IDENTIFICATION OF A XANTHOMONAS EUVESICATORIA RESISTANCE GENE FROM PEPPER (CAPSICUM ANNUUM) AND METHOD FOR GENERATING PLANTS WITH RESISTANCE
The present invention relates to the identification of the xcv-1 gene, which is responsible for a recessive resistance to Xanthomonas euvesicatoria, by genetic mapping-based cloning from Capsicum annuum. In addition, the invention relates to methods for generating plants resistant to an abiotic or biotic factor, in particular to Xanthomonas euvesicatoria, and the plants themselves, in particular tomato plants.
The present invention relates to the identification of the xcv-1 gene, which is responsible for a recessive resistance to Xanthomonas euvesicatoria, by genetic mapping-based cloning from Capsicum annuum. In addition, the invention relates to methods for generating resistant plants, in particular plants resistant to Xanthomonas euvesicatoria.
BACKGROUND OF THE INVENTION AND PRIOR ARTIt is well-known that in arable plant production, sensitive crops are infested by various pathogens (viruses, bacteria, fungi etc.) the serious consequences of which include yield reduction or yield loss. Therefore, one fundamental requirement for advanced and competitive plant varieties is to show resistance to pests and pathogens causing major yield losses. Resistant plants allow cheaper and more environmentally friendly production because no spray liquids containing hazardous substances and toxins are released into nature. The environmentally friendly production technology of resistant plants also increases yield safety; produces of higher quality and quality can be harvested at lower costs.
Identification, isolation and characterisation of the genes providing resistance is of vital importance for both theory and practice: the process can be understood by exploring the genes involved in the infection processes, their products and the functions thereof, and this forms the indispensable basis for developing control strategies.
As regards the molecular processes of plant protection, two basic mechanisms can be distinguished. The first one is dominant resistance and the second one is recessive resistance. In case of a dominant protective mechanism, a signal molecule (typically, a protein) from the pathogen initiates a self destruction process (apoptosis) resulting in the death of the infected cells and their neighbours thereby halting the spreading of the pathogen (Pontier D. et al., C R Acad Sci III. 321:721-34, 1998.). The result of the cell destruction, i.e., programmed cell death, is a dried and discoloured necrotic patch, which is the manifestation of the so-called hypersensitive reaction (HR) (Klement Z. et al., Phytopathology 54: 474-477, 1964). Dominant resistance is race-specific and can be easily abolished as new races may break the resistance.
In case of recessive resistance, a mutation causes a loss of a function that is indispensable for the development of virulence. As yet, the steps of the development of recessive resistance, as well as the functions thereof and the plant genes of such functions are mostly unknown, however, it was found that more than one gene is involved in the development of resistance on the part of both the bacterium and plant host. Recessive resistance genes can be identified when a difference exists between the genes involved in the process induced by the pathogen and causing the disease (virulence) and the wild-type genes. Genes providing recessive resistance have been identified from a few plants, including among others the RRS1-R gene from Arabidopsis thaliana (Deslandes L. et al., PNAS 99: 2404-2409, 2002) and a gene against Xanthomonas oryzae from rice (Iyer-Pacuzzi A. S., Pathosystem. Mol. Plant Microb. Interaction 20:731-739, 2007). A number of genes providing dominant resistance have been identified from pepper on the basis of mutant phenotypes, but no genes providing recessive resistance. In general, recessive resistance is not race-specific and is more difficult; therefore, it is more stable.
In the field growing of edible and spice pepper, the bacterium Xanthomonas campestris pv. vesicatoria (Xcv), recently renamed as Xanthomonas euvesicatoria (Xe) causes the most significant damage (Jones J. B. et al., System. Appl. Microbiol. 27: 755-762, 2004). The Xe bacterium is mediated mostly by water and enters the plant through wounds and leaf gaps. Under warm and moist climatic conditions, the bacterium spreads rapidly. The symptoms of the disease mostly appear on the leaves. Scar-like patches develop on the back of the leaves and later become necrotic areas on the face of the leaves. The infected leaves of sensitive plants die and fall off within one to two weeks, and the yield is burnt by the sun.
Similar to a group of plant and animal bacteria, Xanthomonas euvesicatoria is also capable of growing so-called pili through the Type Three Secretion System (TTSS), and the ends of these pili extend until the eukaryotic cell membrane. The pilus is permeable for the effector molecules of the bacterium. One or more of the effector molecules create a so-called translocon in the cell membrane through which the effector molecules enter the eukaryotic cytoplasm; in several cases, they also enter the nucleus from the cytoplasm if they comprise a Nuclear Localization Signal (NLS). For their growth, the bacteria use the nutrient molecules present in the plant cells, which are released upon the loss of integrity of the plant cells. Disintegration of the cells is induced by the effector molecules introduced through the Type Three Secretion System of Xe via an infection mechanism the details of which are yet unclear. The proliferation of the bacteria damages plant tissue to such an extent as to cause the majority of the leaves to fall off and the plant to dry out sooner or later.
The complete genome of Xanthomonas euvesicatoria (X. campestris pv. vesicatoria strain 85-10) has been determined (Thieme F. et al., J. Bacteriol. 187:7254-7266, 2005), and the genes of the effector proteins involved in the induction of a dominant hypersensitive reaction from pepper have been identified. Genes for recessive resistance to Xanthomonas euvesicatoria have not been identified from pepper yet.
The literature describes a few pepper varieties resistant to Xe. These plants carry resistance genes including but not limited to Bs1 (Cook A. A. and Stall R. E., Plant Dis. 53:1060-1062, 1963), Bs2 (Cook A. A. and Guevara Y. G., Plant Dis. 68:329-330, 1984), Bs3 (Kim B. S. and Hartmann R. W., Plant Dis. 69:233-235, 1985), Bs4 (Hibberd et al., Phytopathology 77:1304-1307, 1987), bs5 (Jones J. B. et al., System. Appl. Microbiol. 27:755-762, 2004) and bs6 (Vallejos C. E. et al., Theor. Appl. Genet. 121:37-46, 2010). The latter two—bs5 and bs6—are recessive types of resistance genes.
Despite the existing resistant pepper varieties, there is still an extreme need for pepper varieties resistant to Xanthomonas species and for other plant varieties that are resistant to biotic or abiotic factors.
The objective of the present study is to identify and isolate a gene from pepper (Capsicum annuum) providing recessive resistance to Xanthomonas euvesicatoria, which can be used to develop single or double (pyramided) resistance varieties—mostly to Xanthomonas sp., but presumably to other biotic or abiotic factors as well—in sensitive pepper species and other plant species such as tomato, potato, rice, citruses, banana, etc.
SUMMARY OF THE INVENTIONThe above objective could be achieved by the present invention. From a Capsicum annuum carrying a recessive resistance, a gene designated as xcv-1 was isolated using genetic map-based cloning. The sequence of the isolated gene was determined (SEQ ID NO:37) and it was found that the xcv-1 protein (SEQ ID NO:38) encoded by the gene comprises a double Leu deletion at the locations corresponding to positions 87 and 88 of the wild-type Xcv-1 protein (SEQ ID NO:42). The mutant xcv-1 protein is a tail-anchored (TA) transmembrane (TM) protein, more specifically a CYSTM protein, which carries the double leucine deletion in its cysteine-rich transmembrane region (hereinafter referred to as ‘CYSTM region’). This CYSTM region shows structural relatedness to the CYSTM region of other known transmembrane proteins (Venancio T. M. and Aravind L., Bioinformatics 26:149-152, 2010) in that it is a common feature that they are rich in cysteine (comprising at least 3 cysteines), that they are boardered by an amino acid with negative charge (aspartic acid or glutamic acid) or a polar amino acid (asparagine) in position 4 from the C-terminus of the protein, and that the Asp, Glu or Asn is preceded by two hydrophobic amino acids (here: leucine), and less frequently, these positions contain isoleucine, methionine, tryptophan, glycine, alanine, threonine, phenylalanine, valine and cysteine.
It is interesting to note that certain fungi (e.g., Schizosaccharomyces pombe, Saccharomyces cerevisiae) which are resistant to certain abiotic factors such as UV radiation, and certain drugs (e.g., canavanine), lose such resistance and their sporulation capacity if a CYSTM-type protein loses function as a result of a gene mutation (Lee J. K. et al., Biochem. Biophys, Res. Comm. 202:1113-1119, 1994; Lee, J. K. et al., Mol. Gen. Genet. 246:663-670, 1995; Venancio T. M. et al., Mol Biosyst. 6:175-181, 2010; Venancio, T. M. and Aravind, L. Bioinformatics 26:149-152, 2010). Similar to fungi, Arabidopsis thaliana plants also suffer severe disturbances in megasporogenesis in case of a loss of function in its genes homologous to the above CYSTM proteins (WIH1, WIH2 double mutation) (Lieber, D. et al., Current Biology 21:1009-1017, 2011).
It was found that by removing two amino acids from the C-terminal CYSTM region—preferably those in positions 5 and 6 from the C-terminus—of a protein homologous to the wild-type Xcv-1 protein but derived from a plant organism other than pepper (for example, tomato), advantageous properties, primarily recessive resistance can be induced in the plant organism. The deletion of the two codons, i.e., 6 base pairs, encoding these two amino acids in the CYSTM region—which include but is not limited to leucine, isoleucine, methionine, tryptophan and cysteine—is referred to as ‘the desired 6-bp deletion’.
Without being limited to any theory regarding the development of resistance, it is likely that a double Leu deletion in the CYSTM region (the last 13 amino acids in the C-terminus of the protein) of the mutant xcv-1 protein encoded by the xcv-1 gene prevents or reduces the entry of the effector molecules of bacteria with type three secretion system into plant cells. This hypothesis is preliminaryly substantiated by the result of double resistant papper lines carrying Bs2 and xcv-1 in homozygous configuration. These plants upon infection with AvrBs2 containing Xe do not show the HR phenotype characteristic of AvrBs2 effector of the infecting Xe most probably because AvrBs2 is not entering the plant cells, on the other hand the phenotype of this infection very similar to that caused by the xcv-1/xcv-1 containing plants. The above result indicate that xcv-1 is epistatic over Bs2.
The identification and characterization of the xcv1 bacterial spot disease resistance has revealed that this gene contains a six nucleotide in-frame deletion that removes two leucine amino acids from the carboxy terminal portion of the protein. Computational analyses suggest that this protein is a membrane protein with an unknown function. Since this mutation confers resistance to several strains of Xanthomonas that cause disease on pepper and tomato, it would be informative to test whether this mutation affects the type three secretion delivery of type three effector proteins into plant cells. One could use a reporter gene assay to examine the biochemical activity of translational fusion proteins between the N-terminal domains of various type three effector proteins and the reporter gene adenylate cyclase (Direct biochemical evidence for type Ill secretion-dependent translocation of the AvrBs2 effector protein into plant cells. Casper-Lindley C. et al., PNAS 99:8336-8341, 2002). Using this assay, xcv-1 and other plants, including wild-type peppers, tomato, citrus, walnut, lettuce, brassica, soybean, bean, rice, etc., can be tested for their ability to receive type three secreted effector proteins from strains of Xanthomonas euvesicatoria, X. perforans, X. gardneri and other type-three secretion system dependent bacteria. The adenylate cyclase assay will allow a means of monitoring the mechanism of resistance in xcv-1 plants or with combinations of resistance genes.
Accordingly, it was assumed that a transgenic plant having recessive resistance can be generated by the removal of the gene encoding the original CYSTM region through knocking out homologous resident genes of a plant, and by the simultaneous replacement with a mutant CYSTM region comprising a desired 6-bp deletion through transformation. Similarly, it is assumed that if a derivative or derivatives carrying the desired 6-bp deletion is/are generated in a plant by spontaneous or induced mutation of the gene segment encoding the CYSTM region of solitary (one-copy) or multiple-copy (double-, triple-copy etc.) CYSTM proteins, or by nuclease-based “genome editing” methods such as, for example, Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease technique—or CRISPR/Cas nuclease technique in short—, or in any other ways, then plants with recessive resistance may be generated. Presumably, the mutant homologues will show an advantageous property, i.e., will render the plant resistant.
This hypothesis was proven by transgenic technique using Xe-sensitive tomato (Solanum lycopersicum) as follows: recessive resistance to Xe was generated in tomato by inactivating two resident genes (SlXcv-1A, SEQ ID NO:49 and SlXcv-1B, SEQ ID NO:51) homologous to the wild-type Xcv-1 gene and by creating in vitro the 6-bp deletions in the homologous genes at the positions corresponding to the xcv-1 gene (Slxcv-1A, SEQ ID NO:59 and Slxcv-1B, SEQ ID NO:66), followed by the introduction of the mutant genes into the plant cells.
Additionally, without limiting the scope of the invention, three methods using genome editing techniques (TALEN, CRISPR/Cas nuclease and ZFN) are described for creating the desired 6-bp deletion and thereby generating recessive resistance to Xanthomonas species in tomato.
Slxcv-1A and Slxcv-1B, the mutant genes responsible for the resistance, could be isolated from the transgenic tomato plants, their sequences were determined (SEQ ID NO:59 and SEQ ID NO:66, respectively), and they encode proteins Slxcv-1A and Slxcv-1B (SEQ ID NO:60 and SEQ ID NO:67, respectively). The sequences of the latter were compared to the wild-type protein sequences (SEQ ID NO:50 and SEQ ID NO:52), and the deletions were confirmed at the locations corresponding to positions 86 to 87 and 88 to 89.
Accordingly, one object of the present invention is the xcv-1 gene isolated from Capsicum annuum, which is responsible for a recessive resistance to Xanthomonas euvesicatoria and has the nucleotide sequence of SEQ ID NO:37. The present invention also relates to the cDNA of the xcv-1 gene, which has the nucleotide sequence of SEQ ID NO:90.
Another object of the present invention is the xcv-1 CYSTM protein providing the resistance, which is encoded by the xcv-1 gene and its cDNA sequence, and has the amino acid sequence of SEQ ID NO:38, wherein the CYSTM region of the protein carries a double Leu deletion at the locations corresponding to positions 87 and 88 of the wild-type protein of SEQ ID NO:42.
Another object of the present invention is a protein homologous to the xcv-1 CYSTM protein, which comprises a deletion of two amino acids in the CYSTM region in comparison with the CYSTM region of the wild-type homologous protein, and provides resistance. Preferably, the mutant homologous protein carries the deletion of two amino acids in the CYSTM region, at positions 5 and 6 from the C-terminus of the wild-type protein. Preferred examples of such mutant proteins include proteins Slxcv-1A or Slxcv-1B, which have the amino acid sequences of SEQ ID NO:60 or SEQ ID NO:67, respectively.
In addition, the invention relates to the homologues of the xcv-1 gene encoding the above homologous mutant CYSTM proteins. Preferred examples include the Slxcv-1A gene having the nucleotide sequence of SEQ ID NO:59 or its cDNA variant having the nucleotide sequence of SEQ ID NO:88, which encode the Slxcv-1A mutant homologous protein (SEQ ID NO:60), or the Slxcv-1B gene having the nucleotide sequence of SEQ ID NO:66 or its cDNA variant having the nucleotide sequence of SEQ ID NO:89, which encode the Slxcv-1B mutant protein (SEQ ID NO:67).
Furthermore, the present invention relates to engineered nuclease proteins specific to the DNA sequence of a gene homologous to the wild-type Xcv-1 gene (SEQ ID NO:41), which selectively recognise the DNA segment encoding the CYSTM region of the gene homologous to the wild-type Xcv-1 gene, or certain partial sequences thereof. Preferred engineered nuclease proteins include those specific to the DNA sequences of genes SlXcv-1A and SlXcv-1B. Additional preferred engineered nuclease proteins include a ZFN nuclease pair selectively recognising the gene segments represented by SEQ ID NO:86 and 87, or a TALEN nuclease pair selectively recognising the gene segments represented by SEQ ID NO:78 and 79 or 78 and 80, or a sgRNS-CRISPR/Cas nuclease selectively recognising the gene segments represented by SEQ ID NO:81.
The present invention also relates to the genes encoding said engineered nuclease proteins.
The present invention also relates to artificial nucleic acid molecules (amiRNA) for the silencing the SlXcv-1A and SlXcv-1B which are complementer to the CYSTM region of the mRNA of the plant cells. These nucleic acid molecules comprises additional nucleotide sequences for expression. Important to note, that these nucleic acid molecules do not silence those genes carrying the desired 6 bp deletion.
Another object of the present invention is a vector comprising the xcv-1 gene (SEQ ID NO:37), one or more homologues thereof, preferably genes Slxcv-1A (SEQ ID NO:59) and/or Slxcv-1B (SEQ ID NO:66), or the genes of the TALEN, CRISPR/Cas and ZFN nucleases specific to the Xcv-1 genes suitable for genome editing and other nucleic acid molecules described in this invention.
Another object of the invention is a host cell transformed with said vector.
Another object of the present invention is a method for the in vitro preparation of a mutant gene homologous to the xcv-1 gene or its cDNA variant, comprising the steps of: a) identifying a gene homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in a plant; b) preparing in vitro the genomic and cDNA sequences of the gene identified in step a) in the form of a DNA; and c) creating in vitro a deletion of the desired 6 bp in the DNA prepared in step b) in the portion of the gene encoding the CYSTM region. Using the method of the invention, the 6-bp deletion is preferably created in those nucleotides of the CYSTM region of the gene that encode the 5th and 6th amino acids from the C-terminus.
The present invention also relates to mutant plants showing resistance to a biotic or abiotic factor, the genomes of which are modified to contain a 6-bp deletion in the segment encoding the CYSTM region of one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) or its cDNA sequence (SEQ ID NO:91).
Another object of the present invention is a method for generating a transgenic plant resistant to biotic or abiotic factors by transformation, comprising the steps of: a) transforming the cells of a sensitive plant by one or more mutant genes homologous to the xcv-1 gene of the invention in a manner ensuring the functional expression thereof, b) inactivating one or more resident genes homologous to the wild-type Xcv-1 gene, or the mRNA or protein product thereof, in the sensitive transformant plant cells obtained in step a); and c) regenerating the plant from the transformants and selecting the resistant individuals.
Another object of the invention is a method based on genome editing, which comprises a step of creating the desired 6-bp deletion in the wild-type homologous gene using ZFN, TALEN or CRISPR/Cas nucleases specific to the sequence of the wild-type Xcv-1 gene.
Yet another object of the invention is a method based on genome editing, in which the ZFN and TALEN nuclease proteins recognising the DNA sequences homologous and specific to the Xcv-1 gene are introduced into plant-derived host cells using bacteria having type three secretion system but not causing diseases (non-pathogenic bacteria) (see e.g., WO/2005/085417).
Yet another object of the present invention is a plant and its progeny resistant to biotic or abiotic factors, which can be generated by the methods of the invention and carries a deletion of two amino acids in the CYSTM region of one or more of its transmembrane proteins in comparison with the wild-type protein. Preferably, the plant is a tomato plant (Solanum lycopersium), in which recessive resistance to Xanthomonas euvesicatoria has been created.
Furthermore, the invention relates to a method to generate resistant plant by combining (pyramiding) at least two resistance genes against the same pathogene (e.g. Xanthomonas sp.). Accordingly, the present invention relates to a tomato plant containing more than one resistance genes conferring resistance against Xanthomonas euvesicatoria which is generated by one of the procedures described in the invention of which one resistance gene is based on the creation of the desired 6 bp deletion in a gene homologues to xcv-1 and the other resistance gene or genes is/are including but not limited to e.g. Bs2, Bs2, Bs3, Bs4, bs5, bs6 or their combination.
The present invention also relates to a rice plant containing more than one resistance genes conferring resistance against Xanthomonas oryzae pv. oryzae which is generated by one of the procedures described in the invention where said rice plant carries in combination another resistance gene or genes against Xanthomonas oryzae pv. oryzae which is/are including but not limited to e.g. Xa-4+xa-5+Xa-7+xa-13+Xa-21 genes.
The present invention also relates to a citrus plant containing more than one resistance genes conferring resistance against Xanthomonas citri pv. citri, Xanthomonas axonopodis pv. citri which is generated by one of the procedures described in the invention where said citrus plant carries in combination another resistance gene or genes against Xanthomonas citri, Xanthomonas axonopodis strains.
Furthermore, the invention relates to antibodies against the proteins of the invention, which are specific to the mutant CYSTM region of the proteins and bind to the resistant mutant protein but not to the wild-type protein, and are useful as probes in in vitro methods for determining whether a plant carries such resistant mutant proteins or not.
The present invention also relates to genetic probes, which are specific to the mutant region of the xcv-1 gene and of its homologues, and are useful for the identification of resistance genes in an in vitro method.
As used herein, the term “resistance to biotic or abiotic factors” means that the plant is resistant to various biotic factors such as plant-pathogenic bacteria, fungi and viruses, or abiotic factors such as salt stress, drought stress etc.
As used herein, the term “recessive resistance to Xanthomonas sp.” means that the plant is resistant to at least one Xanthomonas species including, but not limited to, Xanthomonas euvesicatoria, Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas oryzae pv. oryzae, Xanthomonas citri pv. citri, Xanthomonas axonopodis pv. citri, Xanthomonas campestris pv. musacearum. In a preferred embodiment of the invention, the plant is resistant to at least Xanthomonas euvesicatoria. In some embodiments of the invention, the plant is resistant to two, three, four, or more Xanthomonas species and preferably, one of the species is Xanthomonas euvesicatoria.
As used herein, the term “plant” means an organism capable of photosynthesising, the parts of which, e.g., root, stem, leaf, flower, fruit etc., the progeny of which after sexual reproduction, e.g., F1, F2, F3 etc. generation after crossing or self-pollination, and progeny after vegetative reproduction, e.g., cloning from root cuttings or stem cuttings, grafting, budding, micropropagation, etc.
As used herein, the term “resident gene” means genes naturally occurring in living organisms not engineered by humans.
As used herein a “tail-anchored protein” (TA protein) refers to a protein the NH2-terminal portion (domain) of which is anchored to the double phospholipid membrane through a single hydrophobic portion located near to its COOH-terminus, as described by Borgese N. et al. (J. Cell Biol. 161: 1013-1019, 2003).
As used herein, the term “transmembrane” (TM in short) means a hydrophobic protein portion spanning the double phospholipid membrane.
As used herein, the term “transmembrane protein” (TM protein) means a protein comprising a transmembrane protein domain.
As used herein, the term “CYSTM protein” means a TA protein having a cysteine-rich TM region close to the COOH-terminus (CYSTM region) in the sense described by Venancio T. M. and Aravind L. (Bioinformatics 26:149-152, 2010).
As used herein, the term “CYSTM region” in relation to proteins means a cysteine-rich TM protein segment in the sense described by Venancio T. M. and Aravind L. (Bioinformatics 26:149-152, 2010).
As used herein, the term “homologous” refers to the situation where nucleic acid or protein sequences are similar because they have a common evolutionary origin.
As used herein, the term “proteins homologous to the wild-type Xcv-1 protein” refers to CYSTM proteins in the sense described by Venancio T. M. and Aravind L. (Bioinformatics 26:149-152, 2010).
As used herein, the term “proteins homologous to the mutant xcv-1 protein” means CYSTM protein variants in which the CYSTM region contains a deletion of 2 amino acids compared to its wild type protein and which provide resistance.
As used herein, the term “genes homologous to the Xcv-1 gene” means gene variants or its cDNA sequence without intron(s) encoding the above “proteins homologous to the Xcv-1 protein”.
As used herein, the term “genes homologous to the xcv-1 gene” means gene variants or its cDNA sequence without intron(s) encoding the above “proteins homologous to the xcv-1 protein”.
Fragments and variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of polynucleotides comprising coding sequences may encode protein fragments that retain biological activity of the full-length or native protein and hence retain the ability to initiate in a plant a hypersensitive response in the presence of a effector protein from a plant pathogen. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the invention.
Polynucleotides that are fragments of a native polynucleotide of the present invention comprise at least 16, 20, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, 3000, or 3500 contiguous nucleotides, or up to the number of nucleotides present in a full-length polynucleotide disclosed herein (for example, 3859 nucleotides for SEQ ID NOS: 79, 80 and 81, respectively).
“Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polynucleotides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a polynucleotide of the invention or can be used in decreasing the level of Xcv-1 or a protein homologous to Xcv-1 in a plant by the methods disclosed herein. Variant polynucleotides further include homologous polynucleotides isolated from other species. Generally, variants of a particular polynucleotide of the invention (for example, SEQ ID NO:37 or 39 or 41 or 49 or 51 or 59 or 66 or 69 or 72 or 73 or 74 or 75 or 76 or 77 or 78 or 79 or 80 or 81 or 86 or 87 or 88 or 89 or 90 or 92), will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.
Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, a polynucleotide that encodes a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 38 or 40 or 42 or 50 or 52 or 60 or 67 are disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
“Variant” protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Such variants also include homologous proteins in other species. Biologically active variants of a protein of the present invention will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. Such biologically active variants include, for example, wild-type Xcv-1 and homologous proteins as well as mutant versions thereof (e.g. xcv-1) that confer to a plant resistance to at least one plant pathogenic Xanthomonas species. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
The polynucleotides of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire (i.e full-length) sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. “Orthologs” is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species.
The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, and, (d) “percentage of sequence identity.”
(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers, E. W. and Miller, W. CABIOS 4:11-17, 1988; the local alignment algorithm of Smith, T. F. et al., Adv. Appl. Math. 2:482, 1981; the global alignment algorithm of Needleman, S. B. and Wunsch, C. D. J. Mol. Biol. 48:443-453, 1970; the search-for-local alignment method of Pearson, W. R. and Lipman, D. J. Proc. Natl. Acad. Sci. 85:2444-2448, 1988; the algorithm of Karlin, S. and Altschul, S. F. Proc. Natl. Acad. Sci. USA 872264, 1990, modified as in Karlin, S. and Altschul, S. F. Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993.
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al., Gene 73:237-244, 1988; Higgins, D. G. et al., CABIOS 5:151-153, 1989; Corpet, F. et al., Nucleic Acids Res. 16:10881-90, 1988; Huang, X. et al., CABIOS 8:155-65, 1992; and Pearson, W. R. et al., Meth. Mol. Biol. 24:307-331, 1994. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul, S. F. et al., J. Mol. Biol. 215:403, 1990 are based on the algorithm of Karlin, S. and Altschul, S. F. (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul, S. F. et al., Nucleic Acids Res. 25:3389, 1997. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul, S. F. et al., (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
GAP uses the algorithm of Needleman, S. B. and Wunsch, C. D. 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. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff, S. and Henikoff, J. G. Proc. Natl. Acad. Sci. USA 89:10915, 1989).
(c) As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. 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. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said 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., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
As used herein, the term “CYSTM region” in relation to nucleic acids (DNA, RNA) is a nucleotide segment extending to 52 and 52 nucleotides into both (5′ and 3′) directions, respectively, from the 2nd nucleotide of the stop codon of the “genes homologous to the Xcv-1 gene” or “genes homologous to the xcv-1 gene”.
As used herein, the term “the desired 6-bp deletion” means a deletion of 6 base pairs in the DNA segment encoding the CYSTM region of genes homologous to the Xcv-1 gene, which results in resistance.
As used herein, the term “6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides” means a nucleotide segment extending 52 and 52 nucleotides into both directions from the 6-bp deletion created in the CYSTM region. The 6-bp deletion is created anywhere in the CYSTM region of the gene or in those nucleotides that encode the 5th and 6th amino acid from the C-terminus.
As used herein, the term “Type Three Secretion System” (TTSS in short) is a system by which certain pathogenic bacteria (e.g., Xanthomonas sp., Pseudomonas sp., Erwinia sp., Ralstonia sp., Escherichia sp., Yersinia sp. etc.) introduce effector molecules through the TTSS-specific pili into eukaryotic host organisms as described by Galan J. E. et al. (Nature 444:567-573, 2006).
As used herein, the term “genome editing” means a method in which a engineered nuclease or engineered nuclease pair performs double-strand breaks (DSB) in a predetermined specific DNA segment in which a DNA repair mechanism referred to as Non-homologous End Joining (NHEJ) creates short deletions or insertions as described by Gaj T. et al. (Trends Biotechnol. 31:397-405, 2013).
As used herein the term “Double Stranded Break” (DSB in short) means that a DNA sequence is cleaved by a specific nuclease or nuclease pair at both DNA-strands like a molecular pair of scissors. Nucleases performing double stranded breaks of the DNA include, among others, ZFN, TALEN and CRISPR/Cas.
As used herein, the term “Non-homologous End Joining” (NHEJ) means a method in which the DNA repair mechanism of the cells joins (ligates) two double-stranded DNA ends.
As used herein, the term “ZFN” means an artificial engineered nuclease recognising DNA sequences and cleaving both strands thereof (see below).
As used herein, the term “TALEN” means an artificial engineered nuclease recognising DNA sequences and cleaving both strands thereof (see below).
As used herein, “CRISPR/Cas” recognises complementary DNA sequences and cleaves both strands thereof with the help of the sgRNA and the Cas nuclease (see below).
As used herein, the term “an artificial nucleic acid molecule” is a non-naturally occurring nucleic acid molecule.
As used herein, the term “a gene” is a nucleotide sequence which comprises of promoter, exon(s), intron(s) in addition to 5′- and 3′-untranslated regions.
As used herein, the term “cDNA” is a nucleotide sequence of the copy of the mRNA of a gene.
As used herein, the term “an artificial nuclease” is an engineered nuclease.
As used herein, the term “engineered nucleases” are artificial restriction enzymes that can be programmed to cut a pre-determined nucleic acid sequence.
The Zinc Finger Nuclease (ZFN in short) is a fusion protein consisting of the part of the FokI restriction endonuclease protein responsible for DNA cleavage and a zinc finger protein which recognises specific, designed genomic sequences and cleaves the double-stranded DNS at those sequences, thereby producing free DNA ends (Urnov F. D. et al., Nat Rev Genet. 11:636-46, 2010; Carroll D., Genetics. 188:773-82, 2011).
The Transcription Activator-Like Effector Nuclease (TALEN in short) is a fusion protein consisting of the part of the FokI restriction endonuclease protein responsible for DNA cleavage, the part of the transcription activator-like effector (TALE) protein responsible for DNA binding, and an amino acid segment responsible for transfer into the nucleus (Nuclear Localization Signal, NLS in short). The DNA binding portion of the protein can be designed to be sequence-specific (Christian M. et al., Genetics 189:757-761, 2010; Mussolino C. et al., Nucleic Acids Res. 39:9283-9293, 2011; Miller J. C. et al., Nat. Biotechnol. 29:143-148, 2011; Cermak T. et al., Nucl. Acids Res. 39:e 82, 2011).
The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease (CRISPR/Cas in short) is an RNA-guided (simple guide RNA, sgRNA in short) DNA endonuclease system performing sequence-specific double-stranded breaks in a DNA segment homologous to the designed RNA. It is possible to design the specificity of the sequence (Cho S. W. et al., Nat. Biotechnol. 31:230-232, 2013; Cong L. et al., Science 339:819-823, 2013; Mali P. et al., Science 339:823-826, 2013; Feng Z. et al., Cell Research: 1-4, 2013).
One advantage of the above techniques is that the transgenes containing the TALEN or ZFN or CRISPR/Cas nuclease can be removed from the progeny of the Xe resistant plant by genetic segregation, that is, non-transgenic plants can be generated from them.
The mutant variants of the CYSTM region of the xcv-1 protein can be designed using computer programmes, and common features of them include their localisation at the C-terminus of the protein, the presence of at least 2 cysteines generally followed by an aspartic acid (D), glutamic acid (E) or asparagine (N) residue, and they are represented by the following sequences:
wherein D=aspartic acid, E=glutamic acid, N=asparagine, X=an amino acid residue compatible with the transmembrane character, mostly a hydrophobic or non-polar amino acid residue, e.g., glycine (G), cysteine (C), leucine (L), isoleucine (I), alanine (A), tryptophan (W), threonine (T), methionine (M), phenylalanine (F) or valine (V).
The xcv-1 gene providing recessive resistance was isolated from pepper using Capsicum annuum Gene Bank Accession No. PI163192. The isolation of the xcv-1 gene enables the designing of genetic markers using the sequence information of the gene and the linked DNA region, and the facilitation of traditional pepper breeding using these markers for Marker Assisted Selection, as well as the generation of resistant pepper and other plant varieties based on the sequence information of the gene by biotechnological methods involving the targeted modification or transforming the cells with a xcv-1 homologues gene prior to knocking-out the resident gene.
According to the invention, the xcv-1 gene was isolated from Capsicum annuum using the following method.
1. Genetic Mapping of the Xcv-1 Gene in Pepper 1.1. Generation of the F2 Segregating PopulationFor the mapping, a new population was created by intraspecies crossing (Capsicum annuum x Capsicum annuum) followed by the self-pollination of the F1 plants. For the crossing, Feherozon (FO), a commercially available Hungarian cultivated variety sensitive to Xanthomonas was used as the father parent and an Xe-resistant plant, Gene Bank Accession No. PI163192 (T1), was used as the mother parent. After the crossing, 45 seeds from the fruit of one of the mother plants were sown and the hybrid character of the resulting plants was confirmed by appropriate molecular DNA markers. Next, the 45 F1 individuals were grown, and the F2 seeds from the self-pollination were collected. The F2 individuals from the self-pollination of the F1 individuals were then used for the genetic mapping of the xcv-1 gene. The objective was to grow as many F2 individuals as possible to allow for the identification of individuals carrying recombination events as close to the xcv-1 gene as possible, and thereby for the narrowing of the genetic and—at the same time—the physical region comprising the xcv-1 gene. Until the identification of the xcv-1 gene, more than 3000 F2 individuals were generated, grown and subjected to xcv-1 phenotyping.
1.2. Xanthomonas Resistance TestPhenotyping the segregating individuals as sensitive or resistant to Xanthomonas is indispensable and of key importance for localising the xcv-1 gene on the genetic map. An incorrect phenotyping makes genetic mapping impossible or extremely difficult. As a result of the biological tests, finally 765 and 2354 F2 plants proved to be resistant and sensitive, respectively.
1.3. Mapping of the Xcv-1 LocusOn the basis of the available genotypes and xcv-1 phenotypes, the locus of the xcv-1 gene was mapped to the third chromosome of pepper. For this, sequences available in gene banks or markers used by others were applied for the mapping of the xcv-1 gene. Specific primer pairs were designed, and the primer pairs were used for PCR amplification; the map location of the markers were determined on the basis of the genotype of the markers using the polymorphism data obtained upon electrophoresis. The mapping identified a genetic marker (CaCY), which mapped to the shortest distance from the xcv-1 locus. The CaCY marker can be genotyped using primers Pr_CaCYF1 (SEQ ID NO:1) and Pr_CaCYR1 (SEQ ID NO:2).
1.4. Chromosome WalkingThe identified marker, CaCY, which is closely linked to the xcv-1 gene (located at a distance of 0.22 centimorgan from xcv-1) allowed the initiation of the chromosome walking. In the first step, the primary pepper BAC clone (Clone No. 279) was identified with the help of the CaCY marker using multiplex PCR. The terminal sequences of the primary BAC clone (No. 279) were determined and primer pairs specific to them were designed. With the help of the specific primer pairs, BAC clones overlapping with BAC Clone No. 279 were identified (Clones No. 632 and 1248), and another set of specific primers were designed for their terminal sequences and additional overlapping clones were identified, then additional clones were isolated in a similar manner. Using the specific primer pairs designed for BAC Clones No. 66, 1191, 50, 877 and 472, the BAC ends were back-mapped to the genetic map thereby verifying the correct direction of the contig building. With the specific primer pair designed for the −40 end of BAC Clone No. 50, we managed to pass a recombination towards the xcv-1 gene, therefore, contig building was only continued into this direction. From BAC Clone No. 50, the contig was extended by another overlapping BAC clone in the above manner, and upon back-mapping marker 472_op, further recombinant individuals delimiting the contig comprising the xcv-1 gene could be identified.
1.5. Sequencing of BAC Clones Overlapping the Xcv-1 Region: Subcloning, Sequencing of the Subclones and Solid SequencingIn the next step, two BAC clones overlapping the xcv-1 region (No. 50 and 472) were sequenced. DNA sequencing was carried out in two ways: by subcloning and sequencing of the subclones from both sides, and by the new-generation Solid sequencing method developed by ABI.
2. Identification of the Xcv-1 Gene 2.1. Determination of the Gene Contents of the BAC ClonesThe resulting DNA sequence data were handled and assembled into contigs giving overlapping segments by various computer programs. This “assembly” did not produce a sequence of the complete length of BAC but more than ten contigs, which were closed by the so-called primer walking technique. The sequences of the resulting partial BAC clones (No. 50 and 472) were determined.
The gene content of the two BAC clones were determined by the BLAST programmes (chiefly the blastn, blastx and blastp programmes) of NCBI (http://ncbi.nlm.nih.gov/BLAST/) and DFCI (http://compbio.dfci.harvard.edu/tgi/plant.html). On the basis of the gene content and gene order information, polymorph markers were prepared using specific primer pairs, and the genes were back-mapped. So far, a total of 13 protein-encoding genes were identified in the two BAC clones. On the basis of the mapping data, it was found that the xcv-1 gene is located between markers Pr6 and Pr4b as a single gene encoding more than 50 amino acids. The primer sequences of the markers are as follows: Pr6F1: SEQ ID NO:33, Pr6R1: SEQ ID NO:34, Pr4bF1: SEQ ID NO:35 and Pr4bR1: SEQ ID NO:36.
Next, the DNA sequence of both the xcv-1 gene and its wild-type counterpart (Xcv-1 gene) was determined in both parents using specific primer pairs; see SEQ ID NO:37 and SEQ ID NO:41, respectively.
Thus, the present invention relates to the xcv-1 gene isolated by the above method, which has the DNA sequence presented in SEQ ID NO:37. The cDNA sequence of the xcv-1 gene—which is the nucleotide sequence of SEQ ID NO:90—was generated, and is also covered by the scope of the invention.
The xcv-1 gene encodes a CYSTM protein, the xcv-1 protein, the amino acid sequence of which is that of SEQ ID NO:38, wherein the cysteine-rich transmembrane region (CYSTM region) of the protein carries a double Leu deletion at the locations corresponding to positions 87 and 88 of the wild-type Xcv-1 protein of SEQ ID NO:42. However, the invention also encompasses all those protein sequences which are proteins homologous to the xcv-1 protein and represent protein variants containing at least 53%, preferably 60% to 73%, more preferably 80% to 93% identical amino acids with respect to the last 13 amino acids of the C-terminus of the xcv-1 protein.
Furthermore, the present invention relates to vectors, preferably expression vectors, comprising the genes isolated and prepared according to the invention in a functional form. Preferred vectors useful for the purpose of the invention include the binary vectors of Agrobacterium tumefaciens, such as the pCAMBIA vector family (http://www.cambia.org/daisy/cambia/585).
Furthermore, the present invention relates to ZFN, TALEN and CRISPR/Cas nucleases specific to the interest sequence of the Xcv-1 gene, which are used to generate the 6-bp deletion or to inactivate (by knock-out) the genes homologous to the wild-type Xcv-1 DNA.
Furthermore, the present invention relates to host cells into which the vectors of the invention were introduced, e.g., by transformation or by genome editing techniques (ZFN, TALEN and CRISPR/Cas nuclease), and by means of which the desired 6-bp deletion was generated in the Xcv-1 homologous genes. Preferred host cells useful for the purpose of the invention include Solanaceae, Oryzae, Citroidieae etc. species.
The present invention also relates to a transformation method of generating a transgenic plant resistant to a biotic or abiotic factor, comprising the steps of: a) identifying one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in said plant, preparing in vitro the genomic and cDNA sequences of the identified gene in a DNA form, generating in vitro a 6-bp mutation corresponding to the deletion of two amino acids in the segments encoding the CYSTM region of the one or more identified genes and transforming the cells of a sensitive plant with the one or more mutant genes thus obtained in a manner ensuring the functional expression thereof, b) inactivating the one ore more resident genes identified in step a) or the mRNA or protein thereof in the transformant plant cells, and c) regenerating the plant from the transformants and selecting the resistant individuals.
In step a) of the above method, genetic engineering methods well-known to those skilled in the art were used to identify the gene(s), to prepare them in a DNA form, to generate the mutations and to transform the plant cells.
In step b) of the above method, the resident genes homologous to the Xcv-1 gene are inactivated (silenced). Inactivation (silencing) of the resident genes is necessary because the function of the wild-type protein (pl. Xcv-1) is dominant over the function of the mutant protein (e.g., xcv-1), i.e., the latter function is recessive. Well-known methods are also available for inactivating the gene, i.e., for eliminating the function of the gene. Examples include but are not limited to the following: inhibiting the expression of the protein products of the resident genes using natural, chemical or insertion mutagenesis, amiRNA (artificial miRNA), RNAi (RNA interference) or other techniques; inactivating the resident gene(s) using nuclease deletion methods generating knock-out mutants. Preferably, the above-described TALEN, ZFN or CRISPR/Cas nuclease technique is used. By expressing the gene of a monoclonal antibody specific to the protein, one can inactivate the Xcv-1, or the homologous proteins.
It is important to note that the amiRNA technique has been successfully used to create resistance against viruses in plants (Niu et al., Nat. Biotechnol. 24:1420-1428, 2006), however, resistance to pathogenic bacteria such as Xe has not been created in plants with the amiRNA technique yet.
In step b) of a preferred embodiment of the method of the invention, the mRNA products of the resident gene(s) are functionally inactivated (silenced) by the amiRNA technique as follows: b1) preparing an amiRNA gene construct for the ribonucleotide sequence complementary to the CYSTM region of the mRNA of one or more genes homologous to the wild-type Xcv-1 gene and identified in the transformant plant cells obtained in step a); b2) cloning the construct obtained in step b1) into an appropriate vector; b3) transforming the transformant plant cells with the vector obtained in step b2) in a manner ensuring the functional expression of the amiRNA and inactivation of the mRNA products of the wild-type CYSTM gene(s); and c) regenerating the transformants obtained in step b3) and selecting the resistant plant.
The nucleases of the invention (engineered nucleases) are used for two genome editing functions in the present invention: on the one hand, for inactivating the resident genes in the transformant plant cells; on the other hand, for creating the desired 6-bp deletion in the gene homologous to the Xcv-1 gene in the genome of a sensitive plant cell. The two functions may also be combined, for example, in tomato, where the desired 6-bp deletion appears in one of the two homologous wild-type genes (SlXcv-1A, SEQ ID NO:49 and SlXcv-1B, SEQ ID NO:51), and the gene is inactivated upon a deletion or insertion in the other.
In a manner obvious to a skilled person, the target sequences of the above nucleases are selected in a manner ensuring that they cover the 6-bp sequence to be deleted in order to prevent the recognition and cleavage of the DNA segment already comprising the deletion, and that the desired 6-bp deletion can be created.
In another preferred embodiment of the method of the invention, the resident gene(s) is/are inactivated by a nuclease deletion (genome editing) method as follows: b1) preparing DNA constructs encoding TALEN-L and TALEN-R or ZFN-L and ZFN-R proteins specific to the gene sequence(s) encoding the CYSTM region of one or more genes homologous to the wild-type Xcv-1 gene and identified in the transformant plant cells obtained in step a), more specifically those specific to the DNA segments corresponding to the 6 nucleotides to be deleted and the surrounding nucleotides as target sequence in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of the TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence; b2) cloning the constructs obtained in step b1) into an appropriate vector; b3) transforming the plant cells with a vector comprising the constructs obtained in step b1) in a manner ensuring the functional expression of the transgenes; c) identifying knock-out deletion or insertion mutations in the genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41); d) selecting the plant cells containing the mutations identified in step c); e) regenerating the plant cells identified in step d) and selecting the resistant plants, and f) removing the transgenes comprising the TALEN or ZFN constructs or the CRISPR/Cas constructs containing the gene of the sgRNS protein by genetic segregation.
The present invention also relates to a method for generating a mutant plant showing an abiotic or biotic resistance using a genome editing method, comprising the steps of: a) identifying one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in a sensitive plant; b) preparing DNA constructs encoding TALEN-L and TALEN-R or ZFN-L and ZFN-R proteins specific to the gene sequence encoding the CYSTM region of one or more genes homologous to the wild-type Xcv-1 gene and identified in step a), more specifically those specific to the 6 nucleotides to be deleted and the surrounding nucleotides as target sequence in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence; c) cloning the construct obtained in step b) into an appropriate vector; d) transforming sensitive plant cells with a vector comprising the construct obtained in step b) in a manner ensuring the functional expression of the transgenes and the generation of the 6-bp deletion in the CYSTM region by the nuclease; e) identifying the transformants carrying mutations showing the 6-bp deletion in the CYSTM region of the genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41); f) regenerating the plant cells having the mutation identified in step e) and selecting the resistant plants; and g) removing the transgenes comprising the TALEN or ZFN constructs or CRISPR/Cas constructs containing the gene of the sgRNS protein by genetic segregation.
Furthermore, the mutant plants showing biotic or abiotic resistance according to the invention can be generated by introducing TALEN or ZFN proteins specific to the CYSTM region into the plant using non-pathogenic bacteria, said method, comprising the steps of: a) identifying one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in a sensitive plant; b) preparing DNA constructs encoding TALEN-L plus TALEN-R or ZFN-L plus ZFN-R proteins specific to the DNA segment encoding the CYSTM region of the gene identified in step a) in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of the TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs; c) cloning the constructs obtained in step b) into an appropriate bacterial vector and introducing them into non-pathogenic bacteria with active type three secretion system using said vector; d) infecting the sensitive plant with bacteria obtained step c); and e) regenerating a resistant plant from the infected plant tissue.
In the methods of the invention, not only the genomic sequences but also the cDNA sequences of the genes homologous to the wild-type Xcv-1 or the mutant xcv-1 gene can be used with identical results since the same protein is expressed from both.
Using the method of the invention, resistance can be created in plants which are sensitive to Xanthomonas species, such as Xanthomonas euvesicatoria, Xanthomonas perforans, Xanthomonas gardneri, Xanthomonas vesicatoria pv. oryzae, and other Xanthomonas species. One such preferred plant is tomato.
One of the most dangerous pathogens of tomato is Xanthomonas euvesicatoria (Xe), which also causes severe damage to pepper. Unlike pepper, tomato has not developed appropriate natural resistance, which would ensure sufficient protection. Thus, tomato production is still highly threatened by Xe infection (Hutton S. F. et al., Theor. Appl. Genet. 121:1275-87, 2010). The xcv-1 gene identified in pepper may also provide resistance to Xe infection in tomato by ensuring the introduction and functional expression of the mutant tomato genes Slxcv-1A and/or Slxcv-1B (SEQ ID NO:59 and/or SEQ ID NO:66), which are homologous to the xcv-1 gene, in the tomato genome followed by the inactivation of the dominant resident genes SlXcv-1A and SlXcv-1B (SEQ ID NO:49 and SEQ ID NO:51) homologous to the wild-type Xcv-1 gene, i.e., by generating non-functional variants thereof.
The tomato plant resistant to Xanthomonas euvesicatoria can be generated using the following methods of the invention:
-
- transforming the cells of the tomato plant with the mutant genes Slxcv-1A and/or Slxcv-1B carrying the double Leu deletion, followed by inhibiting the function of the SlXcv-1A and/or SlXcv-1B genes using the amiRNA technique; or
- transforming the cells of the tomato plant with the mutant genes Slxcv-1A and/or Slxcv-1B carrying the double Leu deletion, followed by inactivating the resident genes SlXcv-1A and/or SLXcv-1B located there using the ZFN nuclease or TALEN nuclease or CRISPR/Cas nuclease technique; or
- transforming the cells of the tomato plant with the mutant genes Slxcv-1A and/or Slxcv-1B carrying the double Leu deletion; followed by identifying the mutations inactivating the SlXcv-1A and/or SlXcv-1B gene using the TILLING or a similar technique upon or without mutagenesis; or
- creating the desired 6-bp deletion in genes SlXcv-1A and/or SlXcv-1B in the genome of the sensitive tomato plant using a specific nuclease-based genome editing method, which provides resistance.
Thus, one object of the present invention is a method for generating a tomato plant resistant to Xanthomonas euvesicatoria, comprising the steps of:
a) identifying the resident genes SlXcv-1A and SlXcv-1B (SEQ ID NO:49 and 51, respectively), which are homologous to the wild-type Xcv-1 gene in the tomato plant; b) preparing in vitro the genomic and cDNA sequences of the identified genes in the form of a DNA; c) creating in vitro the 6-bp deletion in the positions corresponding to the deletions in the xcv-1 gene thereby generating constructs carrying the mutant genes Slxcv-1A and Slxcv-1B (SEQ ID NO:59 and 66, respectively) or their cDNA sequences (SEQ ID NO:88 and 89, respectively); d) cloning the constructs obtained in step c) into an appropriate vector and transforming the cells of the tomato plant with the resulting vector in a manner ensuring the functional expression of the transgenes; e) preparing an amiRNA gene construct for silencing the wild-type genes SlXcv-1A and SlXcv-1B (SEQ ID NO:49 and/or SEQ ID NO:51); f) cloning the construct obtained in step e) into an appropriate vector; g) transforming the plant cells generated in step d) with the vector obtained in step f) in a manner ensuring the expression of the amiRNA and inactivation of the mRNA products of the wild-type CYSTM genes SlXcv-1A and SlXcv-1B; and h) regenerating the transformants obtained in step g) and selecting the resistant plant.
In a preferred embodiment the amiRNS gene construct is specific to the complementary ribonucleotide sequence corresponding to the CYSTM region of the wild type SlXcv-1A and/or SlXcv-1B genes (SEQ ID NO:49 and/or SEQ ID NO:51) more specifically an amiRNA gene construct specific to the 6 nucleotides to be deleted and the surrounding nucleotides (SlXe1-amiRNA, SEQ ID NO: 74). In a preferred embodiment the amiRNA gene construct comprises the nucleic acid molecule according to the invention.
The present invention also relates to another method for generating a tomato plant resistant to Xanthomonas euvesicatoria, comprising the steps of: repeating steps a) to d) above; e) preparing proteins TALEN-L (SEQ ID NO:78) and TALEN-R1 (SEQ ID NO:79) and TALEN-R2 (SEQ ID NO:80) specific to the target sequences (SEQ ID NO:75, 76, 77) specific to the CYSTM region of the wild-type SlXcv-1A and SlXcv-1B gene, or DNA constructs encoding the proteins ZFN-L and ZFN-R specific to SEQ ID NO: 86 and 87 in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of the TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence (SEQ ID NO: 81); f) cloning the construct obtained in step e) into an appropriate vector; g) transforming the plant cells generated in step d) with the vector obtained in step f) in a manner ensuring the functional expression of the above TALEN or ZFN or CRISPR/Cas nuclease transgenes and the functional inactivation of genes SlXcv-1A and SlXcv-1B by them; h) identifying deletion or insertion knock-out mutations in the transformant cells; i) regenerating the plant cells having the mutation identified in step h) and selecting the resistant plants; and f) removing the nuclease transgenes TALEN-L plus TALEN-R or ZFN-L plus ZFN-R or CRISPR/Cas by genetic segregation.
The present invention also relates to yet another method for generating a tomato plant resistant to Xanthomonas euvesicatoria, comprising the steps of: a) identifying the resident genes SlXcv-1A (SEQ ID NO:49) and SlXcv-1B (SEQ ID NO:51), which are homologous to the wild-type Xcv-1 gene in the tomato plant; b) preparing proteins TALEN-L (SEQ ID NO:78) and TALEN-R1 (SEQ ID NO:79) and TALEN-R2 (SEQ ID NO:80) specific to the target sequences (SEQ ID NO:75, 76, 77) specific to the CYSTM region of the wild-type SlXcv4A and SlXcv-1B gene, or DNA constructs encoding the proteins ZFN-L and ZFN-R specific to SEQ ID NO: 86 and 87 in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of the TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence (SEQ ID NO: 81); c) cloning the construct obtained in step b) into an appropriate vector; d) transforming the cells of the tomato plant with the vector obtained in step c) in a manner ensuring the functional expression of the above TALEN or ZFN or CRISPR/Cas nuclease transgenes and the creation of the 6-bp deletion in the CYSTM region of genes SlXcv-1A and SlXcv-1B by them; e) identifying the transformants carrying the 6-bp deletion in the CYSTM region of the wild-type genes SlXcv-1A and SlXcv-1B; f) regenerating the transformants having the mutation identified in step e) and selecting the resistant plant; and g) removing the nuclease transgenes TALEN-L plus TALEN-R or ZFN-L plus ZFN-R or CRISPR/Cas by genetic segregation.
The development of resistant cultivars has been the most effective, economical and environmental friendly strategy to control disease epidemic of cultivated plants. Out of many possibilities, pyramided resistance is far more durable than resistance that is controlled by a single dominant R genes (usually causing HR), because new races of pathogens could easily evolve to overcome or escape the resistance consequently plant resistant trait breaks down. Traditional breeding combined with molecular markers based marker assisted selection has made it possible to identify and pyramid valuable genes of agronomic importance in resistance. In addition to this strategy, transgenic approaches serve further possibility to pyramid resistant genes in plant cultivars. As mentioned above tomato, a close relative of pepper is highly susceptible to Xe. To fight against this pathogen and establish Xe resistant tomato, transgenic tomato plants expressing the Bs2 resistance gene from pepper was constructed recently (Horvath et al., PLoS One.; 7(8):e42036, 2012). In replicated multi-year field trials under commercial type growing conditions demonstrated improved resistance to bacterial spot disease caused by Xe. Taking into account the beneficial impact of pyramided gene configuration the above mentioned tomato Bs2 containing can be a starting material to produce double resistant derivatives by expressing the Slxcv-1A and/or Slxcv-1B gene carrying the beneficial 6 bp deletion as described in this invention. By this way highly resistant and durable Xe resistant cultivars of tomato can be breeded for commercial production. A skilled person would recognize that the resistance based on the expression of Slxcv-1A and/or Slxcv-1B gene can be combined not only with Bs2, but with other genes too, which may confer resistance to Xe in tomato including but not limited to Bs1, Bs3, Bs4, bs5, bs6.
In addition to pepper and tomato, several other plants are severely infected by Xanthomonas species causing disease on rice, potato, citrus, banana, grape, etc. (Dangle et al. Science 341: 746, 2013). The desired 6 bp deletion derivative can also be generated in the Xcv-1 homologous gene(s) of these plants and can be combined with other type of resistance genes against Xanthomonas. Accordingly, one can generated rice plants resistant against Xanthomonas oryzae, or citrus plants resistant against Xanthomonas citri pv. citri or Xanthomonas axonopodis pv. citri or banana plants resistant against Xanthomonas campestris pv. musacearum.
In addition, the method of the invention can be used to create resistance to an abiotic or biotic factor other than Xanthomonas sp. in plants.
The present invention further relates to mutant plants resistant to a biotic or abiotic factor, which carry a deletion of two amino acids in their CYSTM region in comparison with the wild-type plant. Preferably, the mutant plant is a pepper plant (Capsicum annuum), a tomato plant (Solanum lycopersicum), a plant from the Solanaceae family, e.g., potato, eggplant etc., a citrus (Citroideae), e.g., orange (Citrus aurantium), mandarin (Citrus reticulata), lemon (Citrus x medica L.), grapefruit (Citrus x paradisi), pomelo (Citrus maxima or grandis) etc., a plant from the Brassicaceae family, e.g., cabbage (Brassica oleracea convar. capitata var. alba), radish (Raphanus sativus), cauliflower (Brassica oleracea convar. botrytis var. botrytis), rape (Brassica napus) etc., a monocot plant (Monocotyledonae), e.g., rice (Oryzae sp.), maize (Zea mays), wheat (Triticum sp.), rye (Secale sp.), barley (Hordeum vulgare), millet (Panicum sp.), etc., a plant from the Fabaceae or Leguminosae families, e.g., alfafa (Medicago sp.), bean (Phaseolus sp.), pea (Pisum sp.), soy (Glycine sp.), horse bean (Faba sp.), lupine (Lupinus sp.), clover (Trifolium sp.), peanut (Arachis sp.), vicia (Vicia sp.), lathyrus (Lathyrus sp.), lentil (Lens sp.), chick-pea (Cicer sp.), mung bean (Vigna sp.), pigeon pea (Cajanus cajan) etc., a plant from the Cucurbitaceae family, e.g., pumpkin (Cucurbita sp.), cucumber (Cucumis sp.), melons (Citrullus sp.) etc., a plant form the Rosaceae family, e.g., apple (Malus sp.), pear (Pyrus communis), quince (Cydonia oblonga), cherry (Prunus subg. Cerasus), sour cherry (Prunus cerasus), plum (Prunus domestica subsp. domestica), apricot (Prunus armeniaca), peach (Prunus persica), grape (Vitis vinifera), etc., in which resistance has been created.
More preferably, the mutant plant is a mutant tomato plant (Solanum lycopersicum) resistant to Xe.
Another object of the present invention are the seeds and the products of the mutant plants generated by this invention, including but limited to fruits, juice, paste, etc., preferably the seeds and products of the mutant tomato plant and its progeny.
Furthermore, we can raise antibodies against the xcv-1 protein of the invention, which can be used as probes in in vitro methods performed in plant-derived cell lines in order to test whether a given plant is resistant to Xe or not. The methods of raising antibodies and such techniques are well known to those skilled in the art.
The present invention further relates to gene probes, which are specific to the xcv-1 gene or its homologous genes and hybridizing with them under stringent conditions.
Another objects of the present invention are primer pairs, which are specific to the xcv-1 gene or its homologous genes, especially to the Slxcv-1A and/or the SlXcv-1B, and can be used to genotype plants carrying the 6 bp deletion including but not limited to markere assisted selection.
The invention is described in more detail through the following examples without limiting the scope of the invention.
Example 1 Genetic Crosses and Analysis of the F2 Progeny of the Xcv PlantFor the generation of F1 individuals, commercially available C. annuum var. Feherozon sensitive to Xanthomonas euvesicatoria (Xe) was used as the father parent (marked as F0), and Capsicum annuum var. T1/1 carrying Xe resistance—an individual of Gene Bank Accession No. PI163192—was used as the mother parent (T1/1). After the crossing, 45 F1 seeds from the mother plant were sown and F2 plants were grown from them. When plants reached the 8-leaved age, a Xanthomonas euvesicatoria infection test was used to determine the sensitivity of the plants to Xe. Finally, 20 F2 individuals—8 resistant and 12 sensitive individuals (see Table 1)—were selected for the general mapping experiments; on the other hand, more than 3000 F2 individuals were used for the fine-mapping of the Xe resistance gene (xcv-1).
Identification by genetic mapping of molecular markers mapping close to the mutated xcv gene, i.e., those linked to Xcv resistance, was carried out using the 20 F2 progeny mentioned in Example 1. Total DNA from fresh leaves was subjected to PCR amplification using specific primers designed on the basis of pepper sequences available in the databases, and the resulting fragments were subjected to electrophoresis on agarose gels or on so-called SSCP acrylamide gels. In order to visualise the DNA fragments, agarose gels and acrylamide gels were stained using ethidium bromide and silver, respectively. The linkage of markers showing polymorphism on the agarose or SSCP gels was determined by colour mapping (Kiss et al., Acta Biologica Hungarica 49:47-64, 1998) with respect to the xcv/Xcv phenotype after ascertaining the homozygote or heterozygote status. As a result of the systematic mapping, a single marker designated as CaCY showed a distance of 0.22 centimorgan.
The identifiers of the primers of the CaCY marker (Pr_CACY_F1 and Pr_CACY_R1) are SEQ ID NO:1 and SEQ ID NO:2, respectively.
Since other mapped markers were either unlinked to the xcv-1 gene or were located at much greater genetic distances; therefore the so-called chromosome walking was initiated using the CaCY marker to physically the xcv-1 gene.
Example 3 Isolation of BAC Clones Overlapping with the Xcv-1 Mutation, Contig BuildingA primary BAC clone (No. 279) was isolated using the molecular marker showing the strongest linkage to the xcv-1 mutation, i.e., CaCY. The primary and other BAC clones are isolated using multiplex PCR from a BAC library comprising 380,000 BACs, which was prepared from an Xcv resistant pepper (Capsicum annuum) plant and ensures a 22-fold coverage of the pepper genome (Bukovinszki et al., VII. Hungarian Congress on Genetics, Abstract Book, p. 91, 2007).
Both ends of BAC Clone No. 279 were sequenced and additional two BAC clones were identified using primer pairs specific to these sequences: BAC Clone No. 1248 using primers Pr_279 op F1 (SEQ ID NO:3) and Pr_279 op R1 (SEQ ID NO:4), and BAC Clone No. 632 using primer pair Pr_279-40 F1 (SEQ ID NO:5) plus Pr_279-40 R1 (SEQ ID NO:6).
Primer pairs specific to the terminal sequences of BAC No. 1248 were used to identify BAC Clone No. 1191: the pair consisted of primers Pr_1248 op F1 (SEQ ID NO:7) and Pr_1248 op R1 (SEQ ID NO:8), and as control, BAC Clone No. 279 was reidentified using primer pair Pr_1248-40 F1 (SEQ ID NO:9) plus Pr_1248-40 R1 (SEQ ID NO:10).
Primer pairs specific to the terminal sequences of BAC No. 1191 were used to identify BAC Clone No. 877: the pair consisted of primers Pr_1191-40 F1 (SEQ ID NO:11) and Pr_1191-40 R1 (SEQ ID NO:12), and as control, BAC Clone No. 1248 was reidentified using primer pair Pr_1191 op F1 (SEQ ID NO:13) plus Pr_1191 op R1 (SEQ ID NO:14).
Primer pair Pr_877-40 F1 (SEQ ID NO:15) plus Pr_877-40 R1 (SEQ ID NO:16) was designed for the −40 terminal sequence of BAC No. 877, and was used for genetic back-mapping.
Primer pairs specific to the terminal sequences of BAC No. 632 were used to identify BAC Clone No. 66: the pair consisted of primers Pr_632-40 F1 (SEQ ID NO:17) and Pr_632-40 R1 (SEQ ID NO:18), and as control, BAC Clone No. 279 was reidentified using primer pair Pr_632 op F1 (SEQ ID NO:19) plus Pr_632 op R1 (SEQ ID NO:20).
Primer pairs specific to the terminal sequences of BAC No. 66 were used to identify BAC Clone No. 50: the pair consisted of primers Pr_66-40 F1 (SEQ ID NO:21) and Pr_66-40 R1 (SEQ ID NO:22), and as control, BAC Clone No. 632 was reidentified using primer pair Pr_66 op F1 (SEQ ID NO:23) plus Pr_66 op R1 (SEQ ID NO:24).
Primer pairs specific to the terminal sequences of BAC No. 50 were used to identify BAC Clone No. 472: the pair consisted of primers Pr_50 op F1 (SEQ ID NO:25) and Pr_50 op R1 (SEQ ID NO:26), and as control, BAC Clone No. 66 was reidentified using primer pair Pr_50-40 F1 (SEQ ID NO:27) plus Pr_50-40 R1 (SEQ ID NO:28).
On the one hand, primer pairs designed for the terminal sequences of BAC No. 472 were used to reidentify BAC Clone No. 66 as control: the pair consisted of primers Pr_472-40 F1 (SEQ ID NO:29) and Pr_472-40 R1 (SEQ ID NO:30), and primer pair Pr_472 op F1 (SEQ ID NO:31) plus Pr_472 op R1 (SEQ ID NO:32) was used for genetic back-mapping.
Thus, by classifying BAC Clones into contig groups, we could draft the so-called xcv contig (
Determining the location of the xcv-1 gene more precisely within the xcv contig is of key importance since the nearer the given molecular marker is, the lower number of BAC clones has to be sequenced, and this results in fewer candidate genes. More precise determination of the location of the xcv-1 gene within the xcv contig was performed as follows. On the one hand, the number of individuals in the F2 segregation population was increased to allow the analysis of as many recombination events as possible. Finally, a total of 3119 individuals were included in the genetic analysis. On the other hand, molecular markers were developed using terminal sequences of overlapping BACs and were back-mapped using the population segregating the xcv-1 gene. On the basis of the results, it was concluded that the position of the xcv-1 gene is between the −40 end of BAC No. 50 and the op end of BAC No. 472 separated by three and four recombination events, respectively.
Example 5 Subcloning of BAC Clone No. 50 and Sequencing of the SubclonesSubcloning of BAC Clone No. 50 was performed after cleavage by restriction enzymes BamHI, EcoRI and HindIII. Upon purification, the subclones were digested using BamHI, HindIII and EcoRI, and the resulting fragments are cloned into vectors digested with BamHI, HindIII és EcoRI and transformed into Escherichia coli cells. The sequences of the fluorescence-labelled amplificates of the recombinant clones were determined using ABI 373 and ABI 377 automated sequencers (Perkin Elmer Applied Biosystems; 850 Lincoln Centre Drive Foster City, Calif. 94404 USA).
The DNA sequence of BAC Clone No. 50 was also determined using second generation sequencing technologies (SOLID and Iontorrent, Applied Biosystems), as well as using the “primer walking” technique until the complete sequence was obtained.
Example 6 Fine-Mapping of the Xcv-1 RegionPartial sequence data were stored in a computer, and the analysis was started by determining their correct order on the basis of their overlapping sequences. In a manner obvious to those skilled in sequence alignment and sequence analysis, the overlapping terminal sequences of the BAC clones and their subclones generated by restriction digestion provide help for the assembly of the sequences and for the determination of the relative locations of the subclones generated by random and restriction digestion and of the BAC clones. Upon the assembly of the partial sequences, we succeeded in compiling the sequence of BAC Clone No. 50, which was then used to develop genetic markers at various distances from the BAC termini. When these genetic markers were back-mapped in the mapping population, it was revealed that the xcv-1 gene is located between PCR-based markers Pr6 and Pr4b. The primer sequences of markers Pr6 and Pr4b are as follows: Pr6F1: SEQ ID NO:33, Pr6R1: SEQ ID NO:34, and Pr4bF1: SEQ ID NO:35, Pr4bR1: SEQ ID NO:36.
Example 7 Sequence Analysis of the Xcv1 Region and Detailed Assessment of the Xcv-1 GeneUpon obtaining the nucleotide sequence of the DNA segment between markers Pr4b and Pr6, the databases of NCBI (National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/BLAST/), DFCI (http://compbio.dfci.harvard.edu/tgi/plant.html), Medicago HapMap (http://www.medicagohapmap.org/?genome) and Arabidopsis (http://www.arabidopsis.org) were successfully searched for homologous genes. The sequences were evaluated with a view to the homology between the sequences, and the common general characteristics of the gene structures [consensus sequences such as start and stop codons; consensus sequences typical of open reading frames (ORFs), exons and introns such as the GT-AG rule, point of divergence etc.]. Sequence analysis was facilitated by the fact that only one gene encoding for a protein of more than 50 amino acids—which is the xcv-1 gene itself—is present between the two genetic markers located on the right and left side, respectively, of the xcv-1 gene responsible for the phenotype, which can be distinguished by single recombination events. The sequence of the DNA segment comprising the xcv-1 gene is represented by the nucleotide sequence of SEQ ID NO:37.
The databases were successfully searched for DNA sequences similar to the xcv-1 gene, and we noted that homologous cDNA sequences occur in the so-called EST (Expressed Sequence Tags) databanks in the case of C. annuum as well. These sequences are a result of random sequencing by laboratories into cDNA clones from cDNA libraries of various organs, tissues or cells or groups of cells (root, stem, leaves, fruit, flower, pistil, stamen, pollen etc.) without having any information regarding their functions. For example, these include sequence TC17947 (SEQ ID NO:39) from C. annuum found in the DFCI database (http://compbio.dfci.harvard.edu/tgi/plant.html), which is presented in the form of the so-called TC (Tentative Consensus), i.e., the DNA form of the mRNA of a gene. The base composition of the TCs are edited by aligning cDNA sequences of various lengths derived from a single gene and determining the consensus sequence. The protein encoded by sequence TC17947 is shown by the amino acid sequence of SEQ ID NO:40. The mutation responsible for the Xe resistance can be identified by searching for differences in the nucleotide sequence of the genome region (SEQ ID NO:37) comprising the xcv-1 gene from the mutant plant in comparison with the homologous regions of the sensitive pepper plant (SEQ ID NO:41). Such differences can be found by comparing the nucleotide sequence of the genomic region (SEQ ID NO:37) comprising the xcv-1 gene with the genomic (SEQ ID NO:41) sequence from the sensitive pepper (C. annuum var. Feherozon) plant. Upon aligning SEQ ID NO:37 and SEQ ID NO:41, it was found that the sequence from the resistant plant is shorter by 6 bp at a certain location. When the TC17947 sequence (SEQ ID NO:39) is aligned with the sequence (SEQ ID NO:37) from the resistant plant, then the cDNAs can be aligned to the genomic sequence at three different segments (these are the exons) (alignment of the genomic sequence of a gene to its cDNA sequence allows for the determination of the exact location of the exons and introns present in the gene). These three segments are separated by 2 introns of the genomic sequence (see SEQ ID NO:37). While the first and second segment of the TC17947 sequence shows 100% identity to the genomic sequence, the cDNA sequence of the third segment is longer by 6 bases at the very location where the two genomic sequences also show a difference of 6 bases. Since only one sequence typical of a gene encoding a protein [putative promoter segment, 5′ UTR, exons, introns, 3′-UTR, start codon (ATG), stop codon (TGA), conserved exon/intron and intron/exon boundaries recognisable on the basis of the so-called GT-AG rule and by the point of divergence, Poly-A site etc.] is present the DNA segment in question, it is assumed that the protein variant carrying the 6-base deletion in the third exon of the genomic sequence is responsible for the resistance. Thus, as a consequence of this deletion, protein synthesis is normal but the resulting protein is shorter by 2 amino acids (see amino acid sequences SEQ ID NO:38 and SEQ ID NO:42).
The putative first codon of the wild-type Xcv-1 gene is the ATG start codon starting at nucleotide 917 of the genomic fragment, and the stop codon is the TAG stop codon starting at nucleotide 2076. The sequences typical of promoter regions are located in the putative promoter region at the 5′-end of the gene. In addition, a polyA site is present at the 3′-end of the gene.
The activity—i.e., the transcription—of genes Xcv-1 and xcv-1 in the cells is also confirmed by the fact that mRNAs corresponding to the two sequences were detected: mRNA segments lacking the 6-base deletion and mRNA segments containing the 6-base deletion were identified from the sensitive plant and the resistant plant, respectively, upon Solid type mRNA sequencing.
The amino acid sequence of the protein of the Xcv-1 gene can be deduced from the predicted cDNA sequence of the Xcv-1 (see features of SEQ ID NO:41). The longest amino acid sequence containing an open reading frame (ORF) deduced from the Xcv-1 cDNA is indicated by SEQ ID NO:42. The Xcv-1 protein consists of 92 amino acids. This protein contains characteristic sequence portions (e.g., the C-terminal transmembrane region), which can be determined using typical features accumulated in data banks and bioinformatics methods (e.g., TMpred and MPEx programmes). As a result of the bioinformatic analysis, it can be concluded that the intracellular part of the molecule starts with an N-terminus followed by a GYPQ-rich portion of 5 to 6 amino acids which contains a repeat sequence (GYPPE-GYPKD-SYPPP-GYPQQ-GYPQQ-GYPQQ-GYPPQ-GYPPQ-YAPQY), and a linker segment is attached to the a C-terminal portion, which is a so called Tail Anchored (TA) section extending into the membrane (amino acids 72 to 88). The transmembrane region is terminated by amino acids with a negative charge (aspartic acid, glutamic acid) or polar amino acids (asparagine). The transmembrane region of the Xcv-1 protein can be detected on the basis of hydrophobicity using programmes predicting transmembrane regions. Without limitation, these include, for example, the “DAS”—Transmembrane Prediction programme (http://www.sbc.su.se/˜miklos/DAS/), and the Tmpred software (http://www.ch.embnet.org/software/TMPRED_form.html).
Identification of cDNA sequences from various tissues supports the fact that the Xcv-1 gene is expressed in different pepper species and in their various tissues from root to flower [see the ESTs constituting the TC17497 sequence found in the DFCI data bank (SEQ ID NO:39); (http://compbio.dfci.harvard.edu/tgi/plant.html)].
In comparison with the wild-type gene, the protein product of the xcv-1 gene is shorter by 2 amino acids in the TM region. No sequences showing 100% percent identity with the xcv-1 protein were found in the data banks. Consequently, a new protein was identified to which the Xe resistance could be assigned on the basis of the data and experimental results.
Example 8 Intracellular Localisation of Polypeptides Xcv-1 and Xcv-1To determine the intracellular localisation of polypeptides Xcv-1 and xcv-1, the coding sequence of the Green Fluorescent Protein (GFP) (Accession no: AF234298, nucleotides 2 to 757) is cloned in front of genes Xcv-1 and xcv-1, and is introduced into Nicotiana bentamiana, a plant of the Solanaceae family, through Agrobacterium tumefaciens-mediated gene transfer. Transformation is preferably carried out in a manner ensuring a so-called transient expression (Martin K. et al., Plant J. 59:150-62, 2010), as a result of which the GFP-Xcv-1/GFP-xcv-1 constructs are not stably incorporated into the genome of the plant but the transgene is expressed from a DNA in an extrachromosomal state. The experiment was carried out as follows:
First, the structural gene of GFP was cloned into a pGemT-Easy vector as follows: the coding sequence of GFP was amplified using the DNA of the pCambia1302 plasmid (www.cambia.org, Marker Gene Technologies, Inc. www.markergene.com) as template, and SEQ ID NO:43 and SEQ ID NO:44 as primers, and the resulting PCR product was cloned into a pGemT-Easy vector. The resulting plasmid was designated ‘pNcoGFPXba’. In the next step, leaf samples were taken from C. annuum var. T1/1 and C. annuum var. Feherozon plants in the six-leaved stage, and a QIAGEN RNA isolation kit (RNeasy Mini Kit, http://www.qiagen.com) was used to isolate RNA, and the SMART™ cDNA Library Construction Kit (http://www.clontech.com) was used to synthesise double-stranded cDNA. The cDNAs of Xcv-1 and xcv-1 were amplified and cloned in a PCR reaction using the resulting cDNA preparation. To amplify the Xcv-1 allele, cDNA from C. annuum var. Feherozon and primers SEQ ID NO:45 and SEQ ID NO:46 were used for the amplification. The PCR reaction was performed as described in Example 2. The resulting fragment was cloned into a pGemT-Easy plasmid. The resulting plasmid was designated ‘pXcv-1CaFo’. Cloning of the xcv-1 allele was performed in a similar way except for using the cDNA of C. annuum var. T1/1 and the primers represented by SEQ ID NO:45 and SEQ ID NO:46. The resulting plasmid was designated ‘pxcv-1CaT1’. In the third phase, plasmids ‘pXcv-1CaFo’ and ‘pxcv-1 CaT1’ were digested with the enzymes XbaI and BcuI, and were cloned into a ‘pNcoGFPXba’ plasmid digested with XbaI and BcuI. The resulting plasmids were designated ‘pGFP-Xcv-1CaFo’ and ‘pGFP-xcv-1 CaT1’, respectively. In the last step, plasmids ‘pGFP-Xcv-1 CaFo’ and ‘pGFP-xcv-1CaT1’ were digested with SalI, and the plasmid pCambia 1302 was digested with BstEII, and then all three preparations were digested with Mung Bean Nuclease according to manufacturer's (New England Biolabs, Inc, www.neb.com) instructions. The blunt-ended linear molecules ‘pGFP-Xcv-1CaFo’ and pCambia 1302, and ‘pGFP-xcv-1CaT1’ and pCambia 1302 were mixed, and both preparations were digested with the enzyme NcoI, ligated and transformed into E. coli cells. The transformants were grown on kanamycin-containing plates, where only the pCambia1302 derivatives grow but not the pGemT-Easy derivatives. Among the transformant colonies, those comprising the fusion products GFP-Xcv-1 and GFP-xcv-1 were identified. The two products were designated “pCambia-GFP-Xcv-1CaFO” and ‘pCambia-GFP-xcv-1 CaT1’. The sequences of the two fusion products are shown in SEQ ID NO: 47 and SEQ ID NO:48, respectively.
The plasmids ‘pCambia-GFP-Xcv-1 CaFO’ and ‘pCambia-GFP-xcv-1 CaT1’ plasmids were transferred into A. tumefaciens C58 by triparental mating, followed by growing the A. tumefaciens strains comprising the two constructs (‘pCambia-GFP-Xcv-1CaFO’ and ‘pCambia-GFP-xcv-1CaT1’) on solid media and infiltrating into Nicotiana bentamiana leaves in accordance with the method described in the relevant literature. After 48 hours, protoplasts were prepared from the leaf areas giving green fluorescence under UV light, and serial photos were taken of the protoplasts with green fluorescence using confocal microscopy. The pictures clearly demonstrate that that the fusion product comprising the wild-type protein (‘pCambia-GFP-Xcv-1CaFO’) occurs as islands (lipid rafts) in the plasma membrane of the cells, but the mutant protein carrying the double leucine deletion (‘pCambia-GFP-xcv-1 CaT1’) does not form such islands and shows homogeneous distribution in the plasma membrane.
Example 9 Identification of Genes Homologous to the Xcv-1 Gene in Plants and AnimalsComplete or partial nucleotide sequences of the genomes of several viruses, bacteria, fungi and animals were determined in the framework of various genome projects. In most cases, the determination of the DNA sequences did not involve the identification of the function of a given segment; thus, the function of the sequences remains unknown. In relation to the Xcv-1 gene, data bank searches revealed that several sequences encoding proteins with similar structures to the xcv-1 protein can be found in the data banks (Feng et al., Mol Biol Rep DOI 10.1007/s11033-010-0419-1, 2010; Lieber et al., Current Biology 21: 1009-1017, 2011; Li et al., Biotechnol. Lett 31:905-910, 2009; Venancio T. M. and Aravind L. Bioinformatics 26:149-152, 2010). Alignment of the amino acid sequences of these proteins clearly demonstrated their structural similarity. In most cases, the region immersed in the membrane is delimited by a negatively charged amino acid (aspartic acid or glutamic acid) or a polar amino acid (asparagine). Alignment of a part of the CYSTM proteins is shown in Venancio T. M. and Aravind L., Bioinformatics 26:149-152, 2010. For the purposes of the invention, “proteins homologous to the Xcv-1 protein” refer to protein variants which contain at least 53% identical amino acids at their C-termini with respect to the last 15 amino acids of the C-terminus of the Xcv-1 protein (CLAALCCCCLLDACF).
Example 10 Induction of Resistance to Xanthomonas euvesicatoria in Tomato by the amiRNA TechniqueOne of the most dangerous bacterial pathogens of tomato is Xanthomonas euvesicatoria (Xe), i.e., the same bacterium which also causes severe damage to pepper. Unlike pepper, tomato has not developed appropriate natural resistance, which would ensure acceptable protection. The bs4 gene identified in tomato does not provide sufficient protection, and therefore, tomato production is still highly threatened by Xe infection (Hutton et al., Theor. Appl. Genet. 121:1275-87, 2010). It was assumed that the xcv-1 gene identified in pepper could also provide resistance to Xe infection in tomato if the genes homologous to the Xcv-1 gene, i.e., genes SlXcv-1A and SlXcv-1B represented by SEQ ID NO:49 and SEQ ID NO:51, respectively, are inactivated in the tomato genome, and the inactivation is preceded by ensuring the functioning of the tomato genes homologous to the xcv-1 gene, i.e., genes Slxcv-1A and Slxcv-1B represented by a SEQ ID NO:59 and SEQ ID NO:66, respectively (see below). This strategy can be implemented in more than one ways including but not limited to:
1. Tomato is transformed with functional Slxcv-1A and Slxcv-1B genes followed by inactivating the resident genes SlXcv-1A and SlXcv-1B using the “amiRNA” technique.
2. Tomato is transformed with functional Slxcv-1A and Slxcv-1B genes followed by inactivating the resident genes SlXcv-1A and SlXcv-1B using the ZFN nuclease technique.
3. Tomato is transformed with functional Slxcv-1A and Slxcv-1B genes followed by identifying mutations—that inactivated SlXcv-1A and SlXcv-1B—using the TILLING or another similar technique, upon or without mutagenesis.
4. Tomato is transformed with functional Slxcv-1A and/or Slxcv-1B genes followed by inactivating the resident genes SlXcv-1A and SlXcv-1B present there using the TALEN technique.
5. Tomato is transformed with functional Slxcv-1A and/or Slxcv-1B genes followed by inactivating the resident genes SlXcv-1A and SlXcv-1B present there using the CRISPR/Cas technique.
A common feature of the above five strategies is the in vitro preparation of the Slxcv-1A and Slxcv-1B sequences and transformation into tomato in the first step. Since the functioning of the Slxcv-1A and Slxcv-1B genes is recessive in comparison with the wild-type genes, the second step involves inactivating the wild-type genes (SlXcv-1A and SlXcv-1B) with suitable methods—including but not limited to the above-listed three methods—in order to manifest the above functions.
Example 10A Generation of Slxcv-1A and Slxcv-1B Sequences with Double Leucine DeletionThe preparation of the genes comprising the double leucine deletion (Slxcv-1A and Slxcv-1B) involved PCR amplification, cloning of the amplificates into pGemT-Easy vectors, and additional restriction digestions and reclonings—steps well-known to those skilled in genetic engineering. For the cloning, the sequences of two tomato genes, i.e., SlXcv-1A and SlXcv-1B, obtained from data banks were used. The sequences of genes SlXcv-1A and SlXcv-1B are SEQ ID NO:49 and SEQ ID NO:51, respectively.
Preparation of the Slxcv-1A Construct:
a PCR amplification was performed in the presence of a genomic DNA template from tomato using primers ‘SlProm1AF3’ (SEQ ID NO:53) and ‘SlProm1AR3’ (SEQ ID NO:54), and the 1074-bp DNA fragment was cloned into pGEM-T Easy vectors. The resulting plasmid (‘p1A1’) was digested with the enzyme NsiI followed by ligation and transformation to generate plasmid ‘p1A2’. A PCR amplification was performed in the presence of a genomic DNA template from tomato using primers ‘SlTerm1AF3’ (SEQ ID NO:55) and ‘SlTerm1AR3’ (SEQ ID NO:56), and the 283-bp DNA fragment was cloned into pGEM-T Easy vectors to generate plasmid ‘p1A3’. The ‘p1A2’ plasmid was digested with NsiI, and the ‘p1A3’ plasmid was digested with NsiI and PstI; next, the two mixtures were combined and ligated, and—upon transformation—plasmid ‘p1A4’, in which the NsiI end of ‘p1A3’ is positioned towards the genomic sequence in ‘p1A2’, was identified. A PCR amplification on tomato genomic DNA was performed using primers ‘SlMid1AF1’ (SEQ ID NO:57) and ‘SlMid1ABR1’ (SEQ ID NO:58), and the 1137-bp DNA fragment was cloned into pGEM-T Easy vectors to generate plasmid ‘p1A5’. Upon mixing ‘p1A4’ and ‘p1A5’, the plasmids were digested with NsiI and ligated, and—upon transformation—plasmid ‘p1A6’, in which the 1094-bp NsiI fragment was cloned into the NsiI site of the ‘p1A4’ plasmid in the correct orientation, was identified. Finally, this resulted in construct Slxcv-1A (SEQ ID NO:59), which encodes the Slxcv-1A protein (SEQ ID NO:60), a variant with the double leucine deletion.
Preparation of the Slxcv-1B Construct:
A PCR amplification was performed in the presence of a genomic DNA template from tomato using primers ‘SlProm1BF3’ (SEQ ID NO:61) and ‘SlProm1BR3’ (SEQ ID NO:62), and the 1450-bp DNA fragment was cloned into pGEM-T Easy vectors. The resulting plasmid (‘p1B1’) was digested with the enzyme NsiI followed by ligation and transformation to generate plasmid ‘p1B2’. A PCR amplification was performed in the presence of a genomic DNA template from tomato using primers ‘SlTerm1 BF3’ (SEQ ID NO:63) and ‘SlTerm1BR3’ (SEQ ID NO:64), and the 787-bp DNA fragment was cloned into pGEM-T Easy vectors to generate plasmid ‘p1B3’. The ‘p1B2’ plasmid was digested with NsiI, and the ‘p1B3’ plasmid was digested with NsiI and PstI; next, the two mixtures were combined and ligated, and—upon transformation—plasmid ‘p1B4’, in which the NsiI end of ‘p1B3’ is positioned towards the genomic sequence in ‘p1B2’, was identified. A PCR amplification on tomato genomic DNA was performed using primers ‘SlMid1BF1’ (SEQ ID NO:65) and ‘SlMid1ABR1’ (SEQ ID NO:58), and the 1273-bp DNA fragment was cloned into pGEM-T Easy vectors to generate plasmid ‘p1B5’. The ‘p1B4’ plasmid was digested with NsiI but the ‘p1B5’ plasmid was only partially digested with NsiI; next, the two samples were combined and ligated, and—upon transformation—plasmid ‘p1B6’, in which the 1236-bp fragment was cloned into the NsiI site of the ‘p1B4’ plasmid in the correct orientation, was identified. Finally, this resulted in construct Slxcv-1B (see SEQ ID NO:66), which encodes the Slxcv-1B protein (SEQ ID NO:67), a variant with the double leucine deletion.
Upon preparing the two constructs, the Slxcv-1A and Slxcv-1B sequences were cloned head to head into an A. tumefaciens vector pCAMBIA2300 cut by XbaI from the pGemT-Easy vector using NotI-SpeI, and were transformed into E. coll. The resulting plasmid was designated ‘pDSlxcv-1AB’. From the E. coli host, the plasmid was introduced into the A. tumefaciens strain using triparental mating. The resulting strains were designated A. tumefaciens (pDSlxcv-1AB).
Example 10B Transformation of the Slxcv-1A and Slxcv-1B Genes into Tomato Using Agrobacterium tumefaciensA. tumefaciens transformation was carried out as follows: Tomato seeds were immersed in 70% ethanol for 1 minute, and were then transferred into a solution of 5.25% sodium perchlorate (NaClO) and 0.1% Tween 20 and shaken for 30 minutes. Next, the seeds were rinsed with distilled water 8 times and transferred to Petri dishes containing medium A, and were grown for 8 days at 25° C. with 16-hour light cycle. The cotyledons of the plants were cut at the apex and at the base, pricked, placed on medium B, and overlaid with MSO liquid medium containing A. tumefaciens (pDSlxcv-1AB). The MSO liquid medium containing A. tumefaciens (pDSlxcv-1AB) was prepared as follows: the A. tumefaciens (pDSlxcv-1AB) strain stored at −80° C. (prepared as described in Example 11A) was plated onto a medium containing YEP+100 μg/ml rifampicin and incubated at 30° C. One of the colonies was inoculated into 3 ml YEP liquid medium (in a 20-ml test tube) using an inoculation loop, and the bacteria were rotated in a roller to ensure aeration and cultured until reaching the stationary phase (24 hours). The bacteria were collected by centrifugation as previously described, the supernatants were discarded and the cells were suspended in 12 ml MSO liquid medium. The leaves were treated with the Agrobacterium suspension for 20 minutes, then the excess suspension was drawn off and the leaves were co-cultivated with the bacteria for 48 hours. After two days, the leaves were transferred to plates with medium C. The plants were transferred to fresh plates with medium C at two-week intervals. The developing calluses could be cut to smaller pieces, and were transferred to plates with medium D and then to fresh plates at two-week intervals. When the small growths appeared, they were transferred again to plates with medium D. When reaching a length of 2 to 4 cm, the growths were transferred to fresh plates with medium E, and they started to form roots. Plants of 5 cm could already be planted into potting soil. A total of 15 independent T0 transformant plants (T0/xcv1 to 15) were grown.
MSO medium (1000 ml): 4.3 g MS salts, 100 mg myo-inositol, 0.4 ml (1 mg/ml) thiamine-HCl, 20 g saccharose
YEP medium (1000 ml): 10 g yeast extract, 10 g peptone, 5 g NaCl (pH adjusted to 7 with NaOH)
Vitamin solution (per 1000 ml): 50 mg thiamine-HCl, 200 mg glycine, 500 mg nicotine aid, 50 mg pyridoxine-HCl, 50 mg folic acid, 5 mg biotin, 10 g myo-inositol
When the stems and roots of the T0/xcv1-15 transgenic plants were strong enough, DNA was isolated from the leaves, and a PCR reaction was performed to detect the transformation events using the following primer pairs:
- 1. Pr_Sl SlMid1AF1 primer (SEQ ID NO:57);
- Pr_SlTerm1AR3 primer (SEQ ID NO:56);
- length of the expected amplificate: 1400 bp;
- 2. Pr_SlTerm1BF3 primer (SEQ ID NO:62);
- Pr_SlTerm1BR3 primer (SEQ ID NO:63);
- length of the expected amplificate: 787 bp.
The transgene sequence between the primers used for the amplification could be detected in all cases.
The microRNAs (miRNAs) discovered in eukaryotic organisms inhibit the efficient expression of the corresponding genes. This gene inactivation allows for an alternative form of gene regulation through a specific mechanism resulting in the inhibition of the function of the gene, which is of great importance in terms of development and differentiation (Kidner C. A. és Martienssen R. A., Curr. Opin. Plant Biol. 8:38-44, 2005). miRNAs are ribonucleic acid molecules present in eukaryotic cells. The miRNAs are short molecules consisting of 21 to 24 nucleotides in contrast to the long RNA molecules fulfilling other functions (e.g., mRNA, ribosomal RNA). The miRNAs are post-transcriptional inhibitors of the functioning of mRNAs by physically inhibiting protein synthesis on complementary mRNAs, or by causing the degradation of complementary mRNAs upon binding to them (Bartel D. P., Cell 16:281-297, 2004).
Studies of the miRNAs and exploration of the biochemical processes on the molecular level made it possible to extend this specific inhibition mechanism to genes for which no natural miRNAs exist. Artificially prepared gene-specific miRNAs were designated amiRNAs (artificial miRNA) (Ossowski et al., Plant J. 53:674-690, 2008; Park et al., Plant Cell Rep. 28:469-480, 2009; Schwab et al., Methods Mol. Biol. 592:71, 2010; Sablok et al., Biochem. and Biophys. Res. Comm. 406:315-319, 2011). The amiRNA-based gene inactivation has been generated in a number of animal and plant systems (Schwab et al, Plant Cell 18:1121-1133, 2006), and in general terms, the target mRNAs can be inactivated, thereby eliminating the gene function in question, through carefully designed experiments.
An amiRNA gene coding for an amiRNA ribonucleotide consist of the following sequences: promoter, 5′ stem extension, amiRNA*, loop region, the amiRNA and a 3′ stem extension with polyA tail (Schwab et al., Methods Mol. Biol. 592:71, 2010).
Strategic Course of Inducing Resistance in Tomato Plants:
During the PCR amplification and sequencing of the tomato genomic DNA, two sequences homologous to the Xcv-1 gene of pepper (SlXcv-1A and SlXcv-1B) were identified: the nucleotide sequences and the deduced amino acid sequences are shown in the DNA sequences of SEQ ID NO:49 and SEQ ID NO:51, respectively, and the amino acid sequences of SEQ ID NO:50 and SEQ ID NO:52, respectively.
The target mRNA sequence—with which the amiRNA designated ‘SlXe1-amiRNS’ will show partial complementarity (18 of 21 bases)—is the segment which precedes the stop codon of genes SlXcv-1A and SlXcv-1B and encodes the two leucines corresponding to the Xcv-1 gene (see
The coding segment of the pre-SlXe1-amiRNA (SEQ ID NO:73) sequence, i.e., the pre-SlXe1-amiDNA (SEQ ID NO:72) is prepared as follows. Amplification is carried out from tomato genomic DNA using the synthesised primers ‘Pri_SlXe1pre-amiRNA’ (SEQ ID NO:70) and ‘Pr2_SlXe1pre-amiRNA’ (SEQ ID NO:71), and the resulting double-stranded pre-SlXe1-amiDNA (SEQ ID NO:72) coding sequence is cloned into pGemT-Easy vectors, and—upon restriction by EcoRI-SpeI—pKSS vectors, and finally into pC61H vectors through KpnI and XbaI cleavage, as described in Example 3. The HindIII-EcorRI fragment of pCK61H was generated by cloning the EcoRI-HindIII fragment of BIN61S (Silhavy D. et al., EMBO J. 21:3070-3080, 2002) carrying the 35S promoter, polilinker and terminator sequences into the EcoRI-HindIII site of the pCAMBIA1300. The resulting plasmid was designated pC61H-pri-SlXe1-amiRNA and the strain containing the plasmid was designated A. tumefaciens (pC61H-pri-SlXe1-amiRNA). The HindIII-EcorRI fragment of pCK61H-SlXe1-amiRNA carrying the gene encoding SlXe1-ami RNA is shown in SEQ ID NO:92
Example 10D Transformation of Pri-SlXe1-amiRNS Sequences into T0/1-15 Transgenic Plants Containing the Genes Slxcv-1A and Slxcv-1BThe transformation with A. tumefaciens was carried out as described in Example 10B, but the plants to be transformed were the T0/1-15 transgenic plants, the A. tumefaciens (pC61K-pri-SlXe1-amiRNA) strain was used for the transformation and the selection was for hygromycin. At the end of the transformation, seven independent plants (T0/ami1 to 7) were grown.
Pr1_SlXe1 pre-amiRNA (SEQ ID NO:70);
Pr2_SlXe1 pre-amiRNA (SEQ ID NO:71);
length of the expected amplificate: 178 bp.
In addition to DNA isolation, total RNA was isolated from the leaves of the control and transgenic plants using the RNeasy Mini Kit, and the total RNA was run on a 12% carbamide/acrylamide gel, transferred to a Hybond NX membrane (GE Healthcare Amersham) and hybridised with an alpha-32ATP-labelled LNA probe encoding the SlXe1-amiRNA. The autoradiogram obtained upon the hybridisation and the image of the total RNA loaded to the gel are shown in
The transformant plants were grown until the 8-leaved stage, infected with the bacterium Xanthomonas euvesicatoria, and evaluated as described in Example 1. The results of the Xanthomonas euvesicatoria infection are summarised in Table 2.
In a certain prior reverse genetic approach, a plant was first mutagenised, and then the mutation was identified in the gene sought. In most cases, the mutation was identified using T_DNA and transposon insertion mutagenesis, and TILLING (Targeted Induced Local Lesions in Genomes) (Feldman, K. A. The Plant Journal 1:71-82, 1991; McCallum, C. M. et al., Nat. Biotech. 18455-457, 2000). However, this approach was troublesome, time-consuming and uncertain. The RNA interference (RNAi) and artificial microRNA (amiRNA) techniques mentioned in Example 10 are already specific to the desired gene, however, the expression of the gene is often impossible to eliminate completely, that is, null phenotype should be obtained by all means (Schwab et al, Plant Cell 18:1121-1133, 2006). Consequently, methods resulting in genes that are completely knocked out and thus guaranteeing a null phenotype are of vital importance. By now, three methods satisfying the above criteria have been disclosed. These are the above-mentioned ZFN, TALEN and CRISPR/Cas nuclease techniques, which generate gene specific c double stranded cuts in the DNA and following the activity of the repair mechanism of the cells insertions and deletions with the size of one to several tens of base pairs or more in a gene-specific manner [Urnov F. D. et al. Nat Rev Genet. 11:636-46, 2010; Carroll D. Genetics. 188:773-82, 2011; Christian M. et al., Genetics 189:757-761, 2010; Cermak, T. et al., Nucl. Acids Res. 39: e82, 2011; Mussolino C. et al., Nucleic Acids Res. 39:9283-9293, 2011; Miller J. C. et al., Nat. Biotechnol. 29:143-148, 2011; Christian M. et al., G3 (Genes,Genomes,Genetics, Bethesda), doi:10.1534/g3.113.007104, 2013; Cho S. W. et al., Nat Biotechnol. 31:230-232, 2013; Cong L. et al., Science 339:819-823, 2013; Mali P. et al., Science 339:823-826, 2013]. For the purpose of generating the 6-bp deletion in the tomato genes SlXcv-1A and SlXcv-1B, the TALEN, CRISPR/Cas nuclease and the ZFN technique can be equally used. From tomato cells only producing the double leucine deletion proteins Slxcv-1A and/or Slxcv-1B, Xe resistant tomato plants can be generated in the same way as in Example 10.
Example 11A Induction of Resistance to Xanthomonas euvesicatoria in Tomato Using the TALEN TechniqueThe TALEN target sequence to which the TALEN pairs recognize (SlXcv-1AB_TALEN-L, SlXcv-1A_TALEN-R and SlXcv-1B_TALEN-R) should be selected and determined in view of the fact that the 6 bp deletions should located at positions 2277 to 2282 and 2640 to 2645, respectively. Since the applied TALEN nucleases quite often cut within the so-called “spacer” sequences, it is reasonable to chose a size of 18 base pairs for the spacer region, and to chose a TALEN target sequence extending into 17 and 17 base pairs both to the right and to the left in a manner ensuring that the target sequences are preceded by a T/A base pair in all cases [Cermak, T. et al., Nucl. Acids Res. 39:e82, 2011; Christian M. et al., G3 (Genes,Genomes,Genetics, Bethesda)]. Since the sequences of the two genes to the left side of the mutation are identical along an at least 36-bp segment from the left end of the desired deletion, the same left TALEN target sequence should be chosen for both genes (SlXcv-1AB_TAL-L, see SEQ ID NO:75 and
In Variant 1, the desired 6-bp deletion is present in both genes; thus, these plants carry the genes Slxcv-1A and Slxcv-1B, which encode the Slxcv-1A (SEQ ID NO:60) and the Slxcv-1B (SEQ ID NO:67) amino acid sequences (see
In Variant 2, the plants carry the Slxcv-1A gene, which encodes the Slxcv-1A (SEQ ID NO:60) amino acid sequence. In Variant 2, the derivative of the SlXcv-1B gene contains a deletion/insertion which changes the open reading frame (out of frame deletion) (see
In Variant 3, the plants carry the Slxcv-1B gene, which encodes the Slxcv-1B (SEQ ID NO:67) amino acid sequence. In Variant 3, the derivative of the SlXcv-1A gene contains a deletion/insertion which changes the open reading frame (out of frame deletion) consequently the gene is inactivated (see
The above three transformant derivatives (Variants 1, 2 and 3) can be vegetatively propagated and grown to the 8-leaved stage, then infected with Xanthomonas euvesicatoria, and the products resistant to Xanthomonas euvesicatoria can be selected. For a skilled person, it is obvious that the transgenes (T-DNAs) can be removed from the genome of the selected plants resistant to Xanthomonas euvesicatoria by crossing, because the transgenes will be segregated from a part of the progeny, that is, non-transgenic plants can be generated from them.
Example 11B Induction of Resistance to Xanthomonas euvesicatoria in Tomato Using the CRISPR/Cas Nuclease TechniqueThe above-described CRISPR/Cas technology (Cho S. W. et al., Nat. Biotechnol. 31:230-232, 2013; Cong L. et al., Science 339:819-823, 2013; Mali P. et al., Science 339:823-826, 2013; Feng Z. et al., Cell Research: 1-4, 2013) is also useful for generating the desired 6-bp deletion(s), or gene mutations resulting in null phenotypes, in tomato and in other plants. In the first case, ‘single guide RNAs’ (sgRNAs in short) were designed for the wild-type tomato sequences corresponding to the desired 6-bp deletion (see
Upon preparing the vectors, the tomato cells are transformed as described in Example 10, the hygromycin and kanamycin resistant calluses are selected as described in Example 11, and the gene derivatives of SlXcv-1A and SlXcv-1B are evaluated as described in Example 11. As a result of the experiments and tests, similar results are expected as in the case of the three variants described in Example 11, that is, the desired 6-bp deletion is present in both SlXcv-1 genes (SlXcv-1A and SlXcv-1B), which encode the Slxcv-1A (SEQ ID NO:60) and Slxcv-1B (SEQ ID NO:67) amino acid sequences, respectively; or the desired 6-bp deletion is only present in SlXcv-1A and SlXcv-1B, and early stop codon, or an out of frame deletion/insertion giving null phenotype is generated in the other gene.
Upon treatment with the CRISPR/Cas nuclease, the transformant derivatives having the advantageous feature (Variants 1, 2 and 3, see Example 11A.) can be vegetatively propagated and grown to the 8-leaved stage, then infected with Xanthomonas euvesicatoria, and the progeny resistant to Xanthomonas euvesicatoria can be selected. For a skilled person, it is obvious that—in this case too—the transgenes (T-DNAs) can be removed from the genome of the selected plants resistant to Xanthomonas euvesicatoria by crossing, because the transgenes will be segregated from a part of the progeny, that is, non-transgenic plants can be generated from them.
Example 11C Induction of Resistance to Xanthomonas euvesicatoria in Tomato Using the ZFN TechniqueThe above-mentioned ZFN technology (Urnov F. D. et al. Nat Rev Genet. 11:636-46, 2010, Carroll D. Genetics. 188:773-82, 2011) is also useful for generating the desired 6-bp deletion(s), or gene mutations resulting in null phenotypes, in tomato and in other plants. In case of generating the desired 6-bp deletion, the procedure is as follows: the target sequences to be recognised by the two zinc finger (ZF) proteins are selected within the segment from bases 2253 to 2306 (SEQ ID NO:84) of the SlXcv-1A gene sequence (SEQ ID NO:49) and within the segment from bases 2616 to 2669 (SEQ ID NO:85) of the SlXcv-1B gene sequence (SEQ ID NO:51)—as shown in
Upon preparing the vectors, the tomato cells are transformed as described in Example 10, and the procedures described in Examples 11A and 11B are followed thereafter.
The deletion/insertion procedure using the ZNF, TALEN and CRIPSR/Cas nucleases can be performed in the transgenic tomato plants carrying one of the genes (i.e., either Slxcv-1A or Slxcv-1B), or both (Slxcv-1A and Slxcv-1B), wherein knock-out insertion or deletion derivatives are sought in the resident genes SlXcv-1A and SlXcv-1B among the plants treated with the ZFN, TALEN or CRIPSR/Cas nucleases. In fortunate cases, the double null allele variant can also be obtained after performing the transformation, and the second transformation is unnecessary.
For the skilled person, it is obvious and understandable that knock-out null mutants in the SlXcv-1A and SlXcv-1B genes may not only be generated within the segment from bases 2244 to 2318 (SEQ ID NO:84) of the SlXcv-1A gene sequence (SEQ ID NO:49) and within the segment from bases 2607 to 2681 (SEQ ID NO:85) of the SlXcv-1B gene sequence (SEQ ID NO:51)—as shown in
Claims
1. A nucleic acid molecule that is capable of conferring to a plant resistance to Xanthomonas euvesicatoria, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
- (a) a non-naturally occurring nucleotide sequence comprising at least 80% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NO:37, 59, and 66;
- (b) the nucleotide sequence set forth in SEQ ID NO:88, 89, or 90;
- (c) a nucleotide sequence comprising at least 80% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS:88, 89, and 90;
- (d) a nucleotide sequence encoding an artificial protein comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:42, wherein the artificial protein comprises a CYSTM region comprising the deletion of two amino acids relative to the CYSTM region of the Xcv-1 protein having the amino acid sequence set forth in SEQ ID NO:42;
- (e) a nucleotide sequence encoding an artificial protein comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:38, 60, or 67, wherein the artificial protein comprises a CYSTM region with a double Leu deletion at the locations corresponding to positions 87 and 88 of the Xcv-1 protein having the amino acid sequence set forth in SEQ ID NO:42.
2. An artificial xcv-1 CYSTM protein that is capable of conferring to a plant providing resistance to Xanthomonas euvesicatoria, wherein the artificial xcv-1 CYSTM protein is encoded by the nucleic acid molecule of claim 1.
3-11. (canceled)
12. A nucleic acid molecule for silencing the expression of the SlXcv-1A gene and/or the SlXcv-1B gene, said nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
- (a) the nucleotide sequence set forth in SEQ ID NO:74;
- (b) a nucleotide sequence comprising at least 60% identity to the nucleotide sequence set forth in SEQ ID NO:74;
- (d) the nucleotide sequence set forth in SEQ ID NO: 72;
- (e) the nucleotide sequence set forth in SEQ ID NO: 73;
- (f) the nucleotide sequence set forth in SEQ ID NO: 92; and
- (g) the full-length complement of any one of (a)-(f).
13. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule further comprises an additional nucleotide sequence wherein said additional nucleotide sequence is a miRNA backbone sequences which is operably linked to the nucleic acid molecule nucleotide for silencing the expression of the SlXcv-1A gene and/or the SlXcv-1B gene.
14. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule is capable of silencing at least one of the SlXcv-1A and the SlXcv-1B genes when said nucleic acid molecule expressed in a plant.
15. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule does not silence the expression of, the 6 bp deletion mutant of the SlXcv-1A gene which encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 50 and/or the 6 bp deletion mutant of the SlXcv-1B gene which encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 52.
16. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule or part thereof is expressed in a plant as an amiRNA.
17. The nucleic acid molecule of claim 12, further comprising an operably linked promoter that is capable of driving the expression of the nucleic acid molecule in a plant.
18. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule is an artificial nucleic acid molecule.
19. A vector comprising a gene according to claim 1.
20. A host cell transformed with a vector according to claim 19.
21. A method for the in vitro preparation of a mutant gene homologous to the xcv-1 gene or its cDNA variant of claim 1, comprising the steps of:
- a) identifying a gene homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in a plant,
- b) preparing in vitro the genomic and cDNA sequences of the gene identified in step a) in the form of a DNA, and
- c) creating in vitro the desired deletion of 6 bp in the DNA prepared in step b) in the segment of the gene encoding the CYSTM region.
22. The method of claim 21, wherein in step c), the desired 6-bp deletion is created in those nucleotides of the CYSTM region of the gene that encode the 5th and 6th amino acids from the C-terminus.
23. A mutant plant showing resistance to a biotic or abiotic factor, the genome of which is modified to contain a 6-bp deletion in the segment encoding the CYSTM region of one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41).
24. A method for generating a transgenic plant resistant to a biotic or abiotic factor according to claim 23 by transformation, comprising the steps of:
- a) transforming the cells of a sensitive plant by a gene comprising the nucleotide sequence set forth in SEQ ID NO: 37 or 90 or a gene encoding a protein homologous to xcv-1 CYSTM and which comprises a deletion of two amino acids in the CYSTM region in comparison with CYSTM region of the wild-type homologous protein, and provides resistance in a manner ensuring the functional expression thereof,
- b) inactivating one or more resident genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41), or the mRNA or protein thereof, in the sensitive transformant plant cells obtained in step a), and
- c) regenerating the plant from the transformants and selecting the resistant individuals.
25. A method according to claim 24, wherein in step b), the mRNA products of the resident gene(s) are functionally silenced by amiRNA technique or an engineered nuclease deletion method.
26. (canceled)
27. A method for generating a mutant plant showing an abiotic or biotic resistance according to claim 23 using a genome editing method, comprising the steps of:
- a) identifying one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in a sensitive plant,
- b) preparing DNA constructs encoding TALEN-L and TALEN-R or ZFN-L and ZFN-R engineered nuclease proteins specific to the gene sequence encoding the CYSTM region of one or more genes homologous to the wild-type Xcv-1 gene and identified in step a), more specifically those specific to the 6 nucleotides to be deleted and the surrounding nucleotides as target sequence in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas nuclease construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence,
- c) cloning the construct obtained in step b) into an appropriate vector,
- d) transforming sensitive plant cells with a vector comprising the construct obtained in step b) in a manner ensuring the functional expression of the transgenes and the generation of the 6-bp deletion in the CYSTM region by the nuclease,
- e) identifying the transformants carrying mutations showing the 6-bp deletion in the CYSTM region of the genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41),
- f) regenerating the plant cells having the mutation identified in step e) and selecting the resistant plants, and
- g) removing the transgenes comprising the TALEN or ZFN constructs or CRISPR/Cas constructs containing the gene of the sgRNS protein by genetic segregation.
28. (canceled)
29. A method for generating a tomato plant resistant to Xanthomonas euvesicatoria, comprising the steps of:
- a) identifying the resident genes SlXcv-1A and SlXcv-1B (SEQ ID NO:49 and 51, respectively), which are homologous to the wild-type Xcv-1 gene in the tomato plant,
- b) preparing in vitro the genomic and cDNA sequences of the identified genes in the form of a DNA,
- c) creating in vitro the 6-bp deletion in the positions corresponding to the deletions in the xcv-1 gene thereby generating constructs carrying the mutant genes Slxcv-1A and Slxcv-1B (SEQ ID NO:59 and 66, respectively) or their cDNA sequences (SEQ ID NO:88 and 89, respectively),
- d) cloning the constructs obtained in step c) into an appropriate vector and transforming the cells of the tomato plant with the resulting vector in a manner ensuring the functional expression of the transgenes,
- e) preparing an amiRNA gene construct specific for silencing the SlXcv-1A and/or the SlXcv-1B gene
- f) cloning the construct obtained in step e) into an appropriate vector,
- g) transforming the plant cells generated in step d) with the vector obtained in step f) in a manner ensuring the expression of the amiRNA and inactivation of the mRNA products of the wild-type CYSTM genes SlXcv-1A and SlXcv-1B, and
- h) regenerating the transformants obtained in step g) and selecting the resistant plant.
30. The method of claim 29, wherein step (e) comprises preparing an amiRNA gene construct specific to the complementary ribonucleotide sequence corresponding to the CYSTM region of the wild-type genes SlXcv-1A and/or SlXcv-1B (SEQ ID NO:49 and 51, respectively), more specifically an amiRNA gene construct specific to the 6 nucleotides to be deleted and the surrounding nucleotides (SlXe1-amiRNA, SEQ ID NO: 74).
31. The method of claim 29, wherein the amiRNA gene construct comprises the nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
- (a) the nucleotide sequence set forth in SEQ ID NO:74;
- (b) a nucleotide sequence comprising at least 60% identity to the nucleotide sequence set forth in SEQ ID NO:74;
- (d) the nucleotide sequence set forth in SEQ ID NO: 72;
- (e) the nucleotide sequence set forth in SEQ ID NO: 73;
- (f) the nucleotide sequence set forth in SEQ ID NO: 92; and
- (g) the full-length complement of any one of (a)-(f).
32. A method for generating a tomato plant according to claim 23 that is resistant to Xanthomonas euvesicatoria, comprising the steps of:
- a) identifying the resident genes SlXcv-1A and SlXcv-1B (SEQ ID NO:49 and 51, respectively), which are homologous to the wild-type Xcv-1 gene in the tomato plant,
- b) preparing in vitro the genomic and cDNA sequences of the identified genes in the form of a DNA,
- c) creating in vitro the 6-bp deletion in the positions corresponding to the deletions in the xcv-1 gene thereby generating constructs carrying the mutant genes Slxcv-1A and Slxcv-1B (SEQ ID NO:59 and 66, respectively) or their cDNA sequences (SEQ ID NO:88 and 89, respectively),
- d) cloning the constructs obtained in step c) into an appropriate vector and transforming the cells of the tomato plant with the resulting vector in a manner ensuring the functional expression of the transgenes,
- e) preparing engineered nuclease proteins TALEN-L (SEQ ID NO:78) and TALEN-R (SEQ ID NO:79, and 80) specific to the target sequences (SEQ ID NO:75, 76, 77) specific to the CYSTM region of the wild-type SlXcv-1A and SlXcv-1B gene, or DNA constructs encoding engineered nuclease proteins ZFN-L and ZFN-R specific to SEQ ID NO: 86 and 87 in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of the TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas nuclease construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence (SEQ ID NO: 81),
- f) cloning the construct obtained in step e) into an appropriate vector,
- g) transforming the plant cells generated in step d) with the vector obtained in step f) in a manner ensuring the functional expression of the above TALEN or ZFN or CRISPR/Cas nuclease transgenes and the functional inactivation of genes SlXcv-1A and SlXcv-1B by them,
- h) identifying deletion or insertion knock-out mutations in the transformant cells,
- i) regenerating the plant cells having the mutation identified in step h) and selecting the resistant plants, and
- j) removing the nuclease transgenes TALEN-L plus TALEN-R or ZFN-L plus ZFN-R or CRISPR/Cas by genetic segregation.
33. (canceled)
34. The mutant plant of claim 23, in which the resistance is recessive resistance to Xanthomonas sp.
35. The mutant plant of claim 23, which is a monocotyledonous plant or a dicotyledonous plant.
36. (canceled)
37. The mutant plant of claim 23, which is a plant selected from the group consisting of: rice, maize, wheat, rye, barley, millet, banana orange, mandarin, lemon, grapefruit, pomelo, potato, tomato, pepper, eggplant, cabbage, radish, cauliflower, rape, alfalfa, bean, pea, pumpkin, cucumber, melon, apple, quince, cherry, plum, apricot, peach and cotton.
38-43. (canceled)
44. A food product produced from the mutant plant of claim 23 or part thereof.
45. (canceled)
46. Seeds of the mutant plant of claim 23.
47-52. (canceled)
Type: Application
Filed: Oct 30, 2013
Publication Date: May 19, 2016
Patent Grant number: 11041165
Inventors: György Botund Kiss (Szeged), Zoltán Szabó (Gárdony), Carmen E. Illiescu (Gödöllö), Márta Balogh (Száhalombatta)
Application Number: 14/439,525
