Polynucleotides and methods for transferring resistance to asian soybean rust

Abstract

Disclosed herein are compositions and methods for improving or enhancing pathogen resistance in legume plants. Compositions comprising polypeptides encoded by legume-derived nucleotide-binding site-leucine-rich repeat (NB-LRR) genes can be used to improve resistance to Asian Soybean Rust in legumes. Methods using NB-LRR genes can be used to make transgenic resistant legume plants.

Description

[0001] Cross-referencing of related patent applications

[0002] This application claims the benefit of U.S. Provisional Application 62 / 159,718, filed May 11, 2015, the entire contents of which are incorporated herein by reference.

[0003] Merging of sequence lists

[0004] This application contains a sequence list submitted via EFS-Web in ASCII format on May 11, 2015, which contains filename 36446_0235U1_SL, is 47,607 bytes in size, was created on May 11, 2015, contains eight sequences, and is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure relates to compositions and methods for enhancing pathogen resistance in legume plants, and more specifically soybean plants, by providing one or more genes to the plant, said genes being associated with resistance to a pathogen of Asian soybean rust (ASR). This disclosure also relates to polynucleotides capable of enhancing resistance in legumes to ASR, and methods for preparing ASR-resistant transgenic legume plants using these polynucleotide sequences. Background Technology

[0006] Soybean (Glycine max) is a major industrial crop and one of the most important protein sources, and is considered a key food group for disease prevention and health optimization by many public health organizations, including the American Diabetes Association, the American Heart Association, and the American Cancer Society. Asian soybean rust (ASR) is a major crop disease affecting soybeans and can adversely impact growth and yield. It is caused by the obligate live parasitic fungus *Phakopsora pachyrhizi*, and to a lesser extent by the closely related fungus *Phakopsora meibomiae*. This disease can lead to yield losses ranging from 10% to 90%. Summary of the Invention

[0007] This disclosure relates to compositions and methods for identifying rust resistance genes from soybean species and transferring those genes into legume crops or legume crop species, such as soybean, to generate ASR-resistant plants.

[0008] This invention discloses isolated polynucleotides comprising a nucleotide sequence encoding a soybean-derived NB-LRR polypeptide having at least 90% amino acid sequence identity with the soybean sequence disclosed herein. In one aspect, plants transformed with the polynucleotides exhibit enhanced resistance to Asian soybean rust compared to susceptible and / or untransformed plants. This invention also discloses recombinant DNA constructs comprising the polynucleotides described herein.

[0009] This document discloses available polypeptides that may include polynucleotides or sequences of SEQ ID NO: 1-8 and their variants, which constitute or encode them.

[0010] This article discloses a method for conferring disease resistance in a legume crop species (e.g., soybean), the method comprising transforming the legume crop species with an NB-LRR gene derived from a heterologous legume, the gene conferring resistance to diseases of the legume crop species (e.g., ASR).

[0011] This document discloses transgenic legume crop plants stably transformed using a recombinant DNA construct. In one aspect, the recombinant DNA construct comprises the polynucleotide disclosed herein, which encodes one or more legume-derived NB-LRR resistance genes capable of conferring resistance to plant diseases, such as ASR. In another aspect, the polynucleotide comprises one or more non-legume-derived NB-LRR resistance genes and / or non-NB-LRR resistance genes capable of conferring resistance to plant diseases. The polynucleotides described herein may also comprise any combination of resistance genes. The transgenic legume crop plants may contain one or more agronomic traits. It is also envisioned that seeds be obtained from such transgenic legume crop plants. Furthermore, this disclosure is characterized by stably transformed transgenic legume crop plants containing legume-derived NB-LRR polynucleotides, which have at least 90% sequence identity with the sequences described herein.

[0012] This article discloses a method for identifying one or more resistance genes that confer resistance to plant diseases (e.g., ASR).

[0013] This document discloses a method for producing ASR-resistant plants (e.g., soybean species). In one aspect, the method includes transforming plant cells with a legume-derived NB-LRR resistance gene. The method may also include regenerating the transformed plant from the transformed plant cells. In another aspect, the method includes growing the transformed plant such that expression of the legume-derived NB-LRR resistance gene produces transformed plants exhibiting enhanced resistance to ASR diseases.

[0014] This article discloses a method for producing legume plants. The legume plants are offspring of crosses with legume plants containing the NB-LRR resistance gene of legume origin described herein.

[0015] This article discloses a method for determining the resistance of legumes to plant diseases (e.g., ASR). In one aspect, the method includes exposing a portion of a legume to a plant pathogen (e.g., soybean rust); measuring the symptoms of the plant disease on the exposed legume; and comparing the plant disease symptoms with a reference standard used for resistance.

[0016] This document discloses a method for enhancing plant resistance to ASR diseases. In one aspect, the method includes conferring resistance to ASR pathogens (e.g., soybean rust) by incorporating a legume-derived NB-LRR resistance gene into germplasm (e.g., a soybean crop species) during an ASR-resistant breeding program. The method is characterized by a legume-derived NB-LRR resistance gene encoding an NB-LRR polypeptide. In one aspect, the NB-LRR polypeptide comprises an amino acid sequence having at least 90% homology with the legume-derived NB-LRR polypeptide disclosed herein. The method is further characterized by plants transformed with said polypeptide exhibiting enhanced resistance to ASR compared to susceptible plants. Attached Figure Description

[0017] Figure 1 The CcRpp1 gene region in pigeon pea (Cajanus cajan) is shown to be homolinear with genomic regions from soybean chromosomes 12 and 9. The marker dCAPS140555, which is tightly linked to CcRpp1 in G119-99, is located close to a single colinear NB-LRR gene (Glyma12g01420) in soybean (denoted as "*"). Similarly, the CAPS20006 marker located in the pigeon pea gene is located in the homologous soybean gene Glyma12g01420.

[0018] Figure 2A-2B The identified CcRpp1 locus is shown. Figure 2A ) and four NB-LRR paralogous genes ( Figure 2B The physical and high-resolution gene map regions of the gene pool. The CcRpp1 genomic region in variety G119-99 contains four NB-LRR paralogous genes ( Figure 2AThe R gene region was narrowed to a 154 kb region encompassing markers dCAPS52491 and SSR2152. This was the most informative recombinant obtained after screening 1141 isolated F2 plants (2282 gametes). The functional gain region was delineated by markers dCAPS52491 and dCAPS239615, and the loss-of-function region by markers dCAPS52491 and SSR2152. BAC 3F carries three paralogs (-1, -2, and -3), and BAC 6G carries four paralogs (-1 to -4). Figure 2B The most closely associated marker, dCAPS140555, was designed from a gene containing an IQ calmodulin binding motif. Detailed Implementation

[0019] Crop diseases cause serious crop management problems and can sometimes lead to crop failure. Asian soybean rust is a threat to soybean production worldwide and is currently being addressed through the use of foliar fungicides. Stable and reliable genetic resistance in commercial plant lines is an important characteristic associated with soybean crop yield, and commercially grown soybean varieties with complete resistance to Asian soybean rust caused by soybean rust fungus are not currently available. The pathogens of ASR, soybean rust fungus and vesicular rust fungus, infect a wide range of foliar tissues in leguminous plants (at least 31 species in 17 genera; Slaminko et al., (2008) Plant Dis., 92: 797-771; and at least 42 species in 19 genera; Frederick et al., (2002) Mycology, 92: 217-227). In summary, another 152 species in other genera have been described as potential hosts for soybean rust (Bonde et al., (2008) Plant Dis., 92:30-38; Goellner et al., (2010) Molecular Plant Pathology, 11:169-177; Ono et al., (1992) Mycol. Res., 96(10):825-850; and Slaminko et al., (2008) Plant Dis., 92:797-771). Currently, fungicide application is the only available method to mitigate ASR.

[0020] Currently, commercially viable soybean (Glycine max) varieties with complete resistance to soybean rust are not available. Resistance to soybean rust is rare in soybeans; the USDA evaluated the entire U.S. soybean germplasm resource and found that less than 5% possessed resistance or partial resistance to soybean rust. Furthermore, the genes available in these soybean varieties only provide isolate-specific resistance; therefore, these resources cannot provide durable resistance under field conditions, such as in the presence of multiple strains.

[0021] Given that ASR is a major threat to soybean production, it is advantageous to identify the source of resistance genes and incorporate these transgenic genes into legume germplasm such as soybean to enhance protection. To identify novel resistance genes, several non-soybean legume species were screened to obtain variants resistant to soybean rust. Dominant resistance genes in several legumes were identified and confirmed to be well-characterized types of resistance (R) genes, members of the nucleotide-binding domain-rich leucine repeat (NB-LRR) gene family. When, for example, these resistance genes are transferred to soybean as single genes, multiple genes, or in the form of multi-gene cassettes, they can provide resistance to soybean rust via heterologous expression.

[0022] Although attempts have been made to transfer resistance genes between plant species, they have largely been unsuccessful or associated with adaptation barriers (Tai et al., (1999) PNAS, 96: 14153-14158; Ren et al., (1997) Euphatica, 93: 353-360; Day et al., (2005) Plant Cell 17: 1292-1305; Banerjee et al., (2001) Genetics 158: 439-450; Tian et al., (2003) Nature 423: 74-77; and Frost et al., (2004) MPMI, 17: 224-232). Furthermore, although successful transfer of resistance has been achieved in some plants (Halterman D. et al., (2008) Plant Disease 92: 339-343 (potato); Foster SJ et al., (2009) Mol Plant Microbe Interact 22: 589-600 (potato); Brunner S et al., (2012) Plant Biotechnology Journal 10: 398-409 (wheat); and Horvath DM et al., (2012) PLoS ONE 7: e42036 (tomato)), resistance to soybean rust fungus has not been previously achieved in soybeans through R gene transfer.

[0023] Plants can protect themselves through various cellular mechanisms. Currently, it should be understood that the plant immune system consists of extracellular (often referred to as first-level immunity) and intracellular (often referred to as second-level immunity) receptors. Both sets of receptors can detect and respond to pathogens. The first level responds to the key components of the pathogen, leading to the activation of pathogen-associated molecular pattern (PAMP)-triggered immunity. Successful pathogens overcome PAMP-triggered immunity by secreting molecules called “effect proteins” or “effectons,” which are located in the plant apoplast or absorbed within plant cells. Effectors regulate host cell function to suppress the host immune response in order to facilitate the establishment of infection or otherwise enhance the growth conditions of the pathogen, such as by ensuring the availability of nutrients. In some cases, plants have evolved second-level immunity, where the R gene product recognizes the activity of specific effectors, leading to effector-triggered immunity.

[0024] Resistance (R) genes in the plant genome produce R proteins that recognize specific pathogen effectors. The largest class of R genes encodes proteins containing a nucleotide-binding (NB) domain and a leucine-rich repeat (LRR) domain, named "NB-LRR," and protects the intracellular environment. A second class of R genes encodes proteins consisting of an extracellular membrane-anchored LRR domain coupled to a receptor-like domain, known as receptor-like proteins (RLPs). RLPs are typically coupled to receptor-like kinases and monitor the cell's external environment. R proteins transmit resistance to pathogens through direct binding to pathogen effectors or through indirect action of pathogen effectors on host cell targets.

[0025] Due to the "arms race" between the host and the pathogen, pathogen effectors can be either non-toxic or toxic. The toxic activity of effectors is associated with manipulating normal host cell function or suppressing the host immune response to establish a successful infection. In the non-toxic case, recognition via corresponding plant R proteins activates the host's immune or defensive response, leading to programmed cell death and resistance to the pathogen.

[0026] Major gene resistance, relying on a one-to-one correspondence between pathogen effectors and plant resistance genes (“gene-to-gene relationships”), has been widely used in breeding methods. However, such resistance based on the introduction of a single R gene is often species-specific and easily overcome by a single mutation in the pathogen’s avr gene, a result of diversity selection to avoid host recognition. Therefore, the durability of such qualitative resistance is a concern. Attempts have been made to introduce novel antimicrobial / antifungal genes or modify the expression of endogenous defense-related genes in transgenic plants. However, in many cases, the effects are only partial or short-lived and may come at the expense of plant yield and / or vigor. Therefore, the effective use of R genes remains one of the most efficient ways to engineer resistance. Furthermore, while a single R gene can be rapidly overcome by pathogens, the simultaneous successful incorporation of several R genes can provide durable species-independent resistance to pathogen isolates. For example, using gene stacking, combining two or more genes of interest into a single plant, may be an effective strategy for providing resistance. An example of successful R gene superposition is the incorporation of the Cf-9 resistance locus gene into tomatoes in the 1970s, which effectively prevented tomato leaf mold caused by Cladosporium fulvum. However, the effective "superposition" of multiple R genes in crops using classical breeding is often hampered by the dominant nature of the R gene and its availability in crops such as soybeans. 。

[0027] The nucleic acids and peptides disclosed herein can be used for methods of conferring, enhancing, or increasing fungal resistance in plants (e.g., legume crop species). The methods and compositions disclosed herein may comprise the following peptide and polynucleotide sequences:

[0028] SEQ ID NO: 1 CcRpp1 gene, from pigeon pea (polynucleotide sequence) (NB-LRR-2)

[0029] SEQ ID NO: 2: CcRppl (peptide sequence) (NB-LRR-2)

[0030] SEQ ID NO: 3: NB-LRR-1 (polynucleotide sequence)

[0031] SEQ ID NO: 4: NB-LRR-1 (peptide sequence)

[0032] SEQ ID NO: 5: NB-LRR-3 (polynucleotide sequence)

[0033] SEQ ID NO: 6: NB-LRR-3 (peptide sequence)

[0034] SEQ ID NO: 7NB-LRR-4 (polynucleotide sequence)

[0035] SEQ ID NO: 8: NB-LRR-4 (peptide sequence)

[0036] The compositions and methods disclosed herein can be used to protect plants from fungal pathogens. The interaction between the host and pathogen can be described on a continuum from “immunity” to “partial resistance” to “susceptibility.” The term “immunity” or “immune” is used herein to mean the absence of any macroscopically visible symptoms of disease. The term “partial resistance” is used herein to mean the presence of macroscopically visible lesions with or without limited spore formation and / or a reduction in the extent or degree of any symptom of disease and / or a delay in progression, and may manifest, for example, as a reduction in the number of lesions or lesions with reduced spore formation. As used herein, the term “susceptibility” or the phrase “lack of resistance” for ASR refers to the presence of lesions at a spore formation level equal to or higher than that observed in reference standards (such as, for example, varieties Williams or Peking).

[0037] The term "resistance" is used herein to refer to the absence or reduction of symptoms of one or more diseases caused by plant pathogens in a plant. Resistance can refer to a reduction, minimization, or decrease in disease symptoms, such as lesion number, defoliation, and associated yield loss, compared to a susceptible plant or a plant that does not contain an effective resistance gene (e.g., the NB-LRR gene) that reduces the symptoms of one or more diseases. Additionally, resistance can include prevention or delay of pathogen (e.g., fungal) proliferation. More broadly, the term "resistance" includes immunity and partial resistance as defined above.

[0038] The terms "plant pathogen" or "fungal pathogen" may be used herein to refer to fungal pathogens, for example, those of the genus *Phakopsora* (including species of soybean rust and *Phakopsora*). These species are known to cause ASRs in plants. Plant diseases or legume crop diseases can be, for example, ASRs.

[0039] The methods disclosed herein can be used to, for example, provide enhanced resistance in soybean to the obligate live parasitic fungus *Soybean rust* (a major pathogen of ASR) or to *Laminaria japonica*. For example, increased or enhanced resistance to fungal pathogens can be compared to the response of susceptible plants, such as, for example, Williams or Peking. Resistance can be variable and correlated with the proportion (i.e., percentage) of disease symptoms (e.g., lesions) observed on the plant or plant part (e.g., leaf). Numerical scores or values ​​for immunity, resistance, and susceptibility can be given. For example, a numerical score of resistance indicates the degree of resistance exhibited by the plant to a plant disease (e.g., ASR). Numerical scores can also be used to compare the degree of resistance between, for example, the plant of interest (e.g., a transgenic legume crop) and susceptible plants (e.g., Williams or Peking) or a reference standard.

[0040] The methods and compositions for resistance disclosed herein involve isolating one or more resistance genes from a legume species and subsequently transferring one or more of these resistance genes into another plant, such as soybean, via homologous or heterologous expression to provide resistance to *A. spp.* rust. The terms “resistance gene” or “resistance gene” are used herein to denote a gene encoding a protein or polypeptide capable of enhancing or improving a defensive or immune system response in a plant. One aspect of this disclosure includes transferring a functional R gene into sexually compatible or incompatible species to produce resistance. The polypeptides and R genes described herein (e.g., the NB-LRR polypeptide and the NB-LRR gene) may be used alone or in combination with other R genes or in combination with non-R genes (including non-NB-LRR resistance genes) to provide resistance to ASR.

[0041] Therefore, the transgenic methods disclosed herein can be used alone or in combination with other strategies to generate or confer ASR resistance in plants. Other available strategies include, but are not limited to, blocking the functional activity of effectors, inhibiting the uptake of pathogens or pathogenic factors (e.g., fungi) into host cells (e.g., plant cells), and / or conventional resistance breeding.

[0042] The methods disclosed herein can provide or enhance plant resistance, preventing the pathogens of diseases such as ASR from reproducing. The term "enhance" means to improve, increase, amplify, reproduce, elevate, and / or boost, thereby reducing one or more disease symptoms. Thus, plants (e.g., soybeans) exhibit increased resistance to diseases (e.g., ASRs) compared to plants susceptible to or tolerant to *A. rust* species. In one aspect, the methods described herein can reduce one or more symptoms (i.e., disease symptoms) of legume diseases (e.g., ASRs). One method may include exposing transgenic legume crop plants (e.g., soybeans) to legume diseases, resulting in transgenic legume crop plants with enhanced resistance to the plant diseases. In some aspects, the transgenic legume crop plants contain one or more legume-derived NB-LRR polynucleotides. The one or more legume-derived NB-LRR polynucleotides may have at least 90% sequence identity with sequences as disclosed herein.

[0043] The term "plant" is used herein to include any plant, tissue, or organ (e.g., plant part). Plant parts include, but are not limited to, cells, stems, roots, flowers, ovules, stamens, seeds, and leaves that can be cultured into a whole plant. Plant cells are cells of a plant that are obtained directly from a seed or plant, or derived from a culture of cells obtained from a plant. Progeny, variants, and mutants of regenerated plants are within the scope of this disclosure, provided that these parts contain introduced polynucleotides.

[0044] In one aspect, the plant, plant part or plant cell is derived from a plant, including but not limited to alfalfa, clover, pea, common bean, lentil, lupin, bean tree, carob tree, soybean, peanut and tamarind.

[0045] In one respect, the plant is a legume. In another respect, the NB-LRR polypeptide, NB-LRR polynucleotide, and / or NB-LRR resistance gene (or NB-LRR gene) originate from legumes. Examples of legumes include, but are not limited to, the genus *Phaseolus* (e.g., French bean, green bean, common bean (*Phaseolus vulgaris*), cotton bean (*Phaseolus lunatus*), broadleaf bean (*Phaseolusacutifolius*), red-flowered bean (*Phaseolus coccineus*)); the genus *Glycine* (e.g., wild soybean (*Glycinesoja*), soybean (*Glycine max (L.))); peas (e.g., podless pea (sometimes called smooth-skinned pea or round pea; common pea (*Pisum sativum*)); wrinkled peas (*Pisum sativum*), sugar-podded peas (*Pisum sativum*), which are also called snow peas, edible-podded peas, or tender peas (*Pisum grandanda*)); peanuts (*Arachis hypogaea*); and clover (*Trifolium* spp.).), alfalfa (Medicago), kudzu (Pueraria lobata), common alfalfa, alfalfa (Medicago sativa), chickpea (Cicer), lentil (Lens culinaris), lupinus; wild pea (Vicia), broad bean, broad bean (Viciafaba), conjoined grass (Lathyrus) (e.g., Lathyrus sativus, Lathyrus tuberosus); cowpea (e.g., Vigna aconitifolia, Vigna angularis, Vigna mungo, Vigna radiata, Vigna subterrane, Vigna umbellata, cowpea (Vigna) *Vigna unguiculata* (also known as cowpea); *Cajanus cajan*; *Macrotyloma* (e.g., peanut *Macrotyloma geocarpum*, broad bean *Macrotyloma uniflorum*); winged bean *Psophocarpus tetragonolobus*, African yam bean *Sphenostylis stenocarpa*, Egyptian black bean, magpie bean *Lablab purpureus*, yam bean *Pachyrhizus erosus*, guar bean *Cyamopsis tetragonolobus*; and / or *Canavalia* (e.g., dwarf canavalia *Canavalia ensiformis*), canavalia gladiata. 。

[0046] The compositions and methods described herein can result in agronomically desired lines or varieties. Agronomical characteristics or traits include, but are not limited to, herbicide tolerance, increased yield, pest and disease control, weed control, insect pest control, pathogen resistance (e.g., fungi, viruses, bacteria), high protein production, germination and seedling growth control, enhanced nutrition, resistance to environmental stress, increased digestibility, male sterility, flowering time, or transformational traits such as cell cycle regulation and / or gene targeting.

[0047] This disclosure provides methods for screening or determining legume plants that are resistant, immune, or susceptible to plant diseases. The determination of plant resistance, immunity, or susceptibility to specific pathogens is known to those skilled in the art. Methods for screening or determining legume plants that are resistant, immune, or susceptible to plant diseases include exposing plant cells, tissues, or organs (e.g., leaves) to a pathogen (e.g., soybean rust) and then determining and / or measuring the degree of resistance, immunity, and / or susceptibility in the exposed plant to a plant disease (e.g., ASR) caused by the pathogen. The methods may also include measuring any observable symptoms of the plant disease on the exposed plant and then comparing the plant disease symptoms to a reference standard to determine the degree or extent of resistance.

[0048] Methods for exposing plant cells, tissues, or organs to pathogens are known in the art. Methods for measuring, comparing, and determining levels of resistance, immunity, and / or susceptibility (e.g., plant disease symptoms) to diseases caused by pathogens, such as ASR, are also well known in the art. Exposed plants can also be evaluated to isolate polynucleotide, amino acid sequences, and / or genetic markers associated with, relating to, and / or conferring said resistance, immunity, or susceptibility to a particular pathogen or disease. Further evaluation includes, but is not limited to, the isolation of polynucleotides, nucleic acids, or amino acid sequences from exposed plants, and the determination 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. Information collected by such methods can be used, for example, in breeding programs.

[0049] In this disclosure, “nucleic acid” means deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, it includes known analogs (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides in a manner similar to that of naturally occurring nucleotides, which hybridize to single-stranded nucleic acids.

[0050] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acids, and also to natural amino acid polymers. The polypeptides of this disclosure can be generated from the nucleic acids disclosed herein, or generated using standard molecular biology techniques. For example, the truncated proteins of this disclosure can be generated by expressing recombinant nucleic acids of this embodiment in suitable host cells, or alternatively by a combination of in vitro methods, such as protease digestion and purification.

[0051] The term "encoding" is used herein to indicate that a nucleic acid contains the desired information specified by using codons to guide the translation of a nucleotide sequence (e.g., a legume sequence) into a specific protein. Nucleic acids encoding proteins may contain untranslated sequences (e.g., introns) within the translated region of the nucleic acid or may lack such intervening untranslated sequences (e.g., as in cDNA).

[0052] This disclosure covers isolated or substantially purified polynucleotide or protein compositions. The “isolated” or “purified” polynucleotide or protein, or its biologically active portion, is substantially or substantially free of components that are normally present in the natural environment of the polynucleotide or protein and that accompany or interact with it. Therefore, the isolated or purified polynucleotide or protein, when produced by recombinant technology (e.g., PCR amplification), is substantially free of other cellular material or culture medium, or when synthesized by chemical methods, is substantially free of chemical precursors or other chemicals. Preferably, the “isolated” polynucleotide does not contain sequences naturally located flanking the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) (e.g., protein-coding sequences). For example, in some embodiments of this disclosure, the isolated polynucleotide may 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 sequences naturally located flanking the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Proteins that are essentially free of cellular material include protein formulations having less than about 30%, about 20%, about 10%, about 5%, or about 1% (by dry weight) of contaminating proteins. When the protein of the embodiment or its biologically active portion is produced by recombinant methods, the culture medium preferably has less than about 30%, about 20%, about 10%, about 5%, or about 1% (by dry weight) of chemical precursors or chemicals of non-protein of concern.

[0053] Fragments and variants associated with encoded nucleotide sequences and proteins are within the scope of this disclosure. A “fragment” refers to a portion of a nucleotide sequence or the amino acid sequence encoded therein, as well as a portion of a protein. A fragment of a nucleotide sequence may encode a protein fragment that retains the biological activity of the native protein and has the ability to confer resistance to plants (i.e., antifungal properties). Alternatively, a nucleotide sequence fragment that can be used as a hybridization probe may not necessarily encode a biologically active fragment protein. Therefore, fragments of nucleotide sequences may range from at least about 15 nucleotides, about 50 nucleotides, about 100 nucleotides, and at most, a full-length nucleotide sequence encoding a polypeptide of the present disclosure.

[0054] Fragments of nucleotide sequences encoding the biologically active portion of the disclosed polypeptide may encode at least about 15, about 25, about 30, about 40, or 45, about 50 consecutive amino acids, or up to the total number of amino acids present in the full-length polypeptide of this embodiment (e.g., 925 amino acids for the peptide encoded by SEQ ID NO: 1). Fragments of nucleotide sequences that can be used as hybridization probes or PCR primers generally do not need to encode the biologically active portion of the protein.

[0055] When referring to a specific polynucleotide, the term "full-length sequence" refers to the entire nucleic acid sequence having a natural sequence. "Natural sequence" is used herein to denote an endogenous sequence, i.e., a non-engineered sequence present in the genome of an organism.

[0056] Therefore, fragments of the nucleotide sequences disclosed herein may encode the biologically active portion of a polypeptide, or they may be fragments that can be used as hybridization probes or PCR primers using the methods disclosed below. The biologically active portion of the resistant polypeptide can be prepared by isolating a portion of one of the nucleotide sequences of the embodiments, expressing the coding portion of the protein, and assessing the ability of the coding portion of the protein to confer or enhance fungal resistance in plants. Nucleic acid molecules containing fragments of the nucleotide sequences belonging to the embodiments comprise at least about 15, about 20, about 50, about 75, about 100, or about 150 nucleotides, or at most the number of nucleotides present in the full-length nucleotide sequence disclosed herein (e.g., 2,778 nucleotides for SEQ ID NO: 1).

[0057] The term "variant" refers to substantially similar sequences. For polynucleotides, a variant comprises the deletion and / or addition of one or more nucleotides at one or more sites in a native polynucleotide, and / or the substitution of one or more nucleotides at one or more sites in a native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. Those skilled in the art will recognize that the nucleic acid variants of this embodiment will be configured such that open reading frames are preserved. For polynucleotides, conserved variants include those sequences that, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of this embodiment. Natural allelic variants (such as these) can be identified using known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques described below. Variant polynucleotides also include polynucleotides of synthetic origin, such as those produced, for example, by site-directed mutagenesis but still encoding the protein of this embodiment. Typically, as determined by sequence alignment procedures well known in the art, variants of the specific polynucleotide disclosed herein may 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 higher sequence identity with the specific polynucleotide.

[0058] Variants of a specific polynucleotide (i.e., a reference polynucleotide) in this embodiment can also be evaluated by comparing the percentage of sequence identity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The percentage of sequence identity between any two polypeptides can be calculated using sequence alignment procedures known in the art. In any given polynucleotide pair of this disclosure evaluated by comparing the percentage of sequence identity shared by the two polypeptides they encode, the percentage of 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 higher.

[0059] "Variant protein" refers to a protein derived from a natural protein by deleting or adding one or more amino acids at one or more sites in the natural protein and / or replacing one or more amino acids at one or more sites in the natural protein. Variant proteins covered by some aspects of this disclosure are biologically active, meaning they retain the desired biological activity of the natural protein, namely the ability to confer or enhance plant resistance (i.e., resistance to plant fungal pathogens) as described herein. Such variants can be obtained, for example, through genetic polymorphism or human manipulation. As determined by sequence alignment procedures known in the art, biologically active variants of the natural protein of this embodiment may 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 higher sequence identity with the amino acid sequence of the natural protein. Biologically active variants of the protein disclosed herein may differ from the protein by as little as about 1-15 amino acid residues, as little as about 1-10 (e.g., about 6-10), as little as 5, as little as 4, 3, 2, or even 1 amino acid residue.

[0060] The proteins disclosed herein can be modified, for example, by means of amino acid substitutions, deletions, truncations, and insertions. Methods of such manipulation are known in the art. For example, amino acid sequence variants and fragments of resistance proteins can be prepared by mutations in DNA. Methods of mutagenesis and polynucleotide alteration are known in the art.

[0061] Variant polynucleotides and proteins also encompass sequences and proteins obtained by mutagenesis and recombination procedures, including but not limited to procedures such as DNA shuffling. Libraries of recombinant polynucleotides can be generated from a group of related sequence polynucleotides containing sequence regions with substantial sequence identity and capable of homologous recombination in vitro or in vivo. For example, using this method, sequence motifs encoding domains of interest can be shuffled between the disclosed protein genes and other known protein genes to obtain new genes encoding proteins with improved properties of interest, such as increased conferral or enhancement of plant resistance to fungal pathogens. Such DNA shuffling strategies are known in the art.

[0062] The polynucleotides described herein can be used to isolate corresponding sequences from other organisms, particularly other plants. In this way, methods such as PCR or hybridization can be used to identify such sequences based on their sequence homology with the sequences described herein. This disclosure covers sequences isolated based on their sequence identity with the entire sequence shown herein or with its variants and fragments. Such sequences include sequences that are orthologs of the disclosed sequences. The term "ortholog" refers to a gene derived from a common ancestral gene and present in different species due to speciation. Genes present 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. The function of orthologs is often highly conserved across species. Therefore, this disclosure covers isolated polynucleotides that encode proteins that confer or enhance resistance to fungal plant pathogens and hybridize with the sequences disclosed herein, or with their variants or fragments.

[0063] In PCR methods, oligonucleotide primers can be designed for 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 the following literature: Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Plainview, NY). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, and partially mismatched primers.

[0064] In hybridization techniques, all or part of a known polynucleotide is used as a probe, which selectively hybridizes with other corresponding polynucleotides present in a set of cloned genomic DNA or cDNA fragments (i.e., a genome or cDNA library) from a selected organism. The hybridization probe can be a genomic DNA fragment, cDNA fragment, RNA fragment, or other oligonucleotide, and can utilize detectable groups such as 3... 2 P or any other detectable marker. Therefore, for example, hybridization probes can be prepared by labeling synthetic oligonucleotides of polynucleotides based on this embodiment. Methods for preparing hybridization probes and constructing cDNA and genomic libraries are known in the art.

[0065] Various procedures can be used to examine the presence or absence of specific sequences in DNA, RNA, or proteins. These include, for example, Southern blotting, Northern blotting, Western blotting, and ELISA analysis. These techniques are well known in the art.

[0066] The compositions and methods disclosed herein can be used to regulate the content of one or more proteins in plants. The term "regulate" is used herein to mean an increase or decrease in the protein content in a genetically altered (i.e., transformed) plant relative to the protein content from a corresponding untransformed plant (i.e., a plant not genetically altered according to the methods of this disclosure).

[0067] As used herein, the terms “inhibition,” “reduction,” etc., refer to any reduction in the expression or function of a target gene product, including any relative reduction in expression or function and at most, including the complete elimination of the expression or function of the target gene product. 。

[0068] The terms “increase,” “enhancement,” etc., are used herein to indicate any promotion or gain or increase in the expression, function, or activity of a target gene (e.g., the R gene) product compared to a susceptible plant, thereby providing increased resistance to one or more pathogens (e.g., *Laminaria*) or diseases (e.g., ASR). Additionally, as used herein, the terms “cause” or “increase” may indicate higher expression of a target gene product relative to cells or plants lacking the target gene or protein of this disclosure, such that said level is increased by 10% or more, 50% or more, or 100%.

[0069] As used herein, the term “expression” refers to the biosynthesis or process that produces the polynucleotide, including transcription and / or translation of the gene product. For example, the polynucleotides of this disclosure may be transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the transcribed mRNA may subsequently be translated into a polypeptide or protein. The term “gene product” may refer, for example, to transcripts and encoded polypeptides. Inhibition (or increase) of the expression or function of a gene product (i.e., the gene product of interest) may be in a comparative setting between any two plants, such as the expression or function of a gene product in a genetically modified plant relative to the expression or function of that gene product in a corresponding but susceptible wild-type plant or other susceptible plant. The expression level of the gene product in the wild-type plant may be absent. For example, a “wild-type” plant may be a plant, plant cell, or plant part that does not express exogenous NB-LRR nucleic acid or exogenous NB-LRR protein.

[0070] Alternatively, the inhibition (or enhancement) of the expression or function of a target gene product may take place in a comparative setting between plant cells, organelles, tissues, or plant parts within or between the same plant, and includes comparisons between developmental or time stages within or between the same plant. Any method or composition that de-regulates the expression of a target gene product at the transcriptional or translational level, or de-regulates the functional activity of a target gene product, can be used to achieve inhibition of the expression or function of a target gene product. Similarly, any method or composition that induces or increments the expression of a target gene product at the transcriptional or translational level, or increases or activates or increments the functional activity of a target gene product, can be used to achieve increased expression or function of a target gene or protein. Methods for inhibiting or enhancing gene expression are well known in the art.

[0071] The genes and polynucleotides disclosed herein include naturally occurring sequences as well as mutant or altered forms. The proteins disclosed herein also encompass naturally occurring proteins as well as their variants, fragments, and modified forms. Such variants and fragments will continue to possess the desired ability to confer or enhance resistance to plant fungal pathogens. In one aspect, mutations made in the DNA encoding their variants or fragments generally do not place the sequence outside the reading frame and, optimally, will not produce complementary regions that could generate secondary mRNA structures.

[0072] One or more genes disclosed herein can be expressed transgenic to produce plants resistant to ASR. The use of different promoters described herein or known to those skilled in the art will allow gene expression to be regulated under different conditions (i.e., the promoter can be selected based on the desired outcome). For example, higher expression levels in a particular tissue system or organ (e.g., leaf) may be required to enhance resistance. The entire gene can be inserted in transgenic form (e.g., both the natural promoter and the coding sequence), thereby allowing for rapid combination with other traits such as insect resistance or herbicide resistance.

[0073] In some aspects of this disclosure, nucleic acid sequences can be superimposed with any combination of polynucleotide sequences of interest to form plants with desired phenotypes. This superposition can be achieved by combining genes within a DNA construct or by hybridizing one or more transgenic plants with another plant line containing the desired combination. For example, polynucleotides of this disclosure or fragments thereof can be superimposed with any other polynucleotides or other genes of this disclosure. The resulting combination may also include multiple copies of any of the polynucleotides of interest. Polynucleotides of this disclosure can also be superimposed with any other genes or combinations of genes to produce plants with a variety of desired combinations of traits, including but not limited to traits desired for animal feed, such as high-oil genes, balanced amino acids, increased digestibility, insect resistance, disease resistance or herbicide resistance, non-toxic and disease-resistant genes, agronomic traits (e.g., male sterility, flowering time), and / or transformational traits (e.g., cell cycle regulation or gene targeting).

[0074] These stacked combinations can be generated by any method, including but not limited to hybridization breeding of plants through any conventional or known method or genetic transformation. If the trait is stacked through genetically transformed plants, the target polynucleotide sequences can be combined at any time and in any order. For example, a transgenic plant containing one or more desired traits can be used as a target to introduce more traits through subsequent transformations. In co-transformation schemes, the trait and the polynucleotide of interest can be introduced simultaneously, provided by any combination of transformation cassettes. For example, if two sequences are to be introduced, they can be contained in separate transformation cassettes (trans) or in the same transformation cassette (cis). The expression of the sequences can be driven by the same promoter or different promoters. In some cases, it is desirable to introduce a transformation cassette that inhibits the expression of the polynucleotide of interest. This can be combined with any combination of other repression or overexpression cassettes to generate the desired combination of traits in the plant.

[0075] This disclosure is characterized by a method for introducing polynucleotides into plants. As used herein, the term "introduction" means providing a plant with, for example, a polynucleotide. In some aspects of this disclosure, the polynucleotide can be present in a manner that allows the sequence to enter the interior of plant cells, including its potential insertion into the plant's genome. The methods of this disclosure are independent of the specific method of introducing the sequence into the plant, as long as the polynucleotide enters the interior of at least one cell of the plant. Methods for introducing polynucleotides into plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0076] The term "transformation" is used herein to refer, for example, the transfer of a nucleic acid fragment into the genome of a host organism, thereby acquiring genetically stable heritability. A host organism containing the transformed nucleic acid fragment is called a "transgenic" organism. The term "host cell" refers to a cell in which the transformation of a recombinant DNA construct occurs and may include yeast cells, bacterial cells, and / or plant cells. Examples of methods for plant transformation include Agrobacterium-mediated transformation and particle bombardment, which can then be used to regenerate the transformed plant using methods known to those skilled in the art.

[0077] Polynucleotides can be introduced transiently or stably into host cells and can remain unintegrated, for example, in plasmid form. "Stable transformation" or "stable conversion" refers to the integration of the introduced nucleotide construct into the plant's genome, which is then inherited by its offspring. As used herein, "transient transformation" means the introduction of a polynucleotide into a plant that does not integrate into the plant's genome, or the introduction of a polypeptide into a plant.

[0078] The transformation method, and the method of introducing polynucleotide sequences into plants, may depend on the type of plant or plant cell to be transformed, i.e., monocot or dicot. Suitable methods for introducing polypeptides or polynucleotides into plant cells include, but are not limited to, microinjection, electroporation, direct gene transfer, Lec1 transformation, and ballistic particle acceleration. As newer methods become available, they may also be applied to this disclosure, since the method of transformation or transfection is not critical.

[0079] Transformed cells can be cultured into plants using conventional methods. These plants can then be grown and pollinated with the same or different transformant lines to identify progeny with constitutive expression of the desired phenotypic trait. Two or more generations of plants can be cultured to ensure the stable maintenance and heritability of the desired phenotypic trait expression. Seeds are then harvested to ensure that the desired phenotypic trait expression has been achieved. In some aspects of this disclosure, transformed seeds or transgenic seeds with nucleotide constructs or expression cassettes are stably incorporated into their genome.

[0080] In one aspect, this disclosure covers seeds comprising the polynucleotide sequences disclosed herein, which can develop into or be used to develop one or more plants that, compared to, for example, wild-type plant seeds, have enhanced resistance to pathogens (e.g., fungi) or infections caused by pathogens. In another aspect, this disclosure is characterized by seeds from transgenic legume crop plants, wherein said seeds comprise the polynucleotides disclosed herein.

[0081] This disclosure can be used to transform any plant species, including but not limited to monocots and dicots. Examples of plants of interest include, but are not limited to, maize (Zea mays), Brassica species (e.g., Brassica napus, Brassica rapa, Brassica juncea), especially those Brassica species that can be used as seed oil sources, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., Pennisetum glaucum, Panicum miliaceum, Setaria italica, Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), and cotton (Gossypium sea island). The following fruits and vegetables are listed: barbadense, upland cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffeaspp.), coconut (Cocos nucifera), pineapple (Ananascomosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangiferaindica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), apricot (Prunus amygdalus), sugar beet (Betavulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamental plants, and conifers.

[0082] In one aspect, plants of interest include leguminous crop species, including but not limited to alfalfa (Medicagosativa); clover or trifoliate grass (species of the genus Trifolium); peas, including (pea (Pisum satinum)), pigeon pea (Gajanus cajan), cowpea (Vigna unguiculata) and pea spp. (Lathyrus spp.); common bean (Fabaceae or Leguminosae); lentil (Lens culinaris); lupin (Lupinus spp.); bean tree (Prosopis spp.); long bean (Ceratonia siliqua), soybean (Glycine max), peanut (Arachis hypogaea) or tamarind (Tamarindus indica). The terms “leguminous species” and “leguminous crop species” are used herein to refer to plants and may, for example, plants of interest. In some aspects, a leguminous species or leguminous crop species is a plant, a plant part, or a plant cell.

[0083] The term “transgenic” as used herein refers to plants, including any part of a plant such as cells, tissues, or organs, in which exogenous nucleic acids (e.g., recombinant constructs, vectors, or expression cassettes comprising one or more nucleic acids) are integrated into the genome through genetic engineering methods, such as Agrobacterium transformation. This involves the stable integration of exogenous nucleic acids into chromosomes through genetic engineering methods, enabling the next generation to also be transgenic. As used herein, “transgenic” also encompasses biological treatments, including plant hybridization and / or natural recombination.

[0084] In one respect, the construct, vector, or expression cassette is not present in the genome of the original plant or is present in the genome of the transgenic plant, but not at the natural locus of the genome of the original plant.

[0085] The compositions disclosed herein can be generated or maintained by gene incorporation. When the method is repeated two or more times, gene incorporation is sometimes referred to as "backcrossing." In gene incorporation or backcrossing, the "donor" parent refers to a parent plant that has the desired gene or locus to be infiltrated. The "recipient" parent (used once or multiple times) or "recurrent" parent (used twice or more) refers to a parent plant in which the gene or locus has been incorporated. The initial hybridization produces the F1 generation; the term "BC1" refers to the second use of the recurrent parent, and "BC2" refers to the third use of the recurrent parent, and so on.

[0086] Therefore, one aspect of this disclosure is a method for enhancing plant resistance to plant diseases such as ASR. The method may include conferring resistance to pathogens, such as those causing ASR, by incorporating a legume-derived NB-LRR resistance gene into the germplasm during a breeding program (i.e., an ASR-resistant breeding program).

[0087] The term "germstone" is used herein to refer to genetic material derived from an individual (e.g., a plant), a group of individuals (e.g., a plant lineage, variety, or family), or a clone derived from a lineage, variety, species, or culture. Germplasm can be a part of an organism or cell, or can be isolated from an organism or cell. Germplasm provides genetic material having a specific molecular structure that provides the physical basis for some or all of the genetic properties of an organism or cell culture. In the context of this disclosure, germstone includes cells, seeds, or tissues from which new plants can be generated, or plant parts such as leaves, stems, pollen, or cells that can be cultured into a whole plant.

[0088] This disclosure includes methods for identifying germplasm (including, but not limited to, germplasm from one or more of the following genera) as sources of resistance: soybean, cowpea, and lentil.

[0089] As described in this paper, the NB-LRR resistance gene from legumes confers different responses to soybean rust fungi. Previous studies of six Australian species of soybean rust fungi can be distinguished, for example, based on their corresponding compatibility or incompatibility with several species of wild-type soybean (Burdon and Marshall (1981) Journal of Ecology, 69: 381-390; Burdon and Marshall (1981) Plant Disease, 65: 44-45; Burdon and Speer (1984) Euphatica, 33: 891-896; Burdon (1987) Ocecologia, 73(2): 257-267; Burdon (1988) Theor. Appl. Genet., 75: 923-928; and Jarosz and Burdon (1990) Heredity, 64: 347-353). Therefore, in one respect, legume crop species or genes of legume originate from the genus *Glycine*. Examples of *Glycine* species include, but are not limited to, *Glycinearenaria*, *Glycine argyrea*, *Glycine cyrtoloba*, *Glycine canescens*, *Glycine clandestine*, *Glycine curvata*, *Glycine efalcata*, *Glycine latifolia*, *Glycine microphylla*, *Glycine pescadrensis*, *Glycine stenophita*, *Glycine syndetica*, *Glycine soja*, *Glycine tabacina*, and *Glycine tomentella*.

[0090] Other genera, such as *Vigna* and *Lysimachia*, also show different responses to soybean rust fungus. Therefore, in one respect, legume crop species or genes of legume originate from the genus *Vigna*. *Vigna* is a pantropical genus containing approximately 100 species. It is further divided into the following subgenera: *Vigna*, *Haydonia*, *Plectotropis* (Africa), *Ceratotropis* (Asia), *Sigmoidotropis*, and *Lasiopron*. This genus includes economically related species such as *Vigna unguiculata* (L.) Walp (cowpea), *Vigna radiata* (L.) Wilczek (mung bean), *Vigna angularis* (adzuki bean) Ohwi and Ohashi (red bean), *Vigna mungo* (L.) Hepper (black bean), and *Vigna umbellata* (adzuki bean) Ohwi and Ohashi (rice bean). Four subspecies of cowpea are well-known: *dekindtiana*, a cultivated subspecies closely related to the wild; *cylindrica*, a cultivated black cowpea; *sesquipedalis*, a cultivated long cowpea; and *unguiculata*, a cultivated black-eyed cowpea. The genus *unguiculata* is further divided into the following varietal groups: cowpea, cultivated as a legume; *Biflora* or *Cilindrica* (black cowpea), primarily used as forage; long cowpea (yardlong or cowpea), cultivated as a vegetable; *Textilis*, cultivated for its long inflorescence stalks and fiber; and *Melanophthalmus* (black-eyed cowpea). The susceptibility of several cowpea species, including mung bean (Vigna radata), black bean (Vigna mungo), and cowpea (Vigna unguiculata), to soybean rust fungus has been recorded under field and greenhouse conditions.

[0091] In one respect, the genes of legume crop species or legume-derived species originate from the genus *Lysimachia*. Bean (Lablabpurpureus (L.) Sweet) (also known as Dolichos benghalensis Jacq., Dolichos lablab L., Dolichos purpureus L., Lablab niger Medikus, Lablab purpurea (L.) Sweet, Lablabvulgaris (L.) Savi, Vigna aristata Piper) is a legume species native to Asia and Africa (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) (Pengelly and Maass, (2001) Gen.resour.cropev.48: 261-272). It is commonly known as magpie bean, lentil, purple lentil, broad bean, Egyptian bean, poor man's bean, Tonga bean (British), and at least 20 additional common names. It is cultivated as a legume crop or as a green vegetable in Africa, Asia, and the Caribbean (Duke et al., (1981) Handbook of Legumes of World Economic Importance, pp. 102-106, Plenum Press, New York, USA and London, UK); and Pengelly and Maass, (2001) Gen. resource. crop v. 48: 261-272).Lablabpurpureus has been reported as an alternative host for soybean rust (Pérez-Hernández, (2007) Alternative hosts of Phakopsora pachyrhizi in the Americas: An analysis of their role in the epidemiology of Asian soybean rust in the continental USMSc.thesis. Iowa State University. Ames, Iowa. USA; Vakili (1981) Plant Dis. 65: 817-819; and Poonpolgul and Surin, (1980) Soybean Rust Newsletter, 3: 30-31).

[0092] In one respect, the genes of leguminous crop species or leguminous origins derive from the genera *Chickpea*, *Pistacia*, *Alfalfa*, *Phragmites*, *Vicia*, *Pueraria*, or *Trifolium*. Examples of *Chickpea* species include, but are not limited to, chickpea, *Cicerechinospermum*, *Cicer reticulatum*, and *Cicer pinnatfidum*. Examples of *Pistacia* species include, but are not limited to, pigeon pea. Examples of *Alfalfa* species include, but are not limited to, medicago truncatula and alfalfa (*Medicago sativa*). Examples of *Phragmites* species include, but are not limited to, common bean (*Phaseolus vulgaris*), cotton bean (*Phaseolus lunatus*), broadleaf bean (*Phaseolus acutifolius*), or red-flowered bean (*Phaseoluscoccineus*). Examples of *Vicia* species include, but are not limited to, *Pisumabyssinicum*, green pea (*Pisum sativum*), *Pisum elatius*, *Pisum fullvum*, *Pisum transcaucasium*, or *Pisum humile*. Examples of species in the genus *Pueraria* include, but are not limited to, kudzu root. Examples of species in the genus *Trifolium* include, but are not limited to, yellow clover (*Trifolium ureum*) and *Trifolium occidentale*.

[0093] This disclosure may also include the sequences described herein, which may be provided in expression cassettes or DNA constructs for expression in plants of interest. In one aspect, the expression cassette may include 5′ and 3′ heterologous regulatory sequences operatively linked to the sequences disclosed herein. The term “operatively linked” is used herein to mean linking a nucleic acid to be expressed to a regulatory sequence, including promoters, terminators, enhancers, and / or other expression control elements (e.g., polyadenylation signals), in a manner that allows nucleic acid expression (i.e., in the host plant cell when the vector is introduced into the host plant cell). Such regulatory sequences are well known in the art and include those that constitutively express nucleotide sequences directly in a variety of host cells, and those that directly express nucleotide sequences in a specific host cell or under specific conditions. The design of the vector may depend, for example, on the type of host cell to be transformed, or the desired level of nucleic acid expression. The expression cassette may contain one or more additional genes to be co-transformed into the plant. Furthermore, any additional gene may be provided on multiple expression cassettes.

[0094] The expression cassette disclosed herein may include multiple restriction sites for insertion of the nucleotide sequence to be under transcriptional regulation in a regulatory region. The expression cassette may also contain a selective marker gene.

[0095] The expression cassette may also include, in the 5′–3′ transcriptional direction, transcription and translation initiation regions that function in plants, the DNA sequence disclosed herein, and transcription and translation termination regions. The transcription initiation region, or promoter, relative to the plant host, may be natural or similar, or foreign or heterologous. Additionally, the promoter may be a natural sequence or alternatively a synthetic sequence. The term "heterologous" means that the transcription initiation region is not present in the natural plant in which it is introduced. As used herein, chimeric genes contain coding sequences operatively linked to a transcription initiation region that is heterologous to that coding sequence. Examples of promoters include, but are not limited to, cauliflower mosaic virus 35S and soybean ubiquitin 6.

[0096] While heterologous promoters are preferred for expressing the sequence, homologous promoters or natural promoter sequences can also be used. Such constructs will alter the expression levels in the host cell (i.e., plant or plant cell). Therefore, the phenotype of the host cell (i.e., plant or plant cell) is altered.

[0097] Termination regions can naturally contain transcription initiation regions, naturally contain operablely ligable DNA sequences of interest, or originate from another source. Easily available termination regions can be obtained from Ti plasmids of *Agrobacterium tumefaciens*, such as the termination regions of octopaline synthase and carmine synthase.

[0098] In one aspect, endogenous or source gene resistance orthologs can be altered by homologous or non-homologous recombination methods, such as, for example, genome editing. Such alterations, when compared to their unmodified sequences, refer to nucleotide sequences having at least one modification, and include, for example: (i) substitution 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).

[0099] In some embodiments, genome editing technology can be used to introduce the NB-LRR polynucleotide composition disclosed in this invention into the genome of a plant, or genome editing technology can be used to edit NB-LRR polynucleotides previously introduced into the plant genome.

[0100] Genome editing can be achieved using any available gene editing method. For example, gene editing can be achieved by introducing a polynucleotide modification template (sometimes called a gene repair oligonucleotide) into a host cell, wherein the polynucleotide modification template includes targeted modifications to genes within the host cell's genome. The polynucleotide modification template can be single-stranded or double-stranded. See, for example, U.S. Patent Publication 2013 / 0019349.

[0101] In some implementations, gene editing can be performed by introducing a double-strand break (DSB) at a defined location in the genome near the desired change. DSBs can be induced using any available DSB inducer, including but not limited to TALENs, homing endonucleases, zinc finger nucleases, Cas9-gRNA systems (based on bacterial CRISPR-Cas9 systems), etc. In some implementations, the introduction of DSBs can be combined with the introduction of a polynucleotide modification template.

[0102] Methods for editing gene sequences may include combining a DSB with a polynucleotide modification template and generally further include: 1) providing a host cell with a DSB-inducer, or a nucleic acid encoding a DSB-inducer, wherein the DSB-inducer recognizes a target sequence in a chromosomal sequence and is thus capable of inducing a DSB in the gene sequence; and 2) one or more polynucleotide modification templates comprising one or more nucleotide changes compared to the nucleotide sequence to be edited. The polynucleotide modification template may also comprise a nucleotide sequence flanking one or more nucleotide changes, wherein the flanking sequence is substantially homologous to the chromosomal region flanking the DSB. Genome editing techniques using DSB-inducers such as Cas9-gRNA complexes are known in the art (see, for example, U.S. Patent Application 14 / 463,687, filed August 20, 2014; PCT Patent Application PCT / US14 / 51781, filed August 20, 2014; and U.S. Patent Application 62 / 036,652, filed August 13, 2014; all of which are incorporated herein by reference). Guide RNA / Cas endonuclease systems are also known in the art (see, for example, U.S. Patent Application 14 / 463,691, filed August 20, 2014, which is incorporated herein by reference). Additional uses of guide RNA / Cas endonuclease systems are described in U.S. Patent Applications 14 / 463,687 and 14 / 463,691, filed August 20, 2014, and include, but are not limited to, modification or substitution of the nucleotide sequence of interest (e.g., regulatory elements), insertion of the polynucleotide of interest, gene knockout, gene knock-in, modification of the binding site and / or introduction of alternative binding sites, modification of the nucleotide sequence encoding the protein of interest, amino acid and / or protein fusion, and gene silencing by expressing an inverted repeat sequence into the gene of interest.

[0103] As needed, one or more genes can be optimized to increase their expression in transformed plants. In other words, plant-preferred codons can be used to synthesize genes to improve expression. Methods for synthesizing plant-preferred genes are known in the art.

[0104] Other sequence modifications are known to enhance gene expression in the host cell. These modifications include the removal of sequences encoding pseudopolyadenylation signals, exon-intron splicing sites, transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The GC content of the sequence can be adjusted to the average level of a given host cell, calculated by referencing known genes expressed in the host cell. When possible, the sequence is modified to avoid predictable hairpin secondary mRNA structures.

[0105] Expression cassettes can contain an additional 5′ leader sequence within the expression cassette construct. Such leader sequences can enhance translation. Translation leader sequences are known in the art and include: microRNA virus leader sequences, such as the EMCV leader sequence (5′ untranslated region of encephalomyocarditis); potato virus group leader sequences, such as the TEV leader sequence (tobacco mosaic virus) and human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequences from the coat protein mRNA of alfalfa mosaic virus (AMVRNA 4); tobacco mosaic virus leader sequence (TMV); and corn scorched spot virus leader sequence (MCMV) (Lommel et al., (1991) Virology 81:382385). Other known methods for enhancing translation, such as introns, can also be used.

[0106] Various DNA fragments can be manipulated during expression cassette preparation to ensure the DNA sequence is correctly oriented and, depending on the case, within the appropriate reading frame. For this purpose, adaptors or linkers can be used to ligate the DNA fragments. Alternatively, other manipulations can be used to provide convenient restriction sites, remove redundant DNA, or remove restriction sites altogether. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, and re-displacement (e.g., transition and transversion) may be involved.

[0107] Generally, expression cassettes may contain selective marker genes for selecting transformed cells. Selective marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicides such as glufosinate, bromosulfuron, imidazolinone, and 2,4-dichlorophenoxyacetic acid (2,4-D). The above list of selective marker genes is not intended to be limiting. Any selective marker gene may be used in this disclosure.

[0108] To express the target genes and / or proteins (e.g., one or more NB-LRR genes and / or one or more R proteins) of this disclosure in plants or plant cells, the methods described herein include transforming plants or plant cells with a polynucleotide encoding the target R protein (e.g., as disclosed herein). The polynucleotide described herein may be operatively linked to a promoter that drives expression in plant cells. Any promoter known in the art may be used in the methods of this disclosure, including but not limited to constitutive promoters, pathogen-inducible promoters, wound-inducible promoters, tissue-preferred promoters, and chemically regulated promoters. The selection of the promoter may depend on the desired expression time and location in the transformed plant, as well as other factors known to those skilled in the art. The transformed cells or plants may be grown or cultivated to form a plant containing one or more of the polynucleotides introduced into the cells or plant, for example, encoding the R protein.

[0109] A variety of promoters can be used to implement this disclosure. Promoters can be selected based on the desired outcome. That is, nucleic acids can be combined with constitutive promoters, tissue-preferred promoters, or other promoters to express them in the host cells of interest. Such 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 6,072,050; the core CaMV 35S promoter; rice actin; ubiquitin; pEMU; MAS; ALS, etc. Other constitutive promoters include, for example, those described in the following U.S. patents: 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142, and 6,177,611, which are known in the art and are contemplated for use in this disclosure.

[0110] Generally speaking, gene expression from inducible promoters, especially pathogen-inducible promoters, may be advantageous. Such promoters include those derived from pathogenesis-associated proteins (PR proteins), which are induced after pathogen infection, such as PR proteins, SAR proteins, β-1,3-glucanase, chitosanase, etc.

[0111] Promoters locally expressed at or near the pathogen infection site are of interest. Additionally, since pathogens can enter plants through wounds or insect lesions, wound-inducible promoters can be used in the constructs disclosed herein. Such wound-inducible promoters include the potato proteinase inhibitor (pinII) gene, wun1 and wun2, win1 and win2, systemin, WIP1, and the MPI gene, among others.

[0112] Chemically regulated promoters can be used to modulate gene expression in plants by applying exogenous chemical regulators. Depending on the target, the promoter can be a chemically inducible promoter, where the application of a chemical substance induces gene expression, or a chemically repressive promoter, where the application of a chemical substance inhibits gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the corn In2-2 promoter (activated by a benzenesulfonamide herbicide safener), the corn GST promoter (activated by a hydrophobic electrophilic compound used as a protamine herbicide), and the tobacco PR-1a promoter (activated by salicylic acid). Other chemically regulated promoters of interest include steroid-responsive promoters (e.g., glucocorticoid-inducible promoters, tetracycline-inducible promoters, and tetracycline-repressive promoters).

[0113] Tissue-preferred promoters can be used to target and enhance the expression of target genes or proteins (e.g., polynucleotide sequences encoding NB-LRR polypeptides derived from legumes) within specific plant tissues. Such tissue-preferred promoters include, but are not limited to, leaf-preferred, root-preferred, seed-preferred, and stem-preferred promoters. Preferred promoters for tissue selection include Yamamoto et al., (1997) Plant J.12(2): 255-265 (Yamamoto et al., 1997, Plant Journal, Vol. 12, No. 2, pp. 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): 133l-134l; Van Camp et al., (1996) Plant Physiol. 112(2): 525-535; Canevascini et al., (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al., (1994) Plant Cell Physiol. 35(5): 773-778; Lam, (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al., (1993) Plant MolBi0l. 23(6): 1129-1138; Matsuoka et al., (1993) Proc Natl. Acad. Sci. USA 90(20): 9586-9590; and Guevara-Garcia et al., (1993) Plant J. 4(3): 495-505. Such promoters can be modified.

[0114] Leaf-specific promoters are known in the art. See, for example, Yamamoto et al., (1997) Plant J.12(2):255-265; Kwon et al., (1994) Plant Physiol.105:357-67; Yamamoto et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor et al., (1993) Plant J.3:509-18; Orozco et al., (1993) Plant Mol.Biol.23(6):1129-1138; and Matsuoka et al., (1993) Proc.Natl.Acad.Sci.USA90(20):9586-9590.

[0115] “Seed-preferred” promoters include “seed-specific” promoters (those that are active during seed development, such as promoters of seed storage proteins) and “seed germination” promoters (those that are active during seed germination). Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced signaling), cZ19B1 (19kDa zein), mi1ps (inositol-1-phosphate synthase), and celA (cellulose synthase) (see WO 00 / 11177, which is incorporated herein by reference). γ-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicotyledonous plants, seed-specific promoters include, but are not limited to, the bean β-bean protein gene promoter, the rapeseed napin gene promoter, the β-conglobulin gene promoter, the soybean lectin gene promoter, and the cruciferous protein gene promoter. For monocotyledonous plants, seed-specific promoters include, but are not limited to, 15kDa zeatin, 22kDa zeatin, 27kDa zeatin, g-zeatin, waxy, shrunken 1, shrunken 2, and globulin 1. See also WO00 / 12733, which discloses seed-preferred promoters from the end1 and end2 genes; this patent is incorporated herein by reference.

[0116] The expression of the polynucleotides disclosed herein may involve the use of the complete natural R gene, wherein the expression is driven by a homologous 5′ upstream promoter sequence. Alternatively, expression may be generated using a construct assembled with a 5′ transcriptional control sequence provided by a heterologous NB-LRR resistance gene expressed in a host legume. Those skilled in the art will be able to identify genes encoding NB-LRR proteins, evaluate their expression levels, and select preferred promoter sequences that can be used to express the R gene of interest. The use of homologous or heterologous NB-LRR promoter sequences provides options for regulating protein expression to avoid or minimize any potentially undesirable outcomes associated with inappropriate or unwanted expression and plant defense activation.

[0117] Specific soybean promoters include, but are not limited to, soybean ubiquitin (subi-1), elongation factor 1A, and S-adenosylmethionine synthase and Rpp4, RPG1-B for constitutive expression, as well as promoters included in gene models, such as the Glyma promoter known to those skilled in the art for more tightly regulating expression provided by the NB-LRR gene promoter.

[0118] This disclosure also includes kits for the assays described herein. Peptide sequences and polynucleotides may also be packaged as components of the kit along with instructions for use in performing the assays disclosed herein. Kits of this disclosure may include any combination of the peptides and / or polynucleotides described herein and suitable instructions (written and / or provided as audio, visual, or audiovisual materials). In one embodiment, the kit relates to a DNA detection kit for identifying an R gene (e.g., the NB-LRR gene) or R protein for ASR resistance. Kits are provided for identifying the anti-ASR efficacy of any of the sequences disclosed herein for transgenic events (e.g., CcRpp1) in plants. For example, the kit may include a specific probe having a sequence corresponding to or complementary to a sequence having sequence identity between 80% and 100% with a specific region of the transgenic event. The kit may include any reagents and materials required to perform the assay or detection method.

[0119] Example

[0120] Example 1: Location and Cloning of CcRpp1

[0121] Germplasm of non-soybean species that are susceptible to soybean rust fungusVarieties of Brazilian pigeon pea (Cajanus cajan), originally obtained from the International Centre for Semi-Arid Tropical Research (ICRISAT) and introduced to Brazil, were screened for desired agronomic traits (Godoy et al., (2005) Rev. Bras. Zootec; 34: 7-19; and Provazi et al., (2007) Rev. Bras. Zootec; 36: 328-334). Pigeon pea is a diploid legume with a genome size of approximately 830 Mbp (Varshney et al., (2012) Nat. Biotechnol., 30: 83-89), is self-pollinating, and has an interseed life cycle of 2-3 months. 。

[0122] The different responses of these varieties to soybean rust fungus allow for the identification of resistant and susceptible varieties (Noriega, (2007) Resistência de plantas hospedeiras e de genes diferencialmenteexpressos na soja-Phakopsora pachyrhizi.M.Sc.Thesis.UniversidadeFederal de (Brazil). Plants from resistant varieties were crossed with those from susceptible varieties, and the resulting F1 plants were self-pollinated. The resistance and susceptibility of the resulting F2 offspring were screened. The CcRpp (pigeonpea resistance to soybean rust) gene of genotypes G119-99 (source of the resistance gene CcRpp1), G59-95, G146-97, G108-99, G127-97, and G184-97 were selected for further characterization. Different responses of several pigeonpea varieties to the soybean rust isolate PPUFV02 were measured. The response ranged from resistance, partial resistance with spots, reddish-brown lesions without spore formation, reddish-brown lesions with spore formation to susceptibility with "brownish" lesions and abundant spore formation on the leaflet axis. The 3:1 segregation of resistance in the F2 population of genotype G119-99 indicated that it was controlled by a single dominant gene. G108-99 is an exception and shows a 15:1 segregation ratio in F2, indicating that both R genes can provide resistance.

[0123] CcRpp1 genetic region in pigeon pea To genetically map the CcRpp1 locus, mRNA was extracted from 50 susceptible and 50 resistant pigeon pea F2 plant systems derived from the G119-99×G48-95 hybrid. SMART TMThe kit and SMARTIV's patented oligonucleotides (Clontech) were used for first-strand cDNA synthesis. The cDNA generated from the resistant and susceptible systems was normalized using a dual-specificity nuclease method to facilitate the identification and analysis of rare transcripts and enhance gene discovery rates in the cDNA library (Zhulidov et al., (2004) Nucleic Acids Res. 32: e37; and Zhulidov et al., (2005) Bioorg Khim. 31: 186-94). The normalized cDNA was then digested with SfiI and granularized using a CHROMA spin-1000 column to select transcript fragments larger than 1 kb. The resistant and susceptible ontological cDNA was subsequently cleaved using Covaris S20 to obtain an average fragment size of 200 bp. These cleaved cDNAs were used to form a library, which was sequenced using an Illumina Ga2 sequencer.

[0124] Several single nucleotide polymorphisms were identified between susceptible and resistant cDNA ontologs linked to the CcRpp1 locus. These polymorphisms were used to form markers for fine mapping. The marker dCAPS 140555 was linked to the CcRpp1 locus in 2282 gametes analyzed (1141 F2 plant species).

[0125] Because no reference pigeon pea genome was available when generating the mapping data, a homolinearity analysis was performed between pigeon pea and alfalfa and soybean. To this end, the BLAST algorithm was used to align the sequences of seven genetically linked markers to CcRpp1 with the alfalfa genome (Alfalfa Sequencing Resource - Mt3.0) and the soybean genome (Phytozome v5.0). Soybean is an ancient allotetraploid, and chromosomes 9 and 12 are homologous. Interestingly, the seven linked markers identified in pigeon pea showed strong homolinearity with two regions in the soybean genome; one on chromosome 12 and one on chromosome 9. Homologous regions on chromosome 4 of alfalfa were also identified. Additional polymorphisms and insertions / deletions developed for marker identification used a 2Mb region starting from the telomere region on chromosome 12 containing the identified homologous regions as a reference. This reference was used to compare transcriptional IIIumina data from the resistant parent variety G119-99 of pigeon pea, which was combined with readings generated from resistant strains of 50 F2 lines and compared with readings obtained from susceptible strains. Using this method, five polymorphic and insertion / deletion sequences were obtained, providing additional markers for fine mapping of CcRpp1 (dCAPS3978, SSR10581, dCAPS52491, SSR2152, dCAPS239615). Figure 1Most of these marker sequences are located within a 1.5 Mb region on chromosome 12 of soybean. Notably, the dCAPS140555 marker (currently linked to the CcRpp1 gene in pigeon pea) is located 6 Kb from the classic CC-NB-LRR gene (Glyma12g01420) in soybean and lies within a 106 Kb genetic region defined by the dCAPS52491 and SSR2152 marker sequences. Figure 1 ).

[0126] Sequencing of a BAC clone from the reference pigeon pea variety “Asha” revealed that the CcRpp1 locus contains one or more genes from the NB-LRR gene family. To this end, a BAC library was screened from the sequenced reference variety “Asha” using probes derived from the marker dCAPS140555 and identified as 97 kb (Varshney et al., (2009) Nat. Biotechnol., 30: 83-89). The clone was cut to obtain an average fragment size of 5 kb using Sanger sequencing. Readings were then assembled using Phred, Phrap, and Consed data analysis. Sequence annotation of the BAC revealed the presence of four highly homologous NB-LRR gene paralogs, three full-length genes, and one truncated gene.

[0127] High-resolution mapping revealed that CcRpp1 is located between markers dCAPS52491 and SSR2152 and is very closely linked to dCAPS140555. Using more than 1114 individual F2 plants with SSR, CAP, and dCAP markers, the CcRpp1 locus was mapped to chromosome 5 in G119-99. The region containing CcRpp1 was narrowed down to a <154 kb region encompassing the dCAPS52491 (6 recombinants / 2282 gametes) and SSR2152 (1 recombinant / 2282 gametes) markers. Figure 2A These markers were tightly linked to CcRpp1, with dCAPS52491 and SSR2152 located at loci of 0.26 cM and 0.04 cM, respectively. Figure 2A Two functional gain recombinants were observed with side-mounted markers dCAPS52491 and SSR12872, and one functional loss recombinant was observed with side-mounted markers CAPS20006 and SSR2152. Therefore, the functional gain range is defined by markers dCAPS52491 and dCAPS239615, and the functional loss range is defined by markers dCAPS52491 and SSR2152. Figure 2A This confirms that the CcRpp1 locus is located in a region containing the NB-LRR gene.

[0128] Example 2: The CcRpp1 locus from G119-99 contains four members of the NB-LRR family.

[0129] To determine the gene organization of the CcRpp1 locus in G119-99, G119-99 BAC libraries were generated and screened using two probes derived from markers dCAPS140555 and SSR2152, respectively. Two positive BAC clones (3F and 6G) ​​were identified, which together spanned the entire region between dCAPS52491 and SSR2152. Figure 2B The integrity of multiple clones containing BAC 3F (145 kb) and BAC 6G (130 kb) was tested via DNA fingerprinting using the restriction enzyme HindIII. Furthermore, the BAC size of these clones was verified by pulse-field gel electrophoresis of NotI-digested DNA. One clone of each 3F and 6G that passed the above quality control was sequenced using PacBio and Illumina MiSeq to enable rapid and accurate assembly of each BAC sequence (Koren et al. (2012) Nature Biotechnology 30: 693-970). Two BAC sequences were assembled into a large contig of 205,344 kb. Figure 2B Four candidate NB-LRR gene sequences were identified in this contiguous group; BAC 3F carries three NB-LRR gene sequences, NB-LRR-1, NB-LRR-2, and NB-LRR-3 (SEQ ID NO: 3, 1, and 5, respectively), and BAC 6G carries four NB-LRR gene sequences, NB-LRR-1 through NB-LRR-4 (SEQ ID NO: 3, 1, 5, and 7). Using Illumina data of transcripts from the non-microbial attack genotype G119-99, re-sequencing with Trinity alone revealed the full-length transcript of NB-LRR-2. Southern blot analysis showed that the CcRpp1 locus in G119-99 contains four members of the NB-LRR gene family, thus confirming the BAC gene annotation.

[0130] Example 3: Transformation of soybean with pigeon NB-LRR-2 gene (SEQ ID NO: 1)

[0131] The plant transformation construct was designed to provide high-level constitutive expression of NB-LRR-2 (SEQ ID NO: 1) in soybean. A 2775 bp SfiI fragment containing the NB-LRR-2 coding region is ligated at the 5′ end to a 1948 bp soybean ubiquitin promoter + intron I fragment, and at the 3′ end to an 888 bp Arabidopsis ubiquitin terminator fragment. The entire promoter coding region terminator cassette is located at... The attR1 and attR2 recombination sites are located in the plant expression vector. In addition to the aforementioned elements, the vector also contains a hygromycin resistance gene for bacterial selection and a herbicide-resistant soybean ALS gene as a marker of plant selectivity.

[0132] The final NB-LRR-2 plant expression vector was electroporated into *Escherichia coli*. Transformants were then selected, and pDNA was isolated using standard micro-preparation methods. Transformants were characterized by diagnostic restriction enzyme digestion of the micro-prepared DNA. Positive clones containing the expected pattern of digestion bands were selected, and the isolated DNA was submitted for gene gun transformation.

[0133] Gene gun transformation of soybeans was performed using a BIORAD Biolistic PDS1000 / He instrument and a particle gun bombardment method for plasmids or fragment DNA (US Patent No. 4,945,050) to generate transgenic soybean lines. The following stock solutions and culture media were used for the transformation and regeneration of soybean plants.

[0134] Original solution Sulfate 100X stock solution (37.0g MgSO4·7H2O, 1.69g MnSO4) 4 .H 2 O, 0.86g ZnSO 4 .7H2O, 0.0025g CuSO4.5H2O); Halides 100X stock solution (30.0g CaCl2.2H2O, 0.083g KI, 0.0025g CoCl2.6H2O); P, B, Mo 100X stock solution (18.5g KH2PO4, 0.62g H3BO3, 0.025g Na2MoO4.2H2O); FeEDTA 100X stock solution (3.724g Na2EDTA, 2.784g FeSO4.7H2O); 2,4D stock solution (10mg / mL) and Vitamin B5, 1000X stock solution (100.0g inositol, 1.0g nicotinic acid, 1.0g pyridoxine HCl and 10g thiamine HCl).

[0135] Culture medium (per liter)SB199 solid medium (1 packet of MS salt (Gibco / BRL; catalog number 11117-066), 1 mL of Vitamin B5 1000X stock solution, 30 g sucrose, 4 mL of 2,4-D (40 mg / L final concentration), pH 7.0, 2 g gellan gel); SB1 solid medium (1 packet of MS salt (Gibco / BRL; catalog number 11117-066), 1 mL of Vitamin B5 1000X stock solution, 31.5 g glucose, 2 mL of 2,4-D (20 mg / L final concentration), pH 5.7, 8 g TC agar); SB196 (10 mL each of the above stock solutions 1-4, 1 mL of Vitamin B5 stock solution, 0.463 g (NH4)2SO4, 2.83 g KNO3, 1 mL of 2,4-D stock solution, 1 g asparagine, 10 g sucrose, pH 7.0, 2 g gellan gel); 5.7); SB71-4 (Gamborg B5 salt, 20g sucrose, 5g TC agar, pH 5.7); SB103 (1pk Murashige & Skoog salt mixture, 1mL B5 vitamin stock solution, 750mg MgCl2 hexahydrate, 60g maltose, 2g gellan gum). TM (pH 5.7); and SB166 (supplemented with SB103 containing 5 g / L activated carbon).

[0136] Initiation of soybean germination suspension culture. 45-55 days after planting, select pods with immature seeds from usable soybean plants, shell them, and place them in a sterile, deep red box. Place the soybean seeds in a solution containing 1 drop of Ivory. TM 5% of soap Solution (i.e., 95 mL of autoclaved distilled water plus 5 mL of...) Sterilize the seeds by shaking them for 15 minutes with 1 drop of soap (mix thoroughly). Wash the seeds with 2L of sterile distilled water, placing seeds smaller than 3mm on a separate microscope slide. Cut off the smaller end of the seed and squeeze out the cotyledon from the seed coat. Transfer the cotyledons to plates containing SB199 medium (25-30 cotyledons per plate) for 2 weeks, then transfer them to SB1 for 2-4 weeks. Wrap the plates with fiber tape. After this time, cut off the secondary embryos and place them in SB196 liquid medium for 7 days.

[0137] Culture conditions: Soybean embryogenesis suspension cultures were kept in 50 mL of liquid SB196 medium on a rotary shaker at 100-150 rpm and 26℃. The photoperiod was 16:8 hours day / night, and the light intensity was 80-100 μE / m². 2 / s. Every 7–14 days, culture is subcultured by inoculating up to 1 / 2 coin-sized amounts of tissue (tissue clumps clustered together) into 50 mL of fresh liquid SB196.

[0138] Preparation of DNA for bombardment. In a particle gun bombardment procedure, purified whole plasmid DNA or a fragment containing only one or more recombinant DNA expression cassettes of interest may be used. For every seventeen bombardment transformations, prepare an 85 μL suspension containing 1 to 90 picograms (pg) of plasmid DNA per base pair for each DNA plasmid. Co-precipitate the DNA plasmid or fragment onto gold particles as follows: Add the DNA from the suspension to 50 μL of a 10–60 mg / mL 0.6 μm gold particle suspension, then mix with 50 μL of CaCl2 (2.5 M) and 20 μL of spermidine (0.1 M). Vortex the mixture for 5 seconds, briefly centrifuge for 5 seconds in a microcentrifuge, and then remove the supernatant. Wash the DNA-coated particles once with 150 μL of 100% ethanol, vortex, and briefly centrifuge again in a microcentrifuge, then resuspend in 85 μL of anhydrous ethanol. Then, five microliters of DNA-coated gold particles were loaded onto each giant carrier disk.

[0139] Tissue preparation and DNA bombardment. Approximately 100 mg of two-week-old suspension culture was placed in an empty 60 mm × 15 mm culture dish, and residual liquid was removed from the tissue using a pipette. The tissue was placed approximately 3.5 inches from the retaining screen, and each plate was bombarded once. The membrane rupture pressure was set to 650 psi, and the chamber was evacuated to a vacuum of 28 inches of mercury. After bombardment, the tissue from each plate was aliquoted into two culture flasks, returned to liquid culture medium, and cultured as described above.

[0140] Selection of transformed embryos and plant regeneration. After bombardment, tissues from each bombarded plate were aliquoted into two culture flasks on SB196 liquid culture maintenance medium (tissue per unit plate bombarded). Seven days after bombardment, the liquid culture medium in each flask was replaced with fresh SB196 culture maintenance medium (selection medium) supplemented with 100 ng / ml of selectant. For the selection of transformed soybean cells, the selectant used was a sulfonylurea (SU) compound, chemically named 2-chloro-N-((4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl)benzenesulfonamide (other commonly used names include DPX-W4189 and chlorsulfuron). Chlorsulfuron is a DuPont sulfonylurea herbicide. The active ingredient in the medium was selected. The selection medium containing SU was changed every two weeks for 8 weeks. After the 8-week selection period, islands of green transformed tissue were observed growing from untransformed necrotic embryogenic clusters. These putative transgenic events were isolated and maintained in SB196 liquid medium containing 100 ng / ml SU for another 5 weeks, with the medium changed every 1-2 weeks, to produce new asexually propagated transformed embryogenic suspension cultures. The embryos were in contact with SU for a total of approximately 13 weeks. The suspension cultures were then subcultured and maintained as immature embryonic clusters, and also regenerated into complete plants by allowing individual somatic embryos to mature and germinate.

[0141] After four weeks on maturation medium (one week on SB166, followed by three weeks on SB103), somatic embryos become suitable for germination. They are then removed from the maturation medium and dried in empty petri dishes for up to seven days. The dried embryos are then seeded into SB71-4 medium, where they are allowed to germinate under the same light and temperature conditions as described above. The germinated embryos are then transferred to potting substrate and grown to maturity to produce seeds.

[0142] Example 4: Testing the anti-ASR efficacy of transgenic plants

[0143] The efficacy of the NB-LRR-2 gene against ASR was tested by transforming the plant expression construct into soybean, inoculating the transgenic plants with soybean rust fungus, and scoring the plant disease symptoms.

[0144] A total of three transgenic events were recovered from soybean transformation experiments and confirmed by qPCR to include the NB-LRR-2 gene (SEQ ID NO: 1). All three events were further visualized by RT-PCR to express a diagnostic 543 bp fragment of the NB-LRR-2 transcript.

[0145] Preliminary tests were conducted on the primary transformants to assess the effect of the NB-LRR-2 transgene on ASR infection. For this purpose, T0 plant material was spray-inoculated with soybean rust fungus at 1 × 10⁵ spores / mi. The inoculum from control and T0 transgenic plants was incubated, and disease symptoms were scored 12 days post-inoculation. The presence of lesions in the plants was visually evaluated, and the presence of uredinia was assessed using microscopy.

[0146] No sporulation was observed on leaves from three plants representing two independent events (5.1 and 7.1), which were confirmed to express full-length NB-LRR-2. Interestingly, one transgenic event (6.1) exhibited complete susceptibility to ASR and included brownish, severe sporulation lesions. Further analysis revealed that in this particular transformant, the integrity of the inserted DNA was impaired, resulting in the synthesis of truncated transcripts, thus excluding NB-LRR-2 expression in this plant. Because NB-LRR-2 was able to provide resistance to soybean rust in events 5.1 and 7.1, it was renamed CcRppl for pigeon pea resistance to soybean rust 1, and both events 5.1 and 7.1 were advanced in T1 plants for further testing.

[0147] The efficacy of CcRpp1 against soybean rust fungus was tested using a T1 transgenic assay. Seeds from selected T1 events were grown in a growth chamber under growth chamber conditions and allowed to mature for 17 days until vitamin C. Plants were sampled by qPCR to determine transgenic copy number and inoculated with a suspension of soybean rust fungus spores. Inoculation was performed using urediniospores collected from susceptible varieties and stored at -80°C. After retrieval from storage, the spores were suspended in a 0.01% Tween 20 aqueous solution, heat-shocked at 40°C for 5 minutes, and thoroughly mixed; then the spore concentration was adjusted to 2 × 10⁻⁶ using a hemocytometer. 4 sp / ml. Plants were spray-inoculated with a urediniospore suspension and incubated in the dark at 100% relative humidity for 24 hours, then transferred to a growth chamber (22°C, 70% RH, 16hr photoperiod) to grow and develop symptoms for 15–29 days. New growths were periodically detached and removed to maintain single leaves during the experiment.

[0148] To evaluate the effects of CcRppl, plants were qualitatively graded as resistant (R; no lesions), partially resistant (PR; reddish-brown (RB; low sporulation lesions), and susceptible (S; brownish-red; high sporulation lesions); and quantitatively graded by excising and scanning leaves, followed by lesion assessment. Most empty samples were scored 15 days post-inoculation, while hemizygous and homozygous plants were scored on day 29 post-inoculation. To determine the gene's effect, transgenic plants were compared with empty plants from the same event.

[0149] The results of ASR infection assays are summarized in Table 1. These results show that CcRppl in homozygous samples provides resistance to ASR. Disease was rarely observed; when averaged across all homozygous plants, there was a low prevalence per leaf area unit (cm²). 2 The number of lesions decreased by >99%. Hemizygous plants showed partial resistance, with reddish-brown lesions, and indicated a reduction of >99% in the number of lesions per cm. 2The number of lesions is reduced by 55-70%. Empty plants contain brown high-spore-forming lesions and are usually susceptible to pathogens.

[0150] These ASR infection assays show that the CcRpp1 gene can provide resistance to soybean rust when transgenic from host legumes (pigeonpea to soybean plants).

[0151] Table 1: Traits of two events carrying CcRppl were measured. Conjugability was used as the transgene copy number (empty = 0, Hemizygote = 1, homozygote = 2); R = resistance, PR = partial resistance, S = susceptibility; Avg LC / cm 2 = Area per unit (cm²) 2 ) Mean lesion count .

[0152] event Adhesion n reaction lesion type <![CDATA[Average (LC / cm 2 ) <!-- 22 -->]]> 5.1 homozygous 10 R resistance 0.01 half-liquid 26 PR RB, low degree of sporulation 3.79 null 16 S brown 8.92 7.1 homozygous 27 R resistance 0.01 half-liquid 48 PR RB, low degree of sporulation 2.83 null 30 S brown 9.5

[0153] Example 5: Identification of ASR resistance gene in pigeon pea variety G108-99

[0154] Two hundred and ninety-two F2 plants from population CG 8-1 (G48-95 × G108-99) were screened using isolate PPUFV01. After inoculation, 266 plants were classified as resistant and 24 as susceptible. This observed segregation ratio indicated the presence of two independent dominant loci. Using markers 101581 and 239615 at the CcRpp1 locus, 53 resistant plants homozygous for the susceptible allele at the CcRpp1 locus were selected. These selected F2 plants were self-pollinated to obtain F3 seeds. Resistance segregation independent of the CcRpp1 locus was observed in multiple F2:3 families, confirming the presence of a novel resistance locus in variety G108-99. This variety was sequenced using an Illumina HiSeq2000 (20x coverage), and the data were used to identify 84,535 single nucleotide polymorphisms (SNPs) between G108-99 and the susceptible parent variety G48-95 (previously sequenced). Sequenom was used. SNP genotyping on the iPLEX platform identified regions associated with novel resistance in G108-99. Within the scope of this disclosure, these resistant plants were used as sources for identifying R genes conferring resistance to soybean rust. Symptomatic varieties were used to generate the segregating populations needed to map and clone genes conferring resistance to soybean rust in the corresponding resistant varieties.

[0155] Example 6: Identification of germplasm as a source of resistance in the genus Vigna

[0156] A total of 89 cowpea varieties from different sources were screened using the vesicular isolate PPUFV02. Initially, 55 cowpea varieties from the Brazilian breeding program were screened (Table 2). Challenging these varieties with the vesicular isolate PPUFV02 allowed the identification of three resistant varieties and several that developed disease symptoms. A notable exception was Vu32, which also developed disease symptoms in mature leaves. In several experiments, varieties Vu3, Vu7, and Vu21 consistently showed resistance to soybean rust. Screening of 16 additional cowpea varieties from USDA-GRIN revealed resistance in plants of varieties Vun_00002, Vun_00008, Vun_00094, Vun_00095, and Vun_00145, while Vun_00001 and Vun_00135 showed a lack of resistance (Table 3). Next, 18 varieties of various cowpea species from AusPGRIS were selected. The latter group included seven cowpea varieties, two varieties each of *Vigna dalzelliana* and *Vigna oblongifolia*, and one variety each of the following: *Vigna parkeri*, *Vigna affinicalis var. filicaulis*, *Vigna kirkii*, *Vigna luteola*, *Vigna radiata*, *Vigna trilobata*, and other cowpea species. Varieties ARG 88 (*Vigna luteola*), ATF 2361, ATF2364 (*Vigna oblongifolia*), ATF 2073 (*Vigna*), AJP 004 (*Vigna parkeri*), and CPI 121683 (cowpea) exhibited strong disease symptoms. In contrast, varieties ATF 2783 (Vigna dalzelliana), ATF 2363 (cowpea), and NI 456 (cowpea subspecies mensensis) are resistant to the disease.

[0157] Within the scope of this disclosure, varieties Vu3, Vu7, Vu21, Vun_00002, Vun_00008, Vun_00094, Vun_00095, Vun_00145, ATF 2783, ATF 2363, and NI 456 are used as sources for identifying the R gene conferring resistance to soybean rust. Symptomatic varieties Vu32, Vun_00001, Vun_00135, ARG 88, ATF 2361, ATF 2364, ATF2073, AJP 004, and CPI 121683 can be used to form the segregating populations required for mapping and cloning the gene conferring resistance to soybean rust in the corresponding resistant varieties. The F1 population is obtained by crossing cowpea varieties with a control phenotype. The F2 population from the cross between Vun_00135×Vun_00094 and Vu32×Vu21 segregated at a ratio of 3:1, indicating resistance similar to that in pigeon pea, where resistance is conferred by a dominant resistance locus.

[0158] Table 2. Sources of cowpea varieties from Brazil resistant to soybean rust fungus PPUFV02. Based on lesion size and... The affected leaf area was used to score the disease symptoms using a scale from 0 (resistance) to 4 (lack of resistance). .

[0159]

[0160]

[0161] Table 3. Sources of cowpea varieties from USDA-GRIN that are resistant to soybean rust fungus PPUFV02. (Based on lesion size...) The size and affected leaf area were used as the basis for disease assessment, with scores ranging from 0-3 (different resistance levels) to 4 (lack of resistance). Symptoms were scored. Scoring was performed separately for the trifoliate and cotyledonous forms. .

[0162]

[0163]

[0164] Example 7: Identification of germplasm as a source of resistance in the genus *Lysimachia*

[0165] Fifty-three varieties of Lablab purpureus from AusPGRIS were screened using the single vesicle isolate PPUFV02 (Table 4). The isolates were obtained at a ratio of 5 × 10⁻⁶. 4Plants with two trifoliate leaves were inoculated with a suspension in water containing 0.01% Tween-80 at a density of 1 well / ml. The inoculated plants were kept in complete darkness in a humid chamber for 24 h before being transferred to a greenhouse. Symptoms were scored using a scale from 0 (resistance) to 4 (lack of resistance) based on lesion size and affected leaf area. Trifoliate and cotyledon types were scored separately. Two varieties (IBS 059 and IBS837) were identified as showing resistance to the isolates of this fungus; however, all other varieties developed disease symptoms. This disclosure envisions using these two varieties as sources of resistance to soybean rust. The symptomatic varieties represent an important tool for using mapping-based cloning to degrade the individual NB-LRR gene, and the cloned gene can provide effective field resistance, similar to transgenic strains in soybean.

[0166] F1 populations derived from crosses between IBS 059 and IBS 837 and several symptomatic varieties (including Tamely, CPI 51565, CPI52508, IBS 879, CPI 40167, Tamely Early, CPI 18662, RJW 5117, CPI 36903, Cor Branca, and several other varieties) were obtained for mapping and cloning of the corresponding resistance genes. For mapping and cloning of the corresponding resistance genes, and based on flowering time and seed production, [the following data was obtained from...]. The F2 population resulting from the cross between ×IBS 837 and Tamely ×IBS059 will be the focus of the next set of experiments.

[0167] Segregation analysis indicated that resistance in the two F2 populations exhibited a 3:1 segregation against PPUFV02, thus indicating that resistance to soybean rust is conferred by dominant loci. This disclosure proposes to map and clone the functional genes conferring soybean resistance at these loci.

[0168] Table 4. Sources of soybean rust resistant to PPUFV02 from AusPGRIS soybean varieties. (Based on lesion size...) The size and affected leaf area were used to assess disease symptoms using a score ranging from 0-3 (resistance) to 4 (lack of resistance). Scoring is done separately for the trifoliate form and cotyledons. .

[0169]

[0170]

[0171] *AusTRCF reference number not found.

[0172] Example 8: Identification of germplasm as a resistance source in the genus *Phaseolus*

[0173] The use of *Phaseolus vulgaris* (common bean) was tested to identify sources of resistance to soybean rust. Common bean has been described as a host for soybean rust under field conditions (Du Preez et al., (2005) Plant Dis. 89: 206; and Lynch et al., (2006) Plant Dis. 7: 970). Furthermore, different responses of 16 common bean cultivars to soybean rust, as well as the severity of cultivar-isolated interactions and sporulation, have been reported (Miles et al., (2007) Plant Dis. 91: 698-704). As a legume crop, it is unique in that it has had two parallel domestication events, one in Central America and one in the Andes (Bitocchi et al., (2013) Mesoamerica and the Andes. New Phytologist 197: 300-313). As a result, the wild original varieties spanned a large geographical area and consisted of two distinct gene pools (Kwak and Gepts, (2009) Theoretical and Applied Genetics 118.5: 979-992). Thirteen varieties of the genus *Vigna* from Brazil were screened using the same scoring scale as for cowpea (see Table 3) (Table 5), and their different responses to PPUFV02 were identified. Populations with segregating resistance phenotypes from crosses between contrasting genotypes were generated and used for genetic studies and gene mapping.

[0174] This disclosure proposes to identify the efficacy of the NB-LRR type resistance gene in the genus Common Vaccinium against soybean rust fungus in soybean.

[0175] Table 5: Response of Brazilian bean varieties to soybean rust isolate PPUFV02

[0176]

[0177] Example 9: Identification of germplasm as a source of resistance in the genus *Pistacia*

[0178] Soybean rust was screened in pea varieties from the USDA / Grin Pisum sativum group using the Brazilian herpes simplex isolate (PPUFV-02) (Table 6). Interestingly, different responses were observed in 72 tested pea varieties 21 days after inoculation with soybean rust. Two partially resistant varieties (PI271118 and PI220189) were selected for further investigation. Microscopy and FITC-WGA staining were performed, followed by fluorescence microscopy to monitor pathogen growth over time. These studies showed that while the pathogen could colonize to a certain extent, its growth was then inhibited.

[0179] In addition, several varieties were identified to identify resistant isolates, which showed a lack of resistance upon visual inspection. Two lines (PI341888 and PI198735) were evaluated in more detail, exhibiting uredinia formation and sporulation. Plants from resistant varieties were crossed with varieties lacking resistance and allowing sporulation, and the resulting F1 plants were self-pollinated. The F1 plants from the PI341888 × PI220189 (and crossover) hybrids were used to construct the first mapping population. The resulting F2 progeny (500 plants in total) were screened for resistance and lack of resistance, showing a 15:1 ratio, indicating that the resistance was regulated by two dominant loci. Interestingly, multiple phenotypes were observed in the F2 population: a resistant phenotype (type 0), two types of partial resistance (red-brown (RB) lesions; types 2 and 3), and a clearly resistance-deficient (type 4) F2 progeny. The segregation pattern was in a 9:3:3:1 ratio (9 resistances; 6 different RB-type resistances (partial resistances) and 1 lacking resistance). These results indicate that the two resistance loci present in this population function in a complementary manner, and that both resistances are required to transmit resistance.

[0180] Table 6: Sources of resistance to soybean rust fungus PPUFV02 in pea varieties from Asia. Disease symptoms are recorded as resistant. Sex (0), partial resistance (1-2), and lack of resistance with uredinia (3-4; see table description). Screening for each variety. The changes in disease symptom scores of the two plants indicate intravarietal variations. .

[0181]

[0182]

[0183] *Disease Score. 0 = Resistance; no macroscopic or microscopic symptoms. 1 = Partial Resistance; small (≤250 μm) reddish-brown necrotic patches caused by hyphal growth, visible under a fluorescence microscope after FITC-wheat germ lectin (WGA) staining. 2 = Partial Resistance; infected patches ≤1000 μm in size, reddish-brown necrotic, caused by hyphal growth, visible under a fluorescence microscope after FITC-WGA staining. 3 = Lack of Resistance; obvious infected structures and / or uredinia and hyphae visible under a bright-field microscope, without spore formation. 4 = Lack of Resistance; lesions with or without necrosis, with fully formed uredinia and spore formation.

[0184] Example 10: Testing the CcRpp1 transgenic strain using an additional soybean rust isolate.

[0185] To assess the effectiveness of CcRpp1 against additional current U.S. field isolates, homozygous and empty plantlets from Event 7.1 were independently inoculated with GA15 (Georgia) and AR15 (Arkansas) isolates at 20,000 sp / ml. These *Laminaria* isolates were obtained from infected leaves harvested from soybean fields in Decatur, Georgia, and Mundby, Alaska, respectively. Inoculation and incubation were performed as previously described, except that fresh spores were collected from susceptible varieties, thus eliminating the need for heat shock; scoring was performed 15 days post-inoculation. The experiment showed high severity in empty plants and susceptible control plants, exhibiting multiple brown lesions; however, homozygous plants showed immunity to both isolates, as outlined in Table 7. However, the sample size was too small for statistical analysis.

[0186] Table 7: Traits obtained from inoculating two field isolates with CcRpp1. Conjugability was used as the transgenic copy number. (Empty = 0, Hemizygous = 1, Homozygous = 2); R = Resistance, PR = Partial Resistance, S = Susceptibility; Avg LC / cm 2 = Per unit surface Area (cm) 2 (mean lesion count) .

[0187]

[0188]

[0189] These data show that transgenic plants carrying CcRppl in a homozygous state are resistant, and hemizygous plants show partial resistance to at least three field isolates of soybean rust.

[0190] Example 11: Transforming soybeans using the CcRpp1 builder PHP74119

[0191] As disclosed in Example 3, the CcRpp1 transgenic event was isolated via DNA gene gun delivery. Then, the transgenic and marker DNA were inserted into the soybean genome using a targeted integration method. This site-specific integration (SSI) procedure relies on the FLP / FRT recombination system, is well-known to those skilled in the art of plant transformation, and is described in Li et al. (2009) Plant Physiol. 151:1087. The transgenic event in Example 3 was generated by bombarding the 93B86-5.1 transformation line with DNA. The additional SSI event was obtained by delivering DNA to the 93B86-TB5 line, which differs from the previous line in the chromosomal location of the integration site. In this experiment, a new CcRpp1 transformation construct (PHP74119) was assembled by linking a 2791 bp CcRpp1 coding region with a 1959 bp fragment containing a soybean ubiquitin promoter + intron I and an 880 bp Arabidopsis ubiquitin terminator fragment.

[0192] Gene gun transformation of soybean line 93B86-TB5. The transgenic soybean line was obtained by delivering DNA (PHP74119 and FLP recombinase constructs) into germinal suspension callus culture (93B86-TB5) using particle bombardment with a BIORAD Biolistic PDS1000 / He instrument (US Patent No. 4,945,050). Site-specific integration of the GmUbi-CcRppl gene into the soybean genome was achieved via recombinase-mediated cassette exchange (RMCE), as described by Li et al., 2009. Transgenic events were identified after selection with 100 ppb chlorsulfuron, and somatic embryo regeneration was performed to produce T0 plantlets for T1 seed production. Identification and characterization of RMCE events were performed using qPCR, as described by Li et al., 2009.

[0193] The PHP74119 transgenic event in 93B86-TB5s was tested. A total of three transgenic events were recovered from two transformation experiments (soybean 5342 and soybean 5861), which were identified by qPCRF as high-quality single-copy insertions containing the CcRppl transgene. The Tl test for ASR resistance was performed on all three events using the method described in Example 4.

[0194] As described in Example 4, T1 seeds from three events were planted, sampled, and inoculated; plants were scored 15 days post-inoculation. The effect of CcRpp1 was observed and confirmed in plants from two events (soybean 5342.11.1 and soybean 5342.11.2). Hemizygous plants showed partial resistance with reduced amounts of RB lesions, while homozygous plants were resistant. In this case, the construct had an effect of approximately 98% on reducing the severity in hemizygous plants, a more significant effect than previously detected in the 5.1 background. The results of the validation experiments are summarized in Table 8. However, the third event (soybean 5861.1.1) did not show a difference in disease severity between transgenic and empty plants. To identify the differences between effective and ineffective events, transcript levels in all three events were evaluated by qRT-PCR. The results showed higher expression in the two effective events, however, event soybean 5861.1.1 showed lower expression of CcRpp1 transcripts.

[0195] Table 8: Measured traits of the two transformation events of GmUbi-CcRpp1 in the TB5 background. Conjugation was used as a criterion for transformation. Gene copy number (empty = 0, hemizygote = 1, homozygote = 2); R = resistance, PR = partial resistance, S = susceptibility; Avg LC / cm 2 = Area per unit (cm²) 2 (mean lesion count) .

[0196]

[0197] These results confirm the efficacy of the CcRpp1 gene against soybean rust fungus GA05. In these experiments, a second overexpressing CcRpp1 construct (PHP74119) was independently generated and introduced into an additional soybean line (93B86-TB5), and high-quality transformation events were isolated and tested. ASR infection assays provided further validation of the resistant CcRpp1 gene present in homozygotes, and strong but partial resistance was observed in hemizygotes.

[0198] The sequence referred to as SEO.ID NO: l-8 is submitted with this application as a text file and is incorporated herein in its entirety.

[0199] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” used herein include multiple referents. Thus, for example, reference to “a cell” includes multiple such cells, and reference to “the protein” includes reference to one or more proteins and their equivalents known to those skilled in the art, etc. Unless otherwise expressly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0200] As used in the specification and claims, the term "comprising" may include aspects of "consisting of" and "substantially consisting of". "Comprising" may also mean "including but not limited to".

[0201] As used in this article, the word “or” refers to any one member of a specific list, and also includes any combination of members in that list.

[0202] As used in this article, the term "soybean" refers to soybean and any plant variety bred or cultivated using soybean.

[0203] A range herein may be expressed as from “about” one specific value and / or to “about” another specific value. When expressing such a range, other aspects include from one specific value and / or to other specific values. Similarly, when a value is expressed as an approximation, it should be understood that the specific value forms another aspect by using the antecedent “about”. It should also be understood that each endpoint in the range is both significantly related to and independent of the other endpoint. It should also be understood that there are multiple values ​​disclosed herein, and each value is also disclosed herein in the form of “about” that specific value, in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0204] All publications and patent applications mentioned in this specification indicate the level of expertise suitable for a person skilled in the art. All publications and patent applications are incorporated herein by reference as if each individual publication or patent application were specifically and independently indicated to be incorporated herein by reference.

[0205] Although the foregoing invention has been described in detail by way of example and illustration for the purpose of clear understanding, some variations and modifications may be implemented within the scope of the appended claims.

[0206] This application relates to the following implementation scheme:

[0207] 1. An isolated polynucleotide comprising a nucleotide sequence wherein the nucleotide sequence encodes a nucleotide-binding site-rich leucine repeat (NB-LRR) polypeptide derived from legumes, having at least 90% amino acid sequence identity with the legume sequences disclosed herein.

[0208] 2. The isolated polynucleotide according to embodiment 1, wherein plants transformed with said polynucleotide show enhanced resistance to Asian soybean rust compared to susceptible plants.

[0209] 3. A recombinant DNA construct comprising the polynucleotide according to embodiment 1, said recombinant DNA construct being operatively linked to a regulatory element.

[0210] 4. The recombinant DNA construct according to embodiment 3, wherein the regulatory element is a heterologous promoter.

[0211] 5. The recombinant DNA construct according to embodiment 4, wherein the promoter drives the expression of operatively linked genes in a plant.

[0212] 6. A method for conferring disease resistance in a legume crop species, the method comprising transforming the legume crop species with a nucleotide-binding site-rich leucine repeat (NB-LRR) gene derived from a heterologous legume, said gene conferring resistance to diseases of the legume crop species.

[0213] 7. The method according to embodiment 6, wherein the legume crop species disease is caused by a plant pathogen.

[0214] 8. The method according to embodiment 7, wherein the plant pathogen is Phakopsora pachyrhizi or Phakopsora meibomiae.

[0215] 9. The method according to implementation plan 6, wherein the legume crop disease is Asian soybean rust.

[0216] 10. The method according to embodiments 6, 7, 8 or 9, wherein the legume crop species is alfalfa, clover, pea, common bean, lentil, lupin, bean tree, carob, soybean, peanut or tamarind.

[0217] 11. The method according to any one of embodiments 6, 7, 8, 9 or 10, wherein the legume crop species is soybean.

[0218] 12. The method according to any one of embodiments 6, 7, 8, 9, 10 or 11, wherein the NB-LRR gene of the legume originates from the genera *Chickpea*, *Pistacia*, *Glycine*, *Lysimachia*, *Alfalfa*, *Phragmites*, *Vitis*, *Pueraria*, *Trifolium* or *Vigna*.

[0219] 13. The method according to embodiment 12, wherein the chickpea species is *Cicerarietinum*, *Cicer echinospermum*, *Cicer reticulatum*, or *Cicerpinnatifidum*.

[0220] 14. The method according to embodiment 12, wherein the soybean species is Glycinearenaria, Glycine argyrea, Glycine cyrtoloba, Glycine canescens, Glycine clandestine, Glycine curvata, Glycine efalcata, Glycine latifolia, Glycine microphylla, Glycine pescadrensis, Glycine stenophita, Glycine syndetica, Glycine sooja, Glycine tabacina, or Glycine tomentella.

[0221] 15. The method according to embodiment 12, wherein the lentil species is Lablabpurpureus.

[0222] 16. The method according to embodiment 12, wherein the alfalfa species is Medicago truncatula or Medicago sativa.

[0223] 17. The method according to embodiment 12, wherein the species of the genus *Phaseolus* is common bean (*Phaseolus vulgaris*), cotton bean (*Phaseolus lunatus*), broadleaf common bean (*Phaseolusacutifolius*), or red-flowered common bean (*Phaseolus coccineus*).

[0224] 18. The method according to embodiment 12, wherein the pea species is Pisumabyssinicum, Pisum sativum, Pisum elatius, Pisumfulvum, Pisum transcaucasium, or Pisumhumile.

[0225] 19. The method according to embodiment 12, wherein the kudzu species is kudzu root (Pueraria lobata).

[0226] 20. The method according to embodiment 12, wherein the clover species is yellow clover (Trifolium ureum) or Trifolium occidentale.

[0227] 21. The method according to embodiment 12, wherein the cowpea species is cowpea (Vigna unguiculata), cowpea adalzelliana, cowpea oblongifolia, cowpea parkeri, cowpea affinicaulis, cowpea kirkii, cowpea luteola, cowpea radiata, cowpea trilobata, cowpea luteola, or cowpea mungo.

[0228] 22. A transgenic legume crop plant transformed with a recombinant DNA construct according to embodiment 3, wherein the polypeptide encodes a legume-derived NB-LRR resistance gene, the gene conferring resistance to plant diseases.

[0229] 23. The genetically modified legume crop plant according to embodiment 22, wherein the plant disease is Asian soybean rust.

[0230] 24. The genetically modified legume crop plant according to embodiment 22 or 23, wherein the genetically modified legume crop plant is soybean.

[0231] 25. A transgenic legume crop plant according to any one of embodiments 22, 23 or 24, wherein the NB-LRR resistance gene of the legume originates from the genera *Chickpea*, *Pistacia*, *Glycine*, *Lysimachia*, *Alfalfa*, *Phaseolus*, *Vitis*, *Pueraria*, *Trifolium* or *Vigna*.

[0232] 26. The genetically modified legume crop plant according to embodiment 25, wherein the soybean species are sand soybean, silver-haired soybean, curved-lobed soybean, gray-haired soybean, Penghu soybean, curved-pod soybean, sickle-pod soybean, broad-leaved soybean, small-leaved soybean, Glycine pescadrensis, Glycine stenophita, Glycine syndetica, wild soybean, tobacco soybean, or short-fibered wild soybean.

[0233] 27. The transgenic legume crop plant according to embodiment 25, wherein the lentil species is lentil.

[0234] 28. The transgenic legume crop plant according to embodiment 25, wherein the alfalfa species is alfalfa trifida or alfalfa var. aurantiaca.

[0235] 29. The genetically modified legume crop plant according to embodiment 25, wherein the species of the genus *Phaseolus* is *Phaseolus vulgaris*, *Phaseolus spp. ... or *Phaseolus spp.*

[0236] 30. The transgenic legume crop plant according to embodiment 25, wherein the pea species is Pisumabyssinicum, pea, Pisum elatius, Pisumfulvum, Pisum transcaucasium, or Pisumhumile.

[0237] 31. The genetically modified legume crop plant according to embodiment 25, wherein the kudzu species is kudzu root.

[0238] 32. The transgenic legume crop plant according to embodiment 25, wherein the clover species is yellow clover or Trifolium occidentale.

[0239] 33. The transgenic legume crop plant according to embodiment 25, wherein the species of the genus Vigna are cowpea, Vigna dalzelliana, long-leaved cowpea, long-rain bean, Vignafilicaulis, Vigna kirkii, long-leaved cowpea, mung bean, three-lobed cowpea, long-leaved cowpea, or black bean.

[0240] 34. The transgenic legume crop plant according to any one of embodiments 22-33 further contains one or more resistance genes.

[0241] 35. The genetically modified legume crop plant according to any one of embodiments 22-33 further includes agronomic traits.

[0242] 36. A seed from a transgenic legume crop plant according to any one of embodiments 22-35, wherein the seed has a DNA construct.

[0243] 37. A method for reducing one or more symptoms of a legume disease, the method comprising exposing a transgenic legume crop plant according to any one of embodiments 22-36 to the legume disease, wherein the transgenic legume crop plant has enhanced resistance to the plant disease.

[0244] 38. The method according to embodiment 37, wherein the transgenic legume crop plant comprises the NB-LRR polypeptide of legume origin according to embodiment 1.

[0245] 39. The method according to embodiment 37, wherein the plant disease is Asian soybean rust.

[0246] 40. A method for producing Asian soybean rust-resistant plants, the method comprising transforming plant cells with a nucleotide-binding site-rich leucine repeat (NB-LRR) resistance gene derived from soybeans.

[0247] 41. The method according to embodiment 40 further includes regenerating the transformed plant from the transformed plant cells.

[0248] 42. The method according to embodiment 41 further includes the step of growing the transformed plant, wherein the expression of the legume-derived NB-LRR resistance gene results in enhanced resistance to Asian soybean rust in the transformed plant.

[0249] 43. The method according to any one of embodiments 40-42, wherein the Asian soybean rust-resistant plant is a legume species.

[0250] 44. A legume plant, which is the offspring of a cross between a transgenic legume plant containing a nucleotide-binding site-rich leucine repeat (NB-LRR) resistance gene of legume origin disclosed herein and a similar legume plant not transformed with the nucleotide-binding site-rich leucine repeat (NB-LRR) resistance gene.

[0251] 45. The plant according to embodiment 44, wherein the legume is alfalfa, clover, pea, common bean, lentil, lupin, bean tree, carob, soybean, peanut or tamarind species.

[0252] 46. ​​A method for determining the resistance of legumes to plant diseases, the method comprising exposing a portion of the legume to a plant pathogen; measuring the symptoms of the plant disease on the legume exposed to the plant pathogen; and comparing the symptoms of the plant disease with a reference standard for resistance.

[0253] 47. The method according to implementation scheme 46, wherein the plant disease is Asian soybean rust.

[0254] 48. The method according to embodiment 46, wherein the plant disease is formed by a plant pathogen.

[0255] 49. The method according to embodiment 48, wherein the plant pathogen is formed by soybean rust fungus or vesicular rust fungus.

[0256] 50. A method for enhancing plant resistance to Asian soybean rust (ASR) disease, the method comprising conferring resistance to the ASR pathogen by incorporating a legume-derived nucleotide-binding site-rich leucine repeat (NB-LRR) resistance gene into the germplasm during an ASR-resistant breeding program.

[0257] 51. The method according to embodiment 50, wherein the NB-LRR resistance gene derived from legumes encodes an NB-LRR polypeptide.

[0258] 52. The method according to embodiment 51, wherein the polypeptide comprises an amino acid sequence having at least 90% homology with the legume-derived nucleotide-binding site-rich leucine repeat (NB-LRR) polypeptide disclosed herein.

[0259] 53. The method according to embodiment 50, wherein the germplasm is a legume crop species.

[0260] 54. The method according to embodiment 52, wherein the plant transformed with the polypeptide exhibits enhanced resistance to ASR compared to susceptible plants.

[0261] 55. The method according to embodiment 53, wherein the legume crop species is alfalfa, clover, pea, common bean, lentil, lupin, bean tree, carob, soybean, peanut or tamarind species.

[0262] 56. The method according to embodiment 55, wherein the legume species is soybean.

[0263] 57. The method according to embodiment 50, wherein the ASR is formed by soybean rust fungus or vesicular rust fungus.

[0264] 58. The method according to any one of embodiments 50-57, wherein the resistance gene derived from the legume is derived from the genera *Chickpea*, *Pistacia*, *Glycine*, *Lysimachia*, *Alfalfa*, *Phaseolus*, *Vitis*, *Pueraria*, *Trifolium*, or *Vigna*.

[0265] 59. The method according to embodiment 58, wherein the species of the genus *Cicer* is *Cicer chinospermum*, *Cicer reticulatum*, or *Cicer pinnatifidum*.

[0266] 60. The method according to embodiment 58, wherein the soybean species is sand soybean, silver-haired soybean, curved-lobed soybean, gray-haired soybean, Penghu soybean, curved-pod soybean, sickle-pod soybean, broad-leaved soybean, small-leaved soybean, Glycinepescadrensis, Glycine stenophita, Glycinesyndetica, wild soybean, tobacco soybean, or short-haired wild soybean.

[0267] 61. The method according to embodiment 58, wherein the lentil species is lentil.

[0268] 62. The method according to embodiment 58, wherein the alfalfa species is alfalfa trifleum or alfalfa purpurea.

[0269] 63. The method according to embodiment 58, wherein the species of the genus *Vigna* is *Vigna angularis*, *Vigna pubescens*, *Vigna lataniae*, or *Vigna rubra*.

[0270] 64. The method according to embodiment 58, wherein the pea species is Pisumabyssinicum, Pisum elatius, Pisum fullvum, Pisum transcaucasium, or Pisum humile.

[0271] 65. The method according to embodiment 58, wherein the kudzu species is kudzu root.

[0272] 66. The method according to embodiment 58, wherein the clover species is yellow clover or Trifolium occidentale.

[0273] 67. The method according to embodiment 58, wherein the cowpea species is cowpea, Vignadalzelliana, long-leaved cowpea, long-rained cowpea, Vignafilicaulis, Vigna kirkii, long-leaved cowpea, mung bean, three-lobed cowpea, long-leaved cowpea, or black bean.

[0274] 68. The recombinant DNA construct according to embodiment 3 further comprises one or more NB-LRR polynucleotides or fragments thereof disclosed herein.

[0275] 69. The recombinant DNA construct according to embodiment 3 further comprises one or more resistance genes.

[0276] 70. The recombinant DNA construct according to embodiment 69 further comprises one or more polynucleotide sequences of interest.

[0277] 71. A seed comprising a recombinant DNA construct according to any one of embodiments 68, 69 or 70.

[0278] 72. A plant comprising a recombinant DNA construct according to any one of embodiments 68, 69 or 70.

Claims

1. A recombinant DNA construct comprising a heterologous regulatory element and a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a legume-derived nucleotide-binding site-rich leucine repeat sequence (NB-LRR) polypeptide of SEQ ID NO: 2, wherein said heterologous regulatory element is operatively linked to the polynucleotide, and wherein soybean plants transformed with said recombinant DNA construct exhibit enhanced resistance to Asian soybean rust compared to susceptible plants.

2. The recombinant DNA construct according to claim 1 further comprises one or more resistance genes, or further comprises one or more target polynucleotide sequences.

3. The recombinant DNA construct according to claim 1 further comprises one or more resistance genes.

4. The recombinant DNA construct according to claim 1 further comprises one or more target polynucleotide sequences.

5. A method for reducing one or more symptoms of Asian soybean rust in transgenic soybean plant cells, said transgenic soybean plant cells being stably transformed with a recombinant DNA construct according to claim 1 or a polynucleotide sequence encoding a legume-derived nucleotide-binding site-rich leucine repeat (NB-LRR) polypeptide of SEQ ID NO: 2, said method comprising exposing the transgenic soybean plant to Asian soybean rust, and the NB-LRR polypeptide of SEQ ID NO: 2 conferring resistance to the plant disease.

6. The method of claim 5, wherein exposing the transgenic soybean plants to Asian soybean rust comprises exposing the transgenic soybean plants to soybean rust fungus (… Phakopsora pachyrhizi ).

7. The method of claim 5, wherein exposing the transgenic soybean plants to Asian soybean rust comprises exposing the transgenic soybean plants to sphaerophytes (Laminaria japonica). Phakopsora meibomiae ).

8. The method of claim 5, wherein the transgenic soybean plant further comprises one or more additional disease resistance genes.

9. A method for determining the resistance of soybean plants to plant diseases, the method comprising exposing a portion of the soybean plant to Asian soybean rust, the plant comprising a recombinant DNA construct according to claim 1 or a polynucleotide sequence encoding a legume-derived nucleotide-binding site-rich leucine repeat (NB-LRR) polypeptide of SEQ ID NO: 2; measuring plant disease symptoms on the soybean plant exposed to the Asian soybean rust; and comparing the plant disease symptoms with a reference standard for resistance.

10. The method of claim 9, wherein the Asian soybean rust is formed by soybean rust fungus.

11. The method of claim 9, wherein the Asian soybean rust is formed by the rust fungus *Pseudomonas spp.*

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