PLANTS AND METHODS FOR CONTROLLING FUNGAL PLANT PATHOGENS
Provided herein are plants that reduce growth of a fungal pathogen, increase resistance of the plant to a fungal pathogen, or a combination thereof. The plant includes a polynucleotide that reduces expression of a coding region present in a fungal pathogen, such as Sclerotinia sclerotiorum or Botrytis cinerea. The polynucleotide can be present on the surface of the plant, expressed by a plant, or a combination thereof. Also provided are methods of making and methods of using the plants.
This application is a divisional of pending U.S. patent application Ser. No. 16/098,387 filed Nov. 1, 2018 which is the § 371 U.S. National Stage of International Application No. PCT/IB2017/052578, filed May 3 2017, which claims the benefit of U.S. Provisional Application No. 62/331,026, filed May 3, 2016, the disclosures of which are incorporated by reference herein in their entireties.
SEQUENCE LISTINGThis application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an ASCII text file entitled “2021-07-19-0352-000037US02-SeqList_ST25.txt” having a size of 135 kilobytes and created on Jul. 19, 2021. The information contained in the Sequence Listing is incorporated by reference herein.
BACKGROUNDCanola is an important oilseed crop, contributing billions of dollars the the economies of many countries (www.canolacouncil.org). Canola crops are threatened by a variety of fungal pathogens, but the most damaging is Sclerotinia sclerotiorum. This fungus occurs throughout the world including all canola growing regions of Canada (Baharlouei et al., 2011, African J Biotech 10: 5785-5794; Litholdo et al., 2010, Genetics Molec Res 10: 868-877; Hu et al., 2011, Can J Microbiology 57: 539-546), and causes stem rot of canola plants. Annual losses due to this fungus are highly variable, ranging from 5 to 100%; in 2010, 90% of Canadian canola crops showed some level of Sclerotinia infection and the loss to growers was estimated at $600 million (www.gov.mb.ca). Damage from Sclerotinia is mitigated primarily by crop rotations and foliar fungicides, but under damp climatic conditions, such methods are often insufficient to control the disease.
SUMMARY OF THE APPLICATIONIncreasing public concern over the risk that chemicals pose to the environment and human health are compelling reasons to find safer (fungal or species-specific) alternatives to control necrotrophic fungal pathogens.
Provided herein are plants. In one embodiment, a plant includes a polynucleotide that reduces expression of a coding region. The coding region includes a sequence that has at least 80% identity to SS1G_06487, SS1G_01703, SS1G_05899, SS1G_02495, SS1G_11912, SS1G_07873, or SS1G_09665, or a homolog of SS1G_06487, SS1G_01703, SS1G_05899, SS1G_02495, SS1G_11912, SS1G_07873, or SS1G_09665, where the coding region is present in a fungal pathogen, and wherein the polynucleotide is on a surface of the plant. In one embodiment, growth of the fungal pathogen on or in the plant is reduced compared to a control plant. In one embodiment, resistance of the plant to the fungal pathogen is increased compared to a control plant. In one embodiment, lesion size after administration of the fungal pathogen to leaves of the plant is reduced by at least 3% compared to a control plant that does not include the polynucleotide. In one embodiment, the plant pathogen is Sclerotinia sclerotiorum or Botrytis cinerea. In one embodiment, the polynucleotide includes a double stranded RNA (dsRNA), where the dsRNA includes a nucleotide sequence that is substantially identical to a series of nucleotides of the coding region. In another embodiment, the polynucleotide includes a single stranded RNA (ssRNA), wherein the ssRNA includes a nucleotide sequence that is substantially complementary to a series of nucleotides of the coding region. In one embodiment, the polynucleotide includes at least 15 nucleotides.
In one embodiment, the polynucleotide reduces expression of the coding region in the fungal pathogen by at least 5% when the polynucleotide is introduced into the fungal pathogen and compared to a control fungal pathogen that does not include the polynucleotide. In one embodiment, the polynucleotide reduces expression of the coding region in the fungal pathogen present on the plant by at least 5% compared to a control fungal pathogen present on a control plant that does not include the polynucleotide. In one embodiment, the surface is a leaf, a flower, a fruit, a seed, a vegetable, or a combination thereof. In one embodiment, the plant further including a second polynucleotide that reduces expression of a coding region selected from SS1G_06487, SS1G_01703, SS1G_05899, SS1G_02495, SS1G_11912, SS1G_07873, or SS1G_09665, or a homolog thereof, and the second polynucleotide is on a surface of the plant.
In one embodiment, the polynucleotide is a first polynucleotide, the plant further including a second polynucleotide that reduces expression of a coding region selected from SS1G_01703, SS1G_09665, or SS1G_08218, or a homolog thereof, and the second polynucleotide is expressed by the plant. In one embodiment, the plant is canola, mustard, flax, sunflower, corn, oat, cotton, camelina, crambe, safflower, rice, sunflower, soybean, peanut, rapeseed, coconut, oil palm, borage, potato, pea, bean, lentil, chickpea, or a forage legume.
Also provided is a method for making a plant. In one embodiment, the method includes applying a composition to a surface of the plant, wherein the composition includes the polynucleotide. In one embodiment, the applying includes spraying the plant, for instance, to a leaf, a flower, a fruit, a seed, a vegetable, or a combination thereof, of the plant. In one embodiment, the composition is applied at a dose of 0.1 nanogram polynucleotide per square millimeter (ng/mm2).
Further provided herein is a transgenic plant and a cell of a transgenic plant. The transgenic plant includes a polynucleotide that reduces expression of a coding region having a sequence that has at least 80% identity to SS1G_01703, SS1G_09665, or SS1G_08218, or a homolog of SS1G_01703, SS1G_09665, or SS1G_08218, wherein the coding region is present in a fungal pathogen. In one embodiment, growth of the fungal pathogen on or in the plant is reduced compared to a control plant. In one embodiment, resistance of the plant to the fungal pathogen is increased compared to a control plant. In one embodiment, lesion size after administration of the fungal pathogen to leaves of the plant is reduced by at least 3% compared to a control plant that does not include the polynucleotide. In one embodiment, the plant pathogen is Sclerotinia sclerotiorum or Botrytis cinerea. In one embodiment, the polynucleotide includes a double stranded RNA (dsRNA), wherein the dsRNA includes a nucleotide sequence that is substantially identical to a series of nucleotides of the coding region. In one embodiment, the polynucleotide includes a single stranded RNA (ssRNA), wherein the ssRNA includes a nucleotide sequence that is substantially complementary to a series of nucleotides of the coding region. In one embodiment, the polynucleotide is at least 15 nucleotides.
In one embodiment, the polynucleotide reduces expression of the coding region in the fungal pathogen by at least 5% when the polynucleotide is introduced into the fungal pathogen and compared to a control fungal pathogen that does not include the polynucleotide. In one embodiment, the polynucleotide reduces expression of the coding region in the fungal pathogen present on the plant by at least 5% compared to a control fungal pathogen present on a control plant that does not include the polynucleotide. In one embodiment, the plant includes a vector that encodes the polynucleotide. In one embodiment, the vector is integrated into to plant genomic DNA. In one embodiment, the polynucleotide is a first polynucleotide, and the plant further includes a second polynucleotide that reduces expression of a coding region having a sequence that has at least 80% identity to SS1G_06487, SS1G_01703, SS1G_05899, SS1G_02495, SS1G_11912, SS1G_07873, or SS1G_09665, or a homolog of SS1G_06487, SS1G_01703, SS1G_05899, SS1G_02495, SS1G_11912, SS1G_07873, or SS1G_09665, wherein the second polynucleotide is on a surface of the plant. In one embodiment, the polynucleotide is a first polynucleotide, and the plant further includes a second polynucleotide that reduces expression of a coding region having a sequence that has at least 80% identity to SS1G_01703, SS1G_09665, or SS1G_08218, or a homolog of SS1G_01703, SS1G_09665, or SS1G_08218, wherein the second polynucleotide is on a surface of the plant, wherein the second polynucleotide is expressed by the plant.
Also provided herein is a method for making a transgenic plant. In one embodiment, the includes transforming a cell of a plant with a polynucleotide to obtain a recombinant plant cell, and generating a transgenic plant from the recombinant plant cell, where the plant includes a polynucleotide that reduces expression of a coding region having a sequence that has at least 80% identity to SS1G_01703, SS1G_09665, or SS1G_08218, or a homolog of SS1G_01703, SS1G_09665, or SS1G_08218, wherein the coding region is present in a fungal pathogen.
The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
It is understood that wherever embodiments are described herein with the language “include,” “includes,” or “including,” and the like, otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided.
Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
Described herein is the use of polynucleotides, such as double-stranded RNAs (dsRNAs), to inhibit the growth of plant parasitic fungi. Certain polynucleotides, when absorbed by fungal cells, will interfere with mRNA molecules and selectively reduce the expression of one or more essential fungal genes, thereby reducing fungal growth, infection, or pathogenicity. The polynucleotides described herein are designed to target fungus genes and not affect the host plant genes. The polynucleotides can be applied to plants in at least two ways: 1) by a transgenic method, whereby the plant expresses a polynucleotide to inhibit the growth or infection of the fungus in the plant; or 2) the polynucleotide can be applied topically to plant leaves, such that the when fungal spores adhere to the plant and attempt to infect the plant cells, they encounter the polynucleotide and their growth is inhibited or the fungal infection is reduced.
Pathogenic fungi infect many commercial crops and can cause devastating impacts on crop yields. Chemical fungicides are routinely applied to plant seeds and as foliar sprays on young plants' leaves to minimize fungal infections. These chemical fungicides are broad-spectrum in their activity, and can adversely affect many fungi, including beneficial fungi in the soil that are important in maintaining nutrient cycling. The efficacy of the fungicides are also dependent on climate, as rains following the aerial spraying can wash off the fungicide, leaving the plants vulnerable to fungal spores.
The technology described herein provides a new class of anti-fungal agents that are designed to be specific for pathogenic fungi and will not adversely affect other fungi, plants, or animals. The technology is inherently sequence-specific, capable of reducing gene expression of individual genes, and consequently, it can be designed to be species-limited, targeting gene sequences within the pathogenic fungus, and not affecting others.
Provided herein is a method for reducing growth of a fungal plant pathogen. As used herein, “reduced growth” refers to reducing the ability of the fungus to replicate, to undergo metabolism, to infect a plant (e.g., colonize a plant, a step that may occur prior to disease), to be pathogenic (e.g., cause disease), or a combination thereof “Reduced growth” includes a reduction in the ability of a fungal plant pathogen to replicate, undergo metabolism, or a combination thereof, for instance, and is similar to the result of using a fungistatic agent. In one embodiment, “reduced growth” refers to death of the fungal plant pathogen, and is similar to the result of using a funigicidal agent.
Also provided herein is a method for increasing the resistance of a plant to a fungal pathogen. As used herein, “resistance” refers to reduced signs of disease caused by a plant pathogen. Resistance can be the result of reduced growth of the plant pathogen, or the result of a reduced ability of the plant pathogen to cause signs of disease despite being present on or in the plant in substantial levels.
As used herein, unless otherwise noted “fungal plant pathogen,” “fungal pathogen,” “fungal cell,” and “fungus” are used interchangeably and refer to a fungus that causes a disease in a plant. Examples of fungal pathogens include members of the family Sclerotiniaceae, such as members of the genus Sclerotinia. Examples of members of the genus Sclerotinia include S. borealis, S. bulborum, S. homoeocarpa, S. minor, S. ricini, S. sclerotiorum, S. spermophila, S. sulcata, S. trifoliorum, and S. veratri. In one embodiment, the fungal pathogen is S. sclerotiorum. Other examples of fungal pathogens include members of the family Trichomaceae, such as members of the genus Aspergillus (for example, A. fumigatus, A. fischerianus (also known as Neosartorya fischeri), A. flavus, A. oryzae, A. clavatus, A. nidulans, A. terreus); members of the Botrytis genus (for example, B. cinerea); members of the family Plectosphaerellaceae, such as members of the genus Verticillium (for example, V. alfalfa, V. tenerum, V. dahlia); members of the family Nectriaceae, such as members of the genus Fusarium (for example, F. gaminearum, F. eumartii, F. martii, F. solani, F. striatum, Nectria cancri, Nectria haematococca); members of the family Glomerellaceae, such as members of the genus Colletotrichum (for example, C. gloeosporioides); and members of the genus Leptosphaeria (for example, L. maculans, L. biglobosa). Other fungal pathogens include Eutypa lata, Glarea lozoyensis, Neurospora crassa, Glomerella cingulate, Marssonina brunnea, Pyrenophora triticirepentis, Coniosporium apollinis, Endocarpon pusillum.
The methods include introducing into a fungus a polynucleotide. The polynucleotide may be introduced directly into the fungus by exposing the fungus to the polynucleotide, or by having a plant that is susceptible to the fungal pathogen express the polynucleotide.
As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides and/or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A polynucleotide may include nucleotide sequences having different functions, including for instance coding sequences, and non-coding sequences such as regulatory sequences. Coding sequence, non-coding sequence, and regulatory sequence are defined below. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. A polynucleotide disclosed herein may be isolated. An “isolated” polynucleotide is one that has been removed from its natural environment. Polynucleotides that are produced by recombinant, enzymatic, or chemical techniques are considered to be isolated and purified by definition, since they were never present in a natural environment.
In one embodiment, the polynucleotide may be a double stranded RNA (dsRNA) that inhibits expression of a coding region. In another embodiment, the polynucleotide may be a DNA sequence that encodes a dsRNA. In other embodiments, the polynucleotide may be an antisense RNA, or a ribozyme. It should be understood that the sequences disclosed herein as DNA sequences can be converted from a DNA sequence to an RNA sequence by replacing each thymidine nucleotide with a uridine nucleotide.
Without intending to be limited by theory, it is believed that dsRNAs described herein mediate RNA interference (RNAi) of a mRNA. RNAi is the process of sequence-specific, post-transcriptional gene silencing, initiated by dsRNA that includes an antisense strand that is complementary or substantially complementary in sequence to a portion of the silenced mRNA. Silencing can result from degradation of specific mRNA, which often occurs when there is high complementarity between one strand of a dsRNA and a target mRNA. Antisense RNA is a single strand RNA (ssRNA) molecule that is complementary to, or antisense to, sequences found in a specific mRNA in a cell. Introduction of an antisense RNA into a cell results in association of the antisense RNA with the mRNA, and inhibition of translation of the mRNA. A ribozyme (also referred to as RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction, such as the cleavage of the phosphodiester backbone of an mRNA molecule. Ribozymes can be designed to include a polynucleotide sequence that is antisense to a target mRNA and thereby target and cleave the target mRNA.
As used herein, the terms “coding region” and “gene” are used interchangeably. A “coding region” is a nucleotide sequence that encodes an unprocessed preRNA (i.e., an RNA molecule that includes both exons and introns), and is processed to result in an mRNA that is translated into a polypeptide. The boundaries of a coding region are generally determined by a transcription initiation site at its 5′ end and a transcription terminator at its 3′ end.
Coding regions have been identified that, when expression is decreased, result in reduced growth of a fungal pathogen. In one embodiment, examples of coding regions that may be decreased to result in reduced growth encode mRNA sequences available at the Genbank accession numbers XM_001592197.1, XM_001592927.1, XM_001596225.1, or XM_001591197.1 (see Tables 5-7 of the Examples). Additional examples of mRNA sequences of coding regions that, when decreased, are expected to result in reduced growth include, but are not limited to, the mRNA sequences available at the Genbank accession XM_001597459.1, XM_001588982.1, XM_001590428.1, XM_001550999.1, XM_001556707.1, XM_001553668.1, XM_001556287.1, XM_001559983.1 (see Tables 3 and 10 of the Example). Additional examples of mRNA sequences of coding regions that, when decreased, are expected to result in reduced growth include, but are not limited to, the mRNA sequences available at the Genbank accession numbers listed in Example 2.
In one embodiment, the mRNA targeted may be used in methods where a polynucleotide, such as a dsRNA, is introduced into a fungal pathogen that is a member of the family Sclerotiniaceae, such as S. sclerotiorum. In other embodiments, a polynucleotide, such as a dsRNA, may be used with other fungal pathogens, where the dsRNA is directed to an mRNA that is encoded by a coding region that is a homologue.
Coding regions that are homologues are coding regions that share ancestry, e.g., they are both derived from a coding region present in a common ancestor. The skilled person can easily determine if a coding region in a fungal pathogen that is not a member of the family Sclerotiniaceae, or not a member of the genus Sclerotinia, is a homolog of a coding region encoding an mRNA disclosed herein through the use of routine methods. In one embodiment, the skilled person can use the nucleotide sequence of a coding region disclosed herein and design degenerate PCR primers for use in a low stringency PCR. Low stringency PCR is a routine method for identifying homologs of a known coding region. In another embodiment, the skilled person can use readily available databases to identify in another fungal plant pathogen a homolog of a coding region encoding an mRNA disclosed herein. Examples of suitable databases include, but are not limited to, GenBank (available through the world wide web at ncbi.nlm.nih.gov/genbank). (See Table 10 for the Example for examples of B. cinerea homologues of S. sclerotiorum coding regions)
In another embodiment, the skilled person can identify a homolog of a coding region disclosed herein by the level of sequence identity between the coding region encoding an mRNA disclosed herein and another coding region. In one embodiment, when two nucleotide sequences are being compared, percent identities greater than 50% are taken as evidence of possible homology. The E value (Expect value) indicates the number of hits (sequences) in the database searched that are expected to align to the query simply by chance, so an E value less than 0.01 (i.e., less than 1% chance of the sequence occurring randomly), coupled with a percent identity of greater than 50% is considered a suitable score to identify a probable homolog. Methods for determining nucleotide sequence identity between two sequences are readily available and routine in the art. In one embodiment, coding regions in a fungal pathogen that are homologues of a coding region encoding an mRNA or protein disclosed herein may be identified using the BLAST-N or the BLAST-X algorithm respectively against the non-redundant database at NCBI with default parameters. A candidate coding region is considered to be a homologue of a coding region encoding an mRNA disclosed herein if the candidate coding region has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity to a respective coding region disclosed herein.
Decreased expression of one or more of the mRNA disclosed herein may result in reduced growth of the fungus, e.g., reduced ability of the fungus to replicate, undergo metabolism, infect a plant, be pathogenic, or a combination thereof.
In one embodiment, a polynucleotide used in a method described herein includes a sense strand and an anti-sense strand. The sense strand is at least 15 nucleotides in length; however, longer lengths are generally more desirable. Thus, the sense strand may be, but not limited to, at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, or at least 400 nucleotides, for instance. In mammalian cells, dsRNAs greater than 30 nucleotides in length may induce an interferon-mediated cell immune response, leading to cell death. Fungus cells lack this interferon-based response, and longer dsRNAs can be delivered. The sense strand is substantially identical, or identical, to a series of nucleotides (e.g., at least 15 nucleotides, at least 25 nucleotides, etc.) of an mRNA that is targeted by the dsRNA, i.e., a target mRNA. The skilled person will recognize that nucleotides of the sense strand that are substantially identical, or identical, to a target mRNA will not be longer than the length of the target mRNA. As used herein, the term “identical” means the nucleotide sequence of the sense strand has the same nucleotide sequence as a portion of the target mRNA. As used herein, the term “substantially identical” means the sequence of the sense strand differs from the sequence of a target mRNA at some number of nucleotides, but the ability of the complementary antisense strand to bind to and silence expression of the target mRNA is maintained. For instance, the sequence of the sense strand differs from the sequence of a target mRNA at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides, while the remaining nucleotides are identical to the sequence of the target mRNA. In another embodiment, the difference in nucleotides between the sequence of the sense strand and the target mRNA can be no greater than 1%, no greater than 5%, no greater than 10%, no greater than 15%, or no greater than 20% of the nucleotides, while the remaining nucleotides are identical to the sequence of the target mRNA.
The other strand of a dsRNA polynucleotide, referred to herein as the anti-sense strand, is substantially complementary, or complementary to the sense strand, and is therefore substantially complementary, or complementary, to a series of nucleotides (e.g., at least 15 nucleotides, at least 25 nucleotides, etc.) of a target mRNA. The term “complementary” refers to the ability of two single stranded polynucleotides to base pair with each other, where an adenine on one polynucleotide will base pair to a thymine or uridine on a second polynucleotide and a cytosine on one polynucleotide will base pair to a guanine on a second polynucleotide. An antisense strand that is “complementary” to another polynucleotide, such as a target mRNA, means the nucleotides of the antisense strand are complementary to a nucleotide sequence of a polynucleotide, such as a target mRNA. As used herein, the term “substantially complementary” means the antisense strand includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides that are not complementary to a nucleotide sequence of the target mRNA, while the remaining nucleotides are complementary to the sequence of the target mRNA. In another embodiment, the term “substantially complementary” means the antisense strand includes no greater than 1%, no greater than 5%, no greater than 10%, no greater than 15%, or no greater than 20% nucleotides that are not complementary to a nucleotide sequence of the target mRNA, while the remaining nucleotides are identical to the sequence of the target mRNA.
In one embodiment, the antisense strand is a single stranded RNA (ssRNA), and is not associated with a sense strand. Such an ssRNA is useful as an antisense polynucleotide for use in reducing translation of a mRNA that is a target of the antisense polynucleotide.
Also provided herein are the single stranded DNA polynucleotides corresponding to the sense strands and antisense strands disclosed herein. Also provided herein are the double stranded polynucleotides disclosed herein, including the complements of the single stranded polynucleotides.
A dsRNA described herein may include overhangs on one or both strands. An overhang is one or more nucleotides present in one strand of a double stranded RNA that are unpaired, i.e., they do not have a corresponding complementary nucleotide in the other strand of the double stranded polynucleotide. An overhang may be at the 3′ end of a sense strand, an antisense strand, or both sense and antisense strands. An overhang is typically 1, 2, or 3 nucleotides in length. In one embodiment, the overhang is at the 3′ terminus and has the sequence uracil-uracil (or thymine-thymine if it is a DNA). Without intending to be limiting, such an overhang may be used to increase the stability of a dsRNA. In one embodiment, if an overhang is present it is not considered when determining whether a sense strand is identical or substantially identical to a target mRNA, and it is not considered when determining whether an antisense strand is complementary or substantially complementary to a target mRNA.
The sense and antisense strands of a dsRNA described herein may also be covalently attached, for instance, by a spacer made up of nucleotides. Such a polynucleotide is often referred to in the art as a hairpin RNA or a short hairpin RNA (shRNA). Upon base pairing of the sense and antisense strands, the spacer region typically forms a loop. The number of nucleotides making up the loop can vary, and loops between 3 and 23 nucleotides have been reported (Sui et al., 2002, Proc. Nat'l. Acad. Sci. USA, 99:5515-5520; Jacque et al., 2002, Nature, 418:435-438). In one embodiment, an shRNA includes a sense strand followed by a nucleotide loop and the analogous antisense strand. In one embodiment, the antisense strand can precede the nucleotide loop structure and the sense strand can follow.
A dsRNA described herein may be modified. Such modifications can be useful to increase stability of the polynucleotide in certain environments. Modifications can include a nucleic acid sugar, base, or backbone, or any combination thereof. The modifications can be synthetic, naturally occurring, or non-naturally occurring. A dsRNA can include modifications at one or more of the nucleic acids present in the polynucleotide. Examples of backbone modifications include, but are not limited to, phosphonoacetates, thiophosphonoacetates, phosphorothioates, phosphorodithioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids. Examples of nucleic acid base modifications include, but are not limited to, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), or propyne modifications. Examples of nucleic acid sugar modifications include, but are not limited to, 2′-sugar modification, e.g., 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-fluoroarabino, 2′-O-methoxyethyl nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, or 2′-deoxy nucleotides. Polynucletotides can be obtained commercially synthesized to include such modifications (for instance, Dharmacon Inc., Lafayette, Colo.).
Polynucleotides described herein are biologically active. A biologically active polynucleotide causes the post-transcriptional inhibition of expression, also referred to as silencing, of a target-coding region. Without intending to be limited by theory, after introduction of a polynucleotide described herein into a fungal cell, the polynucleotide will hybridize with a target mRNA and signal cellular endonucleases to cleave the target mRNA or to inhibit translation of the target mRNA. The result is the inhibition of expression of the polypeptide encoded by the mRNA. Whether the expression of a target mRNA is inhibited can be determined by, for instance, measuring a decrease in the amount of the target mRNA in the cell, measuring a decrease in the amount of polypeptide encoded by the mRNA, or by measuring a change in a phenotype associated with expression of the polypeptide encoded by the mRNA.
Examples of polynucleotides that may be used to silence the expression of mRNAs disclosed herein include those shown below.
The skilled person will understand that a portion of each of the exemplary dsRNAs may be used to silence expression of a target mRNA, and that longer dsRNAs may also be used. Also understood by the skilled person is the ability to use other dsRNAs to silence expression of the target mRNAs disclosed herein. The skilled person will also recognize that antisense RNA molecules can be produced that target the coding regions listed in Tables 1 and 2, and homologues of the coding regions.
Polynucleotides useful in the methods described herein can be designed using methods that are routine and known in the art. For instance, candidate dsRNA polynucleotides or antisense polynucleotides that inhibit the expression of a coding region encoding an mRNA described herein may be based on a selected series of nucleotides, and may be identified using readily available algorithms. A candidate polynucleotide is the polynucleotide that is being tested to determine if it decreases expression of one of the coding regions encoding an mRNA described herein. The candidate polynucleotide can be identical to nucleotides located in the region encoding the polypeptide, or located in the 5′ or 3′ untranslated regions of the mRNA. Candidate polynucleotides may be screened using publicly available algorithms (e.g., BLAST) to compare the candidate polynucleotide sequences with coding sequences present in the fungal pathogen and/or plants that may be treated to prevent infection by a fungal pathogen. Those that are likely to form a duplex with an mRNA expressed by a non-target coding region are typically eliminated from further consideration. The remaining candidate polynucleotides may then be tested to determine if they inhibit expression of a coding region.
In general, candidate polynucleotides are individually tested by introducing a candidate polynucleotide into a cell that expresses the appropriate mRNA and polypeptide. In one embodiment, the candidate polynucleotides may be prepared in vitro and then introduced into a fungal cell that expresses the target mRNA. Methods for in vitro synthesis include, for instance, chemical synthesis with a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and reagents for such syntheses are well known. Methods for in vitro synthesis also include, for instance, in vitro transcription using a circular or linear vector in a cell free system. In one embodiment, the candidate polynucleotide may be introduced into a cell as a dsRNA or as an antisense ssRNA. In one embodiment, a candidate polynucleotide may be introduced into a cell as a construct that encodes the candidate polynucleotide. The cell may be a fungal pathogen. Such constructs are known in the art and include, for example, a vector encoding and expressing a sense strand and an antisense strand of a candidate polynucleotide, and RNA expression vectors that include the sequence encoding the sense strand and antisense strand of a candidate polynucleotide flanked by operably linked regulatory sequences to drive expression of the candidate polynucleotide. In another embodiment, a polynucleotide that is operably linked to a regulatory sequence encodes the antisense strand, the transcription of which produces a polynucleotide which can form secondary structures that affect expression of a target mRNA.
When evaluating whether a candidate polynucleotide functions to inhibit expression of a target mRNA described herein, the amount of target mRNA in a cell containing a candidate polynucleotide can be measured and compared to a control cell (e.g., the same type of cell that does not contain the candidate polynucleotide). Methods for measuring mRNA levels in a cell are known in the art and routine. Such methods include quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Primers and specific conditions for amplification of a target mRNA can be readily determined by the skilled person. Other methods include, for instance, Northern blotting, and array analysis.
Other methods for evaluating whether a candidate polynucleotide functions to inhibit expression of a polypeptide encoded by a target mRNA includes monitoring the polypeptide. For instance, assays can be used to measure a decrease in the amount of polypeptide encoded by the mRNA, or to measure a decrease in the activity of the polypeptide encoded by the mRNA. Methods for measuring a decrease in the amount of a polypeptide include assaying for the polypeptide present in cells containing a candidate polynucleotide and comparing to a control cell. Whether a cell expresses one of the polypeptides can be determined using methods that are routine and known in the art including, for instance, Western immunoblot, ELISA, immunoprecipitation, or immunohistochemistry.
In one embodiment, a candidate polynucleotide is able to decrease the expression of a target mRNA, or the polypeptide encoded by the target mRNA, by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% when compared to a control cell (e.g., a fungal pathogen that does not include the candidate polynucleotide).
A polynucleotide, such as a dsRNA, described herein can be encoded by a polynucleotide present in a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide described herein employs standard ligation techniques known in the art (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989)). A vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors. In one embodiment, such as when the dsRNA is expressed in a plant cell, a vector may result in integration into a cell's genomic DNA. Typically, a vector is capable of replication in a bacterial host, for instance E. coli, and optionally, in other cells. A polynucleotide described herein can be present in a vector as two separate complementary polynucleotides, each of which can be expressed to yield a sense and an antisense strand of the dsRNA, or as a single polynucleotide containing a sense strand, a loop region, and an anti-sense strand, which can be expressed to yield an RNA polynucleotide having a sense and an antisense strand of the dsRNA.
Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. Suitable host cells for cloning or expressing the vectors herein are prokaryote or eukaryotic cells. Suitable prokaryotes include members of the domain Bacteria and members of the domain Archaea. In one embodiment, a suitable prokaryote is E. coli. An example of a eukaryotic cell is a plant cell.
An expression vector optionally includes regulatory sequences operably linked to the polynucleotide of the present disclosure. Typically, the promoter results in the production of an RNA polynucleotide. Examples of such promoters for use in prokaryotic cells include a T7 promoter, a T3 promoter or an SP6 promoter. Examples of such promoters for use in eukaryotic cells include those that cause binding of an RNA polymerase, such as an RNA polymerase III complex, to initiate transcription of an operably linked polynucleotide of the present disclosure. In one embodiment, examples of such promoters include constitutive promoters such as the CaMV35S promoter from the cauliflower mosaic virus, plant-derived actin promoters and ubiquitin promoters, leaf-localized promoters such as photosynthesis-associated nuclear genes (PhANGS) promoters, the chlorophyll a/b-binding proteins (Cab) promoter, or the small subunit of Rubisco (RbcS) promoter. A promoter may be constitutive or inducible. Another regulatory sequence is a transcription terminator. Suitable transcription terminators are known in the art.
The polynucleotide, such as a dsRNA, is introduced into the fungal pathogen. In one embodiment, the dsRNA is expressed by a transgenic plant. As used herein, the term “transgenic plant” refers to a plant that has been transformed to contain at least one polynucleotide to result in expression of a dsRNA. The term “transgenic plant” includes whole plants, plant parts (stems, roots, leaves, fruit, etc.) or organs, plant cells, seeds, and progeny of same. A transformed plant can be a direct transfectant, meaning that the DNA construct was introduced directly into the plant, such as through Agrobacterium or other methods, or the plant can be the progeny of a transfected plant. The second or subsequent generation plant can be produced by sexual reproduction, i.e., fertilization. Furthermore, the plant can be a gametophyte (haploid stage) or a sporophyte (diploid stage).
Examples of plants include, but are not limited to, herbaceous, succulent plants, particularly flowers and vegetables. Specific examples include, but are not limited to, oil seed crops such as canola, mustard, flax, sunflower, corn, oat, cotton, camelina, crambe, safflower, rice, sunflower, soybean, peanut, rapeseed, coconut, and oil palm. Other plants include borage, potato, pea, bean, lentil, chickpea, and forage legumes.
Transgenic plants described herein may be produced using routine methods (see, for instance, Waterhouse et al., US Patent Application 2006/0272049). Methods for transformation and regeneration are known to the skilled person. Transformation of a plant cell with a polynucleotide described herein to yield a recombinant host cell may be achieved by any known method for the insertion of nucleic acid sequences into a prokaryotic or eukaryotic host cell, including Agrobacterium-mediated transformation protocols, viral infection, whiskers, electroporation, microinjection, polyethylene glycol-treatment, heat shock, lipofection, particle bombardment, and chloroplast transformation. Also provided are transgenic plants expressing one or more of the polynucleotides described herein.
Techniques for the transformation of monocotyledon species include direct gene transfer into protoplasts using PEG or electroporation techniques, particle bombardment into callus tissue or organized structures, as well as Agrobacterium-mediated transformation.
The cells that have been transformed may be grown into plants in accordance with conventional techniques. See, for example, McCormick et al. (1986, Plant Cell Reports, 5:81-84). These plants may then be grown and evaluated for expression of the dsRNA. These plants may be either pollinated with the same transformed strain or different strains, and the resulting hybrid having desired phenotypic characteristics identified. Two or more generations may be grown to ensure that the desired expression of one or more dsRNAs is stably maintained and inherited and then seeds harvested to ensure stability of the desired characteristics have been achieved.
In another embodiment, a polynucleotide, such as a dsRNA, is introduced into the fungal pathogen by topical application of the polynucleotide, such as a dsRNA, to a plant having or at risk of having an infection by a fungal plant pathogen. Application of a polynucleotide to a plant may be by spraying, brushing, or any other method. The application can be to the entire plant or to a portion thereof, such as a leaf, a flower, a fruit, a seed, or a vegetable. Polynucleotides may be applied to a plant in any suitable composition. The composition may be aqueous or non-aqueous. In one embodiment, the composition includes agents to aid in stability of the polynucleotide, ability of the polynucleotide to enter the fungal plant pathogen, and/or ability to remain associated with the surface of the plant. Such an agent may be physically associated with the polynucleotide. The composition may include other agents to aid in the topical application of a dsRNA molecule including, but not limited to, a surfactant (for instance, anionic, cationic, amphoteric, nonionic), a biosurfactant, a wetting agent, a penetrant, a thickener, an emusifier, a spreader, a sticker, an oil, an alkyl polyglucoside, an organosilicate, an inorganic salts, or a combination thereof. Also provided is a composition that includes a polynucleotide described herein.
Also provided by the present disclosure are plants having a polynucleotide described herein on its surface. The polynucleotide can be present on the entire plant or on a part of the plant, such as one or more leaves of the plant. Also provided herein are methods for applying a polynucleotide to a plant, or a part of the plant, with a composition that includes a polynucleotide described herein.
In one embodiment, more than one polynucleotide can be expressed in a transgenic plant or applied to a plant. For instance, multiple polynucleotides targeting different areas of a mRNA can be expressed in a transgenic plant or applied to a plant. In another example, multiple polynucleotides targeting different mRNA molecules can be expressed in a transgenic plant or applied to a plant. The number of different polynucleotides can be at least 2, at least 3, at least 4, at least 5, or at least 6. It is expected there is no upper limit to the number of different polynucleotides that can be expressed in a transgenic plant or applied to a plant, but in one embodiment the number is no greater than 15, or no greater than 10. In one embodiment, one or more polynucleotides can be expressed in a transgenic plant and one or more polynucleotides can be applied to the same plant.
Treatment of a plant can be prophylactic or, alternatively, can be initiated after the development of disease caused by a fungal plant pathogen. Treatment that is prophylactic, for instance, initiated before a plant manifests signs of disease, is referred to herein as treatment of a plant that is “at risk” of having an infection. Treatment can be performed before, during, or after the occurrence of an infection by a fungal plant pathogen. Treatment initiated before the development of disease may result in decreasing the risk of infection by the fungal pathogen. Treatment initiated before development of disease includes applying a polynucleotide described herein to the surface of a plant, use of a transgenic plant that expresses a polynucleotide described herein, or a combination there. Treatment initiated after the development of disease may result in decreasing the severity of the signs of the disease, or completely removing the signs. A sign of disease by a fungal pathogen includes, but is not limited to, one or more lesions on the surface of the plant. The dosage administered to a plant is sufficient to result in decreased risk of infection or decreased severity of the signs of the disease (e.g., reduce number of lesions, reduced size of lesions, or a combination thereof). Decreased risk of infection or decreased severity of the signs of the disease can be the result of reduced growth of the pathogen, increased resistance of the plant to the pathogen, or a combination thereof. Such a dosage can be easily determined by the skilled person.
In one embodiment, decreased risk of infection or decreased severity of signs of disease can be determined by reduction of lesion size. Lesion size on leaves or other parts of a plant can be measured using standard methods. The reduction in lesion size can be at least 3%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 08%, or at least 90% compared to a control plant (e.g., the same type of plant that was not treated with the composition, or the same type of plant that does not express the polynucleotide). In one embodiment, lesions on a plant are not detectable.
In one embodiment, a composition applied to a surface of a plant includes a polynucleotide, such as a dsRNA, at a dose of 0.1 nanogram per square millimeter (ng/mm2) to 15 ng/mm2. For example, dsRNA may be applied at a dose of at least 0.1 nanogram per square millimeter (ng/mm2), at least 0.5 ng/mm2, at least 1 ng/mm2, at least 5 ng/mm2, or at least 10 ng/mm2. For example, dsRNA may be applied at a dose of no greater than 15 ng/mm2, no greater than 10 ng/mm2, no greater than 5 ng/mm2, no greater than 1 ng/mm2, or no greater than 0.5 ng/mm2. In one embodiment, more than one polynucleotide may be expressed by a transgenic plant or applied to a plant.
The present disclosure is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.
EXAMPLE 1A technology that could provide a suite of new options to control Sclerotinia is RNA interference (RNAi). RNAi is a sequence-specific method of reducing or silencing a gene's expression following the introduction of double-stranded RNAs (dsRNAs) into a cell. Once within the cell, the dsRNA mediates the destruction of any RNA with identical sequence, thereby silencing the gene's expression. Using bioinformatics tools, it is possible to design dsRNAs to selectively target just one gene in an organism, and also design dsRNA to target only one species or alternatively, a group of related species. With its incredible ability to silence a gene's expression, RNAi is being considered for a great many applications, including a variety of crop protection technologies [6]. We have previously developed transgenic plants that expressed species-specific insecticidal dsRNAs that targeted genes essential to the insects' growth, and thereby killed insects that fed on the plants [7]. Because of RNAi's sequence-specificity, it can be readily adapted to selectively target any pest or pathogen, without adversely affecting other organisms. RNAi has previously been proven effective in protecting transgenic tobacco plants from the pathogenic fungus Fusarium verticillioides [8]. In this study, we examined the utility of RNAi technologies to suppress S. sclerotiorum growth, with the aim of developing topically-applied dsRNAs as an alternative to transgenic-mediated protection of the plants. We first demonstrated that exogenously-supplied dsRNA can be taken up by the fungus and that gene-specific knockdown of mRNA transcripts occurs. Secondly, we have identified the repertoire of genes that are expressed by S. sclerotiorum during early stages of infection in canola plants. By using moderate through-put screening methods, we have identified which of these fungal genes are the most sensitive RNAi targets, to minimize infection in the plants. RNAi can target a specific gene or a family of genes, and hence, the technology offers the ability to produce a vast number of anti-Sclerotinia dsRNAs, which will provide a rich supply of fungal-control dsRNAs if resistant strains arise. The technology could also be developed to control other fungal pathogens that affect other commercial crops.
Our approach to develop an RNAi-based method of controlling Sclerotinia stem rot in canola included three main components: 1) a proof-of-concept demonstration that topically-applied dsRNAs can induce RNAi and inhibit S. sclerotiorum growth in fungal growth bioassays; 2) identification of additional fungal target genes using transcriptomic analyses and moderate throughput RNAi functional screening and 3) testing of topical application formulations of dsRNAs to assess whether plants can be protected from Sclerotinia infection without using transgenic technologies, which may serve as a safer, ready-to-use, more species-specific alternative to fungicides.
Results1. Transcriptomic analyses of canola infected with S. sclerotiorum have identified thousands of putative RNAi targets.
To identify differentially expressed genes during the infection process, we sequenced rapidly infecting S. sclerotiorum infected canola leaf tissues. We selected two cultivars with varying levels of susceptibility, Westar (susceptible) and ZhongYou821 (tolerant), to compare responses within Sclerotinia. RNA samples were extracted and we performed high-throughput RNA sequencing on the Illumina HiSeq platform. One hundred paired end sequence reads generated by the Illumina Hiseq 2500 were quality filtered through the FASTX toolkit, trimmed using a sliding window and headcrop with the jar program Trimmomatic. Samples were aligned to the S. sclerotiorum reference genome using TopHat2 in high sensitivity mode. Raw counts were used as input to DESeq2 for lists of differentially expressed genes and TopHat to identify splice junctions.
We compared gene lists to fungus grown on plates with that grown on leaves to identify thousands of differentially expressed genes that represent excellent targets for RNAi to impede and suppress fungal growth. Infection on the two cultivars was also compared to identify genes important to infection on canola. We identified 10,003 genes as being present before during and after the infection process. We then used previously obtained gene ontology (GO) terms, BLAST, and protein FASTA files to assign predicted biological function.
2. Transcriptomic analyses can provide information on gene expression levels to enable faster identification of genes expressed during the earliest phases of infection.
To identify genes important to the infection process, we found 1,858 genes up-regulated in response to infection in both cultivars. These genes probably represent ideal targets for RNA interference. These genes significantly up-regulated and considered a minimum false discovery rate of 0.05. GO terms and BLAST queries guided the choice of targets. We also targeted some essential genes listed in publically available databases to extend choosing targets without requiring transcriptomic data and develop a bioinformatics pipeline. Targets chosen for S. sclerotiorum are shown in Table 3.
To synthesize dsRNA for the chosen targets, Sclerotinia gene sequences were acquired from Genbank and primers were designed to PCR amplify gene fragments ranging between 180 and 480 bp in length (shown in Table 4). Each product was ligated into a vector, between two T7 RNA polymerase promoter sequences, which then was used to PCR-amplify the gene fragments. The PCR products were then used as templates to prepare dsRNAs by in vitro transcription using T7 RNA polymerase.
3. S. sclerotiorum fungal hyphae can take in exogenous dsRNA to induce RNA interference over time
DsRNA targeting Sclerotinia gene SS1G_05899 was synthesized as described above. Fungus was grown from a sclerotium on potato dextrose agar (PDA). A plug of about 1 mm in diameter was taken from the actively growing edge of the fungal colony and placed in 3 mL of potato dextrose broth (PDB). The fungus grew for 48 hours before 500 ng of dsRNA was added. Fungus was taken at time points 0, 24, 28, 72, and 96 hours post-inoculation; RNA was extracted; cDNA prepared; and qRT-PCR was used to assess the level of mRNA transcripts for the target gene. A maximum knockdown of 47% was observed after 4 days of incubation (Table 5). The knockdown trend in the SS1G_05899 dsRNA treated tubes opposed the trend observed in the GFP dsRNA treated tubes. The GFP did not have any negative impact on the accumulation of the target transcript and this was due to the application of the target dsRNA. The timing of the RNAi effect was consistent with what is observed in human cell lines and insect experiments.
4. S. sclerotiorum fungal hyphae can take in exogenous dsRNA to elicit RNA interference at various doses
Fungus was grown from a sclerotium on PDA. A plug of about 1 mm in diameter was taken from the actively growing edge of the fungal colony and placed in 3 mL of PDB. Specific doses (100 ng, 200 ng, 500 ng, and 1000 ng) of SS1G_07873 dsRNA were added to the medium. In parallel, SS1G_06487, SS1G_05899, and SS1G_02495 dsRNA were tested at the 500 ng dose level. The fungi grew for 3 days before the tissue was collected, RNA extracted, and cDNA synthesized. qRT-PCR was used to assess the level of mRNA transcripts for target genes. Water (0 ng) was used to compare accumulation levels. All genes showed a significant knockdown at all doses tested with a maximum knockdown of 53% (Tables 6 and 7). This suggests that molecules function in reducing the accumulation of the transcript in recipient Sclerotinia.
5. A topical application of dsRNA on canola leaves can reduce S. sclerotiorum growth.
To mimic field conditions, senescing canola (B. napus) petals were coated with 200 ng of dsRNA, and then 10 μl of fungal spores (˜5000 spores) were applied to each petal. After 3 days, a further application of 200 ng of dsRNA was applied to leaf surfaces and the petals placed onto the leaves. Plants were incubated in high humidity conditions, essential for causing an aggressive infection. Leaves were imaged and the lesion measured using ImageJ software. The majority of the genes tested showed a significant reduction in lesion size up to 85.32% (Table 8). The results suggest that topical application of dsRNAs directly on the leaf surface protects the plant from S. sclerotiorum proliferation.
6. DsRNA expressing A. thaliana plants are protected against Sclerotinia infection
Sclerotinia gene targets used to protect plants using a foliar application were selected to evaluate a transgenic approach for RNAi technology. The primers used to synthesize the dsRNA (Table 4) were reused to develop transgenic plants expressing the same targeting region. Products were amplified using PCR and each product was shuttled into a vector for sequencing before being recombined into a final vector, which produces hairpin RNA (hpRNA). The vectors were transformed into Agrobacterium tumefaciens, which allowed A. thaliana plants to be transformed using established transformation protocols. Transgenic plants were selected using kanamycin MS plates and the plants were grown for 3 weeks. At this stage, approximately 10 μL of 10000 spores suspended in glucose-phosphate buffer [9] were dispensed onto the surface of the leaves. The plants were placed in a humidity chamber and the infection progressed for 4 days before the lesions were assessed using photography and ImageJ. All of the hpRNA expressing plants showed a reduction in lesion size up to 74.5% compared to the wild-type Columbia A. thaliana (Table 9). In general, the transgenic plants showed a higher level of protection compared to the foliar application of dsRNA molecules.
6. Sclerotinia homologs in Botrytis cinerea are effective at reducing growth
With the success of the Sclerotinia targets, homologs of some S. sclerotiorum genes were targeted in B. cinerea to extend our targets beyond Sclerotinia (Table 10). Homologs were identified using the BLASTP (available on the world wide web at blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) and then the Genbank accessions could be found. Similar to S. sclerotiorum, primers were designed to PCR amplify gene fragments ranging between 180 and 480 bp in length (shown in Table 11). Each product was ligated into a vector, between two T7 RNA polymerase promoter sequences, which then was used to PCR-amplify the gene fragments. The PCR products were then used as templates to prepare dsRNAs by in vitro transcription using T7 RNA polymerase.
Once dsRNA was synthesized, canola leaves of approximately 4 weeks of age were taken and placed in large petri plates containing dampened paper towel. 500 ng of dsRNA was spread over a 4 cm2 area. To facilitate infection, a small puncture was made in the middle of the 4 cm2 area and then approximately 1000 spores of B. cinerea were placed on the punctured region. The infection proceeded for 4 days before the lesions were photographed and measured using ImageJ software. Lesion sizes were reduced up to 73% (Table 12) suggesting that homologous dsRNAs could be designed for other pathogenic species and would be effective at controlling other plant diseases.
1. www.canolacouncil.org;
2. Baharlouei et al., 2011, African J Biotech 10: 5785-5794; 3. Litholdo et al., 2010, Genetics Molec Res 10: 868-877; 4. Hu et al., 2011, Can J Microbiology 57: 539-546;5. www.gov.mb.ca;
6. Ali et al. 2010. GM Crops 1: 207-213. 7. Waterhouse et al. 2005, WO 2005/049841. 8. Tinoco et al. 2010, BMC Biology 8: 27. 9. Garg et al. 2010, Annals of Botanyl06:897-908 EXAMPLE 2Other targets for RNA interference were evaluated as described in Example 1. The following coding regions, when targeted for decreased expression, were found to result in reduced growth of S. sclerotiorum: XM_001591197.1 (SS1G_07873, SEQ ID NO:42), XM_001590273.1 (SS1G_09088, SEQ ID NO:43), XM_001596198.1 (SS1G_02468, SEQ ID NO:44), XM_001587661.1 (SS1G_11704, SEQ ID NO:45), XM_001596446.1 (SS1G_02716, SEQ ID NO:46), XM_001586961.1 (SS1G_12040, SEQ ID NO:47), XM_001589214.1 (SS1G_09897, SEQ ID NO:48), XM_001592927.1 (SS1G_05899, SEQ ID NO:49), XM_001592197.1 (SS1G_06487, SEQ ID NO:50), XM_001597459.1 (SS1G_01703, SEQ ID NO:51), XM_001596225.1 (SS1G_02495, SEQ ID NO:52), XM_001586833.1 (SS1G_11912, SEQ ID NO:53), XM_001589938.1 (SS1G_08752, SEQ ID NO:54), XM_001588858.1 (SS1G_10456, SEQ ID NO:55), XM_001588798.1 (SS1G_10396, SEQ ID NO:56), XM_001594135.1 (SS1G_03992, SEQ ID NO:57), XM_001586701.1 (SS1G_11780, SEQ ID NO:58), XM_001591282.1 (SS1G_07958, SEQ ID NO:59), XM_001594183.1 (SS1G_04040, SEQ ID NO:60), XM_001598634.1 (SS1G_00773, SEQ ID NO:61), XM_001598299.1 (SS1G_00435, SEQ ID NO:62), XM_001593489.1 (SS1G_04966, SEQ ID NO:63), XM_001588511.1 (SS1G_10108, SEQ ID NO:64), XM_001594947.1 (SS1G_04805, SEQ ID NO:65), XM_001594144.1 (SS1G_04001, SEQ ID NO:66), XM_001592623.1 (SS1G_06914, SEQ ID NO:67), XM_001591055.1 (SS1G_07730, SEQ ID NO:68), XM_001588997.1 (SS1G_09680, SEQ ID NO:69), XM_001594134.1 (SS1G_03991, SEQ ID NO:70, XM_001588201.1 (SS1G_10698, SEQ ID NO:71), XM_001590641.1 (SS1G_08431, SEQ ID NO:72), XM_001585413.1 (SS1G_13702, SEQ ID NO:73), XM_001595070.1 (SS1G_03208, SEQ ID NO:74), XM_001587072.1 (SS1G_12152, SEQ ID NO:75), XM_001595209.1 (SS1G_03348, SEQ ID NO:76), XM_001585128.1 (SS1G_13746, SEQ ID NO:77), XM_001592539.1 (SS1G_06830, SEQ ID NO:78), XM_001586999.1 (SS1G_12078, SEQ ID NO:79), XM_001553779.1 (SS1G_08022, SEQ ID NO:80), XM_001595070.1 (SS1G_03208, SEQ ID NO:81), XM_001594134.1 (SS1G_03991, SEQ ID NO:82), XM_001593489.1 (SS1G_04966, SEQ ID NO:83), XM_001592539.1 (SS1G_06830, SEQ ID NO:84), XM_001588997.1 (SS1G_09680, SEQ ID NO:85), XM_001586942.1 (SS1G_12021, SEQ ID NO:86), XM_001586603.1 (SS1G_12640, SEQ ID NO:87), XM_001585850.1 (SS1G_12992, SEQ ID NO:88), XM_001585413.1 (SS1G_13702, SEQ ID NO:89), or XM_001585128.1 (SS1G_13746, SEQ ID NO:90).
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
Claims
1. A plant comprising a polynucleotide that reduces expression of a coding region having a sequence that has at least 80% identity to SS1G_11912 (SEQ ID NO:5), or a homolog of SS1G_11912 (SEQ ID NO:5), wherein the coding region is present in a fungal pathogen, and wherein the polynucleotide is on a surface of the plant.
2. The plant of claim 1 wherein growth of the fungal pathogen on the plant is reduced compared to a control plant.
3. (canceled)
4. The plant of claim 1 wherein lesion size after administration of the fungal pathogen to leaves of the plant is reduced by at least 3% compared to a control plant that does not comprise the polynucleotide.
5. The plant of claim 1 wherein the plant pathogen is Sclerotinia sclerotiorum or Botrytis cinerea.
6. The plant of claim 1 wherein the polynucleotide comprises a double stranded RNA (dsRNA), wherein the dsRNA comprises a nucleotide sequence that is substantially identical to a series of nucleotides of the coding region.
7. The plant of claim 1 wherein the polynucleotide comprises a single stranded RNA (ssRNA), wherein the ssRNA comprises a nucleotide sequence that is substantially complementary to a series of nucleotides of the coding region.
8.-10. (canceled)
11. The plant of claim 1 wherein the surface is a leaf, a flower, a fruit, a seed, a vegetable, or a combination thereof.
12.-13. (canceled)
14. The plant of claim 1 wherein the plant is canola, mustard, flax, sunflower, corn, oat, cotton, camelina, crambe, safflower, rice, sunflower, soybean, peanut, rapeseed, coconut, oil palm, borage, potato, pea, bean, lentil, chickpea, or a forage legume.
15. A method for making the plant of claim 1 comprising applying a composition to a surface of the plant, wherein the composition comprises the polynucleotide.
16. The method of claim 15 wherein the applying comprises spraying the plant.
17. The method of claim 15 wherein the applying comprises application of the composition to a leaf, a flower, a fruit, a seed, a vegetable, or a combination thereof, of the plant.
18. The method of claim 15 wherein the composition is applied at a dose of 0.1 nanogram polynucleotide per square millimeter (ng/mm2).
19. A transgenic plant comprising a polynucleotide that reduces expression of a coding region having a sequence that has at least 80% identity to SS1G_11912 (SEQ ID NO:5), or a homolog of SS1G_11912 (SEQ ID NO:5), wherein the coding region is present in a fungal pathogen.
20. (canceled)
21. The plant of claim 19 wherein growth of the fungal pathogen on the plant is reduced compared to a control plant.
22. (canceled)
23. The plant of claim 19 wherein lesion size after administration of the fungal pathogen to leaves of the plant is reduced by at least 3% compared to a control plant that does not comprise the polynucleotide.
24. The plant of claim 19 wherein the plant pathogen is Sclerotinia sclerotiorum or Botrytis cinerea.
25. The plant of claim 19 wherein the polynucleotide comprises a double stranded RNA (dsRNA), wherein the dsRNA comprises a nucleotide sequence that is substantially identical to a series of nucleotides of the coding region.
26. The plant of claim 19 wherein the polynucleotide comprises a single stranded RNA (ssRNA), wherein the ssRNA comprises a nucleotide sequence that is substantially complementary to a series of nucleotides of the coding region.
27. The plant of claim 19 wherein the polynucleotide comprises at least 15 nucleotides.
28.-33. (canceled)
34. A method for making the plant of claim 19 comprising:
- transforming a cell of a plant with a polynucleotide to obtain a recombinant plant cell;
- generating a transgenic plant from the recombinant plant cell, wherein the plant comprises a polynucleotide that reduces expression of a coding region having a sequence that has at least 80% identity to SS1G_11912 (SEQ ID NO:5), or a homolog of SS1G_11912 (SEQ ID NO:5), wherein the coding region is present in a fungal pathogen.
Type: Application
Filed: Jun 29, 2021
Publication Date: Dec 16, 2021
Inventors: Steve Whyard (Winnipeg), Mark Belmonte (Winnipeg)
Application Number: 17/361,888
