WO2022140257A1 - Polynucleotides and methods for transferring resistance to asian soybean rust - Google Patents

Polynucleotides and methods for transferring resistance to asian soybean rust Download PDF

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WO2022140257A1
WO2022140257A1 PCT/US2021/064348 US2021064348W WO2022140257A1 WO 2022140257 A1 WO2022140257 A1 WO 2022140257A1 US 2021064348 W US2021064348 W US 2021064348W WO 2022140257 A1 WO2022140257 A1 WO 2022140257A1
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plant
ccrpp2
sequence
seq
polynucleotide
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PCT/US2021/064348
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English (en)
French (fr)
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Ebony JOHNSON
Shawn Thatcher
Karen E BROGLIE
Peter VAN-ESSE
Cintia Goulart KAWASHIMA
Jonathan Jones
Sergio Hermino BROMMONSCHENKEL
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Pioneer Hi-Bred International, Inc.
Two Blades Foundation
Universidad Federal De Vicosa
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Application filed by Pioneer Hi-Bred International, Inc., Two Blades Foundation, Universidad Federal De Vicosa filed Critical Pioneer Hi-Bred International, Inc.
Priority to EP21911972.4A priority Critical patent/EP4271822A1/en
Priority to US18/259,006 priority patent/US20240043861A1/en
Priority to CN202180086286.8A priority patent/CN116802304A/zh
Priority to BR112023012656A priority patent/BR112023012656A8/pt
Priority to CA3204072A priority patent/CA3204072A1/en
Publication of WO2022140257A1 publication Critical patent/WO2022140257A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Definitions

  • the present disclosure relates to compositions and methods useful in enhancing pathogen resistance in legume plants, and more particularly in soybean plants, by providing to the plants a gene or gene(s) that are associated with resistance to the causal agent of Asian soybean rust (ASR).
  • ASR Asian soybean rust
  • the disclosure further relates to polynucleotides capable of enhancing resistance in legumes to ASR and methods of using these polynucleotide sequences to make a transgenic legume plant that is resistant to ASR.
  • BACKGROUND Soybeans (Glycine max), a major industrial use crop, are also one of the most important protein source crops and are considered a key food group for preventing disease and optimizing health by many public health organizations including the American Diabetes Association, the American Heart Association and the American Cancer Society.
  • Asian soybean rust is a major crop disease affecting soybeans and can negatively affect growth and yield. It is caused by the obligate biotrophic fungus Phakopsora pachyrhizi and, to a lesser extent, the closely related fungus Phakopsora meibomiae. The disease can cause yield losses ranging from 10-90%.
  • SUMMARY [0005] The present disclosure relates to compositions and methods for identifying ASR resistance genes from legume species and transforming those genes into legume crops or a legume crop species, such as Glycine max, to generate plants that are resistant to ASR.
  • isolated polynucleotides comprising a nucleotide sequence that encodes one or more of the legume-derived, binary CcRpp2-R1 and CcRpp2-R3 polypeptides having at least 90% amino acid sequence identity to a legume sequence disclosed herein.
  • the polynucleotide is a recombinant sequence comprising a heterologous promoter operably linked to a nucleotide sequence that encodes one or more of the legume- derived, binary CcRpp2-R1 and CcRpp2-R3 polypeptides.
  • Soybean plants transformed with polynucleotides that express such binary polynucleotides have been demonstrated to display enhanced resistance to Asian soybean rust when compared to a susceptible plant and/or a non- transformed plant.
  • recombinant DNA constructs comprising the polynucleotides described herein, wherein the CcRpp2-R1 and CcRpp2-R3 coding sequences are operably linked to heterologous regulatory elements for expressing the CcRpp2-R1 and CcRpp2-R3 gene products in a plant cell.
  • polynucleotides which can comprise, or alternatively consist of or consist essentially of, a nucleic acid sequence of SEQ ID NOs: 1 or 3, and variants thereof.
  • the polypeptides encoded thereby are capable of functioning as a binary polypeptide and are useful in compositions and methods for conferring resistance in a legume crop to ASR.
  • a legume crop species e.g., soybean
  • the method comprising transforming a legume crop species with nucleic acid sequences that encode heterologous legume-derived binary CcRpp2-R1 and CcRpp2-R3 polynucleotides that confer disease resistance to a legume crop species disease (e.g., ASR).
  • a transgenic plant cell comprising a recombinant polynucleotide that encodes a polypeptide that confers disease resistance to a legume crop species disease (e.g., ASR), wherein the encoded polypeptide has at least 65%, 75%, 85%, 90%, 95% or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 21-36, and/or an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 48-58.
  • ASR legume crop species disease
  • transgenic legume crop plant stably transformed with a recombinant DNA construct that comprises polynucleotides encoding one or more legume- derived CcRpp2-R1 and CcRpp2-R3 genes.
  • the polynucleotide comprises one or more non-legume-derived CcRpp2-R1 and CcRpp2-R3 resistance genes and optionally additional non-CcRpp2-R1 and CcRpp2-R3 resistance genes that confer resistance to a plant disease.
  • the polynucleotides described herein can also comprise any combination of resistance genes.
  • the transgenic legume crop plant can comprise one or more input traits and/or agronomic traits.
  • transgenic legume crop plant that is stably transformed that comprises the legume-derived binary CcRpp2-R1 and CcRpp2-R3 polynucleotides that have at least 90% or 95% sequence identity to a sequence described herein, including for example SEQ ID NOs 1, 3, 5-20 and 37-47.
  • ASR a plant disease
  • detecting CcRpp2- R1 and CcRpp2-R3 resistance genes in a biological sample comprising screening nucleic sequences recovered from the biological sample using primers or probes specific for the CcRpp2-R1 and CcRpp2-R3 resistance gene sequences, optionally wherein the primers and probes hybridize under stringent wash conditions to a nucleic acid sequence selected from SEQ ID NOs 1, 3, 5-20 and 37-47.
  • ASR resistant plant e.g., a legume species
  • the method comprises transforming a plant cell with legume-derived binary CcRpp2-R1 and CcRpp2-R3 resistance genes.
  • the method comprises transforming a plant cell with nucleic acid sequences comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NOs: 5-12 and 14-20 and SEQ ID NO: 13 and a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NOs: 37-46 and SEQ ID NO: 47.
  • the method can further comprise regenerating a transformed plant from the transformed plant cell.
  • the method comprises growing the transformed plant such that the expression of the legume-derived CcRpp2-R1 and CcRpp2- R3 resistance gene produces a transformed plant that displays enhanced resistance to ASR disease.
  • transgenic plants are produced that comprise either one of the CcRpp2-R1 and CcRpp2-R3 resistance genes.
  • an plant exhibiting enhanced resistance to ASR is produced by crossing a first plant that comprises a CcRpp2-R1 gene with a second plant that comprises a CcRpp2-R3 gene and selecting ASR resistant progeny plants that comprise both the CcRpp2-R1 and CcRpp2-R3 resistance genes.
  • a legume plant that is a progeny from a cross with a legume plant comprising legume-derived CcRpp2-R1 and CcRpp2-R3 binary resistance genes described herein, wherein progeny are selected that retain the CcRpp2-R1 and CcRpp2-R3 binary resistance genes.
  • methods of assaying a legume plant for disease resistance to a plant disease e.g., ASR.
  • the method comprises exposing a portion of the legume plant to a plant pathogen (e.g., Phakopsora pachyrhizi); measuring plant disease symptoms on the legume plant exposed to the plant pathogen; and comparing the plant disease symptoms to a reference standard for disease resistance.
  • a plant pathogen e.g., Phakopsora pachyrhizi
  • the method comprises conferring resistance to an ASR pathogen (e.g., Phakopsora pachyrhizi) by introgression of legume-derived CcRpp2-R1 and CcRpp2-R3 binary resistance genes into germplasm (e.g., a legume crop species) in a breeding program for resistance to ASR.
  • the method features legume-derived CcRpp2-R1 and CcRpp2-R3 binary resistance genes that encode CcRpp2-R1 and CcRpp2-R3 polypeptides.
  • the CcRpp2-R1 and CcRpp2-R3 polypeptides comprise an amino acid sequence having at least 90% homology to legume-derived CcRpp2-R1 and CcRpp2-R3 polypeptides disclosed herein.
  • the method described herein also features a plant transformed with the polypeptide that displays enhanced resistance to ASR when compared to a susceptible plant.
  • FIG.1 is a schematic drawing of the Fine-mapping of CcRpp2 with reference C. cajan scaffold LGCc02.
  • plant includes a whole plant and any descendant, cell, tissue, or part of a plant.
  • plant parts include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants).
  • a plant tissue or plant organ may be a seed, callus, or any other group of plant cells that is organized into a structural or functional unit.
  • a plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. In contrast, some plant cells are not capable of being regenerated to produce plants.
  • Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks.
  • a harvestable part of a plant may be any useful part of a plant, including, for example and without limitation: flower; pollen; seedling; tuber; leaf; stem; fruit; seed; and root.
  • a plant cell is the structural and physiological unit of the plant.
  • Plant cells as used herein, includes protoplasts and protoplasts with a cell wall.
  • a plant cell may be in the form of an isolated single cell, or an aggregate of cells (e.g., a friable callus and a cultured cell), and may be part of a higher organized unit (e.g., a plant tissue, plant organ, and plant).
  • a plant cell may be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant.
  • a seed which comprises multiple plant cells and is capable of regenerating into a whole plant, is considered a “plant part” in embodiments herein.
  • protoplast refers to a plant cell that had its cell wall completely or partially removed, with the lipid bilayer membrane thereof naked. Typically, a protoplast is an isolated plant cell without cell walls which has the potency for regeneration into cell culture or a whole plant.
  • native or “natural” define a condition found in nature.
  • a “native DNA sequence” is a DNA sequence present in nature that was produced by natural means or traditional breeding techniques but not generated by genetic engineering (e.g., using molecular biology/transformation techniques).
  • exogenous sequence defines the native form of a polynucleotide, gene or polypeptide in its natural location in the organism or in the genome of an organism.
  • exogenous sequence as used herein is any nucleic acid sequence that has been introduced into a cell wherein at least a portion of the introduced nucleic acid sequence is not native to that host cell.
  • an exogenous DNA sequence may comprise a sequence from another species.
  • heterologous sequence as used herein is any nucleic acid sequence that has been removed from its native location and inserted into a new location altering the sequences that flank the nucleic acid sequence that has been moved.
  • the heterologous sequence may be an exogenous sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a heterologous promoter is a promoter sequence that has been operably linked to a coding sequence not natively linked to the promoter thus forming a recombinant nucleic acid sequence.
  • purified relates to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment and means having been increased in purity as a result of being separated from other components of the original composition.
  • purified nucleic acid is used herein to describe a nucleic acid sequence which has been separated from other compounds including, but not limited to polypeptides, lipids and carbohydrates.
  • polypeptide polypeptide
  • peptide and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • nucleic acid sequence that is complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex. In some embodiments, the nucleic acid sequence is fully complementary having 100% sequence identity.
  • Polynucleotide sequence variants is used herein to refer to a nucleic acid sequence that except for the degeneracy of the genetic code encodes the same polypeptide.
  • sequence identity refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity refers to the value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences, and amino acid sequences) over a comparison window, wherein the portion of the 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 nucleotide 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 comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.
  • Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math.2:482; Needleman and Wunsch (1970) J. Mol. Biol.48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
  • NCBI National Center for Biotechnology Information
  • BLASTTM Basic Local Alignment Search Tool
  • Bethesda, MD National Center for Biotechnology Information
  • Blastn the “Blast 2 sequences” function of the BLASTTM (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method.
  • Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na+ and/or Mg++ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are known to those of ordinary skill in the art, and are discussed, for example, in Sambrook et al.
  • stringent conditions encompass conditions under which hybridization will only occur if there is less than 20% mismatch between the hybridization molecule and a sequence within the target nucleic acid molecule. “Stringent conditions” include further particular levels of stringency.
  • “moderate stringency” conditions are those under which molecules with more than 20% sequence mismatch will not hybridize; conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize; and conditions of “very high stringency” are those under which sequences with more than 5% mismatch will not hybridize.
  • the following are representative, non-limiting hybridization conditions.
  • High Stringency condition detects sequences that share at least 90% sequence identity: Hybridization in 5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 16 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 15 minutes each; and wash twice in 0.5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 20 minutes each.
  • 5x SSC buffer wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.
  • Moderate Stringency condition detects sequences that share at least 80% sequence identity: Hybridization in 5x-6x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65-70 °C for 16-20 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 5-20 minutes each; and wash twice in 1x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 55-70 °C for 30 minutes each.
  • Non-stringent control condition (sequences that share at least 50% sequence identity will hybridize): Hybridization in 6x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature to 55 °C for 16-20 hours; wash at least twice in 2x-3x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature to 55 °C for 20-30 minutes each.
  • 6x SSC buffer wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.
  • a first nucleotide sequence is “operably linked” with a second nucleotide sequence when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked nucleotide sequences are generally contiguous and, where necessary to join two protein-coding regions, in the same reading frame. However, nucleotide sequences need not be contiguous to be operably linked.
  • operably linked when used in reference to a regulatory sequence and a coding sequence, means that the regulatory sequence affects the expression of the linked coding sequence.
  • regulatory sequences regulatory elements
  • control elements refer to nucleotide sequences that influence the timing and level/amount of transcription, RNA processing or stability, or translation of the associated coding sequence.
  • Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; stem-loop structures; repressor binding sequences; termination sequences; polyadenylation recognition sequences; etc.
  • Particular regulatory sequences may be located upstream and/or downstream of a coding sequence operably linked thereto.
  • particular regulatory sequences operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule.
  • operably linked means that the first amino acid sequence is in a functional relationship with at least one of the additional amino acid sequences.
  • Resistance is used herein to mean an absence or reduction of one or more disease symptoms in a plant caused by a plant pathogen. Resistance can mean that disease symptoms, such as, for example, number of lesions, defoliation, and associated yield loss, are reduced, minimized or lessened, when compared to a plant that is susceptible to the disease or a plant that does not contain an effective resistance gene, such as, for example, CcRpp2-Rl and CcRpp2-R3 genes that reduce one or more disease symptom. Further, resistance can include the prevention or delay of proliferation of a pathogen (e.g., fungi).
  • a pathogen e.g., fungi
  • Plant pathogen or "fungal pathogen” can be used herein to mean fungal pathogens of, for example, the genus Phakopsora, including the species Phakopsora pachyrhizi and Phakopsora meibomiae. These species are known to cause ASR in plants.
  • a plant disease or a legume crop species disease for example, can be ASR.
  • the term "disease resistance gene” or “resistance gene” is used herein to mean a gene that encodes a protein or polypeptide capable of enhancing or improving a defense or immune system response in a plant.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.
  • encode is used herein to mean that the nucleic acid comprises the required information, specified by the use of codons to direct translation of the nucleotide sequence (e.g., a legume sequence) into a specified protein.
  • a nucleic acid encoding a protein can comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or can lack such intervening non-translated sequences (e.g., as in cDNA).
  • Aspects of the disclosure encompass isolated or recombinant polynucleotide or protein compositions.
  • An "isolated” or “recombinant” nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in an in vitro or in a heterologous recombinant bacterial or plant host cell.
  • An isolated or recombinant nucleic acid molecule, or biologically active portion thereof, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the terms “increase,” “increasing,” “enhance,” “enhancing” and the like are used herein to mean any boost or gain or rise in the expression, function or activity of a target gene (e.g., TIR gene) product providing an increased resistance to one or more pathogens (e.g., Phakopsora spp.) or to a disease (e.g., ASR) compared to a susceptible plant.
  • a target gene e.g., TIR gene
  • pathogens e.g., Phakopsora spp.
  • ASR e.g., ASR
  • the terms “induce” or “increase” as used herein can mean higher expression of a target gene product, such that the level is increased 10% or more, 50% or more or 100% relative to a cell or plant lacking the target gene or protein of the present disclosure.
  • expression refers to the biosynthesis or process by which a polynucleotide, for example, is produced, including the transcription and/or translation of a gene product.
  • a polynucleotide of the present disclosure can be transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into a polypeptide or protein.
  • gene product can refer to for example, transcripts and encoded polypeptides.
  • Inhibition of (or increase in) expression or function of a gene product can be in the context of a comparison between any two plants, for example, expression or function of a gene product in a genetically altered plant versus the expression or function of that gene product in a corresponding, but susceptible wild-type plant or other susceptible plant.
  • the expression level of a gene product in a wild-type plant can be absent.
  • a "wild-type" plant can be a plant, plant cell or plant part that does not express an exogenous CcRpp2-R1 and/or CcRpp2-R3 nucleic acid or exogenous CcRpp2-R1 and/or CcRpp2-R3 protein.
  • inhibition of (or increase in) expression or function of the target gene product can be in the context of a comparison between plant cells, organelles, organs, tissues, or plant parts within the same plant or between plants, and includes comparisons between developmental or temporal stages within the same plant or between plants. Any method or composition that down-regulates expression of a target gene product, either at the level of transcription or translation, or down-regulates functional activity of the target gene product can be used to achieve inhibition of expression or function of the target gene product.
  • any method or composition that induces or up-regulates expression of a target gene product can be used to achieve increased expression or function of the target gene or protein.
  • Methods for inhibiting or enhancing gene expression are well known in the art.
  • the term "introducing" as used herein defines a process of altering the content of a cell/plant through the use of traditional breeding or recombinant transformation techniques. When using recombinant transformation techniques a nucleic acid or protein is passed across a plant cell membrane or cell wall into the interior of a plant cell.
  • Methods for introducing polynucleotides into plants are known in the art, including procedures resulting in stable transformation methods or transient transformation methods. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, PEG, electroporation, ultrasonic methods (e.g., sonoporation), liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell.
  • “Stable transformation” or “stably transformed” means that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof.
  • Transient transformation as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.
  • transformation is used herein to mean the transfer of, for example, a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms.
  • the term "host cell” refers to the cell into which transformation of the recombinant DNA construct takes place and can include a yeast cell, a bacterial cell, and/or a plant cell. Examples of methods of plant transformation include Agrobacterium-mediated transformation and particle-bombardment. Transformed plant cells can then be used to regenerate a transformed plant by methods known to one skilled in the art.
  • transgenic is used herein to refer to a plant, including any part derived from a plant, such as a cell, tissue, or organ in which an exogenous nucleic acid (e.g., recombinant construct, vector or expression cassette including one or more nucleic acids) is integrated into the genome by a genetic engineering method, such as Agrobacteria transformation.
  • the exogenous nucleic acid is stably integrated into a chromosome, so that successive generations may also be transgenic.
  • transgenic also encompasses biological processes including the crossing of plants and/or natural recombination.
  • EMBODIMENTS [0059] Crop diseases cause serious crop management issues and can sometimes lead to total crop failure. Asian soybean rust is a threat to world soybean production and is currently addressed by the use of foliar fungicides. Stable and reliable genetic resistance in commercial plant lines is an important feature associated with soybean crop yields, and presently, commercially grown soybean cultivars that are fully resistant to Asian soybean rust caused by Phakopsora pachyrhizi, are not available.
  • Dominant resistance genes were identified and confirmed to be members of the TIR-TIR class of resistance (R) genes.
  • the binary CcRpp2-R1 and CcRpp2-R3 resistance genes disclosed herein can provide resistance to Phakopsora pachyrhizi via heterologous expression.
  • Plants can defend themselves through a variety of cellular mechanisms. It is currently understood that the plant immune system is made up of receptors on the outside (often called the first tier immunity) and the inside of a cell (often referred to as the second tier immunity). Both sets of receptors can detect and respond to a pathogen.
  • the first tier responds to primary elements of a pathogen resulting in activation of pathogen-associated molecular pattern (PAMP)-triggered immunity.
  • PAMP pathogen-associated molecular pattern
  • Successful pathogens overcome PAMP-triggered immunity by secreting molecules called "effector proteins” or “effectors” that are either localized to the plant apoplast or are taken up into the plant cell. Effectors manipulate host cell functions to suppress host immune responses in order to facilitate the establishment of infection or to otherwise enhance growth conditions for the pathogen, e.g. by ensuring availability to nutrients.
  • Plants have, in some cases, evolved a second tier of immunity in which R gene products recognize the activity of specific effectors resulting in an effector-triggered immunity.
  • LRRs C-terminal leucine-rich repeats
  • NBS nucleotide-binding site
  • the NBS domain functions as a molecular switch depending on the bound nucleotide: ADP-bound in the resting state and ATP-bound in the active state.
  • the LRR domain is generally thought to be involved in effector recognition and autoinhibition (Ting et al., Immunity, 28 (2008), pp.285-287).
  • Typical plant NLRs almost universally feature the additional coiled-coil (CC) or Toll/interleukin-1 receptor (TIR) N- terminal domain.
  • CC coiled-coil
  • TIR Toll/interleukin-1 receptor
  • TIR domains are used to sort plant NLRs into two main groups termed CNLs (CC-NLRs) and TNLs (TIR-NLRs). Both CC and TIR domains have been demonstrated to play key roles in the formation of dimers and oligomers.
  • TIR-NLR family resistance receptors
  • R proteins resistance receptors
  • TIR domain in R proteins functions as an NAD+-cleaving enzyme to trigger localized cell death, known as the hypersensitive response (HR).
  • TIR-only protein regulates cell death in plants Proceedings of the National Academy of Sciences Mar 2017, 114 (10) E2053-E2062; DOI: 10.1073/pnas.16209731142) and TIR domains of plant immune receptors are NAD+-cleaving enzymes that promote cell death. SCIENCE 23 AUG 2019 : 799-803.
  • the LRR domain of plant R proteins appears to be the major determinant of recognition specificity.
  • the NB domain is shared with mammalian nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which also function as regulators of innate immune responses and apoptosis.
  • NOD nucleotide-binding oligomerization domain
  • NLRs mammalian nucleotide-binding oligomerization domain
  • NB domain can bind and hydrolyse nucleotides, and the presence of bound ATP or ADP may determine whether the R protein is in an active or inactive signaling state.
  • pathogen effectors can have either an avirulence or virulence effect.
  • the virulence activity of effectors is associated with the manipulation of normal host cell functions or the suppression of host immune responses by the pathogen in order to establish successful infection.
  • recognition by the corresponding plant R protein activates a host immune or defense response, resulting in programmed cell death and resistance to the pathogen.
  • nucleic acids and polypeptides disclosed herein are useful in generating transgenic plants exhibiting fungal resistance and in methods for conferring or enhancing or increasing fungal resistance to a plant (e.g., a legume crop species).
  • Methods and compositions disclosed herein may comprise the following polypeptide and polynucleotides sequences: [0066] SEQ ID NO: 1 CcRpp2-R1Aa coding sequence from Cajanus cajan (polynucleotide sequence). [0067] SEQ ID NO: 2: CcRpp2-R1Aa (polypeptide sequence). [0068] SEQ ID NO: 3: CcRpp2-R3Aa coding sequence from Cajanus cajan (polynucleotide sequence).
  • SEQ ID NO: 4 CcRpp2-R3Aa (polypeptide sequence).
  • the CcRpp2-R1 polynucleotides of SEQ ID NOs: 5-20 and the CcRpp2-R3 polynucleotides of SEQ ID NOs: 37-47, and the respective CcRpp2-R1 polypeptides of SEQ ID NOs: 21-36 and CcRpp2-R3 polypeptides of SEQ ID NOs: 48-58 disclosed herein are useful in generating transgenic plants exhibiting fungal resistance and in methods for conferring or enhancing or increasing fungal resistance to a plant (e.g., a legume crop species).
  • a CcRpp2-R1 polypeptide has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to any one of SEQ ID NOs: 2 and 21-36, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.
  • the CcRpp2-R3 polypeptide has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to any one of SEQ ID NOs: 4 and 48-58, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.
  • Polypeptides of the present disclosure can be produced either from a nucleic acid disclosed herein, or by the use of standard molecular biology techniques.
  • a CcRpp2-R1 and/or CcRpp2-R3 protein of the present disclosure can be produced by expression of a recombinant nucleic acid of the embodiments in an appropriate host cell, or alternatively by a combination of ex vivo procedures.
  • Compositions and methods disclosed herein are useful in protecting plants from fungal pathogens.
  • the interactions between a host and a pathogen can be described in a continuum of "immunity” to "susceptibility.”
  • the terms “immunity” or “immune” are used herein to mean the absence of any macroscopically visible disease symptom(s).
  • the term “ resistance” is used herein to mean the presence of macroscopically visible lesions with no or limited sporulation, and/or a reduction in the extent or degree and/or a delay in the progression of any disease symptom(s) and can be, for example, manifested as reduced number of lesions or lesions with a reduction in sporulation.
  • the term "susceptibility" or the phrase “lack of resistance” to ASR refers to the occurrence of lesions with sporulation levels equal to or above the sporulation level observed in a reference standard, such as, for example, cultivars Williams or Peking.
  • Methods of the present disclosure can be carried out, for example, to provide enhanced resistance by Glycine max to the obligate biotrophic fungus Phakopsora pachyrhizi, the main causal agent of ASR, or to Phakopsora meibomiae.
  • increased or enhanced resistance to a fungal pathogen may be compared to the response of a susceptible plant, such as, for example, Williams or Peking.
  • Resistance can vary and is related to the proportion (i.e., percent) of disease symptoms (e.g., lesions) observed on a plant or plant part (e.g., leaf).
  • a numerical score or value for immunity, resistance and susceptibility can be given.
  • a numerical score for resistance represents the degree of resistance a plant exhibits to a plant disease (e.g., ASR).
  • the numerical scores can also be used to compare the degree of resistance, for example, between a plant of interest (e.g., a transgenic legume crop plant) to that of a susceptible plant (e.g., Williams or Peking) or a reference standard.
  • Methods and compositions for resistance disclosed herein relate to the isolation of one or more resistance genes from a legume species, and the subsequent transfer of one or more of these resistance genes to another plant, soybeans, for example, to provide resistance to Phakopsora spp. via homologous or heterologous expression.
  • An aspect of the present disclosure comprises the transfer of functioning TIR genes to a sexually compatible or incompatible species to produce disease resistance.
  • Polypeptides and TIR genes (e.g., CcRpp2- R1 and CcRpp2-R3 polypeptides and CcRpp2-R1 and CcRpp2-R3 genes) described herein can be used alone or in a stack with other resistance genes such as R genes (including NB-LRR resistance genes) or in a stack with non-R genes (including non-NB-LRR resistance genes) to provide resistance to ASR.
  • R genes including NB-LRR resistance genes
  • non-NB-LRR resistance genes including non-NB-LRR resistance genes
  • the transgenic approach of the present disclosure may be used in combination with the transgenic expression of a CcRpp1 polynucleotide (for example SEQ ID NO: 59) and the polypeptide encoded thereby (SEQ ID NO: 60)(See also the NB-LRR2 polynucleotide and the polypeptide encoded thereby as disclosed in U.S. Patent Application Publication No. US2018-0103600, incorporated herein by reference in its entirety).
  • a CcRpp1 polynucleotide for example SEQ ID NO: 59
  • SEQ ID NO: 60 See also the NB-LRR2 polynucleotide and the polypeptide encoded thereby as disclosed in U.S. Patent Application Publication No. US2018-0103600, incorporated herein by reference in its entirety.
  • Methods of the present disclosure can provide or enhance the resistance of a plant, such that the causal agents of a disease, such as ASR, can no longer reproduce.
  • the term "enhance” means to improve, increase, amplify, multiply, elevate and/or raise, thereby reducing one or more disease symptoms. Accordingly, plants (e.g., soybean) exhibit an increased resistance to a disease (e.g., ASR) when compared to plants that are susceptible or tolerant to Phakopsora spp.
  • methods described herein can reduce one or more symptoms (i.e., disease symptoms) of a legume plant disease (e.g., ASR).
  • a method can comprise exposing a transgenic legume crop plant (e.g., soybean) to a legume plant disease resulting in the transgenic legume crop plant having enhanced resistance to the plant disease.
  • the transgenic legume crop plant comprises a CcRpp2-R1 and CcRpp2-R3 polynucleotide.
  • One or more legume-derived CcRpp2-R1 and CcRpp2-R3 polynucleotides may have at least 90% sequence identity to a sequence as disclosed herein.
  • the plant, plant part, or plant cell is derived from a plant including but not limited to, alfalfa, clover, peas, beans, lentils, lupins, mesquite, carob, soybeans, peanuts, and tamarind.
  • Progeny, variants, and mutants of disease resistant plants disclosed herein are within the scope of the present disclosure, provided that these progeny, variants, and mutants comprise the original/modified polynucleotides of the parent plant.
  • the plant is a legume.
  • CcRpp2-R1 and CcRpp2-R3 polypeptides, CcRpp2-R1 and CcRpp2-R3 polynucleotides, and/or CcRpp2-R1 and CcRpp2-R3 resistance genes are derived from a legume.
  • legumes include, but are not limited to, the genus Phaseolus (e.g., French bean, dwarf bean, climbing bean (Phaseolus vulgaris), Lima bean (Phaseolus lunatus), Tepary bean (Phaseolus acutifolius), runner bean (Phaseolus coccineus)); the genus Glycine (e.g., Glycine soja, soybeans (Glycine max (L.))); pea (Pisum) (e.g., shelling peas (sometime called smooth or roundseeded peas; Pisum sativum); marrowfat pea (Pisum sativum), sugar pea (Pisum sativum), also called snow pea, edible-podded pea or mangetout, (Pisum granda)); peanut (Arachis hypogaea ), clover (Trifolium spp.), medick (Medicago), kudzu vine (Pueraria lobata gen
  • compositions and methods described herein can result in an agronomically desirable line or variety.
  • Agronomic characteristics or traits include, but are not limited to, herbicide tolerance, increased yield, insect control, weed control, pest control, pathogen disease resistance (e.g., fungal, virus, bacterial), high protein production, germination and seedling growth control, enhanced nutrition, environmental stress resistance, increased digestibility, male sterility, flowering time, or transformation technology traits such as cell cycle regulation and/or gene targeting.
  • pathogen disease resistance e.g., fungal, virus, bacterial
  • high protein production germination and seedling growth control
  • enhanced nutrition environmental stress resistance
  • increased digestibility male sterility
  • flowering time or transformation technology traits such as cell cycle regulation and/or gene targeting.
  • the present disclosure provides a method for screening or assaying legume plants for resistance, immunity, or susceptibility to a plant disease. General methods for determination of resistance, immunity, or susceptibility of a plant to a particular pathogen are known to one skilled in the art.
  • a method for screening or assaying legume plants for resistance, immunity or susceptibility to a plant disease may comprise exposing a plant cell, tissue or organ (e.g., leaf) to a pathogen (e.g., Phakopsora pachyrhizi) and then determining and/or measuring in the exposed plant, the degree of resistance, immunity and/or susceptibility to a plant disease (e.g., ASR) caused by the pathogen.
  • the method can further comprise measuring any observable plant disease symptoms on the plant exposed to the plant pathogen and then comparing the plant disease symptoms to a reference standard to determine the degree or extent of disease resistance.
  • Methods of exposing a plant cell, tissue or organ to a pathogen are known in the art.
  • Methods of measuring, comparing, and determining the level of resistance, immunity and/or susceptibility (e.g., plant disease symptoms) to a disease, such as, for example, ASR, caused by the pathogen are also known in the art.
  • the exposed plants can be further assessed to isolate polynucleotides, amino acid sequences and/or genetic markers that are associated with, linked to, and/or confer resistance, immunity or susceptibility of a plant to a particular pathogen or disease.
  • Further assessments include, but are not limited to, isolating polynucleotides, nucleic acids, or amino acids sequences from the exposed plant, carrying out an assay of the isolated polynucleotides or nucleic acids, for example, to detect one or more biological or molecular markers associated with one or more agronomic characteristics or traits, including but not limited to, resistance, immunity and/or susceptibility.
  • the information gleaned from such methods can be used, for example, in a breeding program.
  • an isolated or recombinant nucleic acid is provided that is free of sequences (optimally protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated polynucleotide sequence encoding the resistance proteins disclosed herein can contain less than about 5 kb, about 4 kb, about 3 kb, about 2 kb, about 1 kb, about 0.5 kb, or about 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived.
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, about 20%, about 10%, about 5%, or about 1% (by dry weight) of contaminating protein.
  • optimally culture medium represents less than about 30%, about 20%, about 10%, about 5%, or about 1 % (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
  • Fragments and variants relating to the nucleotide sequences and proteins encoded are within the scope of the present disclosure.
  • a "fragment” refers to a portion of the nucleotide sequence or a portion of the amino acid sequence and thus the protein encoded thereby.
  • Fragments of a nucleotide sequence can encode protein fragments that retain the biological activity of the native protein and have the ability to confer resistance (i.e., fungal resistance) upon a plant.
  • fragments of a nucleotide sequence that are useful as hybridization probes do not necessarily encode fragment proteins retaining biological activity
  • fragments of a nucleotide sequence can range from at least about 15 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the polypeptides of the present disclosure.
  • “Functional fragment,” “fragment that is functionally equivalent,” and “functionally equivalent fragment” are used interchangeably herein. These terms refer to a portion or subsequence of a polypeptide sequence of the present disclosure in which its native ability is retained.
  • a fragment of a nucleotide sequence that encodes a biologically active portion of a polypeptide of the present disclosure can encode at least about 15, about 25, about 30, about 40, or 45 to about 50 contiguous amino acids, or up to the total number of amino acids present in a full-length polypeptide of the embodiments (for example, 341 amino acids for the peptide encoded by SEQ ID NO: 2).
  • Fragments of a nucleotide sequence that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a protein.
  • the CcRpp2-R1 polypeptide fragment is an N-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more amino acids from the N-terminus of CcRpp2-R1 polypeptides of SEQ ID NOs: 2 and 21-36.
  • the CcRpp2-R3 polypeptide fragment is an N-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more amino acids from the N-terminus of CcRpp2-R3 polypeptides of SEQ ID NOs: 4 and 48-58.
  • the CcRpp2-R1 polypeptide fragment is an N-terminal and/or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from the N- terminus and/or C-terminus relative to CcRpp2-R1 polypeptides of SEQ ID NOs: 2 and 21-36.
  • the CcRpp2-R3 polypeptide fragment is an N-terminal and/or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from the N- terminus and/or C-terminus relative to CcRpp2-R3 polypeptides of SEQ ID NOs: 4 and 48-58.
  • a CcRpp2-R1 polypeptide comprises an amino acid sequence having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequence of any one of the CcRpp2-R1 polypeptides of SEQ ID NOs: 2 and 21-36, wherein the CcRpp2-R1 polypeptide has fungal resistance activity.
  • a CcRpp2-R3 polypeptide comprises an amino acid sequence having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequence of any one of the CcRpp2-R3 polypeptides of SEQ ID NOs: 4 and 48-58, wherein the CcRpp2-R3 polypeptide has fungal resistance activity.
  • a CcRpp2-R1 polypeptide comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entire length of the amino acid sequence of any one of the CcRpp2-R1 polypeptides of SEQ ID NOs: 2 and 21-36.
  • a CcRpp2-R3 polypeptide comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entire length of the amino acid sequence of any one of the CcRpp2-R3 polypeptides of SEQ ID NOs: 4 and 48-58.
  • the polypeptide fragment is an N-terminal and/or a C- terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more amino acids from the N-terminus and/or C- terminus, by proteolysis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon, and/or insertion of a stop codon.
  • the term "full-length sequence,” when referring to a specified polynucleotide, means having the entire nucleic acid sequence of a native sequence.
  • fragments of the polynucleotide sequences disclosed herein, including SEQ ID NOs: 1 and 3 are provided. Such fragments can be used as hybridization probes or PCR primers, and do not necessarily encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence can range from at least about 15 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the polypeptides of the present disclosure. [0099] In accordance with one embodiment a method of identifying plants comprising a CcRpp2-R1 and/or CcRpp2-R3 gene of the disclosure is provided.
  • the method comprises obtaining a nucleic acid sample from one or more plants, and contacting said nucleic acid sample with a nucleic acid sequence that specifically binds to a CcRpp2-R1 and/or CcRpp2-R3 gene of the disclosure, and detecting the specific binding of the nucleic acid to its target sequence.
  • the method can detect the target sequence through the use of a labeled probe or by conducting a PCR reaction with suitable PCR primers that only produce an amplicon in the presence of the target sequence.
  • the method comprises obtaining a nucleic acid sample from one or more plants, and contacting the nucleic acid sample with either [00100] i) a polynucleotide that comprises a sequence of at least 8 nucleotides that are identical or have at least 90-95% sequence identity to a contiguous sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5-20 and 37-47, or complements thereof; wherein said method further comprises subjecting said sample and said polynucleotide to stringent hybridization conditions; and assaying said sample for hybridization of said polynucleotide to said DNA; or [00101] ii) a pair of PCR primers, wherein a first and second PCR primer each specifically bind to a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5-20 and 37-47, wherein said first and second PCR primers are capable of producing an amplicon when bound to their target complementary sequences and subjected to standard PCR reaction conditions; subjecting said sample
  • fusion proteins are provide comprising a CcRpp2-R1 polypeptide and/or CcRpp2-R3 polypeptide of the disclosure represented by a formula selected from the group consisting of: [00103] R1-L-R2, R2-L- R1, R1- R2 or R2- R1 [00104] wherein R1 is a CcRpp2-R1 polypeptide, chimeric CcRpp2-R1 polypeptide of the disclosure, or a protein of interest and R2 is a CcRpp2-R3 polypeptide, chimeric CcRpp2-R3 polypeptide of the disclosure, or a protein of interest.
  • the R1 polypeptide is fused either directly or through a linker (L) segment to the R2 polypeptide.
  • L represents a chemical bound or polypeptide segment to which both R1 and R2 are fused in frame, most commonly L is a linear peptide to which R1 and R2 are bound by amide bonds linking the carboxy terminus of R1 to the amino terminus of L and carboxy terminus of L to the amino terminus of R2.
  • fused in frame is meant that there is no translation termination or disruption between the reading frames of R1 and R2.
  • the linking group (L) is generally a polypeptide of between 1 and 500 amino acids in length.
  • a fragment of a nucleotide sequence of the present disclosure can encode a biologically active portion of a polypeptide, or it can be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below.
  • a biologically active portion of a polypeptide conferring resistance can be prepared by isolating a portion of one of the nucleotide sequences of the embodiments, expressing the encoded portion of the protein and assessing the ability of the encoded portion of the protein to confer or enhance fungal resistance in a plant.
  • Nucleic acid molecules that are fragments of a nucleotide sequence of the embodiments comprise at least about 15, about 20, about 50, about 75, about 100, or about 150 nucleotides, or up to one less than the total number of nucleotides present in a full-length nucleotide sequence disclosed herein (for example, 5210 nucleotides for SEQ ID NO: 8).
  • One source of polynucleotides that encode CcRpp2-R1 and/or CcRpp2-R3 polypeptides or related proteins is a species selected from, but not limited to, Arachis, Cercis, Cajanus, Glycine, Medicago, Phaseolus, Pisum or Vigna species, which contains a homologous CcRpp2-R1 polynucleotide or CcRpp2-R3 polynucleotide.
  • the polynucleotides of SEQ ID NOs: 1 and 5-20 and 3 and 37-47 can be used to express CcRpp2-R1 and CcRpp2-R3 polypeptides, respectively, in legume host plants that include but are not limited to alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, peanut or tamarind.
  • the polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode CcRpp2-R1 and CcRpp2-R3 polypeptides or related proteins.
  • Such probes can be used to identify homologous or substantially homologous polynucleotides derived from species selected from, but not limited to, Arachis, Cercis, Cajanus, Glycine, Medicago, Phaseolus, Pisum or Vigna.
  • Polynucleotides that encode CcRpp2-R1 and CcRpp2-R3 polypeptides can also be synthesized de novo from a CcRpp2-R1 or CcRpp2-R3 polypeptide sequence.
  • the sequence of the polynucleotide gene can be deduced from a CcRpp2-R1 or CcRpp2-R3 polypeptide sequence through use of the genetic code.
  • CcRpp2-R1 or CcRpp2-R3 polypeptide sequences that can be used to obtain corresponding nucleotide encoding sequences include, but are not limited to the CcRpp2-R1 or CcRpp2-R3 polypeptides of SEQ ID NOs: 2, 4, 21-36 and 48-58.
  • the nucleic acid molecule encoding a CcRpp2-R1 or CcRpp2-R3 polypeptide is a polynucleotide having the sequence set forth in one of SEQ ID NOs: 1, 3, 5-20 and 37-47, and variants, fragments and complements thereof. Nucleic acid sequences that are complementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also encompassed. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants.
  • the nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a plant.
  • the nucleic acid molecule encoding a CcRpp2-R1 or CcRpp2-R3 polypeptide is a non-genomic nucleic acid sequence.
  • the nucleic acid molecule encoding a CcRpp2-R1 or CcRpp2-R3 polypeptide is a non-genomic polynucleotide having a nucleotide sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity, to any one of the nucleic acid sequences of SEQ ID NOs: 1, 3, 5-20 and 37-47, wherein the encoded CcRpp2-R1 or
  • the CcRpp2-R1 polynucleotide encodes a CcRpp2-R1 polypeptide having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to any one of SEQ ID NOs: 2 and 21-36, and has at least one amino acid substitution, deletion, insertion or combination therefore, compared to the native sequence.
  • the CcRpp2-R3 polynucleotide encodes a CcRpp2-R3 polypeptide having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to any one of SEQ ID NOs: 4 and 48-58, and has at least one amino acid substitution, deletion, insertion or combination therefore, compared to the native sequence.
  • the nucleic acid molecule encodes a CcRpp2-R1 polypeptide comprising an amino acid sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entire length of any one of the amino acid sequences of SEQ ID NOs: 2 and 21-36.
  • the nucleic acid molecule encodes a CcRpp2-R3 polypeptide comprising an amino acid sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entire length of any one of the amino acid sequences of SEQ ID NOs: 4 and 48-58.
  • the nucleic acid molecule encodes a CcRpp2-R1 polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 2 and 21-36 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 1011, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more amino acid substitutions, deletions and/or insertions compared to the amino acid at the corresponding position of the respective SEQ ID NO: 2 and 21-36.
  • the nucleic acid molecule encodes a CcRpp2-R3 polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 4 and 48-58 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 1011, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more amino acid substitutions, deletions and/or insertions compared to the amino acid at the corresponding position of the respective SEQ ID NO: 4 and 48-58.
  • the polynucleotide coding sequences can be modified to add a codon at the position following the methionine start codon to create a restriction enzyme site for recombinant cloning purposes and/or for expression purposes.
  • the CcRpp2-R1 and/or CcRpp2-R3 polypeptide further comprises an alanine residue at the position after the translation initiator methionine.
  • "Variant" is intended to mean a protein or polypeptide derived from a native protein or polypeptide by deletion or addition of one or more amino acids at one or more internal sites in the native protein or polypeptide and/or substitution of one or more amino acids at one or more sites in a native protein or polypeptide.
  • variants encompassed by the present disclosure exhibit a biological activity of the native protein or polypeptide sequence.
  • a variant comprises a polynucleotide having a deletion (i.e., truncations) at the 5′ and/or 3′ end and/or a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.
  • variants of the nucleic acids of the embodiments will be constructed such that the open reading frame is maintained.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the embodiments.
  • 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 outline below.
  • variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a protein of the embodiments.
  • variants of a particular polynucleotide of the present disclosure can have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs known in the art.
  • Variants of a particular polynucleotide of the embodiments 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. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs known in the art.
  • any given pair of polynucleotides of the present disclosure is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, wherein the percent sequence identity between the two encoded polypeptides is at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity.
  • "Variant protein” means a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein.
  • Variant proteins encompassed by some aspects of the present disclosure are biologically active, that is they continue to possess the desired biological activity of the native protein, which is, the ability to confer or enhance plant resistance (i.e., plant fungal pathogen resistance) as described herein. Such variants can result, for example, from genetic polymorphism or from human manipulation.
  • Biologically active variants of a native protein of the embodiments can have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs known in the art.
  • a biologically active variant of a protein of the present disclosure can differ from that protein by as few as about 1-15 amino acid residues, as few as about 1-10, such as about 6-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • the proteins disclosed herein can be altered, for example, by including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are known in the art. For example, amino acid sequence variants and fragments of the resistance proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are known in the art.
  • Variant polynucleotides and proteins also encompass sequences and proteins derived from mutagenic or recombinogenic procedures, including and not limited to procedures such as DNA shuffling.
  • Libraries of recombinant polynucleotides can be generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest can be shuffled between the protein gene of the present disclosure and other known protein genes to obtain a new gene coding for a protein with an improved property of interest, such as increased ability to confer or enhance plant resistance to a fungal pathogen.
  • Variants may be made by making random mutations or the variants may be designed. In the case of designed mutants, there is a high probability of generating variants with similar activity to the native polypeptide when amino acid identity is maintained in critical regions of the polypeptide which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. A high probability of retaining activity will also occur if substitutions are conservative.
  • Amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type are least likely to materially alter the biological activity of the variant. Table 1 provides a listing of examples of amino acids belonging to each class.
  • polynucleotides described herewith can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods such as PCR or hybridization can be used to identify such sequences based on their sequence identity to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire sequences set forth herein or to variants and fragments thereof are encompassed by the present disclosure. Such sequences include sequences that are orthologs of the disclosed sequences. The term "orthologs" refers to genes derived from a common ancestral gene and which are found in different species as a result of speciation.
  • orthologs Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode for a protein that confers or enhances fungal plant pathogen resistance and that hybridize to the sequences disclosed herein, or to variants or fragments thereof, are encompassed by the present disclosure.
  • oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest.
  • Methods for designing PCR primers and PCR cloning are known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
  • Known methods of PCR include, and are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially- mismatched primers, and the like.
  • hybridization techniques all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism.
  • the hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and can be labeled with a detectable group such as 32 P, or any other detectable marker.
  • probes for hybridization can be made by labeling synthetic oligonucleotides based on the polynucleotides of the embodiments.
  • compositions and methods of the present disclosure are useful for modulating the levels of one or more proteins in a plant.
  • modulate is used herein to mean an increase or decrease in the level of a protein within a genetically altered (i.e., transformed) plant relative to the level of that protein from the corresponding non-transformed plant (i.e., a plant not genetically altered in accordance with the methods of the present disclosure).
  • the genes and polynucleotides of the present disclosure include naturally occurring sequences as well as mutant or altered forms.
  • the proteins disclosed herein also encompass naturally occurring proteins as well as variations, fragments and modified forms thereof. Such variants and fragments will continue to possess the desired ability to confer or enhance plant fungal pathogen resistance.
  • mutations made in the DNA encoding the variant or fragments thereof generally do not place the sequence out of the reading frame and optimally will not create complementary regions that could produce secondary mRNA structure.
  • the gene or genes of the present disclosure can be expressed as a transgene in order to make plants resistant to ASR.
  • the use of different promoters described herein or known to those of skill in the art will allow the gene's expression to be modulated in different circumstances (i.e., the promoters can be selected based on the desired outcome). For instance, higher levels of expression in a particular tissue system or organ (e.g., leaves) may be desired to enhance resistance.
  • a polynucleotide encoding a polypeptide having at least 85%, 90%, 95% or 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 21-36 and 48-58 is provided, wherein the polypeptide when expressed in the cells of a plant confers resistance to Asian Soybean Rust (ASR) disease for said plant.
  • ASR Asian Soybean Rust
  • polynucleotide is selected from the group consisting of SEQ ID NOs: 1, 3, 5-20 and 37-47 or a polynucleotide having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NOs: 1, 3, 5-20 and 37-47.
  • these polynucleotide sequences can be operably linked to heterologous regulatory elements necessary for expressing the encoded CcRpp2-R1 and CcRpp2-R3 gene products in a plant cell.
  • the regulatory elements can include promoters; translation leader sequences; enhancers; termination sequences; and polyadenylation recognition sequences.
  • a recombinant polynucleotide wherein a heterologous plant promoter is operably linked to a CcRpp2-R1 or CcRpp2-R3 coding sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5-20 and 37-47 or a polynucleotide having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NOs: 1, 3, 5-20 and 37-47.
  • a recombinant polynucleotide wherein a heterologous plant promoter is operably linked to a CcRpp2-R1 or CcRpp2-R3 coding sequence selected from the group consisting of SEQ ID NOs: 1 or 3 or a polynucleotide having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NOs: 1 or 3.
  • a recombinant polynucleotide is provided wherein a heterologous plant promoter is operably linked to a CcRpp2-R1 or CcRpp2-R3 coding sequence selected from the group consisting of SEQ ID NOs: 1 or 3.
  • the nucleic acid sequences can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype.
  • This stacking can be accomplished by a combination of genes within a DNA construct, or by crossing one or more plants having transgenes with another plant line that comprises a desired combination.
  • the polynucleotides of the present disclosure or fragments thereof can be stacked with any other polynucleotides of the disclosure, or with other genes.
  • the combinations generated can also include multiple copies of any one of the polynucleotides of interest.
  • the polynucleotides of the present disclosure can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including and not limited to traits desirable for animal feed such as high oil genes, balanced amino acids, increased digestibility, insect, disease or herbicide resistance, avirulence and disease resistance genes, agronomic traits (e.g, male sterility, flowering time) and/or transformation technology traits (e.g., cell cycle regulation or gene targeting).
  • traits desirable for animal feed such as high oil genes, balanced amino acids, increased digestibility, insect, disease or herbicide resistance, avirulence and disease resistance genes, agronomic traits (e.g, male sterility, flowering time) and/or transformation technology traits (e.g., cell cycle regulation or gene targeting).
  • agronomic traits e.g, male sterility, flowering time
  • transformation technology traits e.g., cell cycle regulation or gene targeting
  • a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation.
  • the traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes.
  • the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis).
  • Expression of the sequences can be driven by the same promoter or by different promoters.
  • the stacked combination includes one or more genes encoding pesticidal proteins including, but not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7:1-13); from Pseudomonas protegens strain CHA0 and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank Accession No. EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric.
  • Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7:1-13)
  • Pseudomonas protegens strain CHA0 and Pf-5 previously fluorescens
  • Pechy-Tarr (2008) Environmental Microbiology 10:2368-2386; Gen
  • the stacked combination includes a polynucleotide encoding resistance to an herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea.
  • genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.7:1241 and Miki, et al., (1990) Theor. Appl. Genet.80:449, respectively. See also, US Patent Numbers 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and 5,378,824; US Patent Application Serial Number 11/683,737 and International Publication WO 1996/33270.
  • the stacked combination includes a polynucleotide encoding a protein for resistance to Glyphosate (resistance imparted by mutant 5-enolpyruvl-3- phosphikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar) genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes).
  • Glyphosate resistance to Glyphosate
  • PAT phosphinothricin acetyl transferase
  • bar Streptomyces hygroscopicus phosphinothricin acetyl transferase
  • ACCase inhibitor-encoding genes
  • Glyphosate resistance is also imparted to plants that express a gene encoding a glyphosate oxido-reductase enzyme as described more fully in US Patent Numbers 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose.
  • glyphosate resistance can be imparted to plants by the over expression of genes encoding glyphosate N-acetyltransferase. See, for example, US Patent Numbers 7,462,481; 7,405,074 and US Patent Application Publication Number US 2008/0234130.
  • a DNA molecule encoding a mutant aroA gene can be obtained under ATCC® Accession Number 39256, and the nucleotide sequence of the mutant gene is disclosed in US Patent Number 4,769,061 to Comai.
  • EP Application Number 0333033 to Kumada, et al., and US Patent Number 4,975,374 to Goodman, et al. disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin.
  • nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in EP Application Numbers 0242246 and 0242236 to Leemans, et al.; De Greef, et al., (1989) Bio/Technology 7:61, describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity.
  • the stacked combination includes a polynucleotide encoding a protein for resistance to herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene).
  • a polynucleotide encoding a protein for resistance to herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene).
  • Przibilla, et al., (1991) Plant Cell 3:169 describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in US Patent Number 4,810,648 to Stalker and DNA molecules containing these genes are available under ATCC® Accession Numbers 53435, 67441 and 67442.
  • the stacked combination includes a polynucleotide encoding a protein for resistance to Acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants (see, e.g., Hattori, et al., (1995) Mol Gen Genet.246:419).
  • genes that confer resistance to herbicides include: a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994) Plant Physiol 106:17), genes for glutathione reductase and superoxide dismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genes for various phosphotransferases (Datta, et al., (1992) Plant Mol Biol 20:619).
  • the stacked combination includes a polynucleotide encoding resistance to an herbicide targeting Protoporphyrinogen oxidase (protox) which is necessary for the production of chlorophyll.
  • the protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in US Patent Numbers 6,288,306; 6,282,83 and 5,767,373 and International Publication WO 2001/12825.
  • the stacked combination includes an aad-1 gene (originally from Sphingobium herbicidovorans) encoding the aryloxyalkanoate dioxygenase (AAD-1) protein.
  • AAD-1 aryloxyalkanoate dioxygenase
  • the trait confers tolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly referred to as “fop” herbicides such as quizalofop) herbicides.
  • the aad-1 gene, itself, for herbicide tolerance in plants was first disclosed in WO 2005/107437 (see also, US 2009/0093366).
  • the aad-12 gene derived from Delftia acidovorans, which encodes the aryloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to 2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides by deactivating several herbicides with an aryloxyalkanoate moiety, including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxy auxins (e.g., fluroxypyr, triclopyr).
  • phenoxy auxin e.g., 2,4-D, MCPA
  • pyridyloxy auxins e.g., fluroxypyr, triclopyr
  • the stacked combination includes a polynucleotide encoding an herbicide resistant dicamba monooxygenase disclosed in US Patent Application Publication 2003/0135879 for imparting dicamba tolerance.
  • the stacked combination includes a polynucleotide encoding bromoxynil nitrilase (Bxn) disclosed in US Patent Number 4,810,648 for imparting bromoxynil tolerance.
  • the stacked combination includes a polynucleotide encoding phytoene (crtl) described in Misawa, et al., (1993) Plant J.4:833-840 and in Misawa, et al., (1994) Plant J.6:481-489 for norflurazon tolerance.
  • the stacked combination includes a polynucleotide encoding a protein that confers or contributes to an altered grain characteristic, such as altered fatty acids, for example, by: [00147] (1) Down-regulation of stearoyl-ACP to increase stearic acid content of the plant. See, Knultzon, et al., (1992) Proc. Natl.
  • HSI2 Sugar-Inducible 2
  • Increasing expression of HSI2 increases oil content while decreasing expression of HSI2 decreases abscisic acid sensitivity and/or increases drought resistance
  • (8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oil content in plant seed, particularly to increase the levels of omega-3 fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (US Patent Application Publication Number 2011/0191904).
  • a feature of the present disclosure are methods comprising introducing a polynucleotide into a plant.
  • the polynucleotide can be presented in such a manner that the sequence gains access to the interior of a cell of the plant, including its potential insertion into the genome of a plant.
  • the methods of the present disclosure do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide gains access to the interior of at least one cell of the plant.
  • a polynucleotide can be transiently or stably introduced into a host cell and can be maintained non-integrated, for example, as a plasmid.
  • Transformation methods as well as methods for introducing polynucleotide sequences into plants can depend on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include, but are not limited to, microinjection, electroporation, direct gene transfer, Lecl transformation and ballistic particle acceleration. As newer methods become available, they can also be applied to the present disclosure as the method of transformation or transfection is not critical.
  • the cells that have been transformed can be grown into plants in accordance with conventional ways. These plants can then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations can be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
  • the transformed seed or transgenic seed having a nucleotide construct or an expression cassette is stably incorporated into their genome.
  • the present disclosure encompasses seeds comprising a polynucleotide sequence disclosed herein that can develop into or can be used to develop a plant or plants with increased or enhanced resistance to a pathogen (e.g., fungi) or infection caused by a pathogen as compared to, for example, a wild-type variety of the plant seed.
  • a pathogen e.g., fungi
  • the present disclosure features seeds from transgenic legume crop plants wherein the seed comprises a polynucleotide disclosed herein.
  • the present disclosure can be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica spp.
  • Brassica napus e.g., Brassica napus, Brassica rapa, Brassica juncea
  • Brassica napus e.g., Brassica napus, Brassica rapa, Brassica juncea
  • those Brassica species useful as sources of seed oil alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypoga
  • plants of interest include, a legume crop species, including, but not limited to, alfalfa (Medicago sativa); clover or trefoil (Trifolium spp.); pea, including (Pisum satinum), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata) and Lathyrus spp.; bean (Fabaceae or Leguminosae); lentil (Lens culinaris); lupin (Lupinus spp.); mesquite (Prosopis spp.); carob (Ceratonia siliqua), soybean (Glycine max), peanut (Arachis hypogaea) or tamarind (Tamarindus indica).
  • legume species and “legume crop species” are used herein to refer to plants, and can be for example, a plant of interest.
  • the legume species or legume crop species is a plant, plant part or plant cell.
  • constructs or vectors or expression cassettes are not present in the genome of the original plant or are present in the genome of the transgenic plant, but not at their natural locus of the genome of the original plant.
  • the compositions disclosed herein can be generated or maintained through the process of introgressing. Introgressing is sometimes called "backcrossing" when the process is repeated two or more times.
  • an aspect of the present disclosure is a method of enhancing plant resistance to a plant disease, such as ASR.
  • the method can comprise conferring resistance to a pathogen, for example, a pathogen that causes ASR, by introgression of legume-derived CcRpp2-R1 and CcRpp2-R3 binary resistance genes, or homologs thereof, into germplasm in a breeding program (i.e., a breeding program for resistance to ASR).
  • a breeding program i.e., a breeding program for resistance to ASR.
  • the term "germplasm” is used herein to mean genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture.
  • the germplasm can be part of an organism or cell, or can be separate from the organism or cell.
  • the germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture.
  • Germplasm in the context of the present disclosure includes cells, seed or tissues from which new plants can be grown, or plant parts, such as leaves, stems, pollen, or cells, that can be cultured into a whole plant.
  • Aspects of the present disclosure comprise methods for identification of germplasm as a source of resistance including, but not limited to, germplasm in one or more of the following genus: Glycine, Vigna, and Lablab.
  • the legume crop species or legume-derived gene is derived from the genus Glycine.
  • Glycine species include, but are not limited to, Glycine arenaria, Glycine argyrea, Glycine cyrtoloba, Glycine canescens, Glycine clandestine, Glycine curvata, Glycine falcata, Glycine latifolia, Glycine microphylla, Glycine pescadrensis, Glycine stenophita, Glycine syndetica, Glycine soja, Glycine tabacina and Glycine tomentella.
  • the legume crop species or legume-derived gene is derived from the genus Vigna. Vigna is a pantropic genus that comprises approximately 100 species.
  • Vigna unguiculata L.) Walp (cowpea)
  • Vigna radiata L.
  • Wilczek mung bean
  • Vigna angularis Willd.
  • Ohwi and Ohashi azuki bean
  • Vigna mungo L.
  • Hepper black gram
  • Vigna umbellata Thunb.
  • Vigna unguiculata dendtiana, a wild relative of cultivated subspecies; cylindrica, cultivated catjang; sesquipedalis, cultivated yardlong bean; and unguiculata, cultivated black-eyed pea.
  • Vigna unguiculata ssp. unguiculata is further divided into cultivar groups Unguiculata, grown as a pulse; Biflora or Cilindrica (catjang), mainly used as a forage; Sesquipedalis (yardlong or asparagus bean), grown as a vegetable; Textilis, cultivated for the fibres of its long floral peduncles; and Melanophthalmus (black-eyed pea).
  • the legume crop species or legume-derived gene is derived from the genus Lablab.
  • Lablab purpureus (L.) Sweet is a leguminous species (Verdcourt (1971) Flora of Tropical East Africa, pp.696-699, Crown Agents, London, UK; and Duke et al. (1981) Handbook of Legumes of World Economic Importance, pp.102-106, Plenum Press, New York, USA and London, UK) native to Asia and Africa (Pengelly and Maass, (2001) Gen.
  • the legume crop species or legume-derived gene is derived from the genus Cicer, Cajanus, Medicago, Phaseolus, Pisum, Pueraria, or Trifolium.
  • Cicer species include, but are not limited to, Cicer arietinum, Cicer echinospermum, Cicer reticulatum and Cicer pinnatifldum.
  • An example of the Cajanus species include, but is not limited to Cajanus cajan.
  • Examples of the Medicago species include, but are not limited to, Medicago truncatula and Medicago sativa.
  • Phaseolus species include, but are not limited to, Phaseolus vulgaris, Phaseolus lunatus, Phaseolus acutifolius and Phaseolus coccineus.
  • Pisum species include, but are not limited to, Pisum abyssinicum, Pisum sativum, Pisum elatius, Pisum fulvum, Pisum transcaucasium and Pisum humile.
  • An example of the Pueraria species includes, but is not limited to, Pueraria lobata.
  • Trifolium species include, but are not limited to, Trifolium aureum and Trifolium occidentale.
  • the present disclosure also comprises sequences described herein that can be provided in expression cassettes or DNA constructs for expression in the plant of interest.
  • the cassette can include 5' and 3' heterologous regulatory sequences operably linked to a sequence disclosed herein.
  • “Operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • Such regulatory sequences are well known in the art and include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence in certain host cells or under certain conditions.
  • the design of the vector can depend on, for example, the type of the host cell to be transformed or the level of expression of nucleic acid desired.
  • the cassette can contain one or more additional genes to be co-transformed into the plant. And, any additional gene(s) can be provided on multiple expression cassettes.
  • Expression cassettes of the present disclosure can include many restriction sites for insertion of the nucleotide sequence to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette can also contain selectable marker genes.
  • An expression cassette can further include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of the disclosure, and a transcriptional and translational termination region functional in plants.
  • the transcriptional initiation region, the promoter can be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter can be the natural sequence or alternatively a synthetic sequence.
  • the term "foreign" means that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • promoters include, but are not limited to, the Cauliflower Mosaic Virus 35S and soybean Ubiquitin 6.
  • heterologous promoters While it may be preferable to express the sequences using heterologous promoters, homologous promoters or native promoter sequences can be used. Such constructs would change expression levels in the host cell (i.e., plant or plant cell). Thus, the phenotype of the host cell (i.e., plant or plant cell) is altered.
  • a termination region can be native with the transcriptional initiation region, native with the operably linked DNA sequence of interest, or derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions.
  • endogenous or transgenic resistance orthologs can be altered by homologous or non-homologous recombinatory methods, such as, for example, by genome editing.
  • Such alterations refer to a nucleotide sequence having at least one modification when compared to its non-modified sequence and include, for example: (i) replacement of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).
  • the disclosed CcRpp2-R1 and CcRpp2-R3 polynucleotide compositions can be introduced into the genome of a plant using genome editing technologies, or previously introduced CcRpp2-R1 and CcRpp2-R3 polynucleotides in the genome of a plant may be edited using genome editing technologies.
  • the disclosed polynucleotides can be introduced into a desired location in the genome of a plant through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like.
  • the disclosed polynucleotides can be introduced into a desired location in a genome using a CRISPR-Cas system, for the purpose of site-specific insertion.
  • the desired location in a plant genome can be any desired target site for insertion, such as a genomic region amenable for breeding or may be a target site located in a genomic window with an existing trait of interest.
  • Existing traits of interest could be either an endogenous trait or a previously introduced trait.
  • Target site refers to a polynucleotide sequence, for example in the genome (including chloroplastic and mitochondrial DNA) of a cell, to which an endonuclease is recruited, and optionally nicks or cleaves the DNA of the target site.
  • the target site can be an endogenous site in the plant genome, or alternatively, the target site can be heterologous to the plant and thereby not be naturally occurring in the genome, or the target site can be found in a heterologous genomic location compared to where it occurs in nature.
  • genome editing technologies may be used to alter or modify the introduced polynucleotide sequences or the endogenous homologs.
  • Site specific modifications that can be introduced into the disclosed CcRpp2-R1 and CcRpp2-R3 polynucleotide compositions include those produced using any method for introducing site specific modification, including, but not limited to, through the use of gene repair oligonucleotides (e.g. U.S.
  • genome editing technologies may be used to position coding sequences for additional ASR resistance proteins in close proximity to the disclosed CcRpp2-R1 and CcRpp2-R3 polynucleotide compositions disclosed herein within the genome of a plant, in order to generate molecular stacks of ASR-resistance proteins.
  • An “altered target site,” “altered target sequence.” “modified target site,” and “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence.
  • Such "alterations” include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).
  • the gene(s) can be optimized for increased expression in the transformed plant as needed. In other words, the genes can be synthesized using plant-preferred codons for improved expression. Methods for synthesizing plant-preferred genes are known in the art. [00185] Additional sequence modifications are known to enhance gene expression in a cellular host.
  • the expression cassettes can additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation.
  • Translation leaders include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), and human immunoglobulin heavy chain binding protein (BiP); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4); tobacco mosaic virus leader (TMV); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81 :382385). Other methods known to enhance translation can also be utilized, such as, introns.
  • picornavirus leaders for example, EMCV leader (Encephalomyocarditis 5' noncoding region); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), and human immunoglobulin heavy chain binding protein (BiP); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4); tobacco mosaic virus leader (TMV
  • the various DNA fragments can be manipulated while preparing the expression cassette, to ensure that the DNA sequences are in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers can be employed to join the DNA fragments.
  • other manipulations can be used to provide for convenient restriction sites, removal of superfluous DNA, or removal of restriction sites.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions can be involved.
  • the expression cassette can comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glyphosate, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • NEO neomycin phosphotransferase II
  • HPT hygromycin phosphotransferase
  • genes conferring resistance to herbicidal compounds such as glyphosate, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present disclosure.
  • a target gene and/or protein e.g., one or more CcRpp2-R1 and CcRpp2-R3 genes and/or one or more CcRpp2-R1 and CcRpp2-R3 proteins
  • the methods described herein comprise transforming a plant or plant cell with a polynucleotide, for example, as disclosed herein, that encodes the target protein.
  • the polynucleotides described herein can be operably linked to a promoter that drives expression in a plant cell.
  • Any promoter known in the art can be used in the methods of the present disclosure including, but not limited to, constitutive promoters, pathogen-inducible promoters, wound-inducible promoters, tissue-preferred promoters, and chemical-regulated promoters.
  • the choice of promoter may depend on the desired timing and location of expression in the transformed plant as well as other factors, which are known to those of skill in the art.
  • Transformed cells or plants can be grown or bred to generate a plant comprising one or more of polynucleotides that were introduced into the cell or plant that, for example, encodes CcRpp2-R1 and CcRpp2-R3 proteins. [00190]
  • a number of promoters can be used in the practice of the disclosure.
  • the promoters can be selected based on the desired outcome. That is, the nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in the host cell of interest.
  • constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No.6,072,050; the core CaMV 35S promoter; rice actin; ubiquitin; pEMU; MAS; ALS; and the like.
  • Other constitutive promoters include, for example, those disclosed in U.S.
  • Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen, e.g., PR proteins, SAR proteins, beta-l,3-glucanase, chitinase, etc.
  • PR proteins pathogenesis-related proteins
  • promoters that are expressed locally at or near the site of pathogen infection. Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter can be used in the constructions of the disclosure. Such wound- inducible promoters include potato proteinase inhibitor (pin II) gene, wunl and wun2, winl and win2, systemin, WIP1, MPI gene, and the like.
  • wound- inducible promoters include potato proteinase inhibitor (pin II) gene, wunl and wun2, winl and win2, systemin, WIP1, MPI gene, and the like.
  • Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter can be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid.
  • tissue-preferred promoters can be utilized to target enhanced expression of the target genes or proteins (e.g., polynucleotide sequences encoding legume-derived CcRpp2-R1 and CcRpp2-R3 polypeptides) within a particular plant tissue.
  • tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed- preferred promoters, and stem-preferred promoters.
  • Tissue-preferred promoters include Yamamoto et al. (1997) Plant J.12(2): 255 -265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res.6(2): 157-168; Rinehart et al. (1996) Plant Physiol.112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al.
  • "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed- germinating” promoters (those promoters active during seed germination).
  • seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message), cZ19Bl (maize 19 kDa zein), milps (myo-inositol-1-phosphate synthase), and celA (cellulose synthase) (see WO 00/11177, herein incorporated by reference).
  • Gama-zein is a preferred endosperm-specific promoter.
  • Glob-1 is a preferred embryo-specific promoter.
  • seed-specific promoters include, but are not limited to, bean ⁇ -phaseolin, napin, ⁇ -conglycinin, soybean lectin, cruciferin, and the like.
  • seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where seed-preferred promoters from endl and end2 genes are disclosed; herein incorporated by reference. [00197] Expression of the polynucleotides of the present disclosure can involve the use of the intact, native CcRpp2-R1 and CcRpp2-R3 genes, wherein the expression is driven by a cognate 5' upstream promoter sequence(s).
  • expression can be generated using constructs assembled with 5' transcriptional control sequences provided by heterologous CcRpp2-R1 and CcRpp2-R3 disease resistance genes expressed in the host legume.
  • CcRpp2-R1 and CcRpp2-R3 disease resistance genes expressed in the host legume.
  • One skilled in the art will be able to identify genes encoding CcRpp2-R1 and CcRpp2-R3 proteins following the teachings of this application, to evaluate their expression level, and to select preferred promoter sequences that can be used for expression of the CcRpp2-R1 and/or CcRpp2-R3 gene of interest.
  • the use of either cognate or heterologous CcRpp2-R1 and CcRpp2-R3 promoter sequences provides an option to regulate protein expression to avoid or minimize any potential undesired outcomes associated with inappropriate or unwanted expression and plant defense activation.
  • transgenic plants expressing polynucleotides and polypeptides disclosed herein may also have one or more fungicides applied to the transgenic plants as a method of further preventing ASR associated damage to a legume crop species.
  • fungicidal compounds may also be applied to supplement the protection of a transgenic legume crop species comprising the CcRpp2-R1 and CcRpp2-R3 resistance gene sequences to a wider variety of undesirable diseases.
  • These fungicides may be formulated or tank-mixed with other fungicide(s) disclosed herein or applied sequentially with the other fungicide(s).
  • Such fungicides may include 2-(thiocyanatomethylthio)-benzothiazole, 2-phenylphenol, 8- hydroxyquinoline sulfate, ametoctradin, aminopyrifen, amisulbrom, antimycin, Ampelomyces quisqualis, azaconazole, azoxystrobin, Bacillus subtilis, Bacillus subtilis strain QST713, benalaxyl, benomyl, benthiavalicarb-isopropyl, benzovindiflupyr, benzylaminobenzene- sulfonate (BABS) salt, bicarbonates, biphenyl, bismerthiazol, bitertanol, bixafen, blasticidin-S, borax, Bordeaux mixture, boscalid, bromuconazole, bupirimate, calcium polysulfide, captafol, captan, carbendazim, carboxin, carpropamid, carvone,
  • kits for the assays described herein The polypeptide sequences and polynucleotides can be packaged as a component of a kit with instructions for completing the assay disclosed herein.
  • the kits of the present disclosure can include any combination of the polypeptides and/or polynucleotides described herein and suitable instructions (written and/or provided as audio-, visual-, or audiovisual material).
  • the kit relates to a DNA detection kit for identifying TIR genes (e.g., CcRpp2-R1 and CcRpp2-R3 genes) or CcRpp2-R1 and CcRpp2-R3 proteins against ASR.
  • kits utilizing any of the sequences disclosed herein for the identification of a transgenic event (e.g., CcRpp2-R1 and CcRpp2-R3) in a plant for efficacy against ASR are provided.
  • the kits can comprise a specific probe having a sequence corresponding to or is complementary to a sequence having between 80% and 100% sequence identity with a specific region of the transgenic event.
  • the kits can include any reagents and materials required to carry out the assay or detection method.
  • an ASR resistance polypeptide is provided selected from: a) a CcRpp2-R1 polypeptide comprising an amino acid sequence having greater than 60% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 2 and 21- 36; or b) a CcRpp2-R3 polypeptide comprising an amino acid sequence having greater than 60% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 4 and 48- 58.
  • an ASR resistance composition is provided comprising a CcRpp2-R1 polypeptide of embodiment 1 and a CcRpp2-R3 polypeptide of embodiment 1.
  • a polynucleotide encoding an ASR resistance polypeptide wherein the encoded polypeptide is selected from: [00204] a) a CcRpp2-R1 polypeptide comprising an amino acid sequence having greater than 60% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 2 and 21-36, optionally wherein the polynucleotide encoding an ASR resistance polypeptide is operably linked to a heterologous regulatory element such as a heterologous plant promoter; or [00205] b) a CcRpp2-R3 polypeptide comprising an amino acid sequence having greater than 60% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 4 and 48-58, optionally wherein the polynucleotide encoding an ASR resistance polypeptide is operably linked to a heterologous regulatory element such as a heterologous plant promoter.
  • the recombinant polynucleotide of embodiment 3 is provided, wherein the recombinant polynucleotide is selected from: [00207] a) a polynucleotide having at least 70% sequence identity to the polynucleotide of any one of SEQ ID NOs: 1 and 5-20; and [00208] b) the polynucleotide having at least 70% sequence identity to the polynucleotide of any one of SEQ ID NOs: 3 and 37-47.
  • a DNA construct comprising, the recombinant polynucleotide of embodiment 3 or 4 and a heterologous regulatory sequence operably linked to the recombinant polynucleotide.
  • a transgenic plant or plant cell is provided comprising the DNA construct of any one of embodiments 1-5.
  • the transgenic plant of claim 6 is provided, wherein the plant is a legume crop plant.
  • a transgenic legume crop plant of embodiment 7 is provided, wherein transgenic legume crop plant is soybean.
  • a method of conferring disease resistance in a legume crop species comprises transforming a legume crop species with a heterologous legume-derived CcRpp2-R1 gene and a heterologous legume- derived CcRpp2-R3 gene that confer disease resistance to a legume crop species disease.
  • a method of embodiment 9 is provided, wherein the legume crop species disease is caused by a plant pathogen.
  • the plant pathogen is Phakopsora pachyrhizi or Phakopsora meibomiae.
  • a method of any one of embodiments 9-11 wherein the legume crop species disease is Asian soybean rust.
  • a method of any one of embodiments 9, 10, 11 or 12 is provided, wherein the legume crop species is an alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, peanut or tamarind.
  • a method of any one of embodiments 9, 10, 11, 12 or 13 is provided, wherein the legume crop species is soybean.
  • a method of any one of embodiments 9, 10, 11, 12, 13 or 14 is provided, wherein the legume-derived CcRpp2-R1 or CcRpp2-R3 genes are derived from genus Arachis, Cercis, Cajanus, Glycine, Medicago, Phaseolus, Pisum or Vigna.
  • a transgenic legume crop plant of any one of embodiments 6-8 is provided, further comprising one or more additional resistance genes, optionally wherein the additional resistance gene is a CcRpp1 gene.
  • a transgenic legume crop plant of any one of embodiments 6-8 or 16 is provided, further comprising an improved agronomic trait.
  • a method of reducing one or more symptoms of a legume plant disease is provided, wherein the method comprises exposing the transgenic legume crop plant of any one of claims 6-8 to the legume plant disease wherein the transgenic legume crop plant has an enhanced resistance to the plant disease.
  • the plant disease is Asian soybean rust.
  • a method of producing an Asian soybean rust resistant plant comprising transforming a plant cell with a legume-derived CcRpp2-R1 gene and a legume-derived CcRpp2-R3 gene.
  • the method of embodiment 21 is provided, further comprising regenerating the transformed plant from the transformed plant cell.
  • the method of embodiment 22 is provided, further comprising the step of growing the transformed plant wherein the expression of the legume-derived CcRpp2-R1 gene and the legume-derived CcRpp2-R3 gene results in enhanced resistance to Asian soybean rust disease in the transformed plant.
  • the method of any one of embodiments 21-23 is provided, wherein the Asian soybean rust resistant plant is a legume species.
  • a legume plant is provided that is a progeny from a cross between a transgenic legume plant comprising a legume-derived CcRpp2-R1 gene and a legume-derived CcRpp2-R3 gene disclosed herein and a similar legume plant that is not transformed with the legume-derived CcRpp2-R1 gene and the legume-derived CcRpp2-R3 gene.
  • the plant of embodiment 24 is provided, wherein the legume plant is an alfalfa, clover, pea, bean, lentil, lupin, mesquite, carob, soybean, peanut or tamarind species.
  • a method of assaying a legume plant for disease resistance to a plant disease comprises exposing a portion of the legume plant comprising a legume-derived CcRpp2-R1 gene and a legume-derived CcRpp2-R3 gene to a plant pathogen; measuring plant disease symptoms on the legume plant exposed to the plant pathogen; and comparing the plant disease symptoms to a reference standard for disease resistance, optionally wherein the plant disease is caused by a plant pathogen, optionally wherein the plant pathogen is caused by Phakopsora pachyrhizi or Phakopsora meibomiae, optionally wherein the plant disease is Asian soybean rust [00232] In accordance with embodiment 27 a method of exposing a portion of the legume plant comprising a legume-derived CcRpp2-R1 gene and
  • the method of embodiments 27 wherein the legume-derived CcRpp2-R1 gene encodes a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 2 and the legume-derived CcRpp2-R3 gene encodes a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 4.
  • the method of any one of embodiments 27 or 28 is provided, wherein the CcRpp2-R1 gene encodes the polypeptide of SEQ ID NO: 2 and the CcRpp2-R3 gene encodes the polypeptide of SEQ ID NO: 4, optionally wherein the germplasm is a legume crop species, optionally wherein the legume crop species is an alfalfa, clover, pea, bean, lentil, lupin, mesquite, carob, soybean, peanut or tamarind species, optionally wherein the legume crop species is soybean.
  • the method of embodiment 29 is provided, wherein a plant transformed with the polypeptide displays enhanced resistance to ASR when compared to a susceptible plant.
  • a recombinant DNA construct of embodiment 5 is provided, further comprising one or more NB-LRR polynucleotides or a fragment thereof.
  • a recombinant DNA construct of embodiment 5 or 31 is provided, The recombinant DNA construct of claim 5, further comprising one or more resistance genes.
  • a seed comprising the recombinant DNA construct of any one of embodiments 5, 31 or 32 is provided.
  • a plant comprising the recombinant DNA construct of any one of embodiments 5, 31 or 32 is provided.
  • a seed of embodiment 32 or a plant of embodiment 33 is provided wherein said seed or plant comprises a nucleic acid sequence encoding a polypeptide selected from: [00241] a) a CcRpp2-R1 polypeptide comprising an amino acid sequence having greater than 90% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 2 and 21-36; or [00242] b) a CcRpp2-R3 polypeptide comprising an amino acid sequence having greater than 90% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 4 and 48-58.
  • the seed or plant of embodiment 33 wherein the CcRpp2-R1 polypeptide is SEQ ID NO: 2 and the CcRpp2-R3 polypeptide is SEQ ID NO: 4, optionally wherein the CcRpp2-R1 polynucleotide is SEQ ID NO: 1 and the CcRpp2-R3 polynucleotide is SEQ ID NO: 3.
  • the following examples are offered by way of illustration and not by way of limitation.
  • Example 1 Mapping and cloning of CcRpp2 genes
  • Pigeon pea (Cajanus cajan) is a diploid legume, with a genome size of approximately 830 Mbp (Varshney et al. (2012) Nat. Biotechnol., 30:83-89). The plant is self- fertile and has a life cycle between 2-3 months seed-to-seed.
  • C. cajan (accession G108-99) was previously characterized as exhibiting resistance to Asian Soybean Rust (ASR) disease.
  • ASR Asian Soybean Rust
  • ASR Asian Soybean Rust
  • CcRpp1 locus other than the known CcRpp1 locus are present in the plant’s genome. More particularly, several segregating populations were generated by crossing C. cajan (accession G108-99) with accessions that show full susceptibility, including Ra, Rb, Rc, Rd, Re and Rf. Segregation analysis indicated that a single major resistance gene confers resistance in these populations, except for the Rd population that showed a 15:1 segregation pattern. Further analysis showed that the resistances observed in these accessions map to the same locus, except for a potential second locus in the Rd population.
  • ASR Asian Soybean Rust
  • At least twelve plants homozygous for the susceptible allele at the interval displayed an immune phenotype (class 0) corroborating the hypothesis on the existence of a new resistance locus in accession G108-99. These 56 F2 plants were selfed to obtain F3 seed.
  • the F3 families were inoculated with isolate PPUFV-02 and genotyped using markers SSR10581 and dCAPS239615. The resistance in some of these F3 families displayed a 3:1 segregation and markers analysis confirmed the presence of G48-95 DNA at CcRpp1 locus, corroborating the segregation of a single resistance gene elsewhere in the Cajanus genome. [00247] Two segregating F3 families were selected for mapping of the new resistance locus.
  • BACs include the markers Rdint_264620 and dCAPS_393933, but they don’t contain the marker Rdint_385686.
  • BACJ17 includes the full BACP6 and the third BAC and neither of the BACs fully covers the interval of 121kb (Fig.1). Using suitable markers one loss and one gain of function recombinant on the distal side (marker position Rdint_264620) and three loss of function recombinants on the proximal side of the interval on the marker position Rdint_385686, were identified narrowing down the interval to 121,252 bp.
  • Rd BAC library were constructed and screened using three markers located in this interval: Rdint_264620; dCAPS_393933 and Rdint_385686.
  • the region between the contig_153610 and contig_135277 harbours a sequence that resembles a TIR-NB-LRR gene (homolog to Glyma14g024500). However, after a more detailed observation by checking the C.
  • the first TIR-TIR domain set on the BAC did not show any polymorphism between the resistant and susceptible transcriptome reads.
  • a clear frameshift caused by a two nucleotide deletion was observed in the TIR-TIR sequence of the susceptible parent.
  • TopHat analysis showed a slight induction in the transcriptome dataset of the resistant parent. Therefore this TIR-TIR sequence was a prime candidate for conferring the CcRpp2 resistance.
  • the second TIR-TIR domain did not show an induction in the resistant transcriptome reads and no polymorphisms were detected between the resistant and susceptible reads in the expressed contigs.
  • a frame shift in the gene variant present in the susceptible parent is intriguing.
  • the TIR-TIR gene found in BACJ17 (BACJ17 position: 63,676 to 66,081) is upregulated in the transcriptome of the resistant parent (G108-99) in relation to the susceptible transcriptome parent (G48-95).
  • the TIR-TIR domains encoded by this gene belong to a rare TIR-2 superfamily in which the number of family members in the dicots is restricted to 2-5 genes per species (Sarris et al., 2016).
  • a CT deletion on the second exon was present only in the susceptible allele (position on the BACJ17: 64,691) causing a frameshift on the gene, which results in an early stop codon, creating a short polypeptide sequence that will most likely not be functional and contains only one TIR domain.
  • This CT deletion found in the susceptible allele S48 is fixed in other four C. cajan accessions that do not convey resistance at the CcRpp2 locus, indicating that this gene is likely to be the CcRpp2 resistant gene candidate.
  • a comparison of the TIR-TIR gene present at the CcRpp2 locus of G108-99 to six other accessions of C. cajan reveals that only G108-99 conveys resistance via the CcRpp1 and CcRpp2 locus.
  • G48-95 is the susceptible mother plant used in the cross.
  • G59-95, G119-99, G127-97 and G146-97 contain resistance at the CcRpp1 locus but do not convey resistance via the CcRpp2 locus.
  • RNA Ligase Mediated Rapid Amplification of cDNA Ends [00252] (RLM-RACE) was conducted on the first TIR-TIR gene present in the resistant accession to obtain the whole TIR-TIR gene sequence including the 5’ and 3’ UTRs.
  • the RNA used for the RACE experiments was isolated from uninfected tissue and from non-etiolated leaf material. Interestingly, two different full length transcripts were observed, with one of them showing a deletion of 51 nucleotides at the end of the second exon.
  • the two splice variants observed in the first TIR-TIR resistant transcript are caused by an alternative splicing event resulting in two variations of peptide sequences, with one losing 17 aa from their sequence but this event did not change the function of these two variants.
  • TopHat was used to align the transcriptome of both parents against the updated version of the BACJ17. The relative abundance of the full TIR-TIR resistant transcript with a coverage of 220 x the number of reads versus 9x the number of reads on the variant carrying the 51 nt deletion.
  • Example 2 Transformation of soybean with the Cajanus cajan genes, CcRpp2- R1Aa (SEQ ID NO: 1) and CcRpp2-R3Aa (SEQ ID NO: 3)
  • a plant transformation construct was designed to provide high-level constitutive expression of CcRpp2-R1Aa (SEQ ID NO: 1) and moderate-level constitutive expression of CcRpp2-R3Aa (SEQ ID NO: 3) in soybean.
  • a slot vector was produced with a 1026 bp SfiI fragment containing the CcRpp2-R1Aa coding region that was ligated at the 5’ end to a 1948 bp soybean ubiquitin promoter + IntronI fragment and on the 3’ end to a 1163 bp phaseolin terminator fragment.
  • the entire promoter-coding region-terminator cassette was flanked by attR1 and attL4 Gateway® recombination sites.
  • a second slot vector was generated with a 1035 bp BamHI + SnaBI fragment containing the CcRpp2-R3Aa coding region that was ligated at the 5’ end to a 2,576 bp maize histone 2B promoter + IntronI fragment and on the 3’ end to a 902 bp soybean ubiquitin 14 (UBQ14) terminator fragment.
  • the entire promoter-coding region-terminator cassette was located between Gateway attL3 and attR2 recombination sites.
  • the final stacked gene construct was created by recombining the two promoter-coding region- terminator cassettes, separated by a 1,531 bp attL2 and attL1 flanked buffer fragment, in a Gateway based plant expression vector between compatible attR4 and attR3 recombination sites.
  • This vector in addition to the above elements, contained a spectinomycin resistance gene for bacterial selection and an herbicide resistant soybean ALS gene as a plant selectable marker.
  • the final CcRpp2Aa binary-containing plant expression vector was electroporated into Escherichia coli. Transformants were then selected and pDNA were isolated by standard miniprep methods. Transformants were characterized by diagnostic restriction enzyme digestions of miniprep DNA.
  • a positive clone containing the expected pattern of digestion bands was selected and subsequently transformed into Agrobacterium tumefaciens. Transformants were selected, sequence verified to contain the binary CcRpp2Aa plant transformation construct, and submitted for Agrobacterium-mediated transformation. [00258] Agrobacterium-mediated transformation of soybean. Transgenic soybean lines were produced from immature seed cultures following the Agrobacterium-mediated transformation protocol (Finer and McMullen 1991; Stewart et al.1996; Cho et al.2015). Briefly, immature seeds were harvested from soybean pods of plants grown in the greenhouse under standard conditions.
  • Seeds were surface sterilized, immature cotyledons were aseptically excised and the cultures were maintained in 250 ml flasks containing 50 ml of liquid media on rotary shakers at 26 °C under cool white fluorescent lights with a 16/8 h day/night photoperiod (Samoylov et al.1998; Cho et al.2011).
  • Agrobacterium tumefaciens carrying plasmids with genes of interest were used to transform the immature cotyledons. Transgenic events were selected and regenerated to maturity. These plants were grown under the same conditions as the wild type plants but in separate growth chambers.
  • Additional molecular stack constructs were assembled to express the genomic and predicted cDNA of both CcRpp2-R1Aa and CcRpp2-R3Aa behind either a soybean ubiquitin promoter or a maize histone 2B promoter for high-level or moderate-level expression, respectively.
  • Events were generated from each of the additional constructs and confirmed to express the CcRpp2 transgenes.
  • Homozygous and hemizygous plants displayed the red-brown (RB) phenotype when challenged with Phakopsora pachyrhizi; however, in contrast to event 1- 2 (see below), the additional construct designs resulted in a reduced resistance profile with no significant decrease in sporulation detected.
  • RB red-brown
  • Example 3 Testing transgenic plants for efficacy against ASR
  • the molecular stack of CcRpp2-R1Aa (SEQ ID NO: 1) and CcRpp2-R3Aa (SEQ ID NO: 3) genes was tested for efficacy against ASR by transformation of the plant expression construct into soybean, followed by inoculation of transgenic plants with Phakopsora pachyrhizi and scoring of plant disease symptoms.
  • One transgenic event, event 1-2 was recovered from the soy transformation experiment and confirmed by qPCR to contain the CcRpp2-R1Aa and CcRpp2-R3Aa genes.
  • T1 transgenic testing for efficacy of binary CcRpp2Aa against Phakopsora pachyrhizi Seeds from one T1 event were planted and grown under growth chamber conditions for 15 days until Vc. The plants were sampled at V1 for qPCR to determine the transgene copy number and inoculated with a suspension of Phakopsora pachyrhizi spores. The inoculation was performed with urediniospores collected from a susceptible variety.
  • Freshly harvested spores were suspended in an aqueous solution of 0.01% Tween 20 and mixed thoroughly; the spore concentration was then adjusted to 4x10 3 sp/ml with a hemocytometer. Plants were spray-inoculated with the urediniospore suspension, incubated at 100% relative humidity in the dark for 22 hours and then transferred to a growth chamber optimized for disease development (23 °C, 70% RH, 16 hr photoperiod) where they were allowed to grow and develop symptoms for 15 days. New growth was excised regularly in order to keep the unifoliates for the duration of the experiment.

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WO2024018016A1 (en) 2022-07-21 2024-01-25 Syngenta Crop Protection Ag Crystalline forms of 1,2,4-oxadiazole fungicides
WO2024068837A1 (en) 2022-09-28 2024-04-04 Syngenta Crop Protection Ag Agricultural methods
WO2024068838A1 (en) 2022-09-28 2024-04-04 Syngenta Crop Protection Ag Fungicidal compositions
WO2024100069A1 (en) 2022-11-08 2024-05-16 Syngenta Crop Protection Ag Microbiocidal pyridine derivatives
WO2024107673A1 (en) * 2022-11-15 2024-05-23 Two Blades Foundation Asian soybean rust resistance genes

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