastp 2248 ZLFS30 Pseudomonas sp. 4062 2625 78.1 globlastp 2249 ZLFS30 Pseudomonas sp. 4063 2625 77.2 globlastp 2250 ZLFS30 Pseudomonas sp. 4064 2625 76.7 globlastp 2251 ZLFS30 Pseudomonas sp. 4065 2625 75.5 globlastp 2252 ZLFS30 Environmental sample 4066 2625 72 globlastp 2253 ZLFS30 Pseudomonas sp. 4067 2625 71 globlastp 2254 ZLFS30 Environmental sample 4068 2625 70.1 globlastp 2033 ZLFS2 Arthrobacter sp. 3847 2600 99.4 globlastp 2034 ZLFS2 Arthrobacter sp. 3848 2600 96.3 globlastp 2035 ZLFS2 Arthrobacter sp. 3849 2600 94.6 globlastp 2036 ZLFS2 Environmental sample 3850 2600 92.7 globlastp 2037 ZLFS2 Environmental sample 3851 2600 92.1 globlastp 2038 ZLFS2 Arthrobacter sp. 3852 2600 90.1 globlastp 2039 ZLFS2 Arthrobacter sp. 3853 2600 89.8 globlastp 2040 ZLFS2 Environmental sample 3854 2600 89.3 globlastp 2041 ZLFS2 Arthrobacter sp. 3855 2600 88.9 globlastp 2042 ZLFS2 Arthrobacter sp. 3856 2600 87.3 globlastp 2043 ZLFS2 Arthrobacter sp. 3857 2600 86.2 globlastp 2044 ZLFS2 Arthrobacter sp. 3858 2600 73.7 globlastp 2045 ZLFS2 Arthrobacter sp. 3859 2600 73.2 globlastp 2046 ZLFS2 Sporosarcina sp. 3860 2600 72.6 globlastp 2047 ZLFS2 Bhargavaea sp. 3861 2600 72.1 globlastp 2048 ZLFS2 Arthrobacter sp. 3862 2600 71.5 globlastp 2049 ZLFS2 Arthrobacter sp. 3863 2600 71.2 globlastp 2050 ZLFS2 Sporosarcina sp. 3864 2600 70.6 globlastp 2051 ZLFS2 Planomicrobium sp. 3865 2600 70.5 globlastp 1071 BLFS72 Lysobacter sp. 2913 2510 95 globlastp 1124 BLFS77 Paenibacillus sp. 2963 2515 98.1 globlastp 1125 BLFS77 Paenibacillus sp. 2964 2515 96.6 globlastp 1126 BLFS77 Paenibacillus sp. 2965 2515 81.1 globlastp 1127 BLFS77 Paenibacillus sp. — 2515 73.95 glotblastn 1128 BLFS77 Paenibacillus sp. — 2515 73.24 glotblastn 1599 BLFS122 Pseudomonas sp. 3424 2557 97.7 globlastp 1600 BLFS122 Pseudomonas sp. 3425 2557 96.9 globlastp 1601 BLFS122 Pseudomonas sp. 3426 2557 96.2 globlastp 1602 BLFS122 Pseudomonas sp. 3427 2557 93.1 globlastp 1603 BLFS122 Pseudomonas sp. 3428 2557 79.4 globlastp 1604 BLFS122 Pseudomonas sp. — 2557 72.18 glotblastn 1605 BLFS122 Pseudomonas sp. 3429 2557 71.1 globlastp 1606 BLFS122 Pseudomonas sp. 3430 2557 70.9 globlastp 970 BLFS38 Brevibacillus sp. 2818 2480 81.2 globlastp 1139 BLFS82 Paenibacillus sp. 2976 2518 99 globlastp 1140 BLFS82 Paenibacillus sp. 2977 2518 98 globlastp 1141 BLFS82 Paenibacillus sp. 2978 2518 97.1 globlastp 1142 BLFS82 Paenibacillus sp. 2979 2518 96.1 globlastp 1143 BLFS82 Paenibacillus sp. 2980 2518 94.1 globlastp 1144 BLFS82 Paenibacillus sp. 2981 2518 93.1 globlastp 1145 BLFS82 Paenibacillus sp. 2982 2518 92.2 globlastp 1146 BLFS82 Paenibacillus sp. 2983 2518 89.4 globlastp 1147 BLFS82 Paenibacillus sp. 2984 2518 88.2 globlastp 1148 BLFS82 Paenibacillus sp. 2985 2518 86.3 globlastp 1149 BLFS82 Paenibacillus sp. 2986 2518 85.3 globlastp 1150 BLFS82 Paenibacillus sp. 2987 2518 84.3 globlastp 1151 BLFS82 Paenibacillus sp. 2988 2518 82.4 globlastp 2075 ZLFS6 Collimonas sp. 3889 2603 95.7 globlastp 2076 ZLFS6 Herbaspirillum sp. 3890 2603 72 globlastp 2077 ZLFS6 Janthinobacterium sp. 3891 2603 71.3 globlastp 2078 ZLFS6 Janthinobacterium sp. 3892 2603 70.7 globlastp 2079 ZLFS6 Oxalobacteraceae sp. 3893 2603 70.6 globlastp 812 BLFS11 Arthrobacter sp. 2664 2454 96.3 globlastp 813 BLFS11 Arthrobacter sp. 2665 2454 80.4 globlastp 814 BLFS11 Arthrobacter sp. 2666 2454 79.9 globlastp 815 BLFS11 Environmental sample 2667 2454 75.7 globlastp 816 BLFS11 Arthrobacter sp. 2668 2454 73 globlastp 817 BLFS11 Arthrobacter sp. 2669 2454 71.2 globlastp 818 BLFS11 Arthrobacter sp. 2670 2454 70.1 globlastp 2114 ZLFS21 Paenibacillus sp. 3928 2617 99.1 globlastp 2115 ZLFS21 Paenibacillus sp. 3929 2617 96.7 globlastp 2116 ZLFS21 Paenibacillus sp. 3930 2617 96.4 globlastp 2117 ZLFS21 Paenibacillus sp. 3931 2617 94.5 globlastp 2118 ZLFS21 Paenibacillus sp. 3932 2617 94.2 globlastp 2119 ZLFS21 Paenibacillus sp. 3933 2617 91.3 globlastp 2120 ZLFS21 Paenibacillus sp. 3934 2617 85 globlastp 2121 ZLFS21 Paenibacillus sp. 3935 2617 84.5 globlastp 2122 ZLFS21 Paenibacillus sp. 3936 2617 84.3 globlastp 2123 ZLFS21 Paenibacillus sp. 3937 2617 82.6 globlastp 2124 ZLFS21 Paenibacillus sp. 3938 2617 81.9 globlastp 2125 ZLFS21 Paenibacillus sp. 3939 2617 81.6 globlastp 2126 ZLFS21 Paenibacillus sp. 3940 2617 72.3 globlastp 839 BLFS17 Multievos46 sp. 2691 2460 96 globlastp 840 BLFS17 Environmental sample 2692 2460 86.4 globlastp 841 BLFS17 Arthrobacter sp. 2693 2460 80 globlastp 842 BLFS17 Arthrobacter sp. 2694 2460 79 globlastp 843 BLFS17 Arthrobacter sp. 2695 2460 78.6 globlastp 844 BLFS17 Arthrobacter sp. 2696 2460 77.5 globlastp 1450 BLFS113 Pseudomonas sp. 3276 2549 99.4 globlastp 1451 BLFS113 Pseudomonas sp. 3277 2549 98.8 globlastp 1452 BLFS113 Pseudomonas sp. 3278 2549 97.6 globlastp 1453 BLFS113 Pseudomonas sp. 3279 2549 97 globlastp 1454 BLFS113 Pseudomonas sp. 3280 2549 96.4 globlastp 1455 BLFS113 Pseudomonas sp. 3281 2549 94.6 globlastp 1456 BLFS113 Pseudomonas sp. 3282 2549 92.2 globlastp 1457 BLFS113 Pseudomonas sp. 3283 2549 91 globlastp 1458 BLFS113 Pseudomonas sp. 3284 2549 90.4 globlastp 1459 BLFS113 Pseudomonas sp. 3285 2549 89.2 globlastp 1460 BLFS113 Pseudomonas sp. 3286 2549 88 globlastp 1461 BLFS113 Erwinia sp. 3287 2549 87.4 globlastp 1462 BLFS113 Pseudomonas sp. 3288 2549 86.2 globlastp 1463 BLFS113 Pseudomonas sp. 3289 2549 85 globlastp 1464 BLFS113 Pseudomonas sp. 3290 2549 84.2 globlastp 1465 BLFS113 Pseudomonas sp. 3291 2549 83 globlastp 1466 BLFS113 Pseudomonas sp. 3292 2549 82 globlastp 1467 BLFS113 Pseudomonas sp. 3293 2549 81.4 globlastp 1468 BLFS113 Pseudomonas sp. 3294 2549 80.1 globlastp 1469 BLFS113 Pseudomonas sp. 3295 2549 79 globlastp 1470 BLFS113 Pseudomonas sp. 3296 2549 78.4 globlastp 1471 BLFS113 Pseudomonas sp. 3297 2549 77.8 globlastp 1472 BLFS113 Environmental sample 3298 2549 76.6 globlastp 1473 BLFS113 Pseudomonas sp. 3299 2549 74.9 globlastp 1474 BLFS113 Pseudomonas sp. 3300 2549 71.4 globlastp 817 BLFS13 Arthrobacter sp. 2669 2456 99.4 globlastp 815 BLFS13 Environmental sample 2667 2456 73.3 globlastp 821 BLFS13 Arthrobacter sp. 2673 2456 71.4 globlastp 822 BLFS13 Arthrobacter sp. 2674 2456 70.3 globlastp 1611 BLFS124 Pseudomonas sp. 3435 2559 99 globlastp 1612 BLFS124 Pseudomonas sp. 3436 2559 93.3 globlastp 1613 BLFS124 Pseudomonas sp. 3437 2559 92.4 globlastp 1614 BLFS124 Pseudomonas sp. 3438 2559 83.8 globlastp 1615 BLFS124 Pseudomonas sp. 3439 2559 82.9 globlastp 1616 BLFS124 Pseudomonas sp. 3440 2559 81 globlastp 1617 BLFS124 Pseudomonas sp. 3441 2559 80 globlastp 1618 BLFS124 Pseudomonas sp. 3442 2559 79 globlastp 1619 BLFS124 Pseudomonas sp. 3443 2559 78.1 globlastp 1620 BLFS124 Pseudomonas sp. 3444 2559 77.3 globlastp 1621 BLFS124 Environmental sample 3445 2559 76.2 globlastp 1622 BLFS124 Pseudomonas sp. 3446 2559 75.2 globlastp 1623 BLFS124 Pseudomonas sp. 3447 2559 74.5 globlastp 1624 BLFS124 Environmental sample 3448 2559 73.3 globlastp 1625 BLFS124 Environmental sample 3449 2559 72.4 globlastp 1626 BLFS124 Environmental sample 3450 2559 71.4 globlastp 1627 BLFS124 Pseudomonas sp. 3451 2559 70.5 globlastp 2147 ZLFS24 Meta sp. 3961 2620 98.8 globlastp 2148 ZLFS24 Environmental sample 3962 2620 98.6 globlastp 2149 ZLFS24 Environmental sample 3963 2620 98.3 globlastp 2150 ZLFS24 Pantoea sp. 3964 2620 98.2 globlastp 2151 ZLFS24 Environmental sample 3965 2620 98 globlastp 2152 ZLFS24 Environmental sample 3966 2620 97.9 globlastp 2153 ZLFS24 Pantoea sp. 3967 2620 93.6 globlastp 2154 ZLFS24 Pantoea sp. 3968 2620 93.3 globlastp 2155 ZLFS24 Environmental sample 3969 2620 92.8 globlastp 2156 ZLFS24 Environmental sample 3970 2620 92.7 globlastp 2157 ZLFS24 Environmental sample 3971 2620 88.7 globlastp 2158 ZLFS24 Pantoea sp. 3972 2620 88.6 globlastp 2159 ZLFS24 Pantoea sp. 3973 2620 87.7 globlastp 2160 ZLFS24 Environmental sample 3974 2620 80.3 globlastp 2161 ZLFS24 Pantoea sp. 3975 2620 80 globlastp 2162 ZLFS24 Pantoea sp. 3976 2620 79.4 globlastp 2084 ZLFS11 Collimonas sp. 3898 2608 81.5 globlastp 2085 ZLFS11 Collimonas sp. 3899 2608 79 globlastp 825 BLFS15 Multievos46 sp. 2677 2458 93.2 globlastp 826 BLFS15 Environmental sample 2678 2458 89.2 globlastp 827 BLFS15 Arthrobacter sp. 2679 2458 84.4 globlastp 828 BLFS15 Arthrobacter sp. 2680 2458 83.6 globlastp 829 BLFS15 Arthrobacter sp. 2681 2458 83.4 globlastp 830 BLFS15 Arthrobacter sp. 2682 2458 83.2 globlastp 831 BLFS15 Arthrobacter sp. 2683 2458 76.9 globlastp 832 BLFS15 Arthrobacter sp. 2684 2458 76.7 globlastp 833 BLFS15 Arthrobacter sp. 2685 2458 76 globlastp 1753 BLFS134 Pseudomonas sp. 3577 2569 98.9 globlastp 1754 BLFS134 Pseudomonas sp. 3578 2569 96.8 globlastp 1755 BLFS134 Pseudomonas sp. 3579 2569 95.8 globlastp 1756 BLFS134 Pseudomonas sp. 3580 2569 93.7 globlastp 1757 BLFS134 Pseudomonas sp. 3581 2569 92.6 globlastp 1758 BLFS134 Pseudomonas sp. 3582 2569 91.6 globlastp 1759 BLFS134 Pseudomonas sp. 3583 2569 90.5 globlastp 1760 BLFS134 Pseudomonas sp. 3584 2569 89.5 globlastp 1761 BLFS134 Environmental sample 3585 2569 88.4 globlastp 1762 BLFS134 Environmental sample 3586 2569 87.4 globlastp 1763 BLFS134 Environmental sample 3587 2569 86.3 globlastp 1764 BLFS134 Pseudomonas sp. 3588 2569 85.3 globlastp 1765 BLFS134 Pseudomonas sp. 3589 2569 84.2 globlastp 1766 BLFS134 Pseudomonas sp. 3590 2569 83.2 globlastp 1767 BLFS134 Pseudomonas sp. 3591 2569 82.1 globlastp 1768 BLFS134 Environmental sample 3592 2569 81.1 globlastp 1769 BLFS134 Environmental sample 3593 2569 80 globlastp 1770 BLFS134 Pseudomonas sp. 3594 2569 79.2 globlastp 1771 BLFS134 Environmental sample 3595 2569 78.1 globlastp 1772 BLFS134 Pseudomonas sp. 3596 2569 77.1 globlastp 1773 BLFS134 Pseudomonas sp. 3597 2569 76 globlastp 1774 BLFS134 Pseudomonas sp. 3598 2569 75 globlastp 1775 BLFS134 Pseudomonas sp. 3599 2569 74 globlastp 1776 BLFS134 Environmental sample 3600 2569 73.7 globlastp 1777 BLFS134 Pseudomonas sp. 3601 2569 72.4 globlastp 1778 BLFS134 Environmental sample 3602 2569 71.6 globlastp 1779 BLFS134 Azotobacter sp. 3603 2569 70.5 globlastp 2163 ZLFS25 Curtobacterium sp. 3977 2621 95.4 globlastp 2164 ZLFS25 Environmental sample 3978 2621 91.3 globlastp 2165 ZLFS25 Curtobacterium sp. 3979 2621 90.1 globlastp 2166 ZLFS25 Curtobacterium sp. 3980 2621 89.9 globlastp 2167 ZLFS25 Environmental sample 3981 2621 76.6 globlastp 2168 ZLFS25 Environmental sample 3982 2621 74.4 globlastp 2169 ZLFS25 Environmental sample 3983 2621 73.8 globlastp 834 BLFS16 Environmental sample 2686 2459 94.7 globlastp 835 BLFS16 Arthrobacter sp. 2687 2459 78.1 globlastp 836 BLFS16 Arthrobacter sp. 2688 2459 78 globlastp 837 BLFS16 Arthrobacter sp. 2689 2459 77.7 globlastp 838 BLFS16 Arthrobacter sp. 2690 2459 77 globlastp 984 BLFS43 Burkholderia sp. 2832 2484 99.3 globlastp 985 BLFS43 Burkholderia sp. 2833 2484 76.8 globlastp 986 BLFS43 Burkholderia sp. 2834 2484 71.4 globlastp 987 BLFS43 Burkholderia sp. 2835 2484 70.7 globlastp 2127 ZLFS22 Paenibacillus sp. 3941 2618 99.1 globlastp 2128 ZLFS22 Paenibacillus sp. 3942 2618 98.2 globlastp 2129 ZLFS22 Paenibacillus sp. 3943 2618 97.7 globlastp 2130 ZLFS22 Paenibacillus sp. 3944 2618 96.4 globlastp 2131 ZLFS22 Paenibacillus sp. 3945 2618 95.9 globlastp 2132 ZLFS22 Paenibacillus sp. 3946 2618 95.5 globlastp 2133 ZLFS22 Paenibacillus sp. 3947 2618 95 globlastp 2134 ZLFS22 Paenibacillus sp. 3948 2618 93.7 globlastp 2135 ZLFS22 Paenibacillus sp. 3949 2618 93.2 globlastp 2136 ZLFS22 Paenibacillus sp. 3950 2618 91.9 globlastp 2137 ZLFS22 Paenibacillus sp. 3951 2618 85.6 globlastp 2138 ZLFS22 Paenibacillus sp. 3952 2618 84.7 globlastp 2139 ZLFS22 Paenibacillus sp. 3953 2618 83.8 globlastp 998 BLFS47 Salinibacillus sp. 2846 2488 90.9 globlastp 999 BLFS47 Cellulosimicrobium sp. 2847 2488 77.3 globlastp 1000 BLFS47 Cellulosimicrobium sp. 2848 2488 71.8 globlastp 1001 BLFS47 Multievos46 sp. 2849 2488 70.2 globlastp 1258 BLFS94 Paenibacillus sp. 3093 2530 99 globlastp 1259 BLFS94 Paenibacillus sp. 3094 2530 98.1 globlastp 1260 BLFS94 Paenibacillus sp. 3095 2530 96.3 globlastp 1261 BLFS94 Paenibacillus sp. 3096 2530 95 globlastp 1262 BLFS94 Paenibacillus sp. 3097 2530 94 globlastp 1263 BLFS94 Paenibacillus sp. 3098 2530 93.1 globlastp 1264 BLFS94 Paenibacillus sp. 3099 2530 74.6 globlastp 1265 BLFS94 Paenibacillus sp. 3100 2530 73.4 globlastp 1266 BLFS94 Paenibacillus sp. 3101 2530 71.7 globlastp 1104 BLFS75 Paenibacillus sp. 2944 2513 99.4 globlastp 1105 BLFS75 Paenibacillus sp. 2945 2513 98.8 globlastp 1106 BLFS75 Paenibacillus sp. 2946 2513 98.6 globlastp 1107 BLFS75 Paenibacillus sp. 2947 2513 98.3 globlastp 1108 BLFS75 Paenibacillus sp. 2948 2513 98 globlastp 1109 BLFS75 Paenibacillus sp. 2949 2513 97.7 globlastp 1110 BLFS75 Paenibacillus sp. 2950 2513 97.1 globlastp 1111 BLFS75 Paenibacillus sp. 2951 2513 92.5 globlastp 1112 BLFS75 Paenibacillus sp. 2952 2513 92.2 globlastp 1113 BLFS75 Paenibacillus sp. 2953 2513 91.9 globlastp 1114 BLFS75 Paenibacillus sp. 2954 2513 88.7 globlastp 1725 BLFS132 Pseudomonas sp. 3549 2567 98.5 globlastp 1726 BLFS132 Pseudomonas sp. 3550 2567 97.1 globlastp 1727 BLFS132 Pseudomonas sp. 3551 2567 95.6 globlastp 1728 BLFS132 Pseudomonas sp. 3552 2567 94.1 globlastp 1729 BLFS132 Pseudomonas sp. 3553 2567 92.6 globlastp 1730 BLFS132 Pseudomonas sp. 3554 2567 91.4 globlastp 1731 BLFS132 Pseudomonas sp. 3555 2567 88.6 globlastp 1732 BLFS132 Pseudomonas sp. 3556 2567 87 globlastp 1733 BLFS132 Pseudomonas sp. 3557 2567 85.5 globlastp 1734 BLFS132 Pseudomonas sp. 3558 2567 84.1 globlastp 1735 BLFS132 Environmental sample 3559 2567 83.1 globlastp 1736 BLFS132 Pseudomonas sp. 3560 2567 82.6 globlastp 1737 BLFS132 Pseudomonas sp. 3561 2567 81.2 globlastp 1738 BLFS132 Pseudomonas sp. 3562 2567 80 globlastp 1739 BLFS132 Environmental sample 3563 2567 79.4 globlastp 1740 BLFS132 Pseudomonas sp. 3564 2567 78.3 globlastp 1741 BLFS132 Pseudomonas sp. 3565 2567 77.9 globlastp 1742 BLFS132 Pseudomonas sp. 3566 2567 76.5 globlastp 1743 BLFS132 Pseudomonas sp. 3567 2567 75 globlastp 1744 BLFS132 Pseudomonas sp. 3568 2567 74 globlastp 1745 BLFS132 Pseudomonas sp. 3569 2567 73 globlastp 1746 BLFS132 Pseudomonas sp. 3570 2567 72 globlastp 1747 BLFS132 Pseudomonas sp. 3571 2567 71 globlastp 1748 BLFS132 Pseudomonas sp. 3572 2567 70.7 globlastp 809 BLFS10 Amycolatopsis sp. 2661 2453 97.4 globlastp 810 BLFS10 Amycolatopsis sp. 2662 2453 94.9 globlastp 804 BLFS10 Amycolatopsis sp. 2656 2453 94 globlastp 600 BLFS10 Amycolatopsis sp. 2452 2453 75.2 globlastp 803 BLFS10 Amycolatopsis sp. 2655 2453 74.4 globlastp 811 BLFS10 Streptomyces sp. 2663 2453 70.9 globlastp 2312 ZLFS37 Serratia sp. 4126 2632 99.1 globlastp 2313 ZLFS37 Environmental sample 4127 2632 97.3 globlastp 2314 ZLFS37 Serratia sp. 4128 2632 96.4 globlastp 2315 ZLFS37 Serratia sp. 4129 2632 95.5 globlastp 2316 ZLFS37 Serratia sp. 4130 2632 94.6 globlastp 2317 ZLFS37 Serratia sp. 4131 2632 93.8 globlastp 2318 ZLFS37 Serratia sp. 4132 2632 92.9 globlastp 2319 ZLFS37 Serratia sp. 4133 2632 91.1 globlastp 2320 ZLFS37 Serratia sp. 4134 2632 84.8 globlastp 2321 ZLFS37 Serratia sp. 4135 2632 83.9 globlastp 2322 ZLFS37 Serratia sp. 4136 2632 83 globlastp 2323 ZLFS37 Yersinia sp. 4137 2632 82.1 globlastp 2324 ZLFS37 Serratia sp. 4138 2632 81.6 globlastp 2325 ZLFS37 Yersinia sp. 4139 2632 81.2 globlastp 2326 ZLFS37 Hafnia sp. 4140 2632 80.5 globlastp 2327 ZLFS37 Yersinia sp. 4141 2632 80.4 globlastp 2328 ZLFS37 Yersinia sp. 4142 2632 79.5 globlastp 2329 ZLFS37 Yersinia sp. 4143 2632 78.6 globlastp 2330 ZLFS37 Enterobacteriaceae sp. 4144 2632 77.9 globlastp 2331 ZLFS37 Rahnella sp. 4145 2632 77.7 globlastp 2332 ZLFS37 Ewinyella sp. 4146 2632 76.8 globlastp 2333 ZLFS37 Rahnella sp. 4147 2632 76.5 globlastp 2334 ZLFS37 Rahnella sp. 4148 2632 75.7 globlastp 2335 ZLFS37 Yersinia sp. 4149 2632 74 globlastp 2336 ZLFS37 Candidatus sp. 4150 2632 71.3 globlastp 2337 ZLFS37 Serratia sp. — 2632 70.54 glotblastn 2338 ZLFS37 Serratia sp. 4151 2632 70.5 globlastp 2339 ZLFS37 Candidatus sp. 4152 2632 70.4 globlastp 2340 ZLFS37 gamma sp. 4153 2632 70.2 globlastp 1038 BLFS62 Erwinia sp. 2881 2502 83.2 globlastp 1039 BLFS62 Pantoea sp. 2882 2502 81.5 globlastp 1040 BLFS62 Environmental sample 2883 2502 79.2 globlastp 1041 BLFS62 Environmental sample 2884 2502 78.2 globlastp 1042 BLFS62 Environmental sample 2885 2502 77 globlastp 1043 BLFS62 Environmental sample 2886 2502 76.2 globlastp 1044 BLFS62 Pantoea sp. 2887 2502 75.2 globlastp 1045 BLFS62 Pantoea sp. 2888 2502 74.1 globlastp 1046 BLFS62 Pantoea sp. 2889 2502 73.5 globlastp 1047 BLFS62 Pantoea sp. 2890 2502 71.5 globlastp 1048 BLFS62 Enterobacter sp. — 2502 70.51 glotblastn 1006 BLFS51 Herbaspirillum sp. — 2492 76.51 glotblastn 1007 BLFS51 Collimonas sp. 2854 2492 73.7 globlastp 1008 BLFS51 Collimonas sp. — 2492 72.89 glotblastn 1009 BLFS51 Herbaspirillum sp. 2855 2492 70.3 globlastp 1701 BLFS131 Pseudomonas sp. 3525 2566 98.4 globlastp 1702 BLFS131 Pseudomonas sp. 3526 2566 94.5 globlastp 1703 BLFS131 Pseudomonas sp. 3527 2566 91.2 globlastp 1704 BLFS131 Pseudomonas sp. 3528 2566 90.1 globlastp 1705 BLFS131 Pseudomonas sp. 3529 2566 89 globlastp 1706 BLFS131 Pseudomonas sp. 3530 2566 88 globlastp 1707 BLFS131 Pseudomonas sp. 3531 2566 87 globlastp 1708 BLFS131 Pseudomonas sp. 3532 2566 86.3 globlastp 1709 BLFS131 Environmental sample 3533 2566 85.2 globlastp 1710 BLFS131 Environmental sample 3534 2566 84.1 globlastp 1711 BLFS131 Environmental sample 3535 2566 83 globlastp 1712 BLFS131 Pseudomonas sp. 3536 2566 82.2 globlastp 1713 BLFS131 Pseudomonas sp. 3537 2566 81.1 globlastp 1714 BLFS131 Pseudomonas sp. 3538 2566 80 globlastp 1715 BLFS131 Environmental sample 3539 2566 79.1 globlastp 1716 BLFS131 Pseudomonas sp. 3540 2566 78.1 globlastp 1717 BLFS131 Pseudomonas sp. 3541 2566 77 globlastp 1718 BLFS131 Pseudomonas sp. 3542 2566 76 globlastp 1719 BLFS131 Pseudomonas sp. 3543 2566 75.3 globlastp 1720 BLFS131 Environmental sample 3544 2566 74.2 globlastp 1721 BLFS131 Pseudomonas sp. 3545 2566 73.1 globlastp 1722 BLFS131 Pseudomonas sp. 3546 2566 72 globlastp 1723 BLFS131 Pseudomonas sp. 3547 2566 71.4 globlastp 1724 BLFS131 Pseudomonas sp. 3548 2566 70.3 globlastp 997 BLFS46 Burkholderia sp. 2845 2487 84.9 globlastp 1072 BLFS73 Paenibacillus sp. 2914 2511 99.2 globlastp 1073 BLFS73 Paenibacillus sp. 2915 2511 98.4 globlastp 1074 BLFS73 Paenibacillus sp. 2916 2511 96.7 globlastp 1075 BLFS73 Paenibacillus sp. 2917 2511 95.9 globlastp 1076 BLFS73 Paenibacillus sp. 2918 2511 95.1 globlastp 1077 BLFS73 Paenibacillus sp. 2919 2511 84.6 globlastp 1078 BLFS73 Paenibacillus sp. — 2511 82.93 glotblastn 1079 BLFS73 Paenibacillus sp. 2920 2511 82.1 globlastp 1080 BLFS73 Paenibacillus sp. 2921 2511 81.3 globlastp 1081 BLFS73 Paenibacillus sp. 2922 2511 78.9 globlastp 1082 BLFS73 Paenibacillus sp. 2923 2511 78 globlastp 1083 BLFS73 Paenibacillus sp. 2924 2511 77.2 globlastp 1084 BLFS73 Environmental sample 2925 2511 76.4 globlastp 1085 BLFS73 Paenibacillus sp. 2926 2511 75 globlastp 1086 BLFS73 Paenibacillus sp. 2927 2511 74 globlastp 1087 BLFS73 Paenibacillus sp. — 2511 73.17 glotblastn 1088 BLFS73 Paenibacillus sp. 2928 2511 72.4 globlastp 1089 BLFS73 Paenibacillus sp. 2929 2511 71.2 globlastp 1652 BLFS127 Pseudomonas sp. 3476 2562 98.3 globlastp 1653 BLFS127 Pseudomonas sp. 3477 2562 98.1 globlastp 1654 BLFS127 Pseudomonas sp. 3478 2562 97.7 globlastp 1655 BLFS127 Pseudomonas sp. 3479 2562 97.4 globlastp 1656 BLFS127 Pseudomonas sp. 3480 2562 96.7 globlastp 1657 BLFS127 Pseudomonas sp. 3481 2562 96 globlastp 1658 BLFS127 Pseudomonas sp. 3482 2562 95.8 globlastp 1659 BLFS127 Pseudomonas sp. 3483 2562 95.2 globlastp 1660 BLFS127 Pseudomonas sp. 3484 2562 86.2 globlastp 1661 BLFS127 Pseudomonas sp. 3485 2562 75.5 globlastp 1662 BLFS127 Pseudomonas sp. 3486 2562 75.3 globlastp 1663 BLFS127 Pseudomonas sp. 3487 2562 75.2 globlastp 1664 BLFS127 Pseudomonas sp. 3488 2562 75.1 globlastp 1665 BLFS127 Pseudomonas sp. 3489 2562 75 globlastp 1666 BLFS127 Pseudomonas sp. 3490 2562 74.8 globlastp 1667 BLFS127 Pseudomonas sp. 3491 2562 73.3 globlastp 1067 BLFS68 Janthinobacterium sp. 2909 2507 98.7 globlastp 1068 BLFS68 Janthinobacterium sp. 2910 2507 90.3 globlastp 1069 BLFS68 Janthinobacterium sp. 2911 2507 89.6 globlastp 1070 BLFS68 Janthinobacterium sp. 2912 2507 88.5 globlastp 1049 BLFS63 Erwinia sp. 2891 2503 85.7 globlastp 1050 BLFS63 Erwinia sp. 2892 2503 83.8 globlastp 1051 BLFS63 Environmental sample 2893 2503 82.9 globlastp 1052 BLFS63 Environmental sample 2894 2503 81 globlastp 1053 BLFS63 Environmental sample 2895 2503 80.3 globlastp 1054 BLFS63 Erwinia sp. 2896 2503 79.4 globlastp 1055 BLFS63 Erwinia sp. 2897 2503 78 globlastp 1056 BLFS63 Pantoea sp. 2898 2503 76.8 globlastp 1057 BLFS63 Meta sp. 2899 2503 75 globlastp 1058 BLFS63 Plautia sp. 2900 2503 74 globlastp 1059 BLFS63 Pantoea sp. 2901 2503 73.2 globlastp 1060 BLFS63 Environmental sample 2902 2503 72 globlastp 1061 BLFS63 Environmental sample 2903 2503 71.8 globlastp 1002 BLFS48 Collimonas sp. 2850 2489 98.9 globlastp 1003 BLFS48 Collimonas sp. 2851 2489 78.9 globlastp 1004 BLFS48 Collimonas sp. 2852 2489 76.5 globlastp 2080 ZLFS7 Collimonas sp. 3894 2604 70.4 globlastp 937 BLFS33 Bacillus sp. 2787 2475 99.1 globlastp 938 BLFS33 Bacillus sp. 2788 2475 98.1 globlastp 939 BLFS33 Bacillus sp. 2789 2475 96.4 globlastp 940 BLFS33 Bacillus sp. 2790 2475 94 globlastp 941 BLFS33 Bacillus sp. 2791 2475 93.1 globlastp 942 BLFS33 Bacillus sp. 2792 2475 92.1 globlastp 943 BLFS33 Bacillus sp. 2793 2475 90.5 globlastp 944 BLFS33 Bacillus sp. 2794 2475 80 globlastp 945 BLFS33 Bacillus sp. 2795 2475 78.1 globlastp 946 BLFS33 Bacillus sp. 2796 2475 76.7 globlastp 947 BLFS33 Bacillus sp. 2797 2475 76 globlastp 948 BLFS33 Bacillus sp. 2798 2475 75.4 globlastp 1431 BLFS110 Environmental sample 3257 2546 83.8 globlastp 1227 BLFS91 Paenibacillus sp. 3062 2527 99.2 globlastp 1228 BLFS91 Paenibacillus sp. 3063 2527 96.8 globlastp 1229 BLFS91 Paenibacillus sp. 3064 2527 96 globlastp 1230 BLFS91 Paenibacillus sp. 3065 2527 95.2 globlastp 1231 BLFS91 Paenibacillus sp. 3066 2527 93.6 globlastp 1232 BLFS91 Paenibacillus sp. 3067 2527 88.8 globlastp 1233 BLFS91 Paenibacillus sp. 3068 2527 87.2 globlastp 1234 BLFS91 Paenibacillus sp. 3069 2527 86.4 globlastp 1235 BLFS91 Paenibacillus sp. 3070 2527 84.8 globlastp 1236 BLFS91 Paenibacillus sp. 3071 2527 81.6 globlastp 1237 BLFS91 Paenibacillus sp. 3072 2527 80.8 globlastp 2255 ZLFS31 Serratia sp. 4069 2626 99.2 globlastp 2256 ZLFS31 Serratia sp. 4070 2626 98.8 globlastp 2257 ZLFS31 Serratia sp. 4071 2626 97 globlastp 2258 ZLFS31 Multievos46 sp. 4072 2626 92.1 globlastp 2259 ZLFS31 Serratia sp. 4073 2626 91.3 globlastp 2260 ZLFS31 Serratia sp. 4074 2626 89 globlastp 2261 ZLFS31 Serratia sp. 4075 2626 88.2 globlastp 2262 ZLFS31 Serratia sp. 4076 2626 83.1 globlastp 2263 ZLFS31 Serratia sp. 4077 2626 82.1 globlastp 2264 ZLFS31 Serratia sp. 4078 2626 81 globlastp 2265 ZLFS31 Yersinia sp. 4079 2626 75.3 globlastp 2266 ZLFS31 Yersinia sp. 4080 2626 74.8 globlastp 2267 ZLFS31 Yersinia sp. 4081 2626 73 globlastp 903 BLFS27 Bacillus sp. 2753 2470 89.4 globlastp 904 BLFS27 Bacillus sp. 2754 2470 87.6 globlastp 905 BLFS27 Bacillus sp. 2755 2470 86.6 globlastp 906 BLFS27 Bacillus sp. 2756 2470 78.6 globlastp 907 BLFS27 Bacillus sp. 2757 2470 75.1 globlastp 1475 BLFS114 Pseudomonas sp. 3301 2550 93.6 globlastp 1476 BLFS114 Pseudomonas sp. 3302 2550 93.1 globlastp 1477 BLFS114 Pseudomonas sp. 3303 2550 92.5 globlastp 1478 BLFS114 Pseudomonas sp. 3304 2550 91 globlastp 1479 BLFS114 Environmental sample 3305 2550 90.2 globlastp 1480 BLFS114 Environmental sample 3306 2550 89 globlastp 1481 BLFS114 Environmental sample 3307 2550 88.4 globlastp 1482 BLFS114 Pseudomonas sp. 3308 2550 87.3 globlastp 1483 BLFS114 Pseudomonas sp. 3309 2550 86.2 globlastp 1484 BLFS114 Pseudomonas sp. 3310 2550 85 globlastp 1485 BLFS114 Pseudomonas sp. 3311 2550 84.4 globlastp 1486 BLFS114 Erwinia sp. 3312 2550 83.2 globlastp 1487 BLFS114 Pseudomonas sp. 3313 2550 82.1 globlastp 1488 BLFS114 Pseudomonas sp. 3314 2550 81 globlastp 1489 BLFS114 Pseudomonas sp. 3315 2550 80.3 globlastp 1490 BLFS114 Environmental sample 3316 2550 79.2 globlastp 1491 BLFS114 Environmental sample 3317 2550 78 globlastp 1492 BLFS114 Pseudomonas sp. 3318 2550 77.3 globlastp 1493 BLFS114 Pseudomonas sp. 3319 2550 76 globlastp 1494 BLFS114 Environmental sample 3320 2550 75.1 globlastp 1495 BLFS114 Environmental sample 3321 2550 74 globlastp 1496 BLFS114 Environmental sample 3322 2550 73.4 globlastp 1497 BLFS114 Pseudomonas sp. 3323 2550 72 globlastp 1498 BLFS114 Environmental sample 3324 2550 71.1 globlastp 1499 BLFS114 Environmental sample 3325 2550 70.5 globlastp 1030 BLFS61 Pseudomonas sp. 2873 2501 99 globlastp 1031 BLFS61 Pseudomonas sp. 2874 2501 96 globlastp 1032 BLFS61 Pseudomonas sp. 2875 2501 95.7 globlastp 1033 BLFS61 Environmental sample 2876 2501 92.5 globlastp 1034 BLFS61 Pseudomonas sp. 2877 2501 85.5 globlastp 1035 BLFS61 Environmental sample 2878 2501 79.2 globlastp 1036 BLFS61 Pseudomonas sp. 2879 2501 78.2 globlastp 1037 BLFS61 Pseudomonas sp. 2880 2501 74.5 globlastp 1577 BLFS121 Pseudomonas sp. 3402 2556 99 globlastp 1578 BLFS121 Pseudomonas sp. 3403 2556 98.8 globlastp 1579 BLFS121 Pseudomonas sp. 3404 2556 98.3 globlastp 1580 BLFS121 Pseudomonas sp. 3405 2556 97.8 globlastp 1581 BLFS121 Pseudomonas sp. 3406 2556 93.2 globlastp 1582 BLFS121 Pseudomonas sp. 3407 2556 92.3 globlastp 1583 BLFS121 Pseudomonas sp. 3408 2556 87 globlastp 1584 BLFS121 Pseudomonas sp. 3409 2556 86 globlastp 1585 BLFS121 Pseudomonas sp. 3410 2556 85 globlastp 1586 BLFS121 Environmental sample 3411 2556 84.1 globlastp 1587 BLFS121 Pseudomonas sp. 3412 2556 83.1 globlastp 1588 BLFS121 Environmental sample 3413 2556 82.1 globlastp 1589 BLFS121 Pseudomonas sp. 3414 2556 81.4 globlastp 1590 BLFS121 Pseudomonas sp. 3415 2556 80 globlastp 1591 BLFS121 Pseudomonas sp. 3416 2556 79.2 globlastp 1592 BLFS121 Environmental sample 3417 2556 78 globlastp 1593 BLFS121 Pseudomonas sp. 3418 2556 77.5 globlastp 1594 BLFS121 Pseudomonas sp. 3419 2556 76.1 globlastp 1595 BLFS121 Polaromonas sp. 3420 2556 75.7 globlastp 1596 BLFS121 Pseudomonas sp. 3421 2556 74.2 globlastp 1597 BLFS121 Pseudomonas sp. 3422 2556 72 globlastp 1598 BLFS121 Pseudomonas sp. 3423 2556 71 globlastp 2092 ZLFS15 Collimonas sp. 3906 2612 89.2 globlastp 2093 ZLFS15 Collimonas sp. 3907 2612 87.2 globlastp 2094 ZLFS15 Collimonas sp. 3908 2612 85.6 globlastp 1115 BLFS76 Paenibacillus sp. 2955 2514 95.4 globlastp 1116 BLFS76 Paenibacillus sp. 2956 2514 94.6 globlastp 1117 BLFS76 Paenibacillus sp. 2957 2514 93.4 globlastp 1118 BLFS76 Paenibacillus sp. 2958 2514 92.7 globlastp 1119 BLFS76 Paenibacillus sp. 2959 2514 90 globlastp 1120 BLFS76 Paenibacillus sp. 2960 2514 82.6 globlastp 1121 BLFS76 Paenibacillus sp. 2961 2514 72.3 globlastp 1122 BLFS76 Paenibacillus sp. — 2514 71.04 glotblastn 1123 BLFS76 Paenibacillus sp. 2962 2514 70.1 globlastp 1530 BLFS116 Pseudomonas sp. 3355 2552 95.8 globlastp 1531 BLFS116 Pseudomonas sp. 3356 2552 90.2 globlastp 1532 BLFS116 Pseudomonas sp. 3357 2552 89.2 globlastp 1533 BLFS116 Environmental sample 3358 2552 88.1 globlastp 1534 BLFS116 Pseudomonas sp. 3359 2552 87.8 globlastp 1535 BLFS116 Pseudomonas sp. 3360 2552 86.3 globlastp 1536 BLFS116 Pseudomonas sp. 3361 2552 85.5 globlastp 1537 BLFS116 Pseudomonas sp. 3362 2552 84.8 globlastp 1538 BLFS116 Pseudomonas sp. 3363 2552 83 globlastp 1539 BLFS116 Pseudomonas sp. 3364 2552 82.1 globlastp 1540 BLFS116 Pseudomonas sp. 3365 2552 81 globlastp 1541 BLFS116 Pseudomonas sp. 3366 2552 80.1 globlastp 1542 BLFS116 Pseudomonas sp. 3367 2552 79.1 globlastp 1543 BLFS116 Pseudomonas sp. 3368 2552 78 globlastp 1544 BLFS116 Pseudomonas sp. 3369 2552 77 globlastp 1545 BLFS116 Pseudomonas sp. 3370 2552 76.4 globlastp 1546 BLFS116 Pseudomonas sp. 3371 2552 75.1 globlastp 1547 BLFS116 Pseudomonas sp. 3372 2552 74 globlastp 1548 BLFS116 Pseudomonas sp. 3373 2552 73.2 globlastp 1549 BLFS116 Pseudomonas sp. 3374 2552 72.3 globlastp 1550 BLFS116 Pseudomonas sp. 3375 2552 71 globlastp 1551 BLFS116 Environmental sample 3376 2552 70.2 globlastp 1344 BLFS103 Pantoea sp. 3179 2539 94.1 globlastp 1345 BLFS103 unknown sp. 3180 2539 89.2 globlastp 1346 BLFS103 Pantoea sp. 3181 2539 86.5 globlastp 1347 BLFS103 Photorhabdus sp. — 2539 74.65 glotblastn 1348 BLFS103 Yersinia sp. — 2539 74.31 glotblastn 1349 BLFS103 Serratia sp. — 2539 73.96 glotblastn 1350 BLFS103 Salmonella sp. — 2539 73.61 glotblastn 1351 BLFS103 Salmonella sp. — 2539 73.26 glotblastn 1352 BLFS103 Serratia sp. — 2539 73.1 glotblastn 1353 BLFS103 Yersinia sp. 3182 2539 71.9 globlastp 1354 BLFS103 Yersinia sp. — 2539 71.53 glotblastn 1355 BLFS103 Serratia sp. — 2539 71.18 glotblastn 1356 BLFS103 Pantoea sp. — 2539 70.14 glotblastn 1668 BLFS128 Pseudomonas sp. 3492 2563 99.3 globlastp 1669 BLFS128 Pseudomonas sp. 3493 2563 95.2 globlastp 1670 BLFS128 Environmental sample 3494 2563 94.3 globlastp 1671 BLFS128 Pseudomonas sp. 3495 2563 93.3 globlastp 1672 BLFS128 Pseudomonas sp. 3496 2563 92.9 globlastp 1673 BLFS128 Pseudomonas sp. 3497 2563 91.7 globlastp 1674 BLFS128 Pseudomonas sp. 3498 2563 89.2 globlastp 1675 BLFS128 Pseudomonas sp. 3499 2563 80.1 globlastp 1676 BLFS128 Pseudomonas sp. 3500 2563 79.5 globlastp 1677 BLFS128 Pseudomonas sp. 3501 2563 78 globlastp 1678 BLFS128 Pseudomonas sp. 3502 2563 77 globlastp 1679 BLFS128 Pseudomonas sp. 3503 2563 76 globlastp 1680 BLFS128 Pseudomonas sp. 3504 2563 75.7 globlastp 1681 BLFS128 Erwinia sp. 3505 2563 73 globlastp 1682 BLFS128 Pseudomonas sp. 3506 2563 72 globlastp 1683 BLFS128 Pseudomonas sp. 3507 2563 71 globlastp 1684 BLFS128 Pseudomonas sp. 3508 2563 70 globlastp 1937 BLFS148 Serratia sp. 3760 2583 99.2 globlastp 1938 BLFS148 Serratia sp. 3761 2583 98.4 globlastp 1939 BLFS148 Serratia sp. 3762 2583 95.2 globlastp 1940 BLFS148 Serratia sp. 3763 2583 89.2 globlastp 1941 BLFS148 Serratia sp. 3764 2583 87.2 globlastp 1942 BLFS148 Serratia sp. 3765 2583 86.4 globlastp 1943 BLFS148 Serratia sp. 3766 2583 85.6 globlastp 1944 BLFS148 Yersinia sp. 3767 2583 84 globlastp 1945 BLFS148 Yersinia sp. 3768 2583 83.2 globlastp 1946 BLFS148 Pantoea sp. 3769 2583 82 globlastp 1947 BLFS148 Yersinia sp. 3770 2583 81.2 globlastp 1948 BLFS148 Pectobacterium sp. 3771 2583 80 globlastp 1949 BLFS148 Pectobacterium sp. 3772 2583 79.2 globlastp 1950 BLFS148 Erwinia sp. 3773 2583 78 globlastp 1951 BLFS148 Environmental sample 3774 2583 77.3 globlastp 1952 BLFS148 Pantoea sp. 3775 2583 76 globlastp 1953 BLFS148 Pantoea sp. 3776 2583 75 globlastp 1954 BLFS148 Buttiauxella sp. 3777 2583 74.2 globlastp 1955 BLFS148 Salmonella sp. 3778 2583 73 globlastp 1956 BLFS148 Proteus sp. 3779 2583 72 globlastp 1957 BLFS148 Enterobacter.2 sp. 3780 2583 71 globlastp 1958 BLFS148 Environmental sample 3781 2583 70.2 globlastp 1959 BLFS148 Citrobacter sp. — 2583 70 glotblastn 859 BLFS19 Bacillus sp. 2711 2462 96.4 globlastp 860 BLFS19 Bacillus sp. 2712 2462 96.1 globlastp 861 BLFS19 Bacillus sp. 2713 2462 95.8 globlastp 862 BLFS19 Bacillus sp. 2714 2462 95.5 globlastp 863 BLFS19 Bacillus sp. 2715 2462 94.7 globlastp 864 BLFS19 Bacillus sp. 2716 2462 92 globlastp 865 BLFS19 Bacillus sp. 2717 2462 91.7 globlastp 988 BLFS44 Burkholderia sp. 2836 2485 99.4 globlastp 2095 ZLFS16 Collimonas sp. 3909 2613 94.5 globlastp 2096 ZLFS16 Collimonas sp. 3910 2613 85.1 globlastp 2097 ZLFS16 Herminiimonas sp. 3911 2613 74 globlastp 2098 ZLFS16 Duganella sp. 3912 2613 73 globlastp 2099 ZLFS16 Janthinobacterium sp. 3913 2613 72 globlastp 2100 ZLFS16 Massilia sp. 3914 2613 71.2 globlastp 2101 ZLFS16 Oxalobacteraceae sp. 3915 2613 70 globlastp 1893 BLFS145 Bacillus sp. 3716 2580 99.2 globlastp 1894 BLFS145 Bacillus sp. 3717 2580 97.9 globlastp 1895 BLFS145 Bacillus sp. 3718 2580 94 globlastp 1896 BLFS145 Bacillus sp. 3719 2580 93.2 globlastp 1897 BLFS145 Bacillus sp. 3720 2580 92.7 globlastp 1898 BLFS145 Bacillus sp. 3721 2580 92.1 globlastp 1899 BLFS145 Bacillus sp. 3722 2580 91.1 globlastp 1900 BLFS145 Bacillus sp. 3723 2580 84.3 globlastp 1901 BLFS145 Bacillus sp. 3724 2580 78.1 globlastp 1902 BLFS145 Bacillus sp. 3725 2580 77.5 globlastp 1903 BLFS145 Bacillus sp. 3726 2580 74.2 globlastp 1904 BLFS145 Bacillus sp. 3727 2580 74 globlastp 1905 BLFS145 Bacillus sp. 3728 2580 73.2 globlastp 1906 BLFS145 Bacillus sp. 3729 2580 71 globlastp 1500 BLFS115 Pseudomonas sp. 3326 2551 98.5 globlastp 1501 BLFS115 Pseudomonas sp. 3327 2551 98.3 globlastp 1502 BLFS115 Pseudomonas sp. 3328 2551 98.1 globlastp 1503 BLFS115 Pseudomonas sp. 3329 2551 97.7 globlastp 1504 BLFS115 Pseudomonas sp. 3330 2551 95.2 globlastp 1505 BLFS115 Pseudomonas sp. 3331 2551 94.1 globlastp 1506 BLFS115 Pseudomonas sp. 3332 2551 93.1 globlastp 1507 BLFS115 Pseudomonas sp. 3333 2551 92 globlastp 1508 BLFS115 Environmental sample 3334 2551 91 globlastp 1509 BLFS115 Pseudomonas sp. 3335 2551 90 globlastp 1510 BLFS115 Pseudomonas sp. 3336 2551 89.7 globlastp 1511 BLFS115 Pseudomonas sp. 3337 2551 88.1 globlastp 1512 BLFS115 Pseudomonas sp. 3338 2551 87.9 globlastp 1513 BLFS115 Pseudomonas sp. 3339 2551 86.2 globlastp 1514 BLFS115 Pseudomonas sp. 3340 2551 85.2 globlastp 1515 BLFS115 Pseudomonas sp. 3341 2551 85.1 globlastp 1516 BLFS115 Pseudomonas sp. 3342 2551 84 globlastp 1517 BLFS115 Pseudomonas sp. 3343 2551 83.4 globlastp 1518 BLFS115 Pseudomonas sp. 3344 2551 82.8 globlastp 1519 BLFS115 Environmental sample 3345 2551 80 globlastp 1520 BLFS115 Environmental sample 3346 2551 79 globlastp 1521 BLFS115 Pseudomonas sp. 3347 2551 78 globlastp 1522 BLFS115 Pseudomonas sp. 3348 2551 77 globlastp 1523 BLFS115 Pseudomonas sp. 3349 2551 76 globlastp 1524 BLFS115 Pseudomonas sp. 3350 2551 75.2 globlastp 1525 BLFS115 Environmental sample 3351 2551 74.2 globlastp 1526 BLFS115 Environmental sample 3352 2551 73 globlastp 1527 BLFS115 Azotobacter sp. 3353 2551 72.1 globlastp 1528 BLFS115 Pseudomonas sp. 3354 2551 71 globlastp 1529 BLFS115 Pseudomonas sp. — 2551 70.48 glotblastn 1780 BLFS135 Erwinia sp. 3604 2570 97.6 globlastp 1781 BLFS135 Pseudomonas sp. 3605 2570 97.2 globlastp 1782 BLFS135 Pseudomonas sp. 3606 2570 97 globlastp 1783 BLFS135 Pseudomonas sp. 3607 2570 96.6 globlastp 1784 BLFS135 Pseudomonas sp. 3608 2570 96.4 globlastp 1785 BLFS135 Pseudomonas sp. 3609 2570 95.2 globlastp 1786 BLFS135 Environmental sample 3610 2570 94.6 globlastp 1787 BLFS135 Pseudomonas sp. 3611 2570 88.6 globlastp 1788 BLFS135 Pseudomonas sp. 3612 2570 87.3 globlastp 1789 BLFS135 Pseudomonas sp. 3613 2570 73 globlastp 1790 BLFS135 Pseudomonas sp. 3614 2570 72.8 globlastp 1791 BLFS135 Pseudomonas sp. 3615 2570 70.9 globlastp 1882 BLFS144 Salinibacillus sp. 3705 2579 99.1 globlastp 1883 BLFS144 Bacillus sp. 3706 2579 96.9 globlastp 1884 BLFS144 Bacillus sp. 3707 2579 95.2 globlastp 1885 BLFS144 Bacillus sp. 3708 2579 94.1 globlastp 1886 BLFS144 Bacillus sp. 3709 2579 88 globlastp 1887 BLFS144 Bacillus sp. 3710 2579 87 globlastp 1888 BLFS144 Bacillus sp. 3711 2579 86.7 globlastp 1889 BLFS144 Bacillus sp. 3712 2579 85.4 globlastp 1890 BLFS144 Bacillus sp. 3713 2579 85 globlastp 1891 BLFS144 Bacillus sp. 3714 2579 84.4 globlastp 1892 BLFS144 Bacillus sp. 3715 2579 84.1 globlastp 1063 BLFS65 Duganella sp. 2905 2505 84.5 globlastp 2424 ZLFS49 Curtobacterium sp. 4233 2640 93.2 globlastp 2425 ZLFS49 Pantoea sp. 4234 2640 85.7 globlastp 1811 BLFS139 Salinibacillus sp. 3634 2574 99.4 globlastp 1812 BLFS139 Salinibacillus sp. 3635 2574 98.8 globlastp 1813 BLFS139 Bacillus sp. 3636 2574 97.9 globlastp 1814 BLFS139 Bacillus sp. 3637 2574 93.8 globlastp 1815 BLFS139 Bacillus sp. 3638 2574 89.5 globlastp 1816 BLFS139 Bacillus sp. 3639 2574 88 globlastp 1817 BLFS139 Bacillus sp. 3640 2574 87.3 globlastp 1818 BLFS139 Bacillus sp. 3641 2574 86.4 globlastp 1819 BLFS139 Bacillus sp. 3642 2574 85.8 globlastp 1820 BLFS139 Bacillus sp. 3643 2574 83.9 globlastp 1821 BLFS139 Bacillus sp. 3644 2574 77.3 globlastp 1822 BLFS139 Bacillus sp. 3645 2574 76.3 globlastp 1823 BLFS139 Bacillus sp. 3646 2574 75.1 globlastp 823 BLFS14 Environmental sample 2675 2457 95.7 globlastp 824 BLFS14 Arthrobacter sp. 2676 2457 84 globlastp 1628 BLFS125 Pseudomonas sp. 3452 2560 96.6 globlastp 1629 BLFS125 Pseudomonas sp. 3453 2560 87.6 globlastp 1630 BLFS125 Pseudomonas sp. 3454 2560 86.5 globlastp 1631 BLFS125 Pseudomonas sp. 3455 2560 85.4 globlastp 1632 BLFS125 Pseudomonas sp. 3456 2560 84.3 globlastp 1633 BLFS125 Environmental sample 3457 2560 83.1 globlastp 1634 BLFS125 Meta sp. 3458 2560 82 globlastp 1635 BLFS125 Pseudomonas sp. 3459 2560 80.9 globlastp 1636 BLFS125 Pseudomonas sp. 3460 2560 79.8 globlastp 1637 BLFS125 Pseudomonas sp. 3461 2560 78.8 globlastp 1638 BLFS125 Pseudomonas sp. 3462 2560 78.7 globlastp 1639 BLFS125 Environmental sample 3463 2560 77.5 globlastp 1640 BLFS125 Environmental sample 3464 2560 76.4 globlastp 1641 BLFS125 Environmental sample 3465 2560 75.3 globlastp 1642 BLFS125 Environmental sample 3466 2560 74.2 globlastp 1643 BLFS125 Environmental sample 3467 2560 73 globlastp 1181 BLFS86 Paenibacillus sp. 3018 2522 95.1 globlastp 1182 BLFS86 Paenibacillus sp. 3019 2522 92.7 globlastp 1183 BLFS86 Paenibacillus sp. 3020 2522 89.2 globlastp 1184 BLFS86 Paenibacillus sp. 3021 2522 89 globlastp 1185 BLFS86 Paenibacillus sp. 3022 2522 87.8 globlastp 1186 BLFS86 Paenibacillus sp. 3023 2522 84.7 globlastp 1336 BLFS102 Environmental sample 3171 2538 98.5 globlastp 1337 BLFS102 Environmental sample 3172 2538 97.1 globlastp 1338 BLFS102 Pantoea sp. 3173 2538 88.2 globlastp 1339 BLFS102 Environmental sample 3174 2538 86.8 globlastp 1340 BLFS102 Pantoea sp. 3175 2538 85.3 globlastp 1341 BLFS102 Pantoea sp. 3176 2538 80.9 globlastp 1342 BLFS102 Environmental sample 3177 2538 76.5 globlastp 1343 BLFS102 Pantoea sp. 3178 2538 72.1 globlastp 909 BLFS31 Bacillus sp. 2759 2473 99.2 globlastp 910 BLFS31 Bacillus sp. 2760 2473 98.4 globlastp 911 BLFS31 Streptococcus sp. 2761 2473 97.7 globlastp 912 BLFS31 Bacillus sp. 2762 2473 96.9 globlastp 913 BLFS31 Bacillus sp. 2763 2473 96.1 globlastp 914 BLFS31 Bacillus sp. 2764 2473 95.3 globlastp 915 BLFS31 Bacillus sp. 2765 2473 93 globlastp 916 BLFS31 Bacillus sp. 2766 2473 89.1 globlastp 917 BLFS31 Bacillus sp. 2767 2473 86.9 globlastp 918 BLFS31 Bacillus sp. 2768 2473 86.2 globlastp 919 BLFS31 Bacillus sp. 2769 2473 85.4 globlastp 1437 BLFS112 Pseudomonas sp. 3263 2548 98.8 globlastp 1438 BLFS112 Pseudomonas sp. 3264 2548 96.8 globlastp 1439 BLFS112 Pseudomonas sp. 3265 2548 94.7 globlastp 1440 BLFS112 Pseudomonas sp. 3266 2548 94.3 globlastp 1441 BLFS112 Pseudomonas sp. 3267 2548 93.1 globlastp 1442 BLFS112 Pseudomonas sp. 3268 2548 91.8 globlastp 1443 BLFS112 Pseudomonas sp. 3269 2548 91.4 globlastp 1444 BLFS112 Pseudomonas sp. 3270 2548 90.2 globlastp 1445 BLFS112 Pseudomonas sp. 3271 2548 89.4 globlastp 1446 BLFS112 Pseudomonas sp. 3272 2548 89 globlastp 1447 BLFS112 Pseudomonas sp. 3273 2548 74.8 globlastp 1448 BLFS112 Pseudomonas sp. 3274 2548 72.9 globlastp 1449 BLFS112 Pseudomonas sp. 3275 2548 71.4 globlastp 2268 ZLFS32 Serratia sp. 4082 2627 99.2 globlastp 2269 ZLFS32 Serratia sp. 4083 2627 97 globlastp 2270 ZLFS32 Serratia sp. 4084 2627 96.2 globlastp 2271 ZLFS32 Serratia sp. 4085 2627 88.1 globlastp 2272 ZLFS32 Serratia sp. 4086 2627 87 globlastp 2273 ZLFS32 Serratia sp. 4087 2627 86.1 globlastp 2274 ZLFS32 Serratia sp. 4088 2627 85 globlastp 2275 ZLFS32 Serratia sp. 4089 2627 84.6 globlastp 2276 ZLFS32 Serratia sp. 4090 2627 81.1 globlastp 2277 ZLFS32 Serratia sp. 4091 2627 80.9 globlastp 2278 ZLFS32 Serratia sp. 4092 2627 79.5 globlastp 2279 ZLFS32 Serratia sp. 4093 2627 78.6 globlastp 2280 ZLFS32 Serratia sp. 4094 2627 74.1 globlastp 2281 ZLFS32 Serratia sp. 4095 2627 73.7 globlastp 2282 ZLFS32 Yersinia sp. 4096 2627 72.4 globlastp 2283 ZLFS32 Pectobacterium sp. 4097 2627 70.1 globlastp 1793 BLFS137 Environmental sample 3617 2572 98.2 globlastp 1794 BLFS137 Rhodococcus sp. 3618 2572 94.7 globlastp 1795 BLFS137 Rhodococcus sp. 3619 2572 94.3 globlastp 1796 BLFS137 Environmental sample 3620 2572 81.1 globlastp 1797 BLFS137 Environmental sample 3621 2572 80.8 globlastp 1292 BLFS99 Environmental sample 3127 2535 98.9 globlastp 1293 BLFS99 Pantoea sp. 3128 2535 97.7 globlastp 1294 BLFS99 Environmental sample 3129 2535 96.6 globlastp 1295 BLFS99 Pantoea sp. 3130 2535 93.2 globlastp 1296 BLFS99 Pantoea sp. 3131 2535 90.9 globlastp 1297 BLFS99 Pantoea sp. 3132 2535 81.1 globlastp 1298 BLFS99 Environmental sample 3133 2535 80 globlastp 1299 BLFS99 Pantoea sp. 3134 2535 75 globlastp 1300 BLFS99 Environmental sample 3135 2535 73.3 globlastp 1301 BLFS99 Pantoea sp. 3136 2535 72.2 globlastp 1302 BLFS99 Pantoea sp. 3137 2535 71.6 globlastp 1303 BLFS99 Pantoea sp. 3138 2535 70.5 globlastp 1027 BLFS60 Streptomyces sp. 2870 2500 75.2 globlastp 1028 BLFS60 Streptomyces sp. 2871 2500 72.3 globlastp 1029 BLFS60 Streptomyces sp. 2872 2500 71.8 globlastp 2102 ZLFS17 Environmental sample 3916 2614 73.5 globlastp 971 BLFS39 Brevibacillus sp. 2819 2481 98.4 globlastp 972 BLFS39 Brevibacillus sp. 2820 2481 98.1 globlastp 973 BLFS39 Brevibacillus sp. 2821 2481 97.8 globlastp 2364 ZLFS39 Serratia sp. 4177 2634 98.8 globlastp 2365 ZLFS39 Serratia sp. 4178 2634 98.5 globlastp 2366 ZLFS39 Serratia sp. 4179 2634 98.3 globlastp 2367 ZLFS39 Serratia sp. 4180 2634 92.6 globlastp 2368 ZLFS39 Serratia sp. 4181 2634 91.4 globlastp 2369 ZLFS39 Multievos46 sp. 4182 2634 91.2 globlastp 2370 ZLFS39 Serratia sp. 4183 2634 91.1 globlastp 2371 ZLFS39 Environmental sample 4184 2634 90.9 globlastp 2372 ZLFS39 Serratia sp. 4185 2634 87.3 globlastp 2373 ZLFS39 Serratia sp. 4186 2634 87.1 globlastp 2374 ZLFS39 Serratia sp. 4187 2634 87 globlastp 2375 ZLFS39 Serratia sp. 4188 2634 86.8 globlastp 2376 ZLFS39 Serratia sp. 4189 2634 86.7 globlastp 2377 ZLFS39 Serratia sp. 4190 2634 86.5 globlastp 2378 ZLFS39 Environmental sample 4191 2634 86.4 globlastp 2379 ZLFS39 Serratia sp. 4192 2634 86.2 globlastp 2380 ZLFS39 Serratia sp. 4193 2634 86.1 globlastp 2381 ZLFS39 Serratia sp. 4194 2634 85.8 globlastp 2382 ZLFS39 Serratia sp. 4195 2634 85.6 globlastp 2383 ZLFS39 Serratia sp. 4196 2634 85.2 globlastp 2384 ZLFS39 Serratia sp. 4197 2634 85 globlastp 2385 ZLFS39 Serratia sp. 4198 2634 84.9 globlastp 2386 ZLFS39 Serratia sp. 4199 2634 84.4 globlastp 2387 ZLFS39 Serratia sp. 4200 2634 79.6 globlastp 2388 ZLFS39 Serratia sp. 4201 2634 79.2 globlastp 2209 ZLFS29 Pseudomonas sp. 4023 2624 95 globlastp 2210 ZLFS29 Pseudomonas sp. 4024 2624 94.1 globlastp 2211 ZLFS29 Pseudomonas sp. 4025 2624 93.1 globlastp 2212 ZLFS29 Pseudomonas sp. 4026 2624 92.2 globlastp 2213 ZLFS29 Pseudomonas sp. 4027 2624 91.6 globlastp 2214 ZLFS29 Pseudomonas sp. 4028 2624 90.8 globlastp 2215 ZLFS29 Pseudomonas sp. 4029 2624 89.1 globlastp 2216 ZLFS29 Environmental sample 4030 2624 88.1 globlastp 2217 ZLFS29 Pseudomonas sp. 4031 2624 87 globlastp 2218 ZLFS29 Pseudomonas sp. 4032 2624 86.4 globlastp 2219 ZLFS29 Pseudomonas sp. 4033 2624 85.7 globlastp 2220 ZLFS29 Pseudomonas sp. 4034 2624 84.1 globlastp 2221 ZLFS29 Pseudomonas sp. 4035 2624 83 globlastp 2222 ZLFS29 Environmental sample 4036 2624 82 globlastp 2223 ZLFS29 Environmental sample 4037 2624 81 globlastp 2224 ZLFS29 Pseudomonas sp. 4038 2624 80.3 globlastp 2225 ZLFS29 Environmental sample 4039 2624 79.1 globlastp 2226 ZLFS29 Pseudomonas sp. 4040 2624 78 globlastp 2227 ZLFS29 Environmental sample 4041 2624 77.4 globlastp 2228 ZLFS29 Pseudomonas sp. 4042 2624 76.1 globlastp 2229 ZLFS29 Pseudomonas sp. 4043 2624 75.3 globlastp 2230 ZLFS29 Pseudomonas sp. 4044 2624 74.4 globlastp 2231 ZLFS29 Pseudomonas sp. 4045 2624 73.4 globlastp 2232 ZLFS29 Pseudomonas sp. 4046 2624 72.1 globlastp 2233 ZLFS29 Pseudomonas sp. 4047 2624 71.1 globlastp 2234 ZLFS29 Pseudomonas sp. 4048 2624 70.2 globlastp 1986 BLFS153 Streptomyces sp. 3803 2588 98.6 globlastp 1987 BLFS153 Streptomyces sp. 3804 2588 93.3 globlastp 1988 BLFS153 Streptomyces sp. 3805 2588 71.7 globlastp 1989 BLFS153 Streptomyces sp. 3806 2588 70.9 globlastp 1990 BLFS153 Streptomyces sp. — 2588 70.56 glotblastn 1991 BLFS153 Streptomyces sp. 3807 2588 70.4 globlastp 1992 BLFS153 Streptomyces sp. — 2588 70.36 glotblastn 1993 BLFS153 Streptomyces sp. 3808 2588 70.1 globlastp 1994 BLFS153 Streptomyces sp. — 2588 70 glotblastn 2086 ZLFS12 Collimonas sp. 3900 2609 81.4 globlastp 2087 ZLFS12 Collimonas sp. 3901 2609 81 globlastp 2088 ZLFS12 Collimonas sp. 3902 2609 73.7 globlastp 1574 BLFS120 Pseudomonas sp. 3399 2555 97.2 globlastp 1575 BLFS120 Pseudomonas sp. 3400 2555 84.8 globlastp 1576 BLFS120 Pseudomonas sp. 3401 2555 75.6 globlastp 2391 ZLFS45 Streptomyces sp. 4204 2637 98.2 globlastp 2392 ZLFS45 Streptomyces sp. 4205 2637 97.3 globlastp 2390 ZLFS45 Streptomyces sp. 4203 2637 96.4 globlastp 2389 ZLFS45 Streptomyces sp. 4202 2637 94 globlastp 2397 ZLFS45 Streptomyces sp. 4209 2637 90.2 globlastp 2394 ZLFS45 Streptomyces sp. 4207 2637 90.2 globlastp 2395 ZLFS45 Streptomyces sp. 4207 2637 90.2 globlastp 2393 ZLFS45 Streptomyces sp. 4206 2637 80.4 globlastp 2423 ZLFS45 Streptomyces sp. 4232 2637 73.2 globlastp 2398 ZLFS45 Streptomyces sp. 4210 2637 72.6 globlastp 2396 ZLFS45 Streptomyces sp. 4208 2637 72.6 globlastp 2399 ZLFS44 Streptomyces sp. 4211 2636 98.7 globlastp 2400 ZLFS44 Streptomyces sp. 4212 2636 98.1 globlastp 2401 ZLFS44 Streptomyces sp. 4213 2636 96.8 globlastp 2402 ZLFS44 Streptomyces sp. 4214 2636 75.8 globlastp 2403 ZLFS44 Streptomyces sp. 4215 2636 75.2 globlastp 2404 ZLFS44 Microtetraspora sp. 4216 2636 74.7 globlastp 2405 ZLFS44 Streptomyces sp. 4217 2636 74.5 globlastp 2406 ZLFS44 Streptomyces sp. 4218 2636 74.1 globlastp 2407 ZLFS44 Streptomyces sp. 4219 2636 73.9 globlastp 2408 ZLFS44 Streptomyces sp. — 2636 73.89 glotblastn 2409 ZLFS44 Streptomyces sp. 4220 2636 73.4 globlastp 2410 ZLFS44 Streptomyces sp. 4221 2636 73.2 globlastp 2411 ZLFS44 Streptomyces sp. 4222 2636 72.8 globlastp 2412 ZLFS44 Streptomyces sp. 4223 2636 72.6 globlastp 2413 ZLFS44 Streptomyces sp. 4224 2636 72.2 globlastp 2414 ZLFS44 Environmental sample 4225 2636 72 globlastp 2415 ZLFS44 Streptomyces sp. — 2636 71.52 glotblastn 2416 ZLFS44 Streptomyces sp. 4226 2636 71.5 globlastp 2417 ZLFS44 Streptomyces sp. 4227 2636 71.3 globlastp 2418 ZLFS44 Streptomyces sp. 4228 2636 71.1 globlastp 2419 ZLFS44 Streptomyces sp. 4229 2636 70.8 globlastp 2420 ZLFS44 Streptomyces sp. 4230 2636 70.7 globlastp 2421 ZLFS44 Streptomyces sp. 4231 2636 70.1 globlastp 2422 ZLFS44 Streptomyces sp. — 2636 70.06 glotblastn 1022 BLFS57 Duganella sp. 2865 2497 99 globlastp 1023 BLFS57 Janthinobacterium sp. 2866 2497 98 globlastp 1024 BLFS57 Oxalobacteraceae sp. 2867 2497 85.3 globlastp 1368 BLFS105 Pantoea sp. 3194 2541 96.2 globlastp 1369 BLFS105 Environmental sample 3195 2541 92.4 globlastp 1370 BLFS105 Environmental sample 3196 2541 91.7 globlastp 1371 BLFS105 Environmental sample 3197 2541 87.9 globlastp 1372 BLFS105 Environmental sample 3198 2541 87.1 globlastp 1373 BLFS105 Environmental sample 3199 2541 86.4 globlastp 1374 BLFS105 Environmental sample 3200 2541 85.7 globlastp 1375 BLFS105 Environmental sample 3201 2541 84.8 globlastp 1376 BLFS105 Environmental sample 3202 2541 84.1 globlastp 1377 BLFS105 Pantoea sp. 3203 2541 82.6 globlastp 1378 BLFS105 Pantoea sp. 3204 2541 79.5 globlastp 1379 BLFS105 Erwinia sp. 3205 2541 75.8 globlastp 1380 BLFS105 Ewingella sp. 3206 2541 74.2 globlastp 1267 BLFS95 Paenibacillus sp. 3102 2531 97.4 globlastp 1268 BLFS95 Paenibacillus sp. 3103 2531 96.1 globlastp 1269 BLFS95 Paenibacillus sp. 3104 2531 90.8 globlastp 1270 BLFS95 Paenibacillus sp. 3105 2531 90.2 globlastp 1271 BLFS95 Paenibacillus sp. 3106 2531 89.5 globlastp 1272 BLFS95 Paenibacillus sp. 3107 2531 88.2 globlastp 1273 BLFS95 Paenibacillus sp. 3108 2531 78.4 globlastp 1274 BLFS95 Paenibacillus sp. 3109 2531 77.8 globlastp 1275 BLFS95 Paenibacillus sp. 3110 2531 75.2 globlastp 967 BLFS37 Brevibacillus sp. 2816 2479 99.1 globlastp 968 BLFS37 Brevibacillus sp. 2817 2479 98.2 globlastp 969 BLFS37 Brevibacillus sp. — 2479 97.35 glotblastn 1570 BLFS119 Pseudomonas sp. 3395 2554 96.2 globlastp 1571 BLFS119 Pseudomonas sp. 3396 2554 95 globlastp 1572 BLFS119 Pseudomonas sp. 3397 2554 92.5 globlastp 1573 BLFS119 Pseudomonas sp. 3398 2554 91.2 globlastp 2284 ZLFS33 Serratia sp. 4098 2628 99.2 globlastp 2285 ZLFS33 Serratia sp. 4099 2628 96.4 globlastp 1025 BLFS58 Janthinobacterium sp. 2868 2498 97.4 globlastp 1026 BLFS58 Oxalobacteraceae sp. 2869 2498 83.5 globlastp 2002 PUB6 Bacillus sp. 3816 2595 98.6 globlastp 2003 PUB6 Bacillus sp. 3817 2595 98.5 globlastp 2004 PUB6 Bacillus sp. 3818 2595 98.4 globlastp 2005 PUB6 Bacillus sp. 3819 2595 97.9 globlastp 2006 PUB6 Bacillus sp. 3820 2595 96.1 globlastp 2007 PUB6 Bacillus sp. 3821 2595 94.7 globlastp 2008 PUB6 Bacillus sp. 3822 2595 94.2 globlastp 2009 PUB6 Bacillus sp. 3823 2595 93.2 globlastp 2010 PUB6 Bacillus sp. 3824 2595 92.6 globlastp 2011 PUB6 Bacillus sp. 3825 2595 92.1 globlastp 792 BLFS5 Streptomyces sp. 2644 2448 76.4 globlastp 793 BLFS5 Streptomyces sp. 2645 2448 75.5 globlastp 794 BLFS5 Kitasatospora sp. 2646 2448 74.5 globlastp 795 BLFS5 Kitasatospora sp. 2647 2448 73.8 globlastp 796 BLFS5 Streptomyces sp. 2648 2448 73.2 globlastp 797 BLFS5 Kitasatospora sp. 2649 2448 72 globlastp 798 BLFS5 Streptomyces sp. 2650 2448 71.1 globlastp 799 BLFS5 Streptomyces sp. 2651 2448 70.1 globlastp 989 BLFS45 Burkholderia sp. 2837 2486 97.9 globlastp 990 BLFS45 Environmental sample 2838 2486 82.3 globlastp 991 BLFS45 Burkholderia sp. 2839 2486 81.1 globlastp 992 BLFS45 Burkholderia sp. 2840 2486 80.2 globlastp 993 BLFS45 Burkholderia sp. 2841 2486 79.9 globlastp 994 BLFS45 Burkholderia sp. 2842 2486 76.1 globlastp 995 BLFS45 Burkholderia sp. 2843 2486 75.2 globlastp 996 BLFS45 Burkholderia sp. 2844 2486 74.6 globlastp 1304 BLFS100 Environmental sample 3139 2536 98.9 globlastp 1305 BLFS100 Environmental sample 3140 2536 97.7 globlastp 1306 BLFS100 Pantoea sp. 3141 2536 96.6 globlastp 1307 BLFS100 Environmental sample 3142 2536 95.4 globlastp 1308 BLFS100 Pantoea sp. 3143 2536 94.3 globlastp 1309 BLFS100 Pantoea sp. 3144 2536 88.5 globlastp 1310 BLFS100 Environmental sample 3145 2536 87.4 globlastp 1311 BLFS100 Pantoea sp. 3146 2536 85.1 globlastp 1312 BLFS100 Pantoea sp. 3147 2536 83.9 globlastp 1313 BLFS100 Pantoea sp. 3148 2536 81.8 globlastp 1314 BLFS100 Pantoea sp. 3149 2536 80.7 globlastp 1315 BLFS100 Pantoea sp. 3150 2536 78.4 globlastp 1316 BLFS100 Pantoea sp. 3151 2536 76.1 globlastp 1317 BLFS100 Pantoea sp. 3152 2536 74.4 globlastp 1318 BLFS100 Plautia sp. 3153 2536 71 globlastp 1319 BLFS100 Erwinia sp. 3154 2536 70.1 globlastp 1218 BLFS90 Paenibacillus sp. 3053 2526 99.3 globlastp 1219 BLFS90 Paenibacillus sp. 3054 2526 98.9 globlastp 1220 BLFS90 Paenibacillus sp. 3055 2526 96.1 globlastp 1221 BLFS90 Paenibacillus sp. 3056 2526 95.4 globlastp 1222 BLFS90 Paenibacillus sp. 3057 2526 93.6 globlastp 1223 BLFS90 Paenibacillus sp. 3058 2526 92.1 globlastp 1224 BLFS90 Paenibacillus sp. 3059 2526 91.4 globlastp 1225 BLFS90 Paenibacillus sp. 3060 2526 90.7 globlastp 1226 BLFS90 Paenibacillus sp. 3061 2526 72.9 globlastp 1011 BLFS55 Janthinobacterium sp. 2857 2495 95.5 globlastp 1012 BLFS55 Duganella sp. 2858 2495 78.5 globlastp 2306 ZLFS35 Serratia sp. 4120 2630 99 globlastp 2307 ZLFS35 Serratia sp. 4121 2630 98.8 globlastp 2308 ZLFS35 Serratia sp. 4122 2630 98.1 globlastp 2309 ZLFS35 Serratia sp. 4123 2630 88.3 globlastp 2310 ZLFS35 Serratia sp. 4124 2630 73.2 globlastp 1152 BLFS83 Paenibacillus sp. 2989 2519 99 globlastp 1153 BLFS83 Paenibacillus sp. 2990 2519 97.4 globlastp 1154 BLFS83 Paenibacillus sp. 2991 2519 97 globlastp 1155 BLFS83 Paenibacillus sp. 2992 2519 96.4 globlastp 1156 BLFS83 Paenibacillus sp. 2993 2519 95 globlastp 1157 BLFS83 Paenibacillus sp. 2994 2519 94.3 globlastp 1158 BLFS83 Paenibacillus sp. 2995 2519 93.2 globlastp 1159 BLFS83 Paenibacillus sp. 2996 2519 92.3 globlastp 1160 BLFS83 Paenibacillus sp. 2997 2519 80.6 globlastp 1161 BLFS83 Paenibacillus sp. 2998 2519 80 globlastp 1162 BLFS83 Paenibacillus sp. 2999 2519 79.6 globlastp 870 BLFS21 Bacillus sp. 2722 2464 99.3 globlastp 871 BLFS21 Bacillus sp. 2723 2464 83.1 globlastp 872 BLFS21 Bacillus sp. 2724 2464 79.7 globlastp 873 BLFS21 Bacillus sp. 2725 2464 71.3 globlastp 874 BLFS21 Bacillus sp. 2726 2464 70.5 globlastp 875 BLFS21 Bacillus sp. — 2464 70.23 glotblastn 876 BLFS21 Bacillus sp. 2727 2464 70.2 globlastp 877 BLFS21 Bacillus sp. — 2464 70.14 glotblastn 1685 BLFS129 Pseudomonas sp. 3509 2564 95.4 globlastp 1686 BLFS129 Pseudomonas sp. 3510 2564 95 globlastp 1687 BLFS129 Pseudomonas sp. 3511 2564 94.5 globlastp 1688 BLFS129 Pseudomonas sp. 3512 2564 93.1 globlastp 1689 BLFS129 Pseudomonas sp. 3513 2564 91.3 globlastp 1690 BLFS129 Pseudomonas sp. 3514 2564 90.8 globlastp 1691 BLFS129 Environmental sample 3515 2564 87.6 globlastp 1692 BLFS129 Pseudomonas sp. 3516 2564 83.9 globlastp 1693 BLFS129 Pseudomonas sp. 3517 2564 79.8 globlastp 1694 BLFS129 Pseudomonas sp. 3518 2564 76.6 globlastp 1695 BLFS129 Pseudomonas sp. 3519 2564 76.1 globlastp 920 BLFS32 Bacillus sp. 2770 2474 99 globlastp 921 BLFS32 Bacillus sp. 2771 2474 98 globlastp 922 BLFS32 Bacillus sp. 2772 2474 97.6 globlastp 923 BLFS32 Bacillus sp. 2773 2474 90.3 globlastp 924 BLFS32 Bacillus sp. 2774 2474 89.3 globlastp 925 BLFS32 Salinibacillus sp. 2775 2474 88.3 globlastp 926 BLFS32 Bacillus sp. 2776 2474 87.6 globlastp 927 BLFS32 Bacillus sp. 2777 2474 81.3 globlastp 928 BLFS32 Bacillus sp. 2778 2474 80.2 globlastp 929 BLFS32 Bacillus sp. 2779 2474 79.5 globlastp 930 BLFS32 Bacillus sp. 2780 2474 76.8 globlastp 931 BLFS32 Bacillus sp. 2781 2474 75.2 globlastp 932 BLFS32 Bacillus sp. 2782 2474 74.7 globlastp 933 BLFS32 Bacillus sp. 2783 2474 73.2 globlastp 934 BLFS32 Cohnella sp. 2784 2474 72.2 globlastp 935 BLFS32 Paenibacillus sp. 2785 2474 71.5 globlastp 936 BLFS32 Bacillus sp. 2786 2474 70.1 globlastp 1798 BLFS138 Bacillus sp. 3622 2573 98.7 globlastp 1799 BLFS138 Bacillus sp. 3623 2573 82.2 globlastp 1800 BLFS138 Bacillus sp. 3624 2573 81 globlastp 1801 BLFS138 Bacillus sp. 3625 2573 80.1 globlastp 1802 BLFS138 Bacillus sp. 3626 2573 79.4 globlastp 1803 BLFS138 Bacillus sp. 3627 2573 78.2 globlastp 1804 BLFS138 Bacillus sp. 3628 2573 77.1 globlastp 1805 BLFS138 Bacillus sp. 3629 2573 76.1 globlastp 1806 BLFS138 Bacillus sp. 3630 2573 74.3 globlastp 1807 BLFS138 Bacillus sp. 3631 2573 73.6 globlastp 1808 BLFS138 Bacillus sp. 3632 2573 72.5 globlastp 1809 BLFS138 Bacillus sp. 3633 2573 71.1 globlastp 1810 BLFS138 Bacillus sp. — 2573 70 glotblastn 2103 ZLFS18 Environmental sample 3917 2615 75.5 globlastp 1999 BLFS158 Bacillus sp. 3813 2593 73.8 globlastp 1857 BLFS142 Bacillus sp. 3680 2577 98.1 globlastp 1858 BLFS142 Bacillus sp. 3681 2577 96.7 globlastp 1859 BLFS142 Bacillus sp. 3682 2577 89 globlastp 1860 BLFS142 Bacillus sp. 3683 2577 88.5 globlastp 1861 BLFS142 Bacillus sp. 3684 2577 85.2 globlastp 1862 BLFS142 Bacillus sp. 3685 2577 84.2 globlastp 1863 BLFS142 Bacillus sp. 3686 2577 83.3 globlastp 1864 BLFS142 Bacillus sp. 3687 2577 82.3 globlastp 1865 BLFS142 Bacillus sp. 3688 2577 80.4 globlastp 1866 BLFS142 Bacillus sp. 3689 2577 79.3 globlastp 1867 BLFS142 Bacillus sp. 3690 2577 77.1 globlastp 1247 BLFS93 Paenibacillus sp. 3082 2529 98.9 globlastp 1248 BLFS93 Paenibacillus sp. 3083 2529 98.3 globlastp 1249 BLFS93 Paenibacillus sp. 3084 2529 97.7 globlastp 1250 BLFS93 Paenibacillus sp. 3085 2529 96 globlastp 1251 BLFS93 Paenibacillus sp. 3086 2529 95.4 globlastp 1252 BLFS93 Paenibacillus sp. 3087 2529 94.9 globlastp 1253 BLFS93 Paenibacillus sp. 3088 2529 93.1 globlastp 1254 BLFS93 Paenibacillus sp. 3089 2529 77 globlastp 1255 BLFS93 Paenibacillus sp. 3090 2529 75.9 globlastp 1256 BLFS93 Paenibacillus sp. 3091 2529 74.1 globlastp 1257 BLFS93 Paenibacillus sp. 3092 2529 72.6 globlastp 2341 ZLFS38 Serratia sp. 4154 2633 98.7 globlastp 2342 ZLFS38 Serratia sp. 4155 2633 96.2 globlastp 2343 ZLFS38 Serratia sp. 4156 2633 95 globlastp 2344 ZLFS38 Serratia sp. 4157 2633 94.3 globlastp 2345 ZLFS38 Serratia sp. 4158 2633 93.1 globlastp 2346 ZLFS38 Serratia sp. 4159 2633 92.5 globlastp 2347 ZLFS38 Pectobacterium sp. 4160 2633 89.3 globlastp 2348 ZLFS38 Hafnia sp. 4161 2633 88.1 globlastp 2349 ZLFS38 Dickeya sp. 4162 2633 87.4 globlastp 2350 ZLFS38 Dickeya sp. 4163 2633 86.2 globlastp 2351 ZLFS38 Enterobacter sp. 4164 2633 85 globlastp 2352 ZLFS38 Achromobacter sp. 4165 2633 84.3 globlastp 2353 ZLFS38 Environmental sample 4166 2633 83 globlastp 2354 ZLFS38 Serratia sp. 4167 2633 82.2 globlastp 2355 ZLFS38 Environmental sample 4168 2633 81.1 globlastp 2356 ZLFS38 Environmental sample 4169 2633 80 globlastp 2357 ZLFS38 Cronobacter sp. 4170 2633 79.5 globlastp 2358 ZLFS38 Enterobacter sp. 4171 2633 78.1 globlastp 2359 ZLFS38 Arsenophonus sp. 4172 2633 77.4 globlastp 2360 ZLFS38 Pantoea sp. 4173 2633 76.1 globlastp 2361 ZLFS38 Vibrio sp. 4174 2633 72 globlastp 2362 ZLFS38 Sodalis sp. 4175 2633 71 globlastp 2363 ZLFS38 Vibrio sp. 4176 2633 70 globlastp 803 BLFS9 Amycolatopsis sp. 2655 2452 92.3 globlastp 804 BLFS9 Amycolatopsis sp. 2656 2452 78.6 globlastp 805 BLFS9 Streptomyces sp. 2657 2452 71.8 globlastp 806 BLFS9 Streptomyces sp. 2658 2452 71.4 globlastp 807 BLFS9 Streptomyces sp. 2659 2452 70.9 globlastp 808 BLFS9 Streptomyces sp. 2660 2452 70.1 globlastp 1090 BLFS74 Paenibacillus sp. 2930 2512 99.4 globlastp 1091 BLFS74 Paenibacillus sp. 2931 2512 99.1 globlastp 1092 BLFS74 Paenibacillus sp. 2932 2512 98.6 globlastp 1093 BLFS74 Paenibacillus sp. 2933 2512 97.4 globlastp 1094 BLFS74 Paenibacillus sp. 2934 2512 97.1 globlastp 1095 BLFS74 Paenibacillus sp. 2935 2512 96 globlastp 1096 BLFS74 Paenibacillus sp. 2936 2512 95.7 globlastp 1097 BLFS74 Paenibacillus sp. 2937 2512 95.4 globlastp 1098 BLFS74 Paenibacillus sp. 2938 2512 92.2 globlastp 1099 BLFS74 Paenibacillus sp. 2939 2512 91.7 globlastp 1100 BLFS74 Paenibacillus sp. 2940 2512 90.5 globlastp 1101 BLFS74 Paenibacillus sp. 2941 2512 89.7 globlastp 1102 BLFS74 Paenibacillus sp. 2942 2512 89.1 globlastp 1103 BLFS74 Paenibacillus sp. 2943 2512 87.1 globlastp 2140 ZLFS23 Curtobacterium sp. 3954 2619 95.8 globlastp 2141 ZLFS23 Curtobacterium sp. 3955 2619 95.4 globlastp 2142 ZLFS23 Curtobacterium sp. 3956 2619 90.5 globlastp 2143 ZLFS23 Curtobacterium sp. 3957 2619 80.5 globlastp 2144 ZLFS23 Environmental sample 3958 2619 76.3 globlastp 2145 ZLFS23 Environmental sample 3959 2619 74.5 globlastp 2146 ZLFS23 Environmental sample 3960 2619 71.8 globlastp 1420 BLFS108 Pantoea sp. 3246 2544 99.1 globlastp 1421 BLFS108 Environmental sample 3247 2544 98.3 globlastp 1422 BLFS108 Klebsiella sp. 3248 2544 76.9 globlastp 1423 BLFS108 Citrobacter sp. 3249 2544 76.1 globlastp 1424 BLFS108 Meta sp. 3250 2544 75.2 globlastp 1425 BLFS108 Environmental sample 3251 2544 74.4 globlastp 1426 BLFS108 Salmonella sp. 3252 2544 73.5 globlastp 1427 BLFS108 Escherichia sp. 3253 2544 72 globlastp 1428 BLFS108 Kluyvera sp. 3254 2544 71.8 globlastp 2311 ZLFS36 Serratia sp. 4125 2631 99.2 globlastp 2170 ZLFS27 Pseudomonas sp. 3984 2622 93 globlastp 2171 ZLFS27 Pseudomonas sp. 3985 2622 92 globlastp 2172 ZLFS27 Pseudomonas sp. 3986 2622 91 globlastp 2173 ZLFS27 Pseudomonas sp. 3987 2622 90.8 globlastp 2174 ZLFS27 Pseudomonas sp. 3988 2622 89 globlastp 2175 ZLFS27 Pseudomonas sp. 3989 2622 88 globlastp 2176 ZLFS27 Pseudomonas sp. 3990 2622 87.1 globlastp 2177 ZLFS27 Pseudomonas sp. 3991 2622 86.1 globlastp 2178 ZLFS27 Pseudomonas sp. 3992 2622 85.1 globlastp 2179 ZLFS27 Pseudomonas sp. 3993 2622 84.1 globlastp 2180 ZLFS27 Pseudomonas sp. 3994 2622 83.1 globlastp 2181 ZLFS27 Pseudomonas sp. 3995 2622 82.4 globlastp 2182 ZLFS27 Pseudomonas sp. 3996 2622 81.9 globlastp 2183 ZLFS27 Pseudomonas sp. 3997 2622 79.1 globlastp 2184 ZLFS27 Pseudomonas sp. 3998 2622 78 globlastp 2185 ZLFS27 Pseudomonas sp. 3999 2622 77.1 globlastp 2186 ZLFS27 Pseudomonas sp. 4000 2622 75.9 globlastp 2187 ZLFS27 Pseudomonas sp. 4001 2622 75.3 globlastp 2188 ZLFS27 Pseudomonas sp. 4002 2622 74.2 globlastp 2189 ZLFS27 Pseudomonas sp. 4003 2622 73.8 globlastp 2190 ZLFS27 Environmental sample 4004 2622 72 globlastp 2191 ZLFS27 Pseudomonas sp. 4005 2622 71.5 globlastp 2192 ZLFS27 Pseudomonas sp. 4006 2622 70.3 globlastp 2193 ZLFS27 Pseudomonas sp. 4007 2622 70.1 globlastp 1824 BLFS140 Salinibacillus sp. 3647 2575 99.4 globlastp 1825 BLFS140 Bacillus sp. 3648 2575 98.4 globlastp 1826 BLFS140 Bacillus sp. 3649 2575 95.6 globlastp 1827 BLFS140 Bacillus sp. 3650 2575 88 globlastp 1828 BLFS140 Bacillus sp. 3651 2575 87.1 globlastp 1829 BLFS140 Bacillus sp. 3652 2575 86.1 globlastp 1830 BLFS140 Bacillus sp. 3653 2575 85.2 globlastp 1831 BLFS140 Bacillus sp. 3654 2575 85 globlastp 1832 BLFS140 Bacillus sp. 3655 2575 84.9 globlastp 1833 BLFS140 Bacillus sp. 3656 2575 84.7 globlastp 1834 BLFS140 Bacillus sp. 3657 2575 81.6 globlastp 1835 BLFS140 Bacillus sp. 3658 2575 81.5 globlastp 1836 BLFS140 Bacillus sp. 3659 2575 74 globlastp 1837 BLFS140 Bacillus sp. 3660 2575 73.8 globlastp 2102 ZLFS48 Environmental sample 3916 2639 79.4 globlastp 982 BLFS42 Burkholderia sp. 2830 2483 97.6 globlastp 983 BLFS42 Burkholderia sp. 2831 2483 97.2 globlastp 1927 BLFS147 Salinibacillus sp. 3750 2582 98.9 globlastp 1928 BLFS147 Bacillus sp. 3751 2582 97.7 globlastp 1929 BLFS147 Bacillus sp. 3752 2582 95.7 globlastp 1930 BLFS147 Bacillus sp. 3753 2582 88 globlastp 1931 BLFS147 Bacillus sp. 3754 2582 87.3 globlastp 1932 BLFS147 Bacillus sp. 3755 2582 86 globlastp 1933 BLFS147 Bacillus sp. 3756 2582 85 globlastp 1934 BLFS147 Bacillus sp. 3757 2582 84.8 globlastp 1935 BLFS147 Bacillus sp. 3758 2582 74.1 globlastp 1936 BLFS147 Bacillus sp. 3759 2582 73.9 globlastp 1170 BLFS85 Paenibacillus sp. 3007 2521 99.4 globlastp 1171 BLFS85 Paenibacillus sp. 3008 2521 98.3 globlastp 1172 BLFS85 Paenibacillus sp. 3009 2521 97.6 globlastp 1173 BLFS85 Paenibacillus sp. 3010 2521 97 globlastp 1174 BLFS85 Paenibacillus sp. 3011 2521 96.6 globlastp 1175 BLFS85 Paenibacillus sp. 3012 2521 95.3 globlastp 1176 BLFS85 Paenibacillus sp. 3013 2521 93.2 globlastp 1177 BLFS85 Paenibacillus sp. 3014 2521 92.7 globlastp 1178 BLFS85 Paenibacillus sp. 3015 2521 91 globlastp 1179 BLFS85 Paenibacillus sp. 3016 2521 90.4 globlastp 1180 BLFS85 Paenibacillus sp. 3017 2521 89.1 globlastp 1552 BLFS118 Pseudomonas sp. 3377 2553 98.8 globlastp 1553 BLFS118 Pseudomonas sp. 3378 2553 98.5 globlastp 1554 BLFS118 Pseudomonas sp. 3379 2553 98.2 globlastp 1555 BLFS118 Pseudomonas sp. 3380 2553 97.7 globlastp 1556 BLFS118 Pseudomonas sp. 3381 2553 97.1 globlastp 1557 BLFS118 Pseudomonas sp. 3382 2553 96.8 globlastp 1558 BLFS118 Pseudomonas sp. 3383 2553 96.2 globlastp 1559 BLFS118 Pseudomonas sp. 3384 2553 88 globlastp 1560 BLFS118 Pseudomonas sp. 3385 2553 85.9 globlastp 1561 BLFS118 Pseudomonas sp. 3386 2553 78 globlastp 1562 BLFS118 Pseudomonas sp. 3387 2553 77.1 globlastp 1563 BLFS118 Pseudomonas sp. 3388 2553 76 globlastp 1564 BLFS118 Environmental sample 3389 2553 75.1 globlastp 1565 BLFS118 Pseudomonas sp. 3390 2553 74 globlastp 1566 BLFS118 Pseudomonas sp. 3391 2553 73 globlastp 1567 BLFS118 Pseudomonas sp. 3392 2553 72.1 globlastp 1568 BLFS118 Environmental sample 3393 2553 71 globlastp 1569 BLFS118 Pseudomonas sp. 3394 2553 70.1 globlastp 1357 BLFS104 Pantoea sp. 3183 2540 99.2 globlastp 1358 BLFS104 Pantoea sp. 3184 2540 98.3 globlastp 1359 BLFS104 Environmental sample 3185 2540 95 globlastp 1360 BLFS104 Pantoea sp. 3186 2540 94.1 globlastp 1361 BLFS104 Pantoea sp. 3187 2540 93.3 globlastp 1362 BLFS104 Pantoea sp. 3188 2540 90.8 globlastp 1363 BLFS104 Environmental sample 3189 2540 89.1 globlastp 1364 BLFS104 Pantoea sp. 3190 2540 84.9 globlastp 1365 BLFS104 Pantoea sp. 3191 2540 78.2 globlastp 1366 BLFS104 Environmental sample 3192 2540 77.3 globlastp 1367 BLFS104 Pantoea sp. 3193 2540 73.9 globlastp 1381 BLFS106 Environmental sample 3207 2542 99 globlastp 1382 BLFS106 Pantoea sp. 3208 2542 98 globlastp 1383 BLFS106 Pantoea sp. 3209 2542 97 globlastp 1384 BLFS106 Pantoea sp. 3210 2542 94.4 globlastp 1385 BLFS106 Environmental sample 3211 2542 92.1 globlastp 1386 BLFS106 Pantoea sp. 3212 2542 88.1 globlastp 1387 BLFS106 Pantoea sp. 3213 2542 86.9 globlastp 1388 BLFS106 Pantoea sp. 3214 2542 83.2 globlastp 1389 BLFS106 Pantoea sp. 3215 2542 82.2 globlastp 1390 BLFS106 Pantoea sp. 3216 2542 80.2 globlastp 1391 BLFS106 Environmental sample 3217 2542 79.2 globlastp 1392 BLFS106 Pantoea sp. 3218 2542 78.2 globlastp 1393 BLFS106 Plautia sp. 3219 2542 78.1 globlastp 1394 BLFS106 Pantoea sp. 3220 2542 77.2 globlastp 1395 BLFS106 Cedecea sp. 3221 2542 76.2 globlastp 1396 BLFS106 Enterobacter sp. 3222 2542 75.2 globlastp 1397 BLFS106 Environmental sample 3223 2542 74.3 globlastp 1398 BLFS106 Environmental sample 3224 2542 73.3 globlastp 1399 BLFS106 Environmental sample 3225 2542 72.3 globlastp 1400 BLFS106 Citrobacter sp. 3226 2542 71.3 globlastp 1401 BLFS106 Enterobacter sp. 3227 2542 70.3 globlastp 1238 BLFS92 Paenibacillus sp. 3073 2528 99.2 globlastp 1239 BLFS92 Paenibacillus sp. 3074 2528 97.7 globlastp 1240 BLFS92 Paenibacillus sp. 3075 2528 97.2 globlastp 1241 BLFS92 Paenibacillus sp. 3076 2528 96.6 globlastp 1242 BLFS92 Paenibacillus sp. 3077 2528 96.1 globlastp 1243 BLFS92 Paenibacillus sp. 3078 2528 94.3 globlastp 1244 BLFS92 Paenibacillus sp. 3079 2528 94 globlastp 1245 BLFS92 Paenibacillus sp. 3080 2528 93.5 globlastp 1246 BLFS92 Paenibacillus sp. 3081 2528 92 globlastp 1868 BLFS143 Salinibacillus sp. 3691 2578 99.4 globlastp 1869 BLFS143 Bacillus sp. 3692 2578 98.2 globlastp 1870 BLFS143 Bacillus sp. 3693 2578 96.1 globlastp 1871 BLFS143 Bacillus sp. 3694 2578 95.2 globlastp 1872 BLFS143 Bacillus sp. 3695 2578 94.6 globlastp 1873 BLFS143 Bacillus sp. 3696 2578 87.5 globlastp 1874 BLFS143 Bacillus sp. 3697 2578 86.9 globlastp 1875 BLFS143 Bacillus sp. 3698 2578 79.1 globlastp 1876 BLFS143 Bacillus sp. 3699 2578 78.5 globlastp 1877 BLFS143 Bacillus sp. 3700 2578 74.1 globlastp 1878 BLFS143 Bacillus sp. 3701 2578 72.9 globlastp 1879 BLFS143 Bacillus sp. 3702 2578 71.3 globlastp 1880 BLFS143 Bacillus sp. 3703 2578 70.4 globlastp 1881 BLFS143 Bacillus sp. 3704 2578 70.1 globlastp 800 BLFS8 Amycolatopsis sp. 2652 2451 98.3 globlastp 801 BLFS8 Amycolatopsis sp. 2653 2451 95.7 globlastp 802 BLFS8 Amycolatopsis sp. 2654 2451 77.1 globlastp 961 BLFS36 Bradyrhizobium sp. 2811 2478 82.9 globlastp 962 BLFS36 Bradyrhizobium sp. 2812 2478 78.3 globlastp 963 BLFS36 Bradyrhizobium sp. — 2478 76.83 glotblastn 964 BLFS36 Bradyrhizobium sp. 2813 2478 76.8 globlastp 965 BLFS36 Bradyrhizobium sp. 2814 2478 75.6 globlastp 966 BLFS36 Bradyrhizobium sp. 2815 2478 74.4 globlastp 1402 BLFS107 Environmental sample 3228 2543 97.9 globlastp 1403 BLFS107 Pantoea sp. 3229 2543 97.4 globlastp 1404 BLFS107 Pantoea sp. 3230 2543 96.8 globlastp 1405 BLFS107 Environmental sample 3231 2543 95.8 globlastp 1406 BLFS107 Pantoea sp. 3232 2543 94.2 globlastp 1407 BLFS107 Pantoea sp. 3233 2543 88.4 globlastp 1408 BLFS107 Environmental sample 3234 2543 86.3 globlastp 1409 BLFS107 Environmental sample 3235 2543 85.8 globlastp 1410 BLFS107 Environmental sample 3236 2543 85.7 globlastp 1411 BLFS107 Environmental sample 3237 2543 85.3 globlastp 1412 BLFS107 Pantoea sp. 3238 2543 84.8 globlastp 1413 BLFS107 Pantoea sp. 3239 2543 84.7 globlastp 1414 BLFS107 Pantoea sp. 3240 2543 78.3 globlastp 1415 BLFS107 Erwinia sp. 3241 2543 74.6 globlastp 1416 BLFS107 Erwinia sp. 3242 2543 73.5 globlastp 1417 BLFS107 Erwinia sp. 3243 2543 72.3 globlastp 1418 BLFS107 Pantoea sp. 3244 2543 72 globlastp 1419 BLFS107 Environmental sample 3245 2543 70.4 globlastp 866 BLFS20 Bacillus sp. 2718 2463 99.1 globlastp 867 BLFS20 Bacillus sp. 2719 2463 98.2 globlastp 868 BLFS20 Bacillus sp. 2720 2463 97.2 globlastp 869 BLFS20 Bacillus sp. 2721 2463 96.3 globlastp 2012 PUB65 Variovorax sp. 3826 2598 98.9 globlastp 2013 PUB65 Variovorax sp. 3827 2598 94.8 globlastp 2014 PUB65 Variovorax sp. 3828 2598 94.5 globlastp 2015 PUB65 Variovorax sp. 3829 2598 91.4 globlastp 2016 PUB65 Variovorax sp. 3830 2598 91.3 globlastp 2017 PUB65 Variovorax sp. 3831 2598 90.6 globlastp 2018 PUB65 Variovorax sp. 3832 2598 90.5 globlastp 2019 PUB65 Variovorax sp. 3833 2598 90.3 globlastp 2020 PUB65 Environmental sample 3834 2598 90.1 globlastp 2021 PUB65 Environmental sample 3835 2598 87.1 globlastp 2022 PUB65 Variovorax sp. 3836 2598 84.5 globlastp 2023 PUB65 Variovorax sp. 3837 2598 84.3 globlastp 2024 PUB65 Environmental sample 3838 2598 78.4 globlastp 2025 PUB65 Environmental sample 3839 2598 77.9 globlastp 2026 PUB65 Variovorax sp. 3840 2598 77.4 globlastp 2027 PUB65 Acidovorax sp. 3841 2598 75.5 globlastp 2028 PUB65 Environmental sample 3842 2598 75.4 globlastp 2029 PUB65 Acidovorax sp. 3843 2598 75.2 globlastp 2030 PUB65 Environmental sample 3844 2598 74.5 globlastp 2031 PUB65 Environmental sample 3845 2598 72.3 globlastp 790 BLFS1 Streptomyces sp. 2642 2444 72.8 globlastp 1976 BLFS150 Pseudomonas sp. — 2585 95.9 glotblastn 1977 BLFS150 Xanthomonas sp. — 2585 77.05 glotblastn 1978 BLFS150 Xanthomonas sp. — 2585 75.41 glotblastn 1979 BLFS150 Xanthomonas sp. — 2585 72.95 glotblastn 1980 BLFS150 Xanthomonas sp. — 2585 71.31 glotblastn 2081 ZLFS8 Collimonas sp. 3895 2605 78.5 globlastp 2082 ZLFS8 Collimonas sp. 3896 2605 78.3 globlastp 952 BLFS35 Bradyrhizohium sp. 2802 2477 96.3 globlastp 953 BLFS35 Mesorhizohium sp. 2803 2477 89.8 globlastp 954 BLFS35 Bradyrhizohium sp. 2804 2477 87 globlastp 955 BLFS35 Bradyrhizohium sp. 2805 2477 86 globlastp 956 BLFS35 Bradyrhizohium sp. 2806 2477 76.8 globlastp 957 BLFS35 Bradyrhizohium sp. 2807 2477 75.5 globlastp 958 BLFS35 Bradyrhizohium sp. 2808 2477 75 globlastp 959 BLFS35 Bradyrhizohium sp. 2809 2477 71.2 globlastp 960 BLFS35 Bradyrhizohium sp. 2810 2477 70.2 globlastp 1429 BLFS109 Pantoea sp. 3255 2545 89.2 globlastp 1430 BLFS109 Pantoea sp. 3256 2545 76.2 globlastp 1320 BLFS101 Environmental sample 3155 2537 95.2 globlastp 1321 BLFS101 Pantoea sp. 3156 2537 94 globlastp 1322 BLFS101 Pantoea sp. 3157 2537 87.3 globlastp 1323 BLFS101 Environmental sample 3158 2537 86.1 globlastp 1324 BLFS101 Environmental sample 3159 2537 85.3 globlastp 1325 BLFS101 Pantoea sp. 3160 2537 84.1 globlastp 1326 BLFS101 Erwinia sp. 3161 2537 79.3 globlastp 1327 BLFS101 Serratia sp. 3162 2537 78.1 globlastp 1328 BLFS101 Erwinia sp. 3163 2537 77.3 globlastp 1329 BLFS101 Environmental sample 3164 2537 76.1 globlastp 1330 BLFS101 Klebsiella sp. 3165 2537 75 globlastp 1331 BLFS101 Yersinia sp. 3166 2537 74.1 globlastp 1332 BLFS101 Environmental sample 3167 2537 73 globlastp 1333 BLFS101 Rahnella sp. 3168 2537 72.1 globlastp 1334 BLFS101 Enterobacter sp. 3169 2537 71 globlastp 1335 BLFS101 Xenorhabdus sp. 3170 2537 70.1 globlastp 1607 BLFS123 Pseudomonas sp. 3431 2558 96.2 globlastp 1608 BLFS123 Pseudomonas sp. 3432 2558 95.9 globlastp 1609 BLFS123 Pseudomonas sp. 3433 2558 79.8 globlastp 1610 BLFS123 Pseudomonas sp. 3434 2558 79.5 globlastp 974 BLFS41 Burkholderia sp. 2822 2482 99.2 globlastp 975 BLFS41 Burkholderia sp. 2823 2482 98.9 globlastp 976 BLFS41 Burkholderia sp. 2824 2482 76.3 globlastp 977 BLFS41 Environmental sample 2825 2482 75.1 globlastp 978 BLFS41 Burkholderia sp. 2826 2482 74 globlastp 979 BLFS41 Burkholderia sp. 2827 2482 73.1 globlastp 980 BLFS41 Burkholderia sp. 2828 2482 72.9 globlastp 981 BLFS41 Burkholderia sp. 2829 2482 71.3 globlastp 1064 BLFS67 Janthinobacterium sp. 2906 2506 98.6 globlastp 1065 BLFS67 Janthinobacterium sp. 2907 2506 93.8 globlastp 1066 BLFS67 Janthinobacterium sp. 2908 2506 92.5 globlastp 1276 BLFS98 Pantoea sp. 3111 2534 96.3 globlastp 1277 BLFS98 Environmental sample 3112 2534 95.3 globlastp 1278 BLFS98 Pantoea sp. 3113 2534 93 globlastp 1279 BLFS98 Environmental sample 3114 2534 91.4 globlastp 1280 BLFS98 Environmental sample 3115 2534 89.8 globlastp 1281 BLFS98 Pantoea sp. 3116 2534 83.3 globlastp 1282 BLFS98 Environmental sample 3117 2534 82.9 globlastp 1283 BLFS98 Pantoea sp. 3118 2534 81.5 globlastp 1284 BLFS98 Environmental sample 3119 2534 79.4 globlastp 1285 BLFS98 Pantoea sp. 3120 2534 77.2 globlastp 1286 BLFS98 Pantoea sp. 3121 2534 76.1 globlastp 1287 BLFS98 Environmental sample 3122 2534 75.3 globlastp 1288 BLFS98 Pantoea sp. 3123 2534 74 globlastp 1289 BLFS98 Environmental sample 3124 2534 72.6 globlastp 1290 BLFS98 Erwinia sp. 3125 2534 71.6 globlastp 1291 BLFS98 Environmental sample 3126 2534 70.2 globlastp 898 BLFS26 Bacillus sp. 2748 2469 99 globlastp 899 BLFS26 Bacillus sp. 2749 2469 98.4 globlastp 900 BLFS26 Bacillus sp. 2750 2469 97.9 globlastp 901 BLFS26 Bacillus sp. 2751 2469 96.3 globlastp 902 BLFS26 Bacillus sp. 2752 2469 72.3 globlastp 1838 BLFS141 Salinibacillus sp. 3661 2576 98.1 globlastp 1839 BLFS141 Bacillus sp. 3662 2576 97.5 globlastp 1840 BLFS141 Bacillus sp. 3663 2576 95.9 globlastp 1841 BLFS141 Bacillus sp. 3664 2576 90.1 globlastp 1842 BLFS141 Bacillus sp. 3665 2576 89 globlastp 1843 BLFS141 Bacillus sp. 3666 2576 88.1 globlastp 1844 BLFS141 Bacillus sp. 3667 2576 87.2 globlastp 1845 BLFS141 Bacillus sp. 3668 2576 86.6 globlastp 1846 BLFS141 Bacillus sp. 3669 2576 86.4 globlastp 1847 BLFS141 Bacillus sp. 3670 2576 83.7 globlastp 1848 BLFS141 Bacillus sp. 3671 2576 82.6 globlastp 1849 BLFS141 Bacillus sp. 3672 2576 77.1 globlastp 1850 BLFS141 Bacillus sp. 3673 2576 76 globlastp 1851 BLFS141 Bacillus sp. 3674 2576 75.6 globlastp 1852 BLFS141 Bacillus sp. 3675 2576 74 globlastp 1853 BLFS141 Bacillus sp. 3676 2576 73.2 globlastp 1854 BLFS141 Bacillus sp. 3677 2576 72.4 globlastp 1855 BLFS141 Bacillus sp. 3678 2576 71.5 globlastp 1856 BLFS141 Paenibacillus sp. 3679 2576 70.4 globlastp 1198 BLFS88 Paenibacillus sp. 3035 2524 99.2 globlastp 1199 BLFS88 Paenibacillus sp. 3036 2524 96.9 globlastp 1200 BLFS88 Paenibacillus sp. 3037 2524 95.3 globlastp 1201 BLFS88 Paenibacillus sp. 3038 2524 95 globlastp 1202 BLFS88 Paenibacillus sp. 3039 2524 94.4 globlastp 1203 BLFS88 Paenibacillus sp. 3040 2524 93.6 globlastp 1204 BLFS88 Paenibacillus sp. 3041 2524 92.5 globlastp 1205 BLFS88 Paenibacillus sp. 3042 2524 88.3 globlastp 1206 BLFS88 Paenibacillus sp. 3043 2524 72.9 globlastp 1010 BLFS53 Dickeya sp. 2856 2494 99 globlastp 878 BLFS22 Bacillus sp. 2728 2465 99.3 globlastp 879 BLFS22 Bacillus sp. 2729 2465 97.8 globlastp 880 BLFS22 Bacillus sp. 2730 2465 97.1 globlastp 881 BLFS22 Bacillus sp. 2731 2465 96.4 globlastp 882 BLFS22 Bacillus sp. 2732 2465 95.6 globlastp 883 BLFS22 Bacillus sp. 2733 2465 94.9 globlastp 819 BLFS12 Arthrobacter sp. 2671 2455 98.9 globlastp 820 BLFS12 Arthrobacter sp. 2672 2455 92.3 globlastp 1960 BLFS149 Serratia sp. 3782 2584 98.8 globlastp 1961 BLFS149 Serratia sp. 3783 2584 97 globlastp 1962 BLFS149 Serratia sp. 3784 2584 93.8 globlastp 1963 BLFS149 Serratia sp. 3785 2584 91.7 globlastp 1964 BLFS149 Serratia sp. 3786 2584 84 globlastp 1965 BLFS149 Serratia sp. 3787 2584 83.8 globlastp 1966 BLFS149 Serratia sp. 3788 2584 82 globlastp 1967 BLFS149 Serratia sp. 3789 2584 81.3 globlastp 1968 BLFS149 Serratia sp. 3790 2584 78 globlastp 1969 BLFS149 Serratia sp. 3791 2584 77.8 globlastp 1970 BLFS149 Yersinia sp. 3792 2584 76 globlastp 1971 BLFS149 Yersinia sp. 3793 2584 75 globlastp 1972 BLFS149 Yersinia sp. 3794 2584 74 globlastp 1973 BLFS149 Yersinia sp. 3795 2584 73.1 globlastp 1974 BLFS149 Edwardsiella sp. 3796 2584 72 globlastp 1975 BLFS149 Environmental sample 3797 2584 71.2 globlastp 1984 BLFS152 Streptomyces sp. 3801 2587 82.9 globlastp 1985 BLFS152 Streptomyces sp. 3802 2587 78.9 globlastp 1995 BLFS154 Streptomyces sp. 3809 2589 98.2 globlastp 1996 BLFS154 Streptomyces sp. 3810 2589 72.2 globlastp 1432 BLFS111 Chromobacterium sp. 3258 2547 97 globlastp 1433 BLFS111 Chromobacterium sp. 3259 2547 89.9 globlastp 1434 BLFS111 Chromobacterium sp. 3260 2547 84.4 globlastp 1435 BLFS111 Chromobacterium sp. 3261 2547 83.4 globlastp 1436 BLFS111 Paludibacterium sp. 3262 2547 71.4 globlastp 791 BLFS2 Alkanindiges sp. 2643 2445 91.7 globlastp 1644 BLFS126 Pseudomonas sp. 3468 2561 98.3 globlastp 1645 BLFS126 Pseudomonas sp. 3469 2561 96.7 globlastp 1646 BLFS126 Pseudomonas sp. 3470 2561 92.6 globlastp 1647 BLFS126 Pseudomonas sp. 3471 2561 91.7 globlastp 1648 BLFS126 Pseudomonas sp. 3472 2561 90.9 globlastp 1649 BLFS126 Pseudomonas sp. 3473 2561 80.3 globlastp 1650 BLFS126 Pseudomonas sp. 3474 2561 75.6 globlastp 1651 BLFS126 Pseudomonas sp. 3475 2561 73.2 globlastp 2286 ZLFS34 Serratia sp. 4100 2629 99 globlastp 2287 ZLFS34 Serratia sp. 4101 2629 98.4 globlastp 2288 ZLFS34 Multievos46 sp. 4102 2629 97 globlastp 2289 ZLFS34 Serratia sp. 4103 2629 96 globlastp 2290 ZLFS34 Serratia sp. 4104 2629 95.9 globlastp 2291 ZLFS34 Serratia sp. 4105 2629 93 globlastp 2292 ZLFS34 Serratia sp. 4106 2629 92.1 globlastp 2293 ZLFS34 Serratia sp. 4107 2629 91.4 globlastp 2294 ZLFS34 Serratia sp. 4108 2629 88.1 globlastp 2295 ZLFS34 Serratia sp. 4109 2629 87 globlastp 2296 ZLFS34 Serratia sp. 4110 2629 84.3 globlastp 2297 ZLFS34 Xenorhabdus sp. 4111 2629 83.5 globlastp 2298 ZLFS34 Xenorhabdus sp. 4112 2629 82 globlastp 2299 ZLFS34 Xenorhabdus sp. 4113 2629 80.6 globlastp 2300 ZLFS34 Yersinia sp. 4114 2629 78.8 globlastp 2301 ZLFS34 Yersinia sp. 4115 2629 75 globlastp 2302 ZLFS34 Environmental sample 4116 2629 74.5 globlastp 2303 ZLFS34 Rahnella sp. 4117 2629 73.1 globlastp 2304 ZLFS34 Ewingella sp. 4118 2629 71.5 globlastp 2305 ZLFS34 Rhodanobacter sp. 4119 2629 70.1 globlastp 1907 BLFS146 Bacillus sp. 3730 2581 98.8 globlastp 1908 BLFS146 Bacillus sp. 3731 2581 97.2 globlastp 1909 BLFS146 Bacillus sp. 3732 2581 96 globlastp 1910 BLFS146 Bacillus sp. 3733 2581 95.4 globlastp 1911 BLFS146 Bacillus sp. 3734 2581 94 globlastp 1912 BLFS146 Bacillus sp. 3735 2581 93.4 globlastp 1913 BLFS146 Bacillus sp. 3736 2581 92.9 globlastp 1914 BLFS146 Bacillus sp. 3737 2581 91.7 globlastp 1915 BLFS146 Bacillus sp. 3738 2581 89.1 globlastp 1916 BLFS146 Bacillus sp. 3739 2581 85 globlastp 1917 BLFS146 Bacillus sp. 3740 2581 84.7 globlastp 1918 BLFS146 Bacillus sp. 3741 2581 84 globlastp 1919 BLFS146 Bacillus sp. 3742 2581 81.2 globlastp 1920 BLFS146 Bacillus sp. 3743 2581 78.2 globlastp 1921 BLFS146 Bacillus sp. 3744 2581 77.9 globlastp 1922 BLFS146 Bacillus sp. 3745 2581 76.4 globlastp 1923 BLFS146 Bacillus sp. 3746 2581 74 globlastp 1924 BLFS146 Bacillus sp. 3747 2581 73.1 globlastp 1925 BLFS146 Bacillus sp. 3748 2581 71.1 globlastp 1926 BLFS146 Bacillus sp. 3749 2581 70.8 globlastp 1749 BLFS133 Pseudomonas sp. 3573 2568 93.2 globlastp 1750 BLFS133 Pseudomonas sp. 3574 2568 91.8 globlastp 1751 BLFS133 Pseudomonas sp. 3575 2568 79.5 globlastp 1752 BLFS133 Pseudomonas sp. 3576 2568 78.1 globlastp 893 BLFS25 Bacillus sp. 2743 2468 99.2 globlastp 894 BLFS25 Bacillus sp. 2744 2468 98.8 globlastp 895 BLFS25 Bacillus sp. 2745 2468 98.4 globlastp 896 BLFS25 Bacillus sp. 2746 2468 98 globlastp 897 BLFS25 Bacillus sp. 2747 2468 70.8 globlastp 2070 ZLFS5 Burkholderia sp. 3884 2602 98.4 globlastp 2071 ZLFS5 Burkholderia sp. 3885 2602 97.7 globlastp 2072 ZLFS5 Burkholderia sp. 3886 2602 96.1 globlastp 2073 ZLFS5 Burkholderia sp. 3887 2602 88.3 globlastp 2074 ZLFS5 Burkholderia sp. 3888 2602 75 globlastp 1981 BLFS151 Stenotrophomonas sp. 3798 2586 78.1 globlastp 1982 BLFS151 Stenotrophomonas sp. 3799 2586 75.7 globlastp 1983 BLFS151 Stenotrophomonas sp. 3800 2586 73.5 globlastp 2000 BLFS159 Xanthomonas sp. 3814 2594 79.9 globlastp 2001 BLFS159 Xanthomonas sp. 3815 2594 76.8 globlastp 2089 ZLFS13 Collimonas sp. 3903 2610 96.7 globlastp 2090 ZLFS13 Collimonas sp. 3904 2610 80.3 globlastp 2032 ZLFS1 Actinomyces sp. 3846 2599 73.5 globlastp 1005 BLFS50 Collimonas sp. 2853 2491 85.1 globlastp 2091 ZLFS14 Collimonas sp. 3905 2611 75 globlastp 2083 ZLFS9 Collimonas sp. 3897 2606 98.6 globlastp “Polyn.” = polynucleotide; “Polyp.” = polypeptide; “Algor.” = algorithm (used for sequence alignment and determination of percent homology); “Hom.”—homology; “iden.”—identity; “glob.”—global. Environmental sample = unidentified organisms obtained from the sample.

The output of the functional genomics approach described herein is a set of genes highly predicted to improve plant resistance to fungal infection.

Example 13: Identification of Domains Comprised within Polypeptides Encoded by the Identified Genes

Polypeptide domains were identified as described in Example 4 hereinabove. The polypeptides of some embodiments of the invention, the expression of which in a may result in fungal control by invoking antifungal effects, can be characterized by specific amino acid domains. According to certain embodiments of the invention, particular domains are conserved within a family of polypeptides as described in Table 17 hereinbelow. Without wishing to be bound by specific theory or mechanism of action, the conserved domain may indicate common function of the polypeptides comprising same. The domains are presented by an arbitrary identifier (*ID). Table 18 provides the details of each domain according to the InterPro Entry.

Table 17 summarizes the domains in each of the “core” polypeptides (e.g., the polypeptides from Table 15) identified by the present inventors as being capable of conferring resistant to fungi and/or Oomycetes infection when its expression is modified in a plant (e.g. enhanced or down-regulated), wherein each of the listed domains is conserved in the representative homologous polypeptides identified by the present inventors exhibiting at least 70% global identity to the “core” polypeptides (as detailed in Table 16 in Example 12 above). As explained above, each domain received an arbitrary ID number wherein description of these arbitrary domain IDs according to the InterPro database is provided in Table 18 below. In addition, the start and end position of each of the domains with respect to the amino acid sequence of the “core” polypeptide was identified, as well as the E-values for each of the conserved domains as indicated by the domain tool used for analyzing these sequences, as part of interproscan programs, e.g., SMART, prosite scans patterns and profiles (not shown). For example, in the case of the Prosite search, the Prosite profiles report normalized scores instead of E-values, which are defined as the base 10 logarithm of the size (in residues) of the database in which one false positive match is expected to occur by chance. The normalized score is independent of the size of the databases searched. The so-called bit scores reported by other database-search programs have a distinct meaning but are also independent of the size of the database searched

For example, for SEQ ID NO:9060, the domain ID “1” appears at amino acid positions 37 through 124. For some domains the e-value was not specified. In these cases, the presence of the domain was verified by ScanRegExp, which is able to verify PROSITE matches using corresponding statistically-significant CONFIRM patterns. The CONFIRM patterns were generated based on the true positive SWISS-PROT PROSITE matches using eMOTIF software with a stringency of 10e-9 P-value. Further details can be found in hypertext transfer protocol://computing.bio.cam.ac.uk/local/doc/iprscan.html.

TABLE 17 Core and homologous polypeptides comprising the same domains Core Polypeptide Gene Homologous Polypeptides (SEQ ID Name Characteristic Domains Comprising the Domains NO) (Core) (Domain identifier) (SEQ ID NOs) 2444 BLFS1 1; 2; 3 in core and homologs 2642 2445 BLFS2 4; 5; 6 in core and homologs 2643 2446 BLFS3 7; 8; 9; 10; 11; 12 in core 2447 BLFS4 6 in core 2448 BLFS5 13; 14 in core and homologs 2644; 2645; 2646; 2647; 2648; 2649; 2650; 2651 2449 BLFS6 2; 3 in core 2450 BLFS7 15; 16 in core 2451 BLFS8 1; 2; 3 in core and homologs 2652; 2653; 2654 2452 BLFS9 2; 1; 3 in core and homologs 2655; 2656; 2657; 2658; 2659; 2660 2453 BLFS10 1; 2; 3 in core and homologs 2452; 2655; 2656; 2661; 2662; 2663 2454 BLFS11 no domains in core 2455 BLFS12 no domains in core 2456 BLFS13 no domains in core 2457 BLFS14 17 in core and homologs 2675; 2676 2458 BLFS15 18; 19; 20 in core and homologs 2677; 2678; 2679; 2680; 2681; 2682; 2683; 2684; 2685 2459 BLFS16 21; 22; 23; 24 in core and homologs 2686; 2687; 2688; 2689; 2690 2460 BLFS17 25; 26 in core and homologs 2691; 2692; 2693; 2694; 2695; 2696 2461 BLFS18 27; 28; 29 in core and homologs 2697; 2698; 2699; 2700; 2701; 2702; 2703; 2704; 2705; 2706; 2707; 2708; 2709; 2710 2462 BLFS19 8; 30 in core and homologs 2711; 2712; 2713; 2714; 2715; 2716; 2717 2463 BLFS20 31; 32; 33 in core and homologs 2718; 2719; 2720; 2721 2464 BLFS21 34; 35; 36; 37; 38 in core and 2722; 2723; 2724; 2725; 2726; 2727 homologs 2465 BLFS22 no domains in core 2466 BLFS23 39; 31; 40; 41; 42; 32; 24 in core and 2734; 2735; 2736; 2737; 2738; 2739; homologs 2740; 2741; 2742 2467 BLFS24 no domains in core 2468 BLFS25 43; 44 in core and homologs 2743; 2744; 2745; 2746; 2747 2469 BLFS26 31; 45; 32 in core and homologs 2748; 2749; 2750; 2751; 2752 2470 BLFS27 46; 47; 48 in core and homologs 2753; 2754; 2755; 2756; 2757 2471 BLFS29 8; 49; 30 in core and homologs 2758 2472 BLFS30 6 in core 2473 BLFS31 50 in core and homologs 2759; 2760; 2761; 2762; 2763; 2764; 2765; 2766; 2767; 2768; 2769 2474 BLFS32 52 in core and homologs 2770; 2771; 2772; 2773; 2774; 2775; 2776; 2777; 2778; 2779; 2780; 2781; 2782; 2783; 2784; 2785; 2786 2475 BLFS33 53; 35; 54 in core and homologs 2787; 2788; 2789; 2790; 2791; 2792; 2793; 2794; 2795; 2796; 2797; 2798 2476 BLFS34 31; 32; 33 in core and homologs 2799; 2800; 2801 2477 BLFS35 55 in core and homologs 2802; 2803; 2804; 2805; 2806; 2807; 2808; 2809; 2810 2478 BLFS36 no domains in core 2479 BLFS37 1; 56; 57 in core and homologs 2816; 2817 2480 BLFS38 58; 59 in core and homologs 2818 2481 BLFS39 6 in core and homologs 2819; 2820; 2821 2482 BLFS41 60; 61; 62; 63; 24; 64; 65; 66; 67 in core 2822; 2823; 2824; 2825; 2826; 2827; and homologs 2828; 2829 2483 BLFS42 60; 68 in core and homologs 2830; 2831 2484 BLFS43 51 in core and homologs 2832; 2833; 2834; 2835 2485 BLFS44 69; 70; 71; 7; 10; 24 in core and 2836 homologs 2486 BLFS45 73 in core and homologs 2837; 2838; 2839; 2840; 2841; 2842; 2843; 2844 2487 BLFS46 31; 74; 32; 75 in core and homologs 2845 2488 BLFS47 1 in core and homologs 2846; 2847; 2848; 2849 2489 BLFS48 35; 52; 37 in core and homologs 2850; 2851; 2852 2490 BLFS49 7; 10 in core 2491 BLFS50 60; 68 in core and homologs 2853 2492 BLFS51 76; 24 in core and homologs 2854; 2855 2493 BLFS52 7; 10 in core 2494 BLFS53 no domains in core 2495 BLFS55 1; 57 in core and homologs 2857; 2858 2496 BLFS56 77; 17; 79 in core and homologs 2859; 2860; 2861; 2862; 2863; 2864 2497 BLFS57 80; 81; 7; 10; 82 in core and 2865; 2866; 2867 homologs 2498 BLFS58 1; 83; 57 in core and homologs 2868; 2869 2499 BLFS59 1; 57 in core 2500 BLFS60 1 in core and homologs 2870; 2871; 2872 2501 BLFS61 84; 85; 86; 87; 88 in core and 2873; 2874; 2875; 2876; 2877; 2878; homologs 2879; 2880 2502 BLFS62 31; 74; 32; 75 in core and homologs 2881; 2882; 2883; 2884; 2885; 2886; 2887; 2888; 2889; 2890 2503 BLFS63 89; 90; 31; 32 in core and homologs 2891; 2892; 2893; 2894; 2895; 2896; 2897; 2898; 2899; 2900; 2901; 2902; 2903 2504 BLFS64 8; 49; 30 in core and homologs 2904 2505 BLFS65 91 in core and homologs 2905 2506 BLFS67 1; 57 in core and homologs 2906; 2907; 2908 2507 BLFS68 92 in core and homologs 2909; 2910; 2911; 2912 2508 BLFS69 93; 94; 95; 96; 97 in core 2509 BLFS70 98 in core 2510 BLFS72 99; 100; 101; 102 in core and 2913 homologs 2511 BLFS73 1 in core and homologs 2914; 2915; 2916; 2917; 2918; 2919; 2920; 2921; 2922; 2923; 2924; 2925; 2926; 2927; 2928; 2929 2512 BLFS74 34 in core and homologs 2930; 2931; 2932; 2933; 2934; 2935; 2936; 2937; 2938; 2939; 2940; 2941; 2942; 2943 2513 BLFS75 103; 104 in core and homologs 2944; 2945; 2946; 2947; 2948; 2949; 2950; 2951; 2952; 2953; 2954 2514 BLFS76 no domains in core 2515 BLFS77 17; 7; 78; 79; 10; 24; 76 in core and 2963; 2964; 2965 homologs 2516 BLFS79 43; 44; 105 in core and homologs 2966; 2967; 2968; 2969; 2970; 2971; 2972; 2973; 2974; 2975 2517 BLFS80 17; 78; 19 in core 2518 BLFS82 no domains in core 2519 BLFS83 106; 79; 107; 108; 17; 66; 78; 109 in 2989; 2990; 2991; 2992; 2993; 2994; core and homologs 2995; 2996; 2997; 2998; 2999 2520 BLFS84 86; 110 in core and homologs 3000; 3001; 3002; 3003; 3004; 3005; 3006 2521 BLFS85 71; 24; 111 in core and homologs 3007; 3008; 3009; 3010; 3011; 3012; 3013; 3014; 3015; 3016; 3017 2522 BLFS86 no domains in core 2523 BLFS87 17; 60; 112; 76; 24; 113; 114 in core 3024; 3025; 3026; 3027; 3028; 3029; and homologs 3030; 3031; 3032; 3033; 3034 2524 BLFS88 no domains in core 2525 BLFS89 24; 113; 76 in core and homologs 3044; 3045; 3046; 3047; 3048; 3049; 3050; 3051; 3052 2526 BLFS90 115; 116 in core and homologs 3053; 3054; 3055; 3056; 3057; 3058; 3059; 3060; 3061 2527 BLFS91 no domains in core 2528 BLFS92 34; 35; 36; 37; 38 in core and 3073; 3074; 3075; 3076; 3077; 3078; homologs 3079; 3080; 3081 2529 BLFS93 117; 24 in core and homologs 3082; 3083; 3084; 3085; 3086; 3087; 3088; 3089; 3090; 3091; 3092 2530 BLFS94 17; 118 in core and homologs 3093; 3094; 3095; 3096; 3097; 3098; 3099; 3100; 3101 2531 BLFS95 no domains in core 2532 BLFS96 7; 10; 6 in core 2533 BLFS97 119 in core 2534 BLFS98 34 in core and homologs 3111; 3112; 3113; 3114; 3115; 3116; 3117; 3118; 3119; 3120; 3121; 3122; 3123; 3124; 3125; 3126 2535 BLFS99 120; 121; 122 in core and homologs 3127; 3128; 3129; 3130; 3131; 3132; 3133; 3134; 3135; 3136; 3137; 3138 2536 BLFS100 120; 121; 122 in core and homologs 3139; 3140; 3141; 3142; 3143; 3144; 3145; 3146; 3147; 3148; 3149; 3150; 3151; 3152; 3153; 3154 2537 BLFS101 52 in core and homologs 3155; 3156; 3157; 3158; 3159; 3160; 3161; 3162; 3163; 3164; 3165; 3166; 3167; 3168; 3169; 3170 2538 BLFS102 no domains in core 2539 BLFS103 no domains in core 2540 BLFS104 no domains in core 2541 BLFS105 123 in core and homologs 3194; 3195; 3196; 3197; 3198; 3199; 3200; 3201; 3202; 3203; 3204; 3205; 3206 2542 BLFS106 124 in core and homologs 3207; 3208; 3209; 3210; 3211; 3212; 3213; 3214; 3215; 3216; 3217; 3218; 3219; 3220; 3221; 3222; 3223; 3224; 3225; 3226; 3227 2543 BLFS107 125 in core and homologs 3228; 3229; 3230; 3231; 3232; 3233; 3234; 3235; 3236; 3237; 3238; 3239; 3240; 3241; 3242; 3243; 3244; 3245 2544 BLFS108 126 in core and homologs 3246; 3247; 3248; 3249; 3250; 3251; 3252; 3253; 3254 2545 BLFS109 127 in core and homologs 3255; 3256 2546 BLFS110 128 in core and homologs 3257 2547 BLFS111 129 in core and homologs 3258; 3259; 3260; 3261; 3262 2548 BLFS112 no domains in core 2549 BLFS113 130; 131 in core and homologs 3276; 3277; 3278; 3279; 3280; 3281; 3282; 3283; 3284; 3285; 3286; 3287; 3288; 3289; 3290; 3291; 3292; 3293; 3294; 3295; 3296; 3297; 3298; 3299; 3300 2550 BLFS114 132 in core and homologs 3301; 3302; 3303; 3304; 3305; 3306; 3307; 3308; 3309; 3310; 3311; 3312; 3313; 3314; 3315; 3316; 3317; 3318; 3319; 3320; 3321; 3322; 3323; 3324; 3325 2551 BLFS115 34; 133; 35; 37 in core and homologs 3326; 3327; 3328; 3329; 3330; 3331; 3332; 3333; 3334; 3335; 3336; 3337; 3338; 3339; 3340; 3341; 3342; 3343; 3344; 3345; 3346; 3347; 3348; 3349; 3350; 3351; 3352; 3353; 3354 2552 BLFS116 86; 110 in core and homologs 3355; 3356; 3357; 3358; 3359; 3360; 3361; 3362; 3363; 3364; 3365; 3366; 3367; 3368; 3369; 3370; 3371; 3372; 3373; 3374; 3375; 3376 2553 BLFS118 31; 32 in core and homologs 3377; 3378; 3379; 3380; 3381; 3382; 3383; 3384; 3385; 3386; 3387; 3388; 3389; 3390; 3391; 3392; 3393; 3394 2554 BLFS119 no domains in core 2555 BLFS120 134; 135 in core and homologs 3399; 3400; 3401 2556 BLFS121 136; 137; 138; 139; 140 in core and 3402; 3403; 3404; 3405; 3406; 3407; homologs 3408; 3409; 3410; 3411; 3412; 3413; 3414; 3415; 3416; 3417; 3418; 3419; 3420; 3421; 3422; 3423 2557 BLFS122 no domains in core 2558 BLFS123 141; 8 in core and homologs 3431; 3432; 3433; 3434 2559 BLFS124 142 in core and homologs 3435; 3436; 3437; 3438; 3439; 3440; 3441; 3442; 3443; 3444; 3445; 3446; 3447; 3448; 3449; 3450; 3451 2560 BLFS125 72 in core and homologs 3452; 3453; 3454; 3455; 3456; 3457; 3458; 3459; 3460; 3461; 3462; 3463; 3464; 3465; 3466; 3467 2561 BLFS126 no domains in core 2562 BLFS127 24 in core and homologs 3476; 3477; 3478; 3479; 3480; 3481; 3482; 3483; 3484; 3485; 3486; 3487; 3488; 3489; 3490; 3491 2563 BLFS128 143; 144; 145 in core and homologs 3492; 3493; 3494; 3495; 3496; 3497; 3498; 3499; 3500; 3501; 3502; 3503; 3504; 3505; 3506; 3507; 3508 2564 BLFS129 146; 82 in core and homologs 3509; 3510; 3511; 3512; 3513; 3514; 3515; 3516; 3517; 3518; 3519 2565 BLFS130 147; 148; 115; 116 in core and 3520; 3521; 3522; 3523; 3524 homologs 2566 BLFS131 83 in core and homologs 3525; 3526; 3527; 3528; 3529; 3530; 3531; 3532; 3533; 3534; 3535; 3536; 3537; 3538; 3539; 3540; 3541; 3542; 3543; 3544; 3545; 3546; 3547; 3548 2567 BLFS132 no domains in core 2568 BLFS133 no domains in core 2569 BLFS134 149 in core and homologs 3577; 3578; 3579; 3580; 3581; 3582; 3583; 3584; 3585; 3586; 3587; 3588; 3589; 3590; 3591; 3592; 3593; 3594; 3595; 3596; 3597; 3598; 3599; 3600; 3601; 3602; 3603 2570 BLFS135 60; 68 in core and homologs 3604; 3605; 3606; 3607; 3608; 3609; 3610; 3611; 3612; 3613; 3614; 3615 2571 BLFS136 150; 151 in core and homologs 3616 2572 BLFS137 152; 153 in core and homologs 3617; 3618; 3619; 3620; 3621 2573 BLFS138 154; 155 in core and homologs 3622; 3623; 3624; 3625; 3626; 3627; 3628; 3629; 3630; 3631; 3632; 3633 2574 BLFS139 31; 32; 8; 30 in core and homologs 3634; 3635; 3636; 3637; 3638; 3639; 3640; 3641; 3642; 3643; 3644; 3645; 3646 2575 BLFS140 156; 157; 158; 159; 24; 160 in core 3647; 3648; 3649; 3650; 3651; 3652; and homologs 3653; 3654; 3655; 3656; 3657; 3658; 3659; 3660 2576 BLFS141 161; 162; 163; 24 in core and 3661; 3662; 3663; 3664; 3665; 3666; homologs 3667; 3668; 3669; 3670; 3671; 3672; 3673; 3674; 3675; 3676; 3677; 3678; 3679 2577 BLFS142 45 in core and homologs 3680; 3681; 3682; 3683; 3684; 3685; 3686; 3687; 3688; 3689; 3690 2578 BLFS143 164; 165; 166 in core and homologs 3691; 3692; 3693; 3694; 3695; 3696; 3697; 3698; 3699; 3700; 3701; 3702; 3703; 3704 2579 BLFS144 72; 167; 73; 168; 169; 170 in core and 3705; 3706; 3707; 3708; 3709; 3710; homologs 3711; 3712; 3713; 3714; 3715 2580 BLFS145 103; 171; 172; 173 in core and 3716; 3717; 3718; 3719; 3720; 3721; homologs 3722; 3723; 3724; 3725; 3726; 3727; 3728; 3729 2581 BLFS146 74; 75 in core and homologs 3730; 3731; 3732; 3733; 3734; 3735; 3736; 3737; 3738; 3739; 3740; 3741; 3742; 3743; 3744; 3745; 3746; 3747; 3748; 3749 2582 BLFS147 174; 143; 39 in core and homologs 3750; 3751; 3752; 3753; 3754; 3755; 3756; 3757; 3758; 3759 2583 BLFS148 52 in core and homologs 3760; 3761; 3762; 3763; 3764; 3765; 3766; 3767; 3768; 3769; 3770; 3771; 3772; 3773; 3774; 3775; 3776; 3777; 3778; 3779; 3780; 3781 2584 BLFS149 175; 60; 61; 62; 63; 24; 64; 17; 65; 66; 3782; 3783; 3784; 3785; 3786; 3787; 67 in core and homologs 3788; 3789; 3790; 3791; 3792; 3793; 3794; 3795; 3796; 3797 2585 BLFS150 no domains in core 2586 BLFS151 86; 110 in core and homologs 3798; 3799; 3800 2587 BLFS152 176; 177 in core and homologs 3801; 3802 2588 BLFS153 34; 178; 179; 180; 181 in core and 3803; 3804; 3805; 3806; 3807; 3808 homologs 2589 BLFS154 182 in core and homologs 3809; 3810 2590 BLFS155 4; 5 in core and homologs 3811; 3812 2591 BLFS156 1; 2; 3 in core 2592 BLFS157 99; 100; 183; 184; 6; 102 in core 2593 BLFS158 185; 186; 187 in core and homologs 3813 2594 BLFS159 no domains in core 2595 PUB6 188; 7; 10; 189 in core and homologs 3816; 3817; 3818; 3819; 3820; 3821; 3822; 3823; 3824; 3825 2596 PUB23 190; 191; 7; 10; 192; 193; 194; 19 in core 2597 PUB31 195; 196; 7; 10; 19 in core 2598 PUB65 197; 15; 7; 198; 10; 82; 199; 200; 201; 3826; 3827; 3828; 3829; 3830; 3831; 202 in core and homologs 3832; 3833; 3834; 3835; 3836; 3837; 3838; 3839; 3840; 3841; 3842; 3843; 3844; 3845 2599 ZLFS1 203; 204; 205 in core and homologs 3846 2600 ZLFS2 206 in core and homologs 3847; 3848; 3849; 3850; 3851; 3852; 3853; 3854; 3855; 3856; 3857; 3858; 3859; 3860; 3861; 3862; 3863; 3864; 3865 2601 ZLFS3 31; 32; 8; 30 in core and homologs 3866; 3867; 3868; 3869; 3870; 3871; 3872; 3873; 3874; 3875; 3876; 3877; 3878; 3879; 3880; 3881; 3882; 3883 2602 ZLFS5 123 in core and homologs 3884; 3885; 3886; 3887; 3888 2603 ZLFS6 207; 208 in core and homologs 3889; 3890; 3891; 3892; 3893 2604 ZLFS7 209 in core and homologs 3894 2605 ZLFS8 210; 211 in core and homologs 3895; 3896 2606 ZLFS9 113; 24 in core and homologs 3897 2607 ZLFS10 no domains in core 2608 ZLFS11 212; 213; 214 in core and homologs 3898; 3899 2609 ZLFS12 215; 216; 217; 218 in core and 3900; 3901; 3902 homologs 2610 ZLFS13 no domains in core 2611 ZLFS14 126 in core and homologs 3905 2612 ZLFS15 219; 52 in core and homologs 3906; 3907; 3908 2613 ZLFS16 220; 221; 103; 171; 173; 172 in core 3909; 3910; 3911; 3912; 3913; 3914; 3915 and homologs 2614 ZLFS17 2; 1; 222 in core and homologs 3916 2615 ZLFS18 99; 100; 102 in core and homologs 3917 2616 ZLFS19 31; 74; 32; 75 in core and homologs 3918; 3919; 3920; 3921; 3922; 3923; 3924; 3925; 3926; 3927 2617 ZLFS21 223; 101; 224 in core and homologs 3928; 3929; 3930; 3931; 3932; 3933; 3934; 3935; 3936; 3937; 3938; 3939; 3940 2618 ZLFS22 225; 31; 32; 226 in core and 3941; 3942; 3943; 3944; 3945; 3946; homologs 3947; 3948; 3949; 3950; 3951; 3952; 3953 2619 ZLFS23 82; 92; 227 in core and homologs 3954; 3955; 3956; 3957; 3958; 3959; 3960 2620 ZLFS24 176; 228; 229; 230; 231 in core and 3961; 3962; 3963; 3964; 3965; 3966; homologs 3967; 3968; 3969; 3970; 3971; 3972; 3973; 3974; 3975; 3976 2621 ZLFS25 176; 232; 177 in core and homologs 3977; 3978; 3979; 3980; 3981; 3982; 3983 2622 ZLFS27 31; 233; 32; 101; 234 in core and 3984; 3985; 3986; 3987; 3988; 3989; homologs 3990; 3991; 3992; 3993; 3994; 3995; 3996; 3997; 3998; 3999; 4000; 4001; 4002; 4003; 4004; 4005; 4006; 4007 2623 ZLFS28 86; 110 in core and homologs 4008; 4009; 4010; 4011; 4012; 4013; 4014; 4015; 4016; 4017; 4018; 4019; 4020; 4021; 4022 2624 ZLFS29 235; 219; 52 in core and homologs 4023; 4024; 4025; 4026; 4027; 4028; 4029; 4030; 4031; 4032; 4033; 4034; 4035; 4036; 4037; 4038; 4039; 4040; 4041; 4042; 4043; 4044; 4045; 4046; 4047; 4048 2625 ZLFS30 136; 140; 236 in core and homologs 4049; 4050; 4051; 4052; 4053; 4054; 4055; 4056; 4057; 4058; 4059; 4060; 4061; 4062; 4063; 4064; 4065; 4066; 4067; 4068 2626 ZLFS31 228; 230; 229; 231 in core and 4069; 4070; 4071; 4072; 4073; 4074; homologs 4075; 4076; 4077; 4078; 4079; 4080; 4081 2627 ZLFS32 237; 238; 239 in core and homologs 4082; 4083; 4084; 4085; 4086; 4087; 4088; 4089; 4090; 4091; 4092; 4093; 4094; 4095; 4096; 4097 2628 ZLFS33 60; 68 in core and homologs 4098; 4099 2629 ZLFS34 240; 241; 242 in core and homologs 4100; 4101; 4102; 4103; 4104; 4105; 4106; 4107; 4108; 4109; 4110; 4111; 4112; 4113; 4114; 4115; 4116; 4117; 4118; 4119 2630 ZLFS35 no domains in core 2631 ZLFS36 no domains in core 2632 ZLFS37 243; 244 in core and homologs 4126; 4127; 4128; 4129; 4130; 4131; 4132; 4133; 4134; 4135; 4136; 4137; 4138; 4139; 4140; 4141; 4142; 4143; 4144; 4145; 4146; 4147; 4148; 4149; 4150; 4151; 4152; 4153 2633 ZLFS38 245; 246; 247; 248; 249 in core and 4154; 4155; 4156; 4157; 4158; 4159; homologs 4160; 4161; 4162; 4163; 4164; 4165; 4166; 4167; 4168; 4169; 4170; 4171; 4172; 4173; 4174; 4175; 4176 2634 ZLFS39 250; 60; 251; 24; 71; 17; 252; 111 in 4177; 4178; 4179; 4180; 4181; 4182; core and homologs 4183; 4184; 4185; 4186; 4187; 4188; 4189; 4190; 4191; 4192; 4193; 4194; 4195; 4196; 4197; 4198; 4199; 4200; 4201 2635 ZLFS42 1; 2; 3 in core and homologs 2637; 2660; 4202; 4203; 4204; 4205; 4206; 4207; 4208; 4209; 4210 2636 ZLFS44 8; 30 in core and homologs 4211; 4212; 4213; 4214; 4215; 4216; 4217; 4218; 4219; 4220; 4221; 4222; 4223; 4224; 4225; 4226; 4227; 4228; 4229; 4230; 4231 2637 ZLFS45 2; 1; 3 in core and homologs 4202; 4203; 4204; 4205; 4206; 4207; 4208; 4209; 4210; 4232 2638 ZLFS47 2; 1; 222; 92 in core 2639 ZLFS48 2; 1; 222 in core and homologs 3916 2640 ZLFS49 4; 74; 5 in core and homologs 4233; 4234 2641 ZLFS51 31; 40; 41; 42; 32; 24 in core and 4235; 4236; 4237; 4238; 4239; 4240; homologs 4241; 4242; 4243; 4244; 4245; 4246; 4247; 4248; 4249; 4250; 4251; 4252

TABLE 18 Details of identified domains presented in Table 17 Domain InterPro Identifier No. Accession No. Description 1 IPR011024 SSF49695 Gamma-crystallin-like 2 IPR015791 G3DSA:2.60.20.30 Antimicrobial/protein inhibitor, gamma-crystallin-like 3 IPR015161 PF09076 Beta/Gamma crystallin Streptomyces killer toxin-like, beta/gamma crystallin 4 IPR001480 PS50927 Bulb-type lectin domain profile. Bulb-type lectin domain 5 IPR036426 G3DSA:2.90.10.10 Bulb-type lectin domain superfamily 6 IPR004991 PF03318 Clostridium epsilon toxin ETX/Bacillus mosquitocidal toxin MTX2 Aerolysin-like toxin 7 IPR035992 SSF50370 Ricin B-like lectins 8 IPR038765 SSF54001 Papain-like cysteine peptidase superfamily 9 IPR013688 PF08481 GBS Bsp-like repeat GBS Bsp-like 10 IPR000772 cd00161 RICIN Ricin B, lectin domain 11 IPR008999 SSF50405 Actin-crosslinking 12 IPR039564 PF13529 Peptidase C39 like family Peptidase C39-like 13 IPR001322 PF00932 Lamin Tail Domain Lamin tail domain 14 IPR036415 SSF74853 Lamin tail domain superfamily 15 IPR032812 PF13205 Bacterial Ig-like domain SbsA, Ig-like domain 16 IPR005077 PF03415 Clostripain family Peptidase C11, clostripain 17 IPR013783 G3DSA:2.60.40.10 Immunoglobulin-like fold 18 IPR003737 PF02585 GlcNAc-PI de-N-acetylase N-acetylglucosaminyl phosphatidylinositol deacetylase-related 19 IPR008979 G3DSA:2.60.120.260 Galactose-binding-like domain superfamily 20 IPR024078 SSF102588 Putative deacetylase LmbE-like domain superfamily 21 IPR008270 PS00953 Glycosyl hydrolases family 25 active sites signature. Glycoside hydrolase, family 25, active site 22 IPR002053 PF01183 Glycosyl hydrolases family 25 Glycoside hydrolase, family 25 23 IPR018077 SM00641 Glycoside hydrolase, family 25 subgroup 24 IPR017853 SSF51445 Glycoside hydrolase superfamily 25 IPR009045 G3DSA:3.30.1380.10 Hedgehog signalling/DD-peptidase zinc-binding domain superfamily 26 IPR003709 PF02557 D-alanyl-D-alanine carboxypeptidase Peptidase M15B 27 IPR012340 SSF50249 Nucleic acid-binding, OB-fold 28 IPR003029 PS50126 S1 domain profile. S1 domain 29 IPR022967 SM00316 RNA-binding domain, S1 30 IPR000064 PF00877 NlpC/P60 family Endopeptidase, NLPC/P60 domain 31 IPR018392 SM00257 LysM domain 32 IPR036779 G3DSA:3.10.350.10 LysM domain superfamily 33 IPR022887 MF_02014 Cell division suppressor protein YneA [yneA], Cell division suppressor protein, YneA 34 IPR009003 SSF50494 Peptidase S1, PA clan 35 IPR036034 SSF50156 PDZ superfamily 36 IPR008763 PF05580 SpoIVB peptidase S55 Peptidase S55, SpoIVB 37 IPR001478 PS50106 PDZ domain profile. PDZ domain 38 IPR014219 TIGR02860 spore_IV_B: stage IV sporulation protein B Peptidase S55, sporulation stage IV, protein B 39 IPR003646 PF08239 Bacterial SH3 domain SH3-like domain, bacterial-type 40 IPR001223 PF00704 Glycosyl hydrolases family 18 Glycoside hydrolase family 18, catalytic domain 41 IPR011583 SM00636 Chitinase II 42 IPR029070 G3DSA:3.10.50.10 Chitinase insertion domain superfamily 43 IPR011330 SSF88713 Glycoside hydrolase/deacetylase, beta/alpha-barrel 44 IPR002509 PS51677 NodB homology domain profile. NodB homology domain 45 IPR011105 PF07486 Cell Wall Hydrolase Cell wall hydrolase, SleB 46 IPR010310 PF06013 Proteins of 100 residues with WXG Type VII secretion system ESAT-6-like 47 IPR037146 G3DSA:3.90.540.10 Colicin/pyocin, DNase domain superfamily 48 IPR036689 SSF140453 ESAT-6-like superfamily 49 IPR001119 PF00395 S-layer homology domain S-layer homology domain 50 IPR019730 PF10794 Protein of unknown function (DUF2606) Protein of unknown function DUF2606 51 IPR013784 SSF49452 Carbohydrate-binding-like fold 52 IPR001915 PF01435 Peptidase family M48 Peptidase M48 53 IPR008915 PF02163 Peptidase family M50 Peptidase M50 54 IPR004387 TIGR00054 TIGR00054: RIP metalloprotease RseP Peptidase M50, putative membrane-associated zinc metallopeptidase 55 IPR025493 PF14326 Domain of unknown function (DUF4384) Domain of unknown function DUF4384 56 IPR015059 PF08964 Beta/Gamma crystallin Calcium-dependent cell adhesion molecule, N-terminal 57 IPR001064 PS50915 Crystallins beta and gamma ‘Greek key’ motif profile. Beta/gamma crystallin 58 IPR036404 G3DSA:2.100.10.30 Jacalin-like lectin domain superfamily 59 IPR001229 PS51752 Jacalin-type lectin domain profile. Jacalin-like lectin domain 60 IPR014756 SSF81296 Immunoglobulin E-set 61 IPR015883 PF00728 Glycosyl hydrolase family 20, catalytic domain Glycoside hydrolase family 20, catalytic domain 62 IPR015882 PF02838 Glycosyl hydrolase family 20, domain 2 Beta- hexosaminidase, bacterial type, N-terminal 63 IPR012291 G3DSA:2.60.40.290 CBM2, carbohydrate-binding domain superfamily 64 IPR029018 G3DSA:3.30.379.10 Beta-hexosaminidase-like, domain 2 65 IPR025705 PR00738 Glycosyl hydrolase family 20 signature Beta-hexosaminidase 66 IPR008965 SSF49384 CBM2/CBM3, carbohydrate-binding domain superfamily 67 IPR004866 PF03173 Putative carbohydrate binding domain Chitobiase/ beta-hexosaminidases, N-terminal domain 68 IPR004302 PF03067 Lytic polysaccharide mono-oxygenase, cellulose- degrading Cellulose/chitin-binding protein, N-terminal 69 IPR013785 G3DSA:3.20.20.70 Aldolase-type TIM barrel 70 IPR002241 PF16499 Alpha galactosidase A Glycoside hydrolase, family 27 71 IPR013780 G3DSA:2.60.40.1180 Glycosyl hydrolase, all-beta 72 IPR025711 PF13670 Peptidase propeptide and YPEB domain PepSY domain 73 IPR011096 PF07504 Fungalysin/Thermolysin Propeptide Motif FTP domain 74 IPR011055 G3DSA:2.70.70.10 Duplicated hybrid motif 75 IPR016047 PF01551 Peptidase family M23 Peptidase M23 76 IPR001547 PF00150 Cellulase (glycosyl hydrolase family 5) Glycoside hydrolase, family 5 77 IPR007484 PF04389 Peptidase family M28 Peptidase M28 78 IPR036116 SSF49265 Fibronectin type III superfamily 79 IPR003961 cd00063 FN3 Fibronectin type III 80 IPR002594 PF01670 Glycosyl hydrolase family 12 Glycoside hydrolase family 12 81 IPR013319 G3DSA:2.60.120.180 Glycoside hydrolase family 11/12 82 IPR013320 SSF49899 Concanavalin A-like lectin/glucanase domain superfamily 83 IPR008816 PF05433 Glycine zipper 2TM domain Glycine zipper 2TM domain 84 IPR011049 SSF51120 Serralysin-like metalloprotease, C-terminal 85 IPR001343 PF00353 RTX calcium-binding nonapeptide repeat (4 copies) RTX calcium-binding nonapeptide repeat 86 IPR029058 G3DSA:3.40.50.1820 Alpha/Beta hydrolase fold 87 IPR003995 PR01488 Gram-negative bacterial RTX toxin determinant A family signature RTX toxin determinant A 88 IPR018511 PS00330 Hemolysin-type calcium-binding region signature. Hemolysin-type calcium-binding conserved site 89 IPR021731 PF11741 AMIN domain AMIN domain 90 IPR002508 cd02696 MurNAc-LAA N-acetylmuramoyl-L-alanine amidase, catalytic domain 91 IPR014262 TIGR02913 HAF_rpt: probable extracellular repeat, HAF family Extracellular HAF 92 IPR006311 PS51318 Twin arginine translocation (Tat) signal profile. Twin-arginine translocation pathway, signal sequence 93 IPR011050 SSF51126 Pectin lyase fold/virulence factor 94 IPR024973 PF13018 Extended Signal Peptide of Type V secretion system ESPR domain 95 IPR008638 TIGR01901 adhes_NPXG: filamentous hemagglutinin family N-terminal domain Filamentous haemagglutinin, N-terminal 96 IPR011493 PF07581 The GLUG motif GLUG 97 IPR012334 G3DSA:2.160.20.10 Pectin lyase fold 98 IPR003540 PF03496 ADP-ribosyltransferase exoenzyme ADP ribosyltransferase 99 IPR036365 SSF47090 PGBD-like superfamily 100 IPR002477 PF01471 Putative peptidoglycan binding domain Peptidoglycan binding-like 101 IPR023346 SSF53955 Lysozyme-like domain superfamily 102 IPR036366 G3DSA:1.10.101.10 PGBD superfamily 103 IPR012338 SSF56601 Beta-lactamase/transpeptidase-like 104 IPR001466 PF00144 Beta-lactamase Beta-lactamase-related 105 IPR014235 cd10948 CE4_BsPdaA_like Peptidoglycan-N-acetylmuramic acid deacetylase PdaA 106 IPR036434 SSF51989 1,4-beta cellobiohydrolase superfamily 107 IPR016288 PF01341 Glycosyl hydrolases family 6 1,4-beta cellobiohydrolase 108 IPR036966 G3DSA:2.60.40.710 Carbohydrate-binding module 3 superfamily 109 IPR001956 PS51172 CBM3 (carbohydrate binding type-3) domain profile. Carbohydrate-binding module 3 110 IPR000073 PF12697 Alpha/beta hydrolase family Alpha/beta hydrolase fold-1 111 IPR006047 SM00642 Glycosyl hydrolase, family 13, catalytic domain 112 IPR016282 PIRSF001043 Glycoside hydrolase, family 5, endoglucanase B 113 IPR018087 PS00659 Glycosyl hydrolases family 5 signature. Glycoside hydrolase, family 5, conserved site 114 IPR005102 PF03442 Carbohydrate binding domain X2 Carbohydrate binding X2 domain 115 IPR001611 PS51450 Leucine-rich repeat profile. Leucine-rich repeat 116 IPR032675 G3DSA:3.80.10.10 Leucine-rich repeat domain superfamily 117 IPR011683 PF07745 Glycosyl hydrolase family 53 Glycosyl hydrolase family 53 118 IPR034641 cd10318 RGL11 Polysaccharide lyase family 11 119 IPR020864 PF01823 MAC/Perforin domain Membrane attack complex component/perforin (MACPF) domain 120 IPR010854 PF07338 Protein of unknown function (DUF1471) Domain of unknown function DUF1471 121 IPR036275 SSF159871 YdgH-like superfamily 122 IPR025543 G3DSA:3.30.1660.10 Dodecin-like 123 IPR009739 PF07007 Lysozyme inhibitor LprI Lysozyme inhibitor LprI, N-terminal 124 IPR010279 PF05957 Bacterial protein of unknown function (DUF883) Inner membrane protein YqjD/ElaB 125 IPR007893 SM00972 Spore coat protein U 126 IPR009241 PF05973 Phage derived protein Gp49-like (DUF891) Toxin HigB-like 127 IPR006914 PF04829 Pre-toxin domain with VENN motif VENN motif-containing domain 128 IPR013728 PF08522 Domain of unknown function (DUF1735) Domain of unknown function DUF1735 129 IPR003848 cd06259 YdcF-like Domain of unknown function DUF218 130 IPR005490 PF03734 L,D-transpeptidase catalytic domain L,D-transpeptidase catalytic domain 131 IPR038063 SSF141523 L,D-transpeptidase catalytic domain-like 132 IPR021109 SSF50630 Aspartic peptidase domain superfamily 133 IPR001940 PR00834 HtrA/DegQ protease family signature Peptidase S1C 134 IPR025425 PF13665 Domain of unknown function (DUF4150) Domain of unknown function DUF4150 135 IPR028917 PF15635 GHH signature containing HNH/Endo VII superfamily nuclease toxin 2 Tox-GHH2 domain 136 IPR002933 PF01546 Peptidase family M20/M25/M40 Peptidase M20 137 IPR001261 PS00758 ArgE/dapE/ACY1/CPG2/yscS family signature 1. ArgE/DapE/ACY1/CPG2/YscS. conserved site 138 IPR017150 cd03885 M20_CPDG2 Peptidase M20, glutamate carboxypeptidase 139 IPR011650 PF07687 Peptidase dimerisation domain Peptidase M20, dimerisation domain 140 IPR036264 SSF55031 Bacterial exopeptidase dimerisation domain 141 IPR000668 PF00112 Papain family cysteine protease Peptidase C1A, papain C-terminal 142 IPR012661 PF09498 Protein of unknown function (DUF2388) Conserved hypothetical protein CHP02448 143 IPR002901 PF01832 Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase Mannosyl-glycoprotein endo-beta-N-acetylglucosamidase- like domain 144 IPR013377 TIGR02541 flagell_FlgJ: flagellar rod assembly protein/muramidase FlgJ Peptidoglycan hydrolase FlgJ 145 IPR019301 PF10135 Rod binding protein Flagellar protein FlgJ, N-terminal 146 IPR014895 PF08787 Alginate lyase Alginate lyase 2 147 IPR029487 PF14496 C-terminal novel E3 ligase, LRR-interacting Novel E3 ligase domain 148 IPR003591 SM00369 Leucine-rich repeat, typical subtype 149 IPR021357 PF11191 Protein of unknown function (DUF2782) Protein of unknown function DUF2782 150 IPR007055 PS50914 BON domain profile. BON domain 151 IPR014004 SM00749 Transport-associated and nodulation domain, bacteria 152 IPR015286 PF09203 MspA Porin family, mycobacterial-type 153 IPR036435 SSF56959 Leukocidin/porin MspA superfamily 154 IPR005320 cd03146 GAT1_Peptidase_E Peptidase S51 155 IPR029062 G3DSA:3.40.50.880 Class I glutamine amidotransferase-like 156 IPR001764 PF00933 Glycosyl hydrolase family 3 N terminal domain Glycoside hydrolase, family 3, N-terminal 157 IPR002772 PF01915 Glycosyl hydrolase family 3 C-terminal domain Glycoside hydrolase family 3 C-terminal domain 158 IPR036962 G3DSA:3.20.20.300 Glycoside hydrolase, family 3, N-terminal domain superfamily 159 IPR019800 PS00775 Glycosyl hydrolases family 3 active site. Glycoside hydrolase, family 3, active site 160 IPR036881 SSF52279 Glycoside hydrolase family 3 C-terminal domain superfamily 161 IPR016714 PIRSF018168 Mannan endo-1,4-beta-mannosidase 162 IPR000805 PR00739 Glycosyl hydrolase family 26 signature Glycoside hydrolase family 26 163 IPR022790 PF02156 Glycosyl hydrolase family 26 Glycosyl hydrolase family 26 domain 164 IPR029045 SSF52096 ClpP/crotonase-like domain superfamily 165 IPR002142 PF01343 Peptidase family S49 Peptidase S49 166 IPR004635 TIGR00706 SppA_dom: signal peptide peptidase SppA, 36K type Peptidase S49, SppA 167 IPR027268 G3DSA:1.10.390.10 Peptidase M4/M1, CTD superfamily 168 IPR001570 PF02868 Thermolysin metallopeptidase, alpha-helical domain Peptidase M4, C-terminal 169 IPR013856 PF01447 Thermolysin metallopeptidase, catalytic domain Peptidase M4 domain 170 IPR023612 PR00730 Thermolysin metalloprotease (M4) family signature Peptidase M4 171 IPR012907 PF07943 Penicillin-binding protein 5, C-terminal domain Peptidase S11, D-Ala-D-Alacarboxypeptidase A, C-terminal 172 IPR018044 PR00725 D-Ala-D-Alacarboxypeptidase 1 (S11) family signature Peptidase S11, D-alanyl-D-alanine carboxypeptidase A 173 IPR001967 PF00768 D-alanyl-D-alanine carboxypeptidase Peptidase S11, D-alanyl-D-alanine carboxypeptidase A, N- terminal 174 IPR007730 PS51724 SPOR domain profile. Sporulation-like domain 175 IPR004867 cd02847 E_set_Chitobiase_C Chitobiase C-terminal domain 176 IPR036514 G3DSA:3.40.50.1110 SGNH hydrolase superfamily 177 IPR013830 PF13472 GDSL-like Lipase/Acylhydrolase family SGNH hydrolase-type esterase domain 178 IPR001316 PR00861 Alpha-lytic endopeptidase serine protease (S2A) signature Peptidase S1A, streptogrisin 179 IPR033116 PS00135 Serine proteases, trypsin family, serine active site. Serine proteases, trypsin family, serine active site 180 IPR004236 PF02983 Alpha-lytic protease prodomain Peptidase S1A, alpha-lytic prodomain 181 IPR001254 PF00089 Trypsin Serine proteases, trypsin domain 182 IPR007921 PF05257 CHAP domain CHAP domain 183 IPR006616 SM00696 DM9 repeat 184 IPR024518 PF11901 Protein of unknown function (DUF3421) Domain of unknown function DUF3421 185 IPR011051 SSF51182 RmlC-like cupin domain superfamily 186 IPR006045 PF00190 Cupin Cupin 1 187 IPR014710 G3DSA:2.60.120.10 RmlC-like jelly roll fold 188 IPR001434 PF01345 Domain of unknown function DUF11 Domain of unknown function DUF11 189 IPR008966 SSF49401 Adhesion domain superfamily 190 IPR036399 G3DSA:2.100.10.10 Pesticidal crystal protein, central domain superfamily 191 IPR005639 PF03945 delta endotoxin, N-terminal domain Pesticidal crystal protein, N-terminal 192 IPR001178 PF00555 delta endotoxin Pesticidal crystal protein, central domain 193 IPR005638 PF03944 delta endotoxin Pesticidal crystal protein, C-terminal 194 IPR036716 SSF56849 Pesticidal crystal protein, N-terminal domain superfamily 195 IPR000909 PF00388 Phosphatidylinositol-specific phospholipase C, X domain Phosphatidylinositol-specific phospholipase C, X domain 196 IPR017946 SSF51695 PLC-like phosphodiesterase, TIM beta/alpha-barrel domain superfamily 197 IPR022409 SM00089 PKD/Chitinase domain 198 IPR000601 PF00801 PKD domain PKD domain 199 IPR036909 SSF46626 Cytochrome c-like domain superfamily 200 IPR015943 G3DSA:2.130.10.10 WD40/YVTN repeat-like-containing domain superfamily 201 IPR009056 PS51007 Cytochrome c family profile. Cytochrome c-like domain 202 IPR035986 SSF49299 PKD domain superfamily 203 IPR036574 G3DSA:3.30.30.10 Knottin, scorpion toxin-like superfamily 204 IPR003614 cd00107 Knot1 Knottin, scorpion toxin-like 205 IPR001542 PF01097 Arthropod defensin Defensin, invertebrate/fungal 206 IPR000834 PS00132 Zinc carboxypeptidases, zinc-binding region 1 signature. Peptidase M14, carboxypeptidase A 207 IPR007048 PF04965 Gene 25-like lysozyme GpW/Gp25/IraD 208 IPR017737 TIGR03357 VI_zyme: type VI secretion system lysozyme-like protein Type VI secretion system-related protein 209 IPR021459 PF11308 Glycosyl hydrolases related to GH101 family, GH129 Glycosyl hydrolases related to GH101 family, GHL1-GHL3 210 IPR003610 SM00495 Carbohydrate-binding module family 5/12 211 IPR036573 SSF51055 Carbohydrate-binding module superfamily 5/12 212 IPR000184 PF01103 Surface antigen Bacterial surface antigen (D15) 213 IPR016035 SSF52151 Acyl transferase/acyl hydrolase/lysophospholipase 214 IPR002641 PS51635 Patatin-like phospholipase (PNPLA) domain profile. Patatin-like phospholipase domain 215 IPR036852 SSF52743 Peptidase S8/S53 domain superfamily 216 IPR030400 PS51695 Sedolisin domain profile. Sedolisin domain 217 IPR023828 PS00138 Serine proteases, subtilase family, serine active site. Peptidase S8, subtilisin, Ser-active site 218 IPR015366 PF09286 Pro-kumamolisin, activation domain Peptidase S53, activation domain 219 IPR011990 SSF48452 Tetratricopeptide-like helical domain superfamily 220 IPR037167 G3DSA:2.60.410.10 D-Ala-D-Ala carboxypeptidase, C-terminal domain superfamily 221 IPR015956 SSF69189 Penicillin-binding protein, C-terminal domain superfamily 222 IPR015201 PD068438 ANTIMICROBIAL PEPTIDE FUNGICIDE 3D-STRUCTURE DEFENSE SEQUENCING DIRECT AMP1 PLANT PRECURSOR Antimicrobial protein MiAMP1 223 IPR023099 G3DSA:3.30.386.10 Glycoside hydrolase, family 46, N-terminal 224 IPR000400 PS60000 Chitosanases families 46 and 80 active sites signature. Glycoside hydrolase, family 46 225 IPR036908 SSF50685 RlpA-like domain superfamily 226 IPR010611 PF06725 3D domain 3D domain 227 IPR000757 PF00722 Glycosyl hydrolases family 16 Glycoside hydrolase family 16 228 IPR005546 PS51208 Autotransporter beta-domain profile. Autotransporter beta-domain 229 IPR017186 PIRSF037375 Lipase, autotransporter EstA 230 IPR036709 SSF103515 Autotransporter beta-domain superfamily 231 IPR001087 PF00657 GDSL-like Lipase/Acylhydrolase GDSL lipase/esterase 232 IPR037460 cd01823 SEST like Streptomyces scabies esterase-like 233 IPR008258 PF01464 Transglycosylase SLT domain Transglycosylase SLT domain 1 234 IPR000189 PS00922 Prokaryotic transglycosylases signature. Prokaryotic transglycosylase, active site 235 IPR030873 MF_00997 Beta-barrel assembly-enhancing protease [bepA]. Beta-barrel assembly-enhancing protease 236 IPR010964 TIGR01887 dipeptidaselike: putative dipeptidase Peptidase M20A, peptidase V-related 237 IPR012341 G3DSA:1.50.10.10 Six-hairpin glycosidase-like superfamily 238 IPR008928 SSF48208 Six-hairpin glycosidase superfamily 239 IPR002037 PR00735 Glycosyl hydrolase family 8 signature Glycoside hydrolase, family 8 240 IPR024077 G3DSA:1.10.1370.10 Neurolysin/Thimet oligopeptidase, domain 2 241 IPR001567 PF01432 Peptidase family M3 Peptidase M3A/M3B catalytic domain 242 IPR034005 cd06456 M3A_DCP Peptidyl-dipeptidase DCP 243 IPR036913 SSF160113 YegP-like superfamily 244 IPR010879 PF07411 Domain of unknown function (DUF1508) Domain of unknown function DUF1508 245 IPR012347 G3DSA:1.20.1260.10 Ferritin-like 246 IPR002024 cd00907 Bacterioferritin Bacterioferritin 247 IPR009040 PS50905 Ferritin-like diiron domain profile. Ferritin-like diiron domain 248 IPR009078 SSF47240 Ferritin-like superfamily 249 IPR008331 PF00210 Ferritin-like domain Ferritin/DPS protein domain 250 IPR011837 TIGR02100 glgX_debranch: glycogen debranching enzyme GlgX Glycogen debranching enzyme, GlgX type 251 IPR004193 PF02922 Carbohydrate-binding module 48 (Isoamylase N-terminal domain) Glycoside hydrolase, family 13, N-terminal 252 IPR022844 MF_01248 Glycogen debranching enzyme [glgX]. Glycogen debranching enzyme, bacterial

Example 14: Cloning of Resistance-Conferring Bacterial Genes for Expression in E. coli

The fungicidal effect of isolated bacterial polypeptides was examined by expressing the polypeptides in E. coli and purifying the expressed proteins. For expression in E. co/i, selected genes were synthesized by Genscript. The original bacterial sequences were modified such that the codons were optimized for protein expression in E. co/i (further details are available at genscript.com/tools/codon-frequency-table), and a 6-Histidine sequence tag was inserted at either the 5′ or the 3′ end.

In cases where the original bacterial sequences included a nucleic acid sequence encoding a native signal peptide, it was removed up to the native cleavage site separating the encoded mature protein (i.e., the protein not including the signal peptide) from the native signal peptide. The polynucleotide encoding the mature protein was further modified by adding an artificial initiator Methionine codon immediately after the cleavage site.

All optimized genes were synthesized with 5′ NcoI and 3′ EcoRI restrictions sites, and in some of the genes as a result of the addition of the restriction site an additional glycine residue codon was added at the 2nd position (after the initiator Methionine codon) in order to maintain the reading frame of the coding sequence.

Genes lacking an original (native) signal peptide were cloned into pET22bd (a modified version of pET22B+ in which the periplasmic signal peptide PelB SEQ ID NO:5 was removed).

Genes having an original (native) signal peptide that was replaced with an artificial signal peptide were cloned into either the pET22bd and/or the pET22B+(purchased from Merck Millipore, merckmillipore.com/INTL/en/product/pET-22b %28%2B %29-DNA---Novagen,EMD_BIO-69744?ReferrerURL=%3A %2F%2Fwww.google.co(dot)i1%2F&bd=1#anchor_Description) by digesting the gene and the vector with NcoI and EcoRI.

The sequence of each gene was verified by Sanger sequencing in each expression vector. All aforementioned modifications are summarized in Table 19 below.

TABLE 19 Sequences synthesized for cloning in E. coli Native Derived Synthesized Synthesized Gene Polyp. Polyp. Polyn. Polyp. Name SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO Modifications BLFS1 2444 4776 4420 4617 Native signal peptide removed; Met & 3′ His-tag added BLFS2 2445 — 4442 4639 Gly & 3′ His-tag added BLFS3 2446 4698 4316 4513 Native signal peptide removed; Met & 3′ His-tag added BLFS4 2447 — 4392 4589 3′ His-tag added BLFS5 2448 4744 4373 4570 Native signal peptide removed; Met & 3′ His-tag added BLFS6 2449 4682 4298 4495 Native signal peptide removed; MetGly & 3′ His- tag added BLFS7 2450 4753 4386 4583 Native signal peptide removed; Met & 3′ His-tag added BLFS8 2451 4771 4414 4611 Native signal peptide removed; MetGly & 3′ His- tag added BLFS9 2452 4757 4394 4591 Native signal peptide removed; MetGly & 3′ His- tag added BLFS10 2453 4690 4306 4503 Native signal peptide removed; MetGly & 3′ His- tag added BLFS11 2454 4669 4284 4481 Native signal peptide removed; MetGly & 3′ His- tag added BLFS12 2455 4788 4435 4632 Native signal peptide removed; Met & 3′ His-tag added BLFS13 2456 4673 4288 4485 Native signal peptide removed; Met & 3′ His-tag added BLFS14 2457 4722 4344 4541 Native signal peptide removed; Met & 3′ His-tag added BLFS15 2458 4678 4294 4491 Native signal peptide removed; MetGly & 3′ His- tag added BLFS16 2459 4681 4297 4494 Native signal peptide removed; MetGly & 3′ His- tag added BLFS17 2460 4671 4286 4483 Native signal peptide removed; Met & 3′ His-tag added BLFS18 2461 — 4265 4462 Gly & 3′ His-tag added BLFS19 2462 4712 4334 4531 Native signal peptide removed; MetGly & 3′ His- tag added BLFS20 2463 4774 4417 4614 Native signal peptide removed; Met & 3′ His-tag added BLFS21 2464 4751 4382 4579 Native signal peptide removed; MetGly & 3′ His- tag added BLFS22 2465 4787 4434 4631 Native signal peptide removed; Met & 3′ His-tag added BLFS23 2466 4653 4259 4456 Native signal peptide removed; Met & 3′ His-tag added BLFS24 2467 — 4395 4592 3′ His-tag added BLFS25 2468 4799 4449 4646 Native signal peptide removed; Met & 3′ His-tag added BLFS26 2469 4784 4430 4627 Native signal peptide removed; Met & 3′ His-tag added BLFS29 2471 4657 4263 4460 Native signal peptide removed; MetGly & 3′ His- tag added BLFS30 2472 — 4408 4605 3′ His-tag added BLFS31 2473 4726 4348 4545 Native signal peptide removed; Met & 3′ His-tag added BLFS32 2474 4752 4384 4581 Native signal peptide removed; Met & 3′ His-tag added BLFS33 2475 4701 4319 4516 Native signal peptide removed; MetGly & 3′ His- tag added BLFS34 2476 4650 4256 4453 Native signal peptide removed; Met & 3′ His-tag added BLFS35 2477 4778 4423 4620 Native signal peptide removed; Met & 3′ His-tag added BLFS36 2478 4772 4415 4612 Native signal peptide removed; MetGly & 3′ His- tag added BLFS37 2479 — 4368 4565 Gly & 3′ His-tag added BLFS38 2480 — 4281 4478 Gly & 3′ His-tag added BLFS39 2481 4732 4356 4553 Native signal peptide removed; MetGly & 3′ His- tag added BLFS41 2482 4781 4427 4624 Native signal peptide removed; MetGly & 3′ His- tag added BLFS42 2483 4763 4404 4601 Native signal peptide removed; MetGly & 3′ His- tag added BLFS43 2484 4683 4299 4496 Native signal peptide removed; MetGly & 3′ His- tag added BLFS44 2485 4713 4335 4532 Native signal peptide removed; Met & 3′ His-tag added BLFS45 2486 4745 4374 4571 Native signal peptide removed; MetGly & 3′ His- tag added BLFS46 2487 4694 4311 4508 Native signal peptide removed; Met & 3′ His-tag added BLFS47 2488 4686 4302 4499 Native signal peptide removed; MetGly & 3′ His- tag added BLFS48 2489 4699 4317 4514 Native lipid anchor removed; added MetGly; 3′ His-tag BLFS49 2490 4790 4438 4635 Native lipid anchor removed; added MetGly; 3′ His-tag BLFS50 2491 4754 4388 4585 Native signal peptide removed; MetGly & 3′ His- tag added BLFS51 2492 4692 4309 4506 Native signal peptide removed; MetGly & 3′ His- tag added BLFS52 2493 4737 4364 4561 Native signal peptide removed; Met & 3′ His-tag added BLFS53 2494 — 4433 4630 Gly & 3′ His-tag added BLFS55 2495 4748 4378 4575 Native signal peptide removed; Met & 3′ His-tag added BLFS56 2496 4651 4257 4454 Native signal peptide removed; Met & 3′ His-tag added BLFS57 2497 4738 4365 4562 Native signal peptide removed; Met & 3′ His-tag added BLFS58 2498 4743 4371 4568 Native signal peptide removed; MetGly & 3′ His- tag added BLFS59 2499 4674 4290 4487 Native signal peptide removed; Met & 3′ His-tag added BLFS60 2500 4730 4353 4550 Native signal peptide removed; Met & 3′ His-tag added BLFS61 2501 — 4325 4522 3′ His-tag added BLFS62 2502 4691 4308 4505 Native signal peptide removed; MetGly & 3′ His- tag added BLFS63 2503 4697 4315 4512 Native signal peptide removed; Met & 3′ His-tag added BLFS64 2504 4654 4260 4457 Native signal peptide removed; Met & 3′ His-tag added BLFS65 2505 4719 4341 4538 Native signal peptide removed; MetGly & 3′ His- tag added BLFS67 2506 4782 4428 4625 Native signal peptide removed; Met & 3′ His-tag added BLFS68 2507 4696 4314 4511 Native signal peptide removed; MetGly & 3′ His- tag added BLFS69 2508 4655 4261 4458 Native signal peptide removed; Met & 3′ His-tag added BLFS70 2509 — 4269 4466 3′ His-tag added BLFS72 2510 — 4278 4475 Gly & 3′ His-tag added BLFS73 2511 — 4312 4509 3′ His-tag added BLFS74 2512 4758 4396 4593 Native signal peptide removed; Met & 3′ His-tag added BLFS75 2513 4688 4304 4501 Native signal peptide removed; MetGly & 3′ His- tag added BLFS76 2514 4710 4329 4526 Native signal peptide removed; Met & 3′ His-tag added BLFS77 2515 4668 4279 4476 Native signal peptide removed; MetGly & 3′ His- tag added BLFS79 2516 4665 4275 4472 Native signal peptide removed; MetGly & 3′ His- tag added BLFS80 2517 4709 4328 4525 Native signal peptide removed; Met & 3′ His-tag added BLFS82 2518 — 4282 4479 Gly & 3′ His-tag added BLFS83 2519 4750 4380 4577 Native signal peptide removed; Met & 3′ His-tag added BLFS84 2520 4663 4273 4470 Native signal peptide removed; MetGly & 3′ His- tag added BLFS85 2521 4765 4406 4603 Native signal peptide removed; Met & 3′ His-tag added BLFS86 2522 4724 4346 4543 Native signal peptide removed; Met & 3′ His-tag added BLFS87 2523 4648 4254 4451 Native signal peptide removed; Met & 3′ His-tag added BLFS88 2524 4786 4432 4629 Native signal peptide removed; MetGly & 3′ His- tag added BLFS89 2525 4652 4258 4455 Native signal peptide removed; Met & 3′ His-tag added BLFS90 2526 — 4376 4573 3′ His-tag added BLFS91 2527 4703 4321 4518 Native signal peptide removed; MetGly & 3′ His- tag added BLFS92 2528 4769 4412 4609 Native signal peptide removed; Met & 3′ His-tag added BLFS93 2529 4756 4391 4588 Native signal peptide removed; Met & 3′ His-tag added BLFS94 2530 4687 4303 4500 Native signal peptide removed; Met & 3′ His-tag added BLFS95 2531 4740 4367 4564 Native signal peptide removed; Met & 3′ His-tag added BLFS96 2532 — 4419 4616 3′ His-tag added BLFS97 2533 — 4443 4640 Gly & 3′ His-tag added BLFS98 2534 4783 4429 4626 Native signal peptide removed; Met & 3′ His-tag added BLFS99 2535 4729 4352 4549 Native signal peptide removed; Met & 3′ His-tag added BLFS100 2536 4746 4375 4572 Native signal peptide removed; Met & 3′ His-tag added BLFS101 2537 4780 4425 4622 Native signal peptide removed; MetGly & 3′ His- tag added BLFS102 2538 4725 4347 4544 Native signal peptide removed; MetGly & 3′ His- tag added BLFS103 2539 — 4331 4528 Gly & 3′ His-tag added BLFS104 2540 4768 4410 4607 Native lipid anchor removed; added MetGly; 3′ His-tag BLFS105 2541 4739 4366 4563 Native signal peptide removed; Met & 3′ His-tag added BLFS106 2542 — 4411 4608 Gly & 3′ His-tag added BLFS107 2543 4773 4416 4613 Native signal peptide removed; Met & 3′ His-tag added BLFS108 2544 — 4399 4596 Gly & 3′ His-tag added BLFS109 2545 4779 4424 4621 Native signal peptide removed; Met & 3′ His-tag added BLFS110 2546 4702 4320 4517 Native lipid anchor removed; added MetGly; 3′ His-tag BLFS111 2547 4793 4441 4638 Native signal peptide removed; MetGly & 3′ His- tag added BLFS112 2548 — 4349 4546 Gly & 3′ His-tag added BLFS113 2549 4672 4287 4484 Native signal peptide removed; Met & 3′ His-tag added BLFS114 2550 4705 4323 4520 Native signal peptide removed; Met & 3′ His-tag added BLFS115 2551 4716 4338 4535 Native signal peptide removed; Met & 3′ His-tag added BLFS116 2552 — 4330 4527 3′ His-tag added BLFS118 2553 4767 4409 4606 Native signal peptide removed; MetGly & 3′ His- tag added BLFS119 2554 4741 4369 4566 Native signal peptide removed; Met & 3′ His-tag added BLFS120 2555 — 4361 4558 3′ His-tag added BLFS121 2556 4707 4326 4523 Native signal peptide removed; Met & 3′ His-tag added BLFS122 2557 — 4280 4477 Gly & 3′ His-tag added BLFS123 2558 — 4426 4623 3′ His-tag added BLFS124 2559 4675 4291 4488 Native signal peptide removed; MetGly & 3′ His- tag added BLFS125 2560 4723 4345 4542 Native signal peptide removed; Met & 3′ His-tag added BLFS126 2561 4795 4445 4642 Native signal peptide removed; Met & 3′ His-tag added BLFS127 2562 4695 4313 4510 Native signal peptide removed; MetGly & 3′ His- tag added BLFS128 2563 — 4332 4529 3′ His-tag added BLFS129 2564 — 4383 4580 Gly & 3′ His-tag added BLFS130 2565 — 4268 4465 Gly & 3′ His-tag added BLFS131 2566 4693 4310 4507 Native signal peptide removed; MetGly & 3′ His- tag added BLFS132 2567 4689 4305 4502 Native signal peptide removed; Met & 3′ His-tag added BLFS133 2568 4798 4448 4645 Native signal peptide removed; Met & 3′ His-tag added BLFS134 2569 4679 4295 4492 Native signal peptide removed; Met & 3′ His-tag added BLFS135 2570 4717 4339 4536 Native signal peptide removed; MetGly & 3′ His- tag added BLFS136 2571 4662 4272 4469 Native signal peptide removed; Met & 3′ His-tag added BLFS137 2572 4728 4351 4548 Native signal peptide removed; MetGly & 3′ His- tag added BLFS138 2573 — 4385 4582 Gly & 3′ His-tag added BLFS139 2574 4721 4343 4540 Native signal peptide removed; Met & 3′ His-tag added BLFS140 2575 4761 4402 4599 Native signal peptide removed; MetGly & 3′ His- tag added BLFS141 2576 4785 4431 4628 Native signal peptide removed; MetGly & 3′ His- tag added BLFS142 2577 4755 4390 4587 Native signal peptide removed; MetGly & 3′ His- tag added BLFS143 2578 4770 4413 4610 Native signal peptide removed; Met & 3′ His-tag added BLFS144 2579 4718 4340 4537 Native signal peptide removed; Met & 3′ His-tag added BLFS145 2580 4715 4337 4534 Native signal peptide removed; Met & 3′ His-tag added BLFS146 2581 4797 4447 4644 Native signal peptide removed; Met & 3′ His-tag added BLFS147 2582 4764 4405 4602 Native signal peptide removed; Met & 3′ His-tag added BLFS148 2583 4711 4333 4530 Native signal peptide removed; MetGly & 3′ His- tag added BLFS149 2584 4789 4437 4634 Native signal peptide removed; MetGly & 3′ His- tag added BLFS150 2585 — 4421 4618 Gly & 3′ His-tag added BLFS151 2586 4664 4274 4471 Native signal peptide removed; MetGly & 3′ His- tag added BLFS152 2587 4791 4439 4636 Native signal peptide removed; Met & 3′ His-tag added BLFS153 2588 4734 4359 4556 Native signal peptide removed; MetGly & 3′ His- tag added BLFS154 2589 4792 4440 4637 Native signal peptide removed; Met & 3′ His-tag added BLFS155 2590 4659 4266 4463 Native signal peptide removed; Met & 3′ His-tag added BLFS156 2591 4660 4267 4464 Native signal peptide removed; Met & 3′ His-tag added BLFS157 2592 4747 4377 4574 Native signal peptide removed; Met & 3′ His-tag added BLFS158 2593 — 4389 4586 Gly & 3′ His-tag added BLFS159 2594 — 4289 4486 Gly & 3′ His-tag added PUB6 2595 — 4372 4569 3′ His-tag added PUB23 2596 — 4355 4552 Gly & 3′ His-tag added PUB31 2597 — 4398 4595 3′ His-tag added PUB65 2598 4775 4418 4615 Native signal peptide removed; Met & 3′ His-tag added ZLFS1 2599 — 4381 4578 3′ His-tag added ZLFS2 2600 4667 4277 4474 Native signal peptide removed; Met & 3′ His-tag added ZLFS3 2601 4649 4255 4452 Native signal peptide removed; Met & 3′ His-tag added ZLFS5 2602 4647 4253 4450 Native signal peptide removed; Met & 3′ His-tag added ZLFS6 2603 — 4283 4480 Gly & 3′ His-tag added ZLFS7 2604 4700 4318 4515 Native signal peptide removed; Met & 3′ His-tag added ZLFS8 2605 4777 4422 4619 Native lipid anchor removed; added MetGly; 3′ His-tag ZLFS9 2606 4794 4444 4641 Native signal peptide removed; MetGly & 3′ His- tag added ZLFS10 2607 4766 4407 4604 Native signal peptide removed; Met & 3′ His-tag added ZLFS11 2608 4677 4293 4490 Native signal peptide removed; Met & 3′ His-tag added ZLFS12 2609 — 4360 4557 Gly & 3′ His-tag added ZLFS13 2610 4706 4324 4521 Native signal peptide removed; Met & 3′ His-tag added ZLFS14 2611 — 4436 4633 Gly & 3′ His-tag added ZLFS15 2612 4708 4327 4524 Native signal peptide removed; Met & 3′ His-tag added ZLFS16 2613 4714 4336 4533 Native signal peptide removed; MetGly & 3′ His- tag added ZLFS17 2614 4731 4354 4551 Native signal peptide removed; MetGly & 3′ His- tag added ZLFS18 2615 — 4387 4584 3′ His-tag added ZLFS19 2616 4658 4264 4461 Native lipid anchor removed; added MetGly; 3′ His-tag ZLFS21 2617 4670 4285 4482 Native signal peptide removed; Met & 3′ His-tag added ZLFS22 2618 4685 4301 4498 Native signal peptide removed; Met & 3′ His-tag added ZLFS23 2619 4759 4397 4594 Native signal peptide removed; Met & 3′ His-tag added ZLFS24 2620 4676 4292 4489 Native signal peptide removed; MetGly & 5' His- tag added ZLFS25 2621 4680 4296 4493 Native signal peptide removed; Met & 3′ His-tag added ZLFS27 2622 4760 4401 4598 Native lipid anchor removed; added MetGly; 3′ His-tag ZLFS28 2623 4656 4262 4459 Native signal peptide removed; Met & 3′ His-tag added ZLFS29 2624 4733 4358 4555 Native signal peptide removed; Met & 3′ His-tag added ZLFS30 2625 4666 4276 4473 Native signal peptide removed; MetGly & 3′ His- tag added ZLFS31 2626 4704 4322 4519 Native signal peptide removed; MetGly & 5' His- tag added ZLFS32 2627 4727 4350 4547 Native lipid anchor removed; added Met; 3′ His-tag ZLFS33 2628 4742 4370 4567 Native signal peptide removed; Met & 3′ His-tag added ZLFS34 2629 4796 4446 4643 Native signal peptide removed; Met & 3′ His-tag added ZLFS35 2630 4749 4379 4576 Native signal peptide removed; Met & 3′ His-tag added ZLFS36 2631 — 4400 4597 Gly & 3′ His-tag added ZLFS37 2632 — 4307 4504 3′ His-tag added ZLFS38 2633 — 4393 4590 Gly & 3′ His-tag added ZLFS39 2634 — 4357 4554 Gly & 3′ His-tag added ZLFS42 2635 4661 4271 4468 Native signal peptide removed; MetGly & 3′ His- tag added ZLFS44 2636 4736 4363 4560 Native signal peptide removed; Met & 3′ His-tag added ZLFS45 2637 4735 4362 4559 Native signal peptide removed; MetGly & 3′ His- tag added ZLFS47 2638 4684 4300 4497 Native signal peptide removed; MetGly & 3′ His- tag added ZLFS48 2639 4762 4403 4600 Native signal peptide removed; MetGly & 3′ His- tag added ZLFS49 2640 4720 4342 4539 Native signal peptide removed; Met & 3′ His-tag added ZLFS51 2641 — 4270 4467 3′ His-tag added “Polyp.”-Polypeptide. “Polyn.”-Polynucleotide

In most cases, following the optimization, the synthesized sequences exhibit at least 70% global identity to the curated sequences from which they were obtained. In several cases replacing the native signal peptide resulted in a polynucleotide and/or polypeptide having less than 7000 global identity to their parent sequence. For the genes presented in Table 20, the optimized synthesized polypeptide sequences exhibit less than 70% global identity to the curated sequences from which they were obtained, due to a replacement of relatively long signal sequence. For example, the polypeptide of SEQ ID NO:4502 exhibits only 48.65% o global identity to SEQ ID NO:2567 which is the original curated sequence of BLFS132. These polypeptides, however, comprise the core amino acid sequence of the parent polypeptides.

TABLE 20 Optimized polypeptides having less that 70% global identity to the parent not optimized polypeptide Native Synthesized Polypeptide Polypeptide Gene Name SEQ ID NO. SEQ ID NO. ZLFS42 2635 4468 ZLFS47 2638 4497 BLFS132 2567 4502 BLFS10 2453 4503 BLFS86 2522 4543 BLFS102 2538 4544 BLFS52 2493 4561 BLFS119 2554 4566 BLFS9 2452 4591 BLFS36 2478 4612 BLFS20 2463 4614 BLFS133 2568 4645

Example 15: Purification of Proteins Expressed in Bacterial Cells

Transformation of Bacterial Cells with the Polynucleotides Encoding the Polypeptides Having the Ability to Kill or Inhibit the Development of Fusarium

Genes encoding unknown toxin candidate proteins of the present invention were cloned in pET22/T7-lac promoter-based vector and coding DNA sequence was confirmed by sequencing. pET-based expression vectors were transformed into BL21(DE3) E. coli host using heat shock method. After overnight growth in Terrific Broth (TB) medium at 37° C. in the presence of Ampicillin (100 μg/mL), 5 mL starter cultures were used to inoculate 100 mL TB culture at OD600 0.03 in 1 L flat bottom flask. The cultures were allowed to grow until OD600 ˜0.5 (2-3 hours at 37° C. with 250 rpm). The incubator shaker temperature was reduced to 22° C. and Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM. The cultures were incubated further for 15 to 18 hours for target protein expression and cells were harvested by centrifuging at 4,000 rpm/4° C./10 minutes. The cell pellet was washed with cold water and stored at −80° C. until used for protein purification.

Bacterial cell pellet was lysed using B-PER™ Bacterial Protein Extraction Reagent (Thermo Fisher Scientific, catalog number: 78248) at room temperature for 1 hour. The supernatant fraction (containing soluble protein) was clarified by centrifugation at 16000 rcf/4° C./20 minutes.

Purification of Expressed Recombinant Polypeptides:

Soluble fractions—The supernatant fraction containing soluble protein was incubated with Ni-NTA beads (washed with binding buffer prior to addition of supernatant fraction: 50 mM potassium phosphate pH 8.0, 600 mM NaCl and 20 mM imidazole) for 1 hour at 4° C. on a rotatory shaker with gentle shaker speed. The Ni-NTA-protein bound beads were collected by centrifugation at 800 rcf/4° C./3 minutes. The Ni-NTA-protein bound beads were washed with washing buffer (50 mM potassium phosphate pH 8.0, 600 mM NaCl, 0.5% Triton and 20 mM imidazole) for 3 times. The bound proteins were eluted with elution buffer (50 mM potassium phosphate pH 8.0, 600 mM NaCl and 250 mM imidazole). The salts in the eluted proteins were removed by dialysis. Samples were loaded on a Pur-A+Lyzer tubes (catalog number: PurR35100) and placed in a beaker containing 0.5 liter of sterile dialysis buffer (16.5 mM Potassium phosphate buffer pH 5.0) for 1 night/weekend in the refrigerator at 8° C. SDS-PAGE analysis was used to quantify protein using known concentrations of bovine serum albumin (BSA) as standard. The known concentrations of toxin candidates were used for bioassay.

Example 16: Effect of Isolated Proteins on Fungal Growth

The spore assay is an in vitro bioassay in a 96 wells plate format, implementing spores from a Fusarium verticillioides (Fv) GFP-labeled strain and the candidate protein purified from bacterial cells as described hereinabove. Mycelia growth of fungi treated with the protein was compared to the growth of untreated fungi and growth inhibition was quantified by detection of GFP fluorescence (475 nm excitation, 509 nm emission). Optical Density (OD) of 620 nm is also measured for QA purposes.

Fresh spores were prepared, quantified and dispensed in each well (500 spores) in 50 μl 2×PDB and incubated for 2-3 hours at 25° C. before protein addition.

Purified clean proteins prepared as described hereinabove were quantified and diluted with buffer phosphate pH 5.0 at the desired concentration and added to each assay well (up to 40 μM) for a final reaction volume of 100 μl. The plates are incubated for 72 hr at 25° C. and responses were measured every 24 hr.

Each protein treatment in each plate was compared to the corresponding spore growth in untreated control by t-test a=0.05 one tailed

Table 21 summarizes the results of two independent assays using purified proteins from bacterial cells over-expressing the polypeptides of some embodiments of the invention.

TABLE 21 Inhibition of Fusarium mycelia growth by proteins of the invention isolated from bacteria First assay Second assay Gene Name General annotation % Inhibition ** % Inhibition ** ZLFS42 Killer toxin like 90% (72 hr) 86% (72 hr) 20 μM 25 μM ZLFS48 Antimicrobial protein MiAMP1 56% (48 hr) 30% (72 hr) 25 μM 20 μM BLFS129 Alginate lyase 85% (48 hr) 69% (72 hr) 6.4 μM 4 μM BLFS139 Peptidoglycan endopeptidase 72% (48 hr) 64% (48 hr) 14 μM 6 μM BLFS128 Peptidoglycan hydrolase flgj 37% (48 hr) 52% (48 hr) 14 μM 3 μM BLFS70 Hypothetical protein 36% (48 hr) 52% (48 hr) 2 μM 3 μM BLFS111 Hypothetical protein) 20% (72 hr) 63% (48 hr) 4.5 μM 10 μM BLFS157 Hypothetical protein 37% (48 hr) 41% (48 hr) 12 μM 10 μM BLFS101 Metalloprotease loiP 47% (24 hr) 70% (48 hr) 25 μM 25 □M BLFS63 n-acetylmuramoyl-1-alanine 44% (48 hr) 55% (48 hr) amidase amib 39 μM 2 μM ZLFS1 Arthropod defensin 29% (48 hr) 32% (48 hr) 13 μM 27 μM BLFS155 Mannose-binding protein 45% (48 hr) 70% (48 hr) 25 μM 44 μM BLFS7 Peptidase C11 clostripain 65% (48 hr) 36% (48 hr) <1 μM 5 μM BLFS140 Uncharacterized lipoprotein ybbd 75% (48 hr) 63% (48 hr) 9 μM 40 μM BLFS103 Toxin tccc3 67% (48 hr) 56% (48 hr) 3 μM 3.75 μM BLFS46 Lysm domain protein 46% (48 hr) 32% (48 hr) 18 μM 40 μM BLFS115 Probable periplasmic serine 39% (48 hr) 39% (48 hr) endoprotease DegP-like 23 μM 40 μM BLFS47 Hypothetical protein 11% (72 hr) 25% (48 hr) 40 μM 0.5 μM BLFS87 Endoglucanase b 25% (48 hr) 27% (48 hr) 40 μM 40 μM BLFS102 Hypothetical protein 30% (48 hr) 44% (48 hr) 40 μM 40 μM BLFS42 Chitin-binding protein 21% (72 hr) 60% (48 hr) 25 μM 40 μM ZLFS29 Beta-barrel assembly-enhancing 36.5% (48 hr) 44% (48 hr) protease [bepA]. 40 μM 40 μM BLFS154 Peptidase s1 52% (48 hr) 33% (48 hr) 40 μM 40 μM BLFS62 Murein hydrolase activator nlpd 53% (24 hr) 27% (48 hr) 40 μM 40 μM BLFS19 Peptidase p60 31% (48 hr) 29% (48 hr) 40 μM 40 μM ZLFS16 d-alanyl-d-alanine 11% (24 hr) 36% (24 hr) carboxypeptidase dacd 41 μM 38 μM ZLFS19 Lipoprotein nlpd/lppb homolog 21% (48 hr) 33% (24 hr) 38 μM 37 μM BLFS1 Antifungal protein 57% (48 hr) 31% (48 hr) 40 μM 40 μM BLFS9 Oxidoreductase 21% (48 hr) 15% (48 hr) 25 μM 18 μM BLFS80 Hypothetical protein 33% (48 hr) 43% (48 hr) 40 μM 40 μM BLFS123 Peptidase c1 17% (72 hr) 1% (48 hr) 20 μM 20 μM BLFS113 Transpeptidase 11% (72 hr) 19% (48 hr) 40 μM 25 μM BLFS121 Glutamate carboxypeptidase 26% (48 hr) 14% (24 hr) 40 μM 25 μM BLFS142 Uncharacterized protein ykvt 34% (48 hr) 31% (48 hr) 21 μM 42 μM ZLFS5 Hypothetical protein 29% (48 hr) 15% (48 hr) 20 μM 40 μM ZLFS32 Endoglucanase 29% (48 hr) 16% (48 hr) 20 μM 40 μM ZLFS22 Hypothetical protein 95% (72 hr) 11% (24 hr) 40 μM 40 μM ZLFS51 Glycosyl hydrolase 36% (24 hr) 3% (24 hr) 40 μM 40 μM ZLFS27 Lytic transglycosylase 28% (48 hr) 87% (48 hr) 40 μM 20 μM ZLFS47 Antimicrobial protein MiAMP1 5% (48 hr) 56% (48 hr) 20 μM 25 μM ZLFS45 Oxidoreductase 63% (24 hr) 81% (72 hr) 20 μM 40 μM ** = Best inhibition value scored at defined time and concentration; α = 0.05 and % inhibition >10%

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A plurality of bacteria, each bacterium comprising a recombinant polynucleotide optimized for expression of at least one recombinant polypeptide in the bacterium, wherein the at least one expressed recombinant polypeptide is at least 70% identical to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2635 or a fragment thereof, wherein said at least one expressed recombinant polypeptide is effective in inhibiting the growth and/or development of at least one plant pathogenic fungi and/or Oomycetes.

2. The plurality of bacteria of claim 1, wherein the at least one expressed recombinant polypeptide comprises its natural signal peptide or a part thereof.

3. The plurality of bacteria of claim 1, wherein the at least one expressed recombinant polypeptide is devoid of its natural signal peptide or a part thereof.

4. The plurality of bacteria of claim 3, wherein the at least one expressed recombinant polypeptide comprises a non-native signal peptide.

5. The plurality of bacteria of claim 4, wherein the origin of the non-native signal peptide is selected from the group consisting of a plant origin and a bacterium species other than the species of said plurality of bacteria.

6. The plurality of bacteria of claim 1, wherein the at least one expressed recombinant polypeptide or the fragment thereof further comprises a tag sequence.

7. The plurality of bacteria of claim 3, wherein the at least one expressed recombinant polypeptide or the fragment thereof further comprises a tag sequence.

8. The plurality of bacteria of claim 7, wherein the at least one expressed recombinant polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4468.

9. A recombinant polypeptide isolated from at least one bacterium of the plurality of bacteria of claim 1, wherein the recombinant polypeptide is at least 70% identical to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2635 or a fragment thereof, and wherein said expressed recombinant polypeptide is effective in inhibiting the growth and/or development of at least one plant pathogenic fungi and/or Oomycetes.

10. The recombinant polypeptide of claim 9, wherein said recombinant polypeptide comprises its natural signal peptide or a part thereof.

11. The recombinant polypeptide of claim 9, wherein said recombinant polypeptide is devoid of its natural signal peptide or a part thereof.

12. The recombinant polypeptide of claim 11, wherein said recombinant polypeptide comprises a non-native signal peptide.

13. The recombinant polypeptide of claim 12, wherein the non-native signal peptide is of an origin selected from the group consisting of a plant origin and a bacterium species other than the species of said plurality of bacteria.

14. The recombinant polypeptide of claim 9, wherein said recombinant polypeptide or the fragment thereof comprises a tag sequence.

15. The recombinant polypeptide of claim 11, wherein said recombinant polypeptide or fragment thereof comprises a tag sequence.

16. The recombinant polypeptide of claim 15, wherein said recombinant polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4468.

17. A fungicidal composition comprising the plurality of bacteria of claim 1.

18. The fungicidal composition of claim 17, wherein said fungicidal composition further comprises at least one agriculturally compatible agent selected from the group consisting of a carrier, a stabilizer, a diluent, a surfactant, a mineral and an adjuvant.

19. A fungicidal composition comprising the recombinant polypeptide of claim 9.

20. The fungicidal composition of claim 19, wherein said fungicidal composition further comprises at least one agriculturally compatible agent selected from the group consisting of a carrier, a stabilizer, a diluent, a surfactant, a mineral and an adjuvant.

Patent History
Publication number: 20230287446
Type: Application
Filed: Dec 13, 2022
Publication Date: Sep 14, 2023
Applicant: EVOGENE LTD. (Rehovot)
Inventors: Tidhar TURGEMAN (Givatayim), Ada VITERBO-FAINZILBER (Rehovot), Eyal EMMANUEL (Rehovot)
Application Number: 18/065,288
Classifications
International Classification: C12N 15/82 (20060101); A01N 63/50 (20060101); C07K 14/415 (20060101);