Asian soybean rust resistance genes and methods of use

Abstract

Disclosed herein are compositions and methods for improving or enhancing pathogen resistance in legume plants. Compositions comprising polypeptides encoded by the nucleotide-biding site leucine-rich repeat (NLR) polynucleotides disclosed herein are useful in improving resistance in legumes to Asian soybean rust (ASR). Methods of using NLR polynucleotides disclosed herein to make transgenic ASR-resistant legume plants are also disclosed.

Description

Docket # 118737-WO-SEC-1 ASIAN SOYBEAN RUST RESISTANCE GENES AND METHODS OF USE REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The official copy of the sequence listing is submitted electronically via Patent Center as an XML formatted sequence listing with a file named 118737_SequenceListing created on January 17, 2024, and having a size of 49,866 bytes and is filed concurrently with the specification. The sequence listing comprised in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety. FIELD

[0002] This disclosure relates to the field of molecular biology, and particularly, relates to compositions and methods useful in enhancing resistance to the causal agent of Asian soybean rust (ASR) in legume plants. The disclosure further relates to polynucleotides capable of enhancing resistance in legumes to ASR, methods of using these polynucleotide sequences to make transgenic legume plants that are resistant to ASR, and methods to identify polynucleotides capable of enhancing resistance in legumes to ASR. BACKGROUND

[0003] Soybean diseases are a major threat for soybean production, resulting in yield losses and decreases in grain quality. Asian soybean rust (ASR), caused by the biotrophic fungus Phakopsora pachyrhizi and, to a lesser extent, the closely related fungus Phakopsora meibomiae, can cause yield losses ranging from 10-90%.

[0004] Accordingly, there is a need to develop compositions and methods for conferring resistance to ASR. This disclosure provides such compositions and methods. SUMMARY

[0005] Provided are polynucleotides encoding NLR polypeptides comprising an amino acid sequence that is at least, or at least about, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 1-6, and variants thereof. In certain embodiments, theDocket # 118737-WO-SEC-1 polypeptides when expressed in the cells of a plant confer resistance to Asian soybean rust (ASR).

[0006] Further provided are nucleic acid constructs and expression cassettes comprising any of the polynucleotides encoding the polypeptides described herein operably linked to at least one regulatory sequence. In certain embodiments, the at least one regulatory sequence is a promoter (e.g., heterologous promoter).

[0007] Also provided are plants, plant cells, plant parts, seeds and grain comprising any of the polynucleotides encoding NLR polypeptides described herein, and plant cells, plant parts, seeds and grain comprising any of the nucleic acid constructs and expression cassettes described herein. The plants or plants grown or generated from the plant cells, plant parts, or seeds have resistance or increased resistance to ASR. In certain embodiments, the plants are legume crop plants such as, for example, soybean.

[0008] Provided are methods for detecting an ASR resistance polypeptide encoding nucleic acid present in plant tissues comprising obtaining a nucleic acid sample from plant tissues; and i) contacting the nucleic acid sample with a polynucleotide that comprises a sequence of at least 8 nucleotides that are identical to a contiguous sequence of any of SEQ ID NOs: 7-14, or complements thereof, subjecting the sample and the polynucleotide to stringent hybridization conditions, and assaying the sample for hybridization of said polynucleotide to said DNA; or ii) contacting said nucleic acid sample with a first and second PCR primer, wherein said first and second PCR primer each specifically bind to any of SEQ ID NOs: 7-14; subjecting said sample to polymerase chain reaction; and assaying for an amplicon generated by said first and second primers.

[0009] Also provided are methods for conferring resistance to ASR in a legume crop species comprising introducing into a regenerable plant cell, optionally of a legume crop species, a polynucleotide encoding any of the polypeptides described herein and generating a plant from the plant cell. The plant produced by the method comprises the polynucleotide and has increased resistance to ASR as compared to a control plant not comprising the polynucleotide.

[0010] Further provided is a method for producing ASR resistant plants, the method comprising crossing a first plant, optionally of a legume crop species, comprising a polynucleotide encoding any of the polypeptides described herein with a second plant, harvesting the seed, isolatingDocket # 118737-WO-SEC-1 harvested seeds comprising the polynucleotide and growing the isolated seeds to produce the resistant plant, wherein the resistant plant comprises the polynucleotide.

[0011] Provided is a method of preventing ASR damage to a legume crop species such as, for example, soybean, comprising planting a field with a seed comprising a polynucleotide encoding any of the polypeptides described herein and optionally treating the field with a fungicide. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0012] The disclosure can be more fully understood from the following detailed description and the accompanying Sequence Listing, which form a part of this application. The sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§1.831-1.835. DETAILED DESCRIPTION

[0013] The present disclosure provides compositions and methods for producing plants having resistance to Asian soybean rust (ASR). The methods involve expressing in plants one or more nucleic acid sequences encoding nucleotide-biding site leucine-rich repeat (NLR) proteins. The methods also involve expressing one or more of said NLR proteins in ASR susceptible plants to provide ASR resistance. NLR proteins are receptors found inside of a cell that function in plant immunity. In particular, pathogens, such as Phakopsora pachyrhizi and Phakopsora meibomiae, which cause ASR, can overcome first-tier immunity of a plant by secreting molecules known as “effector proteins” or “effectors.” Plants have evolved, in certain cases, a second tier of immunity in which R gene products (e.g., NLR proteins) recognize specific effectors resulting in an effector triggered immunity.

[0014] Accordingly, one aspect of the disclosure provides NLR polynucleotides (e.g., isolated polynucleotides and recombinant polynucleotides) encoding NLR polypeptides (e.g., isolated polypeptides and recombinant polypeptides) comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-6, and variants thereof. In certain embodiments, the NLR polynucleotides confer resistance or increased resistance to ASR when expressed in plants.Docket # 118737-WO-SEC-1

[0015] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0016] In the present disclosure, "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

[0017] An "isolated" polynucleotide (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in an in vitro or in a heterologous recombinant bacterial or plant host cell. An isolated polynucleotide, or biologically active portion thereof, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated polynucleotide is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. A “recombinant” polynucleotide (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is in a recombinant bacterial or plant host cell. In some embodiments, an “isolated” or “recombinant” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, “isolated” or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in certain embodiments, the recombinant nucleic acid molecules encoding the NLR polypeptides of the disclosure can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

[0018] As used herein "percent (%) sequence identity" with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieveDocket # 118737-WO-SEC-1 the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions of query sequence ×100).

[0019] Unless otherwise stated, sequence identity / similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al., (1997) Nucleic Acids Res.25:3389-402).

[0020] As used herein "variant" refers to a protein or polypeptide derived from a native protein or polypeptide by deletion or addition of one or more amino acids at one or more internal sites in the native protein or polypeptide and / or a substitution of one or more amino acids at one or more sites in a native protein or polypeptide. Variants encompassed by the present disclosure exhibit a biological activity of the native protein or polypeptide sequence (e.g., increase resistance to ASR). For polynucleotides, a variant comprises a polynucleotide having a deletion (i.e., truncations) at the 5′ and / or 3′ end and / or a deletion and / or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and / or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. One of skill in the art can recognize that variants of the nucleic acids of the embodiments will be constructed such that the open reading frame is maintained. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the embodiments. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, such as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site- directed mutagenesis but which still encode a protein of the embodiments. Generally, variants ofDocket # 118737-WO-SEC-1 a particular polynucleotide of the present disclosure can have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to that particular polynucleotide. Variants of a particular polynucleotide of the embodiments (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.

[0021] For polypeptides, a variant comprises a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and / or a substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present disclosure are biologically active, that is they continue to possess the desired biological activity of the native protein, which is, the ability to confer or enhance plant resistance (i.e., ASR resistance) as described herein. Such variants can result, for example, from genetic polymorphism or from human manipulation. Biologically active variants of a native protein of the embodiments can have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the amino acid sequence for the native protein. A biologically active variant of a protein of the present disclosure can differ from that protein by as few as about 1-15 amino acid residues, as few as about 1-10, such as about 6-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acid residue.

[0022] The proteins disclosed herein can be altered, for example, by including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are known in the art. For example, amino acid sequence variants and fragments of the resistance proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are known in the art.

[0023] Variant polynucleotides and polypeptides also encompass sequences and proteins derived from mutagenic or recombinogenic procedures, including, and not limited to, procedures such as DNA shuffling. Libraries of recombinant polynucleotides can be generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using thisDocket # 118737-WO-SEC-1 approach, sequence motifs encoding a domain of interest can be shuffled between the protein gene of the present disclosure and other known protein genes to obtain a new gene coding for a protein with an improved property of interest, such as increased ability to confer or enhance plant resistance to a fungal pathogen. Strategies for such DNA shuffling are known in the art.

[0024] Variants may be made by making random mutations or the variants may be designed. In the case of designed mutants, there is a high probability of generating variants with similar activity to the native polypeptide when amino acid identity is maintained in critical regions of the polypeptide which account for biological activity or are involved in the determination of three- dimensional configuration which ultimately is responsible for the biological activity. A high probability of retaining activity will also occur if substitutions are conservative. Amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type are least likely to materially alter the biological activity of the variant. Table 1 provides a listing of examples of amino acids belonging to each class. Table 1: Classes of amino acids Class of Amino Acid Examples of Amino Acids

[0025] The polynucleotides described herein can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods, such as PCR or hybridization, can be used to identify such sequences based on their sequence identity to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire sequences set forth herein or to variants and fragments thereof are encompassed by the presentDocket # 118737-WO-SEC-1 disclosure. Such sequences include sequences that are orthologs of the disclosed sequences. The term "orthologs" refers to genes derived from a common ancestral gene, and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and / or their encoded protein sequences share at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode for a protein that confers or enhances ASR resistance and that hybridize to the sequences disclosed herein, or to variants or fragments thereof, are encompassed by the present disclosure.

[0026] As used herein an “amino acid deletion,” “deletion mutation,” or the like, refers to a mutation in which the indicated amino acid residue is removed from the polypeptide sequence, so that, when aligned to the reference sequence (e.g., SEQ ID NO: 1) the mutated sequence does not have an amino acid corresponding to the indicated position of the reference sequence. An “amino acid addition,” “addition mutation,” “amino acid insertion,” “insertion,” or the like, refers to a mutation in which at least one amino acid residue is added to the polypeptide sequence, so that, when aligned to the reference sequence (e.g., SEQ ID NO: 1) the mutated sequence contains an additional amino acid corresponding to the indicated position of the reference sequence.

[0027] An “amino acid substitution,” “substitution mutation,” or the like, refers to a mutation in which the indicated amino acid residue is replaced with a different amino acid residue, so that, when aligned to the reference sequence (e.g., SEQ ID NO: 1) the mutated sequence does not have the same amino acid at the indicated position. When the amino acid residue is substituted for a residue that has similar properties (e.g., size, charge, and / or hydrophobicity) the substitution is referred to as a conservative amino substitution. Conservative amino acid substitutions are well known in the art. Alternatively, when the amino acid residue is substituted for an amino acid that has dissimilar properties the mutation is referred to as a radical amino acid substitution.

[0028] As used herein “encoding,” “encoded,” or the like, with respect to a specified nucleic acid, means comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as inDocket # 118737-WO-SEC-1 cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. However, variants of the universal code, such as is present in some plant, animal and fungal mitochondria, the bacterium Mycoplasma capricolum (Yamao, et al., (1985) Proc. Natl. Acad. Sci. USA 82:2306-9) or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.

[0029] When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledonous plants or dicotyledonous plants as these preferences have been shown to differ (Murray, et al., (1989) Nucleic Acids Res.17:477-98 and herein incorporated by reference).

[0030] In certain embodiments, the NLR polynucleotide encoding the NLR polypeptide comprises a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 7-14.

[0031] In certain embodiments, the NRL polynucleotide encoding the NLR polypeptide is operably linked to at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7 or more) regulatory element. In certain embodiments, the regulatory element is a promoter. In certain embodiments, the regulatory element is a heterologous regulatory element (e.g., heterologous promoter). In certain embodiments, the heterologous regulatory element is heterologous to the polynucleotide sequence encoding the polypeptide. In certain embodiments, in which the polynucleotide operably linked to the heterologous regulatory element is introduced in a cell the regulatory element is heterologous to the cell.

[0032] As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide that is from a species different from the species from which the polynucleotide was derived, or, if from the same / analogous species,Docket # 118737-WO-SEC-1 one or both are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

[0033] As used herein "operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, operably linked is intended that the coding regions are in the same reading frame.

[0034] As used herein “regulatory element” generally refers to a transcriptional regulatory element involved in regulating the transcription of a nucleic acid molecule such as a gene or a target gene. The regulatory element is a nucleic acid and may include a promoter, an enhancer, an intron, expression modulating elements (EMEs), a 5’-untranslated region (5’-UTR, also known as a leader sequence), or a 3’-UTR or a combination thereof. A regulatory element may act in "cis" or "trans", and generally a regulatory element acts in "cis", i.e., it activates expression of genes located on the same nucleic acid molecule, e.g., a chromosome, where the regulatory element is located.

[0035] An “enhancer” element is any nucleic acid molecule that increases transcription of a nucleic acid molecule when functionally linked to a promoter regardless of its relative position. Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9:1685-96) and the enhancers of US Patent Number 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments described herein.

[0036] A “repressor” (also sometimes called herein silencer) is defined as any nucleic acid molecule which inhibits the transcription when functionally linked to a promoter regardless of relative position. The term "cis-element" generally refers to transcriptional regulatory element that affects or modulates expression of an operably linked transcribable polynucleotide, where the transcribable polynucleotide is present in the same DNA sequence. A cis-element mayDocket # 118737-WO-SEC-1 function to bind transcription factors, which are trans-acting polypeptides that regulate transcription.

[0037] An “intron” is an intervening sequence in a gene that is transcribed into RNA but is then excised in the process of generating the mature mRNA. The term is also used for the excised RNA sequences. An “exon” is a portion of the sequence of a gene that is transcribed and is found in the mature mRNA derived from the gene but is not necessarily a part of the sequence that encodes the final gene product. The 5' untranslated region (5’UTR) (also known as a translational leader sequence or leader RNA) is the region of an mRNA that is directly upstream from the initiation codon. This region is involved in the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes. The “3' non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0038] As used herein “promoter” refers to a region of DNA upstream from the start of transcription involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. In certain embodiments, the polynucleotides described herein are operably linked to a promoter that drives expression in a plant cell. Any promoter known in the art can be used in the methods of the present disclosure including, but not limited to, constitutive promoters, pathogen- inducible promoters, wound-inducible promoters, tissue-preferred promoters, and chemical- regulated promoters. The choice of promoter may depend on the desired timing and location of expression in the transformed plant as well as other factors, which are known to those of skill in the art. Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. Patent No.6,072,050; the core CaMV 35S promoter; rice actin; ubiquitin; pEMU; MAS; ALS; and the like. Other constitutive promoters which are known in the art can be contemplated for use in the present disclosure.

[0039] Generally, it can be beneficial to express the gene from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen, e.g., PR proteins, SAR proteins, beta-l,3-glucanase, chitinase, etc.Docket # 118737-WO-SEC-1

[0040] Of interest are promoters that are expressed locally at or near the site of pathogen infection. Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter can be used in the constructions of the disclosure. Such wound- inducible promoters include potato proteinase inhibitor (pin II) gene, wunl and wun2, winl and win2, systemin, WIP1, MPI gene, and the like.

[0041] Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter can be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (e.g., the glucocorticoid-inducible promoter, and tetracycline-inducible and tetracycline-repressible promoters).

[0042] Tissue-preferred promoters can be utilized to target enhanced expression of the target genes or proteins within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Examples of tissue-preferred promoters include those described in Yamamoto et al. (1997) Plant J.12(2): 255 -265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res.6(2): 157-168; Rinehart et al. (1996) Plant Physiol.112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ.20: 181-196; Orozco et al. (1993) Plant Mol Biol.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.

[0043] 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)Docket # 118737-WO-SEC-1 Plant Mol. Biol.23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0044] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seed- germinating" promoters (those promoters active during seed germination). Such seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message), cZ19Bl (maize 19 kDa zein), milps (myo-inositol-1-phosphate synthase), and celA (cellulose synthase) (see WO 00 / 11177). Gama-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00 / 12733, where seed- preferred promoters from endl and end2 genes are disclosed; herein incorporated by reference.

[0045] In certain embodiments, the polynucleotides of the present disclosure can involve the use of the intact, native NLR genes, wherein the expression is driven by a cognate 5' upstream promoter sequence(s).

[0046] Specific soybean promoters include but are not limited to soy ubiquitin (subi-1), elongation factor 1A, and S-adenosyl methionine synthase for constitutive expression and Rpp4, RPG1-B, and promoters contained in gene models such as Glyma promoters.

[0047] Also provided are nucleic acid (e.g., DNA) constructs comprising any of the NLR polynucleotides described herein (e.g., SEQ ID NOs: 7-14). The use of the term "nucleotide constructs" herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules, and sequences. Further, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. SuchDocket # 118737-WO-SEC-1 deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.

[0048] In certain embodiments, the NLR polynucleotides described herein are provided in expression cassettes (e.g., a plasmid, cosmid, virus, autonomously replicating sequence, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleotide sequence) for expression in a plant of interest or any organism of interest. The cassette can include 5' and 3' regulatory sequences operably linked to a NLR polynucleotide. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for insertion of the NLR polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0049] The expression cassette can include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (e.g., a promoter), a NLR polynucleotide (e.g., SEQ ID NO: 8- 13), and a transcriptional and translational termination region (e.g., termination region) functional in plants. The regulatory regions (e.g., promoters, transcriptional regulatory regions, and translational termination regions) and / or the NLR polynucleotide may be native / analogous to the host cell or to each other. Alternatively, the regulatory regions and / or the NLR polynucleotide may be heterologous to the host cell or to each other.

[0050] The termination region may be native with the transcriptional initiation region, with the plant host, or may be derived from another source (i.e., foreign or heterologous) than the promoter, the NLR polynucleotide, the plant host, or any combination thereof.

[0051] The expression cassette may additionally contain a 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include viral translational leader sequences.

[0052] Generally, the expression cassette can comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well asDocket # 118737-WO-SEC-1 genes conferring resistance to herbicidal compounds, such as glyphosate, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present disclosure.

[0053] In preparing the expression cassette, the various DNA fragments may be manipulated, to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0054] In certain embodiments, the nucleic acid constructs or expression cassettes, described herein, are expressed in a host cell, plant or seed. The nucleic acid constructs or expression cassettes disclosed herein may be used for transformation of any plant species. In certain embodiments, the plant or seed is a soybean plant or soybean seed.

[0055] Also provided are host cells that are engineered (e.g., transduced, transformed, or transfected) to express one or more of any of the NLR polynucleotides encoding an NLR polypeptide described herein. The inventive NLR polypeptides, polynucleotides or nucleic acid constructs can be expressed in any organism, including in non-animal cells such as yeast, fungi, bacteria and the like. Details regarding non-animal cell culture can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin, Heidelberg, New York); and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

[0056] Host cells of interest can include, for example, a eukaryotic cell, an animal cell, a protoplast, a tissue culture cell, a prokaryotic cell, a bacterial cell, such as E. coli, B. subtilis, Streptomyces, Salmonella typhimurium, a gram positive bacteria, a purple bacteria, a green sulfur bacteria, a green non-sulfur bacteria, a cyanobacteria, a spirochetes, a thermatogale, a flavobacteria, bacteroides; a fungal cell, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; an insect cell such as Drosophila and Spodoptera frugiperda; a mammalian cell such as CHO, COS, BHK, HEK 293 or Bowes melanoma; an archaebacteria such asDocket # 118737-WO-SEC-1 Korarchaeota, Thermoproteus, Pyrodictium, Thermococcales, Methanogens, Archaeoglobus, and extreme Halophiles.

[0057] Further provided are plants, plant cells, plant parts, seeds, and grain comprising at least one of the NLR polynucleotide sequences, nucleic acid constructs, or expression cassettes described herein, so that the plants, plant cells, plant parts, seeds, and / or grain express any of the NLR polypeptides described herein. In certain embodiments, the plants, plant cells, plant parts, seeds, and / or grain have stably incorporated at least one NLR polynucleotide into its genome.

[0058] For example, in certain embodiments the plant, plant cell, plant part, seed, or grain comprises a NLR polynucleotide encoding a NLR polypeptide comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-6. In certain embodiments, the polynucleotide is operably linked to a regulatory element, such as, for example, a heterologous promoter. In certain embodiments, the plant or a plant generated from the plant cell, plant part, or seed, has resistance to ASR. In certain embodiments, the plant or a plant generated from the plant cell, plant part, or seed, has increased resistance to ASR as compared to a control plant (e.g., a plant of the same genetic background not comprising the polynucleotide).

[0059] In certain embodiments, the plant, plant cell, plant part, seed, or grain comprises two or more of the NLR polynucleotide sequences, nucleic acid constructs, or expression cassettes described herein, so that the plants, plant cells, plant parts, seeds, and / or grain express at least two of the NLR polypeptides described herein.

[0060] For example, in certain embodiments the plant, plant cell, plant part, seed, or grain comprises a NLR polynucleotide encoding a NLR polypeptide comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 and a NLR polynucleotide encoding a NLR polypeptide comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5. In certain embodiments, one or both of the polynucleotides are operably linked to a regulatory element, such as, for example a heterologous promoter. In certain embodiments, the plant or a plant generated from the plant cell, plant part, or seed, has resistanceDocket # 118737-WO-SEC-1 to ASR. In certain embodiments, the plant or a plant generated from the plant cell, plant part, or seed, has increased resistance to ASR as compared to a control plant (e.g., a plant of the same genetic background not comprising the polynucleotide).

[0061] As used herein, the term "resistance" refers to an absence or reduction of one or more disease symptoms in a plant caused by a plant pathogen. Resistance can mean that disease symptoms, such as, for example, number of lesions, defoliation, and associated yield loss, are reduced, minimized or lessened, when compared to a plant that is susceptible to the disease or a plant that does not contain an effective resistance gene. Resistance can also include the prevention or delay of proliferation of a pathogen (e.g., fungi).

[0062] In certain embodiments, the polynucleotides described herein are transiently expressed in the plant, plant cell, plant part, seed or grain using a transient transformation technique. In certain embodiments, the polynucleotides described herein are stably expressed in the plant, plant cell, plant part, seed or grain using a stable transformation technique. In certain embodiments, the polynucleotides are introduced into the plant, plant cell, plant part, seed or grain using a nucleic acid construct or an expression cassette described herein. In certain embodiments, the polynucleotides described herein are introduced into the plant, plant cell, plant part, seed or grain using a genome editing technique.

[0063] In certain embodiments, the plant cells or plant parts are grown into plants. The method for generating the plants from the plant cells or plant parts is not particularly limited. These plants can then be grown, and either pollinated with the same strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations can be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In certain embodiments of the present disclosure, the transformed seed or transgenic seed have a nucleotide construct described herein or an expression cassette described herein stably incorporated into their genome.

[0064] In certain embodiments, the present disclosure encompasses seeds comprising a polynucleotide sequence disclosed herein that can develop into or can be used to develop a plant or plants with increased or enhanced resistance to a pathogen (e.g., ASR) or infection caused by a pathogen as compared to, for example, a wild-type variety of the plant seed. In certainDocket # 118737-WO-SEC-1 embodiments, the present disclosure features seeds from transgenic legume crop plants wherein the seed comprises a polynucleotide disclosed herein.

[0065] In certain embodiments, the plants described herein are elite plant lines (e.g., elite soybean line). In certain embodiments, the plant cells, plant parts, seeds, and grain are isolated from or produced by an elite plant line. As used herein, “elite line” refers to any line that has resulted from breeding and selection for superior agronomic performance that allows a producer to harvest a product of commercial significance. Numerous elite lines are available and known to those of skill in the art of plant breeding (e.g., soybean breeding). An “elite population” is an assortment of elite individuals or lines that can be used to represent the state of the art in terms of agronomically superior genotypes of a given crop species, such as soybean.

[0066] As used herein, the term “plant” includes plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the disclosure, provided that these parts comprise the introduced polynucleotides.

[0067] The plant species of the compositions and methods of the present disclosure can be any plant species for which expression of an NLR polynucleotide or NLR polypeptide described herein is desired, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica spp. (e.g., Brassica napus, Brassica rapa, Brassica juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Per sea americana), fig (Ficus casica), guava (Psidium guajava),Docket # 118737-WO-SEC-1 mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

[0068] In certain embodiments, the plant of the compositions and methods described herein is a legume crop species, including, but not limited to, alfalfa (Medicago sativa); clover or trefoil (Trifolium spp.); pea, including (Pisum satinum), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata) and Lathyrus spp.; bean (Fabaceae or Leguminosae); lentil (Lens culinaris); lupin (Lupinus spp.); mesquite (Prosopis spp.); carob (Ceratonia siliqua), soybean (Glycine max), peanut (Arachis hypogaea) or tamarind (Tamarindus indica). The terms "legume species" and "legume crop species" are used herein to refer to plants, and can be for example, a plant of interest. In certain embodiments, the plant, plant part or plant cell is a legume species or legume crop species.

[0069] In certain embodiments, the plants, plant cells, plant parts, seeds, and grain further comprise a polynucleotide encoding at least one additional ASR resistance gene. In certain embodiments, the at least one additional ASR resistance gene is CcRpp1 (SEQ ID NOs: 1 (nucleic acid) and 2 (polypeptide) of WO2016183130) and / or CcRpp2 (SEQ ID NOs: 1 and / or 3 (nucleic acid) and 2 and / or 4 (polypeptide) of WO2022140257), optionally operably linked to a regulatory element, such as, for example, those described herein (e.g., heterologous regulatory element).

[0070] In certain embodiments of the present disclosure, the polynucleotide sequences can be stacked with any combination of polynucleotide sequences of interest to create plants with a desired phenotype. This stacking can be accomplished by a combination of genes within a DNA construct, or by crossing one or more plants having transgenes with another plant line that comprises a desired combination. For example, the polynucleotides of the present disclosure or variants thereof can be stacked with any other polynucleotides of the disclosure (e.g., stack of genes encoding SEQ ID NOs: 1 and 5), or with other genes. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the present disclosure can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including and not limited to traits desirable for animal feed such as high oil genes, balanced amino acids, increased digestibility, insect, disease or herbicide resistance, avirulence and disease resistance genes, agronomic traitsDocket # 118737-WO-SEC-1 (e.g, male sterility, flowering time) and / or transformation technology traits (e.g., cell cycle regulation or gene targeting).

[0071] These stacked combinations can be created by any method including, but not limited to, cross breeding plants by any conventional or known methodology, genetic transformation, or genome editing. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co- transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that can suppress the expression of the polynucleotide of interest. This can be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant.

[0072] In one embodiment, the stacked combination includes one or more genes encoding pesticidal proteins including, but not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7:1-13); from Pseudomonas protegens strain CHA0 and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank Accession No. EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58:12343-12349) and from Pseudomonas pseudoalcaligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult.89:159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal, 3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro.67:2062-2069); US Patent Number 6,048,838, and US Patent Number 6,379,946; a PIP-1 polypeptide of US 9,688,730; an AfIP-1A and / or AfIP-1B polypeptide of US9,475,847; a PIP-47 polypeptide of US Publication Number US20160186204; an IPD045 polypeptide, an IPD064 polypeptide, an IPD074 polypeptide, an IPD075 polypeptide, and an IPD077 polypeptide of PCT Publication Number WO 2016 / 114973; an IPD080 polypeptide of International Patent Application Publication Number WO2018 / 075350; an IPD078 polypeptide,Docket # 118737-WO-SEC-1 an IPD084 polypeptide, an IPD085 polypeptide, an IPD086 polypeptide, an IPD087 polypeptide, an IPD088 polypeptide, and an IPD089 polypeptide of International Patent Application Publication Number WO2018 / 084936; PIP-72 polypeptide of US Patent Publication Number US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide of US Publication Number US20170166921; an IPD098 polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109 polypeptide of International Patent Application Publication Number WO2018 / 232072; a PtIP-83 polypeptide of US Publication Number US20160347799; a PtIP-96 polypeptide of US Publication Number US20170233440; an IPD079 polypeptide of PCT Publication Number WO2017 / 23486; an IPD082 polypeptide of PCT Publication Number WO 2017 / 105987, an IPD090 polypeptide of International Patent Application Publication Number WO2017 / 192560, an IPD093 polypeptide of International Patent Application Publication Number WO2018 / 111551; an IPD103 polypeptide of International Patent Application Publication Number WO2018 / 005411; an IPD101 polypeptide of International Patent Application Publication Number WO2018 / 118811; an IPD121 polypeptide of International Patent Application Publication Number WO2018 / 208882; and δ-endotoxins including, but not limited to a Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71, and Cry 72 classes of δ-endotoxin polypeptides and the B. thuringiensis cytolytic cyt1 and cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins can be found in Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2011), at lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / which can be accessed on the world-wide web using the "www" prefix).

[0073] In another embodiment, the stacked combination includes a polynucleotide encoding resistance to an herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.7:1241 and Miki, et al., (1990) Theor. Appl. Genet.80:449, respectively. See also, US Patent Numbers 5,605,011; 5,013,659;Docket # 118737-WO-SEC-1 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and 5,378,824; US Patent Application Serial Number 11 / 683,737 and International Publication WO 1996 / 33270.

[0074] In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to Glyphosate (resistance imparted by mutant 5-enolpyruvl-3- phosphikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar) genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes). See, for example, US Patent Number 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSPS which can confer glyphosate resistance. US Patent Number 5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes. See also, US Patent Numbers 6,566,587; 6,338,961; 6,248,876; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 5,094,945, 4,940,835; 5,866,775; 6,225,114; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re.36,449; RE 37,287 and 5,491,288 and International Publications EP 1173580; WO 2001 / 66704; EP 1173581 and EP 1173582, which are incorporated herein by reference for this purpose. Glyphosate resistance is also imparted to plants that express a gene encoding a glyphosate oxido-reductase enzyme as described more fully in US Patent Numbers 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose. In addition, glyphosate resistance can be imparted to plants by the over expression of genes encoding glyphosate N-acetyltransferase. See, for example, US Patent Numbers 7,462,481; 7,405,074 and US Patent Application Publication Number US 2008 / 0234130. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC® Accession Number 39256, and the nucleotide sequence of the mutant gene is disclosed in US Patent Number 4,769,061 to Comai. EP Application Number 0333033 to Kumada, et al., and US Patent Number 4,975,374 to Goodman, et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in EP Application Numbers 0242246 and 0242236 to Leemans, et al.; De Greef, et al., (1989) Bio / Technology 7:61, describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. See also, US Patent Numbers 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616 and 5,879,903, which areDocket # 118737-WO-SEC-1 incorporated herein by reference for this purpose. Exemplary genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop, are the Acc1- S1, Acc1-S2 and Acc1-S3 genes described by Marshall, et al., (1992) Theor. Appl. Genet. 83:435.

[0075] In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell 3:169, describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in US Patent Number 4,810,648 to Stalker and DNA molecules containing these genes are available under ATCC® Accession Numbers 53435, 67441 and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes, et al., (1992) Biochem. J.285:173.

[0076] In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to Acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants (see, e.g., Hattori, et al., (1995) Mol Gen Genet.246:419). Other genes that confer resistance to herbicides include: a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994) Plant Physiol 106:17), genes for glutathione reductase and superoxide dismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genes for various phosphotransferases (Datta, et al., (1992) Plant Mol Biol 20:619).

[0077] In another embodiment, the stacked combination includes a polynucleotide encoding resistance to an herbicide targeting Protoporphyrinogen oxidase (protox) which is necessary for the production of chlorophyll. The protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in US Patent Numbers 6,288,306; 6,282,83 and 5,767,373 and International Publication WO 2001 / 12825.

[0078] In another embodiment, the stacked combination includes an aad-1 gene (originally from Sphingobium herbicidovorans) encoding the aryloxyalkanoate dioxygenase (AAD-1) protein. The trait confers tolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionateDocket # 118737-WO-SEC-1 (commonly referred to as “fop” herbicides such as quizalofop) herbicides. The aad-1 gene, itself, for herbicide tolerance in plants was first disclosed in WO 2005 / 107437 (see also, US 2009 / 0093366). The aad-12 gene, derived from Delftia acidovorans, which encodes the aryloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to 2,4- dichlorophenoxyacetic acid and pyridyloxyacetate herbicides by deactivating several herbicides with an aryloxyalkanoate moiety, including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxy auxins (e.g., fluroxypyr, triclopyr).

[0079] In another embodiment, the stacked combination includes a polynucleotide encoding an herbicide resistant dicamba monooxygenase, such as a polynucleotide disclosed in US Patent Application Publication 2003 / 0135879 for imparting dicamba tolerance.

[0080] In another embodiment, the stacked combination includes a polynucleotide encoding bromoxynil nitrilase (Bxn), such as a polynucleotide disclosed in US Patent Number 4,810,648 for imparting bromoxynil tolerance.

[0081] In another embodiment, the stacked combination includes a polynucleotide encoding CcRpp1 disclosed in U.S.10,842,097 for imparting ASR resistance. In another embodiment, the stacked combination includes a polynucleotide encoding CcRpp2 disclosed in WO2022140257 for imparting ASR resistance.

[0082] In another embodiment, the stacked combination includes a polynucleotide encoding phytoene (crtl) described in Misawa, et al., (1993) Plant J.4:833-840 and in Misawa, et al., (1994) Plant J.6:481-489 for norflurazon tolerance.

[0083] In another embodiment, the stacked combination includes a polynucleotide encoding a protein that confers or contributes to an altered grain characteristic, such as altered fatty acids, for example, by:

[0084] (1) Down-regulation of stearoyl-ACP to increase stearic acid content of the plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA 89:2624 and WO 1999 / 64579 (Genes to Alter Lipid Profiles in Corn).

[0085] (2) Elevating oleic acid via FAD-2 gene modification and / or decreasing linolenic acid via FAD-3 gene modification (see, US Patent Numbers 6,063,947; 6,323,392; 6,372,965 and WO 1993 / 11245).

[0086] (3) Altering conjugated linolenic or linoleic acid content, such as in WO 2001 / 12800.Docket # 118737-WO-SEC-1

[0087] (4) Altering LEC1, AGP, Dek1, Superal1, mi1 ps, and various Ipa genes such as Ipa1, Ipa3, hpt or hggt. For example, see, WO 2002 / 42424, WO 1998 / 22604, WO 2003 / 011015, WO 2002 / 057439, WO 2003 / 011015, US Patent Numbers 6,423,886, 6,197,561, 6,825,397 and US Patent Application Publication Numbers US 2003 / 0079247, US 2003 / 0204870 and Rivera- Madrid, et al., (1995) Proc. Natl. Acad. Sci.92:5620-5624.

[0088] (5) Genes encoding delta-8 desaturase for making long-chain polyunsaturated fatty acids (US Patent Numbers 8,058,571 and 8,338,152), delta-9 desaturase for lowering saturated fats (US Patent Number 8,063,269), Primula delta 6-desaturase for improving omega-3 fatty acid profiles.

[0089] (6) Isolated nucleic acids and proteins associated with lipid and sugar metabolism regulation, in particular, lipid metabolism protein (LMP) used in methods of producing transgenic plants and modulating levels of seed storage compounds including lipids, fatty acids, starches or seed storage proteins and use in methods of modulating the seed size, seed number, seed weights, root length and leaf size of plants (EP 2404499).

[0090] (7) Altering expression of a High-Level Expression of Sugar-Inducible 2 (HSI2) protein in the plant to increase or decrease expression of HSI2 in the plant. Increasing expression of HSI2 increases oil content while decreasing expression of HSI2 decreases abscisic acid sensitivity and / or increases drought resistance (US Patent Application Publication Number 2012 / 0066794).

[0091] (8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oil content in plant seed, particularly to increase the levels of omega-3 fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (US Patent Application Publication Number 2011 / 0191904).

[0092] (9) Nucleic acid molecules encoding wrinkled1-like polypeptides for modulating sugar metabolism (US Patent Number 8,217,223).

[0093] Also provided are methods for providing, conferring, or enhancing resistance of a plant to ASR, such that the causal agents of the disease (e.g., Phakopsora pachyrhizi and Phakopsora meibomiae) can no longer reproduce. As used herein the term "enhance" means to improve, increase, amplify, multiply, elevate and / or raise, thereby reducing one or more disease symptoms. Accordingly, plants (e.g., soybean) exhibit an increased resistance to a disease (e.g., ASR) when compared to plants that are susceptible or tolerant to Phakopsora spp. In certainDocket # 118737-WO-SEC-1 embodiments, methods described herein can reduce one or more symptoms (i.e., disease symptoms) of a legume plant disease (e.g., ASR).

[0094] As used herein "plant pathogen" or "fungal pathogen" can be used herein to mean fungal pathogens of, for example, the genus Phakopsora, including the species Phakopsora pachyrhizi and Phakopsora meibomiae. These species are known to cause ASR in plants.

[0095] In certain embodiments, the method for providing, conferring, or enhancing resistance comprises introducing into a regenerable plant cell at least one of the polynucleotides, nucleic acid constructs, or expression cassettes described herein. In certain embodiments, the plant is a legume crop species (e.g., soybean).

[0096] In certain embodiments of the methods for providing, conferring, or enhancing resistance of a plant to ASR described herein, the method comprises the introduction of a NLR polynucleotide encoding a NLR polypeptide comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-6, optionally operably linked to a regulatory element (e.g., heterologous promoter); and generating a plant from the plant cell wherein the plant comprises the polynucleotide and has enhanced resistance, increased resistance, or resistance to ASR.

[0097] In certain embodiments, the method for providing, conferring, or enhancing resistance of a plant to ASR comprises expressing in a regenerable plant cell a nucleic acid construct or expression cassette comprising a polynucleotide described herein; and generating the plant from the plant cell. In certain embodiments, the polynucleotide is operably linked to at least one regulatory sequence. In certain embodiments, the at least one regulatory sequence is a heterologous promoter. The nucleic acid construct or expression cassette for use in the method may be any nucleic acid construct or expression cassette provided herein. In certain embodiments, the nucleic acid construct or expression cassette is expressed by introducing into a plant, plant cell, plant part, seed, and / or grain the nucleic acid construct or expression cassette, whereby the polypeptide is expressed in the plant, plant cell, plant part, seed, and / or grain. In certain embodiments the nucleic acid construct or expression cassette is incorporated into the genome of the plant.

[0098] Various methods can be used to introduce the NLR sequences into a plant, plant part, plant cell, seed, and / or grain. "Introducing" is intended to mean presenting to the plant, plant cell,Docket # 118737-WO-SEC-1 seed, and / or grain the inventive polynucleotide or resulting polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the disclosure do not depend on a particular method for introducing a sequence into a plant, plant cell, seed, and / or grain, only that the polynucleotide or polypeptide gains access to the interior of at least one cell of the plant.

[0099] "Stable transformation" is intended to mean that the polynucleotide introduced into a plant integrates into the genome of the plant of interest and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant of interest and does not integrate into the genome of the plant or organism, or a polypeptide is introduced into a plant or organism.

[0100] Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Patent No.5,563,055 and U.S. Patent No. 5,981,840), Ochrobacterium-mediated transformation (U.S. Patent Application Publication 2018 / 0216123 and WO20 / 092494) direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Patent Nos.4,945,050; U.S. Patent No.5,879,918; U.S. Patent No.5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO 00 / 28058). D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

[0101] In certain embodiments, the NLR sequences can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the NLR protein directly into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet.202:179-185; Nomura et al. (1986) Plant Sci.44:53-58; Hepler et al. (1994) Proc.Docket # 118737-WO-SEC-1 Natl. Acad. Sci.91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference.

[0102] In other embodiments, the inventive polynucleotides disclosed herein may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the disclosure within a DNA or RNA molecule. It is recognized that the inventive polynucleotide sequence may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters disclosed herein also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221; herein incorporated by reference.

[0103] Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In certain embodiments, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99 / 25821, WO99 / 25854, WO99 / 25840, WO99 / 25855, and WO99 / 25853, all of which are herein incorporated by reference. Briefly, the polynucleotide disclosed herein can be contained in a transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided, and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome. Other methods to target polynucleotides are set forth in WO 2009 / 114321 (herein incorporated by reference), which describes “custom" meganucleases produced to modify plant genomes, in particular the genome of maize. See, also, Gao et al. (2010) Plant Journal 1:176-187.

[0104] One of skill will recognize that after the nucleic acid constructs and / or expression cassettes containing the polynucleotides described herein are stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing.Docket # 118737-WO-SEC-1 Any of several standard breeding techniques can be used, depending upon the species to be crossed.

[0105] Parts obtained from the regenerated plants described herein, such as flowers, seeds, leaves, branches, fruit, and the like are included, provided that these parts comprise cells comprising the inventive polynucleotide. Progeny and variants, and mutants of the regenerated plants are also included, provided that these parts comprise the introduced nucleic acid sequences.

[0106] In certain embodiments, a homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced. Backcrossing to a parental plant and outcrossing with a non-transgenic plant are also contemplated.

[0107] In certain embodiments, the method for providing, conferring, or enhancing resistance of a plant to ASR comprises introducing a polynucleotide encoding a polypeptide described herein into the genome of the regenerable plant cell using genome editing technologies. In certain embodiments, the method comprises editing NLR genes or previously introduced NLR genes of a plant to produce the NLR polynucleotides provided herein using genome editing technologies.

[0108] The genome editing technology for use in the methods and compositions described herein is not particularly limited and may be any genome editing technique that allows for the introduction and / or targeted introduction of the desired polynucleotide.

[0109] In certain embodiments the genome editing technique uses an enzyme selected from the group consisting of a polynucleotide-guided endonuclease, CRISPR-Cas endonucleases, base editing deaminases, zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) or engineered site-specific meganuclease.

[0110] In certain embodiments, the genome modification may be facilitated through the induction of a double-stranded break (DSB) or single-strand break, in a defined position in the genome near the desired alteration. DSBs can be induced using any DSB-inducing agent available, including, but not limited to, TALENs, meganucleases, zinc finger nucleases, Cas9- gRNA systems (based on bacterial CRISPR-Cas systems), guided cpf1 endonuclease systems, and the like. In some embodiments, the introduction of a DSB can be combined with the introduction of a polynucleotide modification template.Docket # 118737-WO-SEC-1

[0111] The process for editing a genomic sequence combining DSB and modification templates generally comprises: providing to a host cell, a DSB-inducing agent, or a nucleic acid encoding a DSB-inducing agent, that recognizes a target sequence in the chromosomal sequence and is able to induce a DSB in the genomic sequence, and at least one polynucleotide modification template comprising at least one nucleotide alteration when compared to the nucleotide sequence to be edited. The polynucleotide modification template can further comprise nucleotide sequences flanking the at least one nucleotide alteration, in which the flanking sequences are substantially homologous to the chromosomal region flanking the DSB.

[0112] The endonuclease can be provided to a cell by any method known in the art, for example, but not limited to, transient introduction methods, transfection, microinjection, and / or topical application or indirectly via recombination constructs. The endonuclease can be provided as a protein or as a guided polynucleotide complex directly to a cell or indirectly via recombination constructs. The endonuclease can be introduced into a cell transiently or can be incorporated into the genome of the host cell using any method known in the art. In the case of a CRISPR-Cas system, uptake of the endonuclease and / or the guided polynucleotide into the cell can be facilitated with a Cell Penetrating Peptide (CPP) as described in WO2016073433.

[0113] TAL effector nucleases (TALEN) are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism (Miller et al. (2011) Nature Biotechnology 29:143–148).

[0114] Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Endonucleases include restriction endonucleases, which cleave DNA at specific sites without damaging the bases, and meganucleases, also known as homing endonucleases (HEases), which like restriction endonucleases, bind and cut at a specific recognition site, however the recognition sites for meganucleases are typically longer, about 18 bp or more. Meganucleases have been classified into four families based on conserved sequence motifs. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganuclease is similar to the convention for other restriction endonuclease. Meganucleases are also characterized by prefix F-, I-, or PI- for enzymes encoded by free-standing ORFs, introns, and inteins, respectively. One step in the recombination process involves polynucleotide cleavage at or near the recognition site. TheDocket # 118737-WO-SEC-1 cleaving activity can be used to produce a double-strand break. For reviews of site-specific recombinases and their recognition sites, see, Sauer (1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:760-7. In some examples the recombinase is from the Integrase or Resolvase families.

[0115] Zinc finger nucleases (ZFNs) are engineered double-strand break inducing agents comprised of a zinc finger DNA binding domain and a double-strand-break-inducing agent domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprises two, three, or four zinc fingers, for example having a C2H2 structure, however other zinc finger structures are known and have been engineered. Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence. ZFNs include an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain, for example nuclease domain from a Type IIs endonuclease such as FokI. Additional functionalities can be fused to the zinc-finger binding domain, including transcriptional activator domains, transcription repressor domains, and methylases. In some examples, dimerization of nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3-finger domain recognized a sequence of 9 contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind an 18-nucleotide recognition sequence.

[0116] Genome editing using DSB-inducing agents, such as Cas9-gRNA complexes, has been described, for example in U.S. Patent Application US 20150082478, WO2015 / 026886, WO2016007347, and WO201625131 all of which are incorporated by reference herein.

[0117] In certain embodiments the genetic modification is introduced without introducing a double strand break using base editing technology, see e.g., Gaudelli et al., (2017) Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551(7681):464- 471; Komor et al., (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, Nature 533(7603):420-4.

[0118] In certain embodiments, base editing comprises (i) a catalytically impaired CRISPR– Cas9 mutant that is mutated such that one of their nuclease domains cannot make DSBs; (ii) a single-strand-specific cytidine / adenine deaminase that converts C to U or A to G within an appropriate nucleotide window in the single-stranded DNA bubble created by Cas9; (iii) a uracil glycosylase inhibitor (UGI) that impedes uracil excision and downstream processes that decreaseDocket # 118737-WO-SEC-1 base editing efficiency and product purity; or (iv) nickase activity to cleave the non-edited DNA strand, followed by cellular DNA repair processes to replace the G-containing DNA strand.

[0119] In certain embodiments of the method for providing, conferring, or enhancing resistance of a plant to ASR, the method comprises crossing a first plant comprising a polynucleotide encoding a polypeptide described herein (e.g., SEQ ID NOs: 1-6) with a second different plant line and harvesting the seed produced thereby and generating a second-generation progeny plant. In certain embodiments, the generated plant comprises the polynucleotide and has resistance or increased resistance to ASR.

[0120] In certain embodiments, the second plant line is susceptible to ASR. In certain embodiments, the second plant line comprises a different ASR resistance polypeptide than the polypeptide expressed in the first plant line, such as, for example CcRpp1 and / or CcRpp2.

[0121] In certain embodiments, the method further comprises backcrossing the second- generation progeny plant to the second plant to produce a backcross progeny plant that comprises the polypeptide and produces backcrossed seed with ASR resistance.

[0122] Introgressing is sometimes called "backcrossing" when the process is repeated two or more times. In introgressing or backcrossing, the "donor" parent refers to the parental plant with the desired gene or locus to be introgressed. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. The initial cross gives rise to the Fl generation; the term "BC1 " then refers to the second use of the recurrent parent, and "BC2" refers to the third use of the recurrent parent, and so on.

[0123] The present disclosure provides a method for screening or assaying legume plants for resistance, immunity, or susceptibility to a plant disease. General methods for determination of resistance, immunity, or susceptibility of a plant to a particular pathogen are known to one skilled in the art. For example, a method for screening or assaying legume plants for resistance, immunity or susceptibility to a plant disease may comprise exposing a plant cell, tissue or organ (e.g., leaf) to a pathogen (e.g., Phakopsora pachyrhizi) and then determining and / or measuring in the exposed plant, the degree of resistance, immunity and / or susceptibility to a plant disease (e.g., ASR) caused by the pathogen. The method can further comprise measuring any observable plant disease symptoms on the plant exposed to the plant pathogen and then comparing the plant disease symptoms to a reference standard to determine the degree or extent of disease resistance.Docket # 118737-WO-SEC-1

[0124] Methods of exposing a plant cell, tissue or organ to a pathogen are known in the art. Methods of measuring, comparing, and determining the level of resistance, immunity and / or susceptibility (e.g., plant disease symptoms) to a disease, such as, for example, ASR, caused by the pathogen are also known in the art. The exposed plants can be further assessed to isolate polynucleotides, amino acid sequences and / or genetic markers that are associated with, linked to, and / or confer resistance, immunity or susceptibility of a plant to a particular pathogen or disease. Further assessments include, but are not limited to, isolating polynucleotides, nucleic acids, or amino acids sequences from the exposed plant, carrying out an assay of the isolated polynucleotides or nucleic acids, for example, to detect one or more biological or molecular markers associated with one or more agronomic characteristics or traits, including but not limited to, resistance, immunity and / or susceptibility. The information gleaned from such methods can be used, for example, in a breeding program.

[0125] In certain embodiments, the plants expressing the polynucleotides and polypeptides disclosed herein may also have one or more fungicides applied to the plants as a method of further preventing ASR associated damage to a legume crop species. These fungicidal compounds may also be applied to supplement the protection of a legume crop species comprising the NLR polynucleotides described herein to a wider variety of undesirable diseases. These fungicides may be formulated or tank-mixed with other fungicide(s) disclosed herein or applied sequentially with the other fungicide(s). Such fungicides may include 2- (thiocyanatomethylthio)-benzothiazole, 2-phenylphenol, 8-hydroxyquinoline sulfate, ametoctradin, aminopyrifen, amisulbrom, antimycin, Ampelomyces quisqualis, azaconazole, azoxystrobin, Bacillus subtilis, Bacillus subtilis strain QST713, benalaxyl, benomyl, benthiavalicarb-isopropyl, benzovindiflupyr, benzylaminobenzene-sulfonate (BABS) salt, bicarbonates, biphenyl, bismerthiazol, bitertanol, bixafen, blasticidin-S, borax, Bordeaux mixture, boscalid, bromuconazole, bupirimate, calcium polysulfide, captafol, captan, carbendazim, carboxin, carpropamid, carvone, chlazafenone, chloroinconazide, chloroneb, chlorothalonil, chlozolinate, Coniothyrium minitans, copper hydroxide, copper octanoate, copper oxychloride, copper sulfate, copper sulfate (tribasic), cuprous oxide, cyazofamid, cyflufenamid, cymoxanil, cyproconazole, cyprodinil, dazomet, debacarb, diammonium ethylenebis- (dithiocarbamate), dichlofluanid, dichlorophen, diclocymet, diclomezine, dichloran, diethofencarb, difenoconazole, difenzoquat ion, diflumetorim, dimethomorph, dimoxystrobin,Docket # 118737-WO-SEC-1 diniconazole, diniconazole-M, dinobuton, dinocap, diphenylamine, dithianon, dodemorph, dodemorph acetate, dodine, dodine free base, edifenphos, enestrobin, enestroburin, epoxiconazole, ethaboxam, ethoxyquin, etridiazole, famoxadone, fenamidone, fenarimol, fenbuconazole, fenfuram, fenhexamid, fenoxanil, fenpiclonil, fenpropidin, fenpropimorph, fenpyrazamine, fentin, fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazinam, fludioxonil, fluindapyr, flumorph, fluopicolide, fluopyram, fluoroimide, fluoxapiprolin, fluoxastrobin, fluquinconazole, flusilazole, flusulfamide, flutianil, flutolanil, flutriafol, fluxapyroxad, folpet, formaldehyde, fosetyl, fosetyl-aluminium, fuberidazole, furalaxyl, furametpyr, guazatine, guazatine acetates, GY-81, hexachlorobenzene, hexaconazole, hymexazol, imazalil, imazalil sulfate, imibenconazole, iminoctadine, iminoctadine triacetate, iminoctadine tris(albesilate), inpyrfluxam, iodocarb, ipconazole, ipfenpyrazolone, iprobenfos, iprodione, iprovalicarb, isofetamide, isoflucypram, isoprothiolane, isopyrazam, isotianil, kasugamycin, kasugamycin hydrochloride hydrate, kresoxim-methyl, laminarin, mancopper, mancozeb, mandipropamid, maneb, mefenoxam, mepanipyrim, mepronil, meptyl-dinocap, mercuric chloride, mercuric oxide, mercurous chloride, metalaxyl, metalaxyl-M, metam, metam- ammonium, metam-potassium, metam-sodium, metconazole, methasulfocarb, methyl iodide, methyl isothiocyanate, metiram, metominostrobin, metrafenone, mildiomycin, myclobutanil, nabam, nitrothal-isopropyl, nuarimol, octhilinone, ofurace, oleic acid (fatty acids), orysastrobin, oxadixyl, oxathiapiprolin, oxine-copper, oxpoconazole fumarate, oxycarboxin, pefurazoate, penconazole, pencycuron, penflufen, pentachlorophenol, pentachlorophenyl laurate, penthiopyrad, phenylmercury acetate, phosphonic acid, phthalide, picoxystrobin, polyoxin B, polyoxins, polyoxorim, potassium bicarbonate, potassium hydroxyquinoline sulfate, probenazole, prochloraz, procymidone, propamocarb, propamocarb hydrochloride, propiconazole, propineb, proquinazid, prothioconazole, pydiflumetofen, pyrametostrobin, pyraoxystrobin, pyraclostrobin, pyraziflumid, pyrazophos, pyribencarb, pyributicarb, pyrifenox, pyrimethanil, pyriofenone, pyroquilon, quinoclamine, quinoxyfen, quintozene, Reynoutria sachalinensis extract, sedaxane, silthiofam, simeconazole, sodium 2-phenylphenoxide, sodium bicarbonate, sodium pentachlorophenoxide, spiroxamine, sulfur, SYP-Z048, tar oils, tebuconazole, tebufloquin, tecnazene, tetraconazole, thiabendazole, thifluzamide, thiophanate- methyl, thiram, tiadinil, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazoxide, tricyclazole, tridemorph, trifloxystrobin, triflumizole, triforine, triticonazole, validamycin,Docket # 118737-WO-SEC-1 valifenalate, valiphenal, vinclozolin, zineb, ziram, zoxamide, Candida oleophila, Fusarium oxysporum, Gliocladium spp., Phlebiopsis gigantea, Streptomyces griseoviridis, Trichoderma spp., (RS)-N-(3,5-dichlorophenyl)-2-(methoxymethyl)-succinimide, 1,2-dichloropropane, 1,3- dichloro-1,1,3,3-tetrafluoroacetone hydrate, 1-chloro-2,4-dinitronaphthalene, 1-chloro-2- nitropropane, 2-(2-heptadecyl-2-imidazolin-1-yl)ethanol, 2,3-dihydro-5-phenyl-1,4-dithi-ine 1,1,4,4-tetraoxide, 2-methoxyethylmercury acetate, 2-methoxyethylmercury chloride, 2- methoxyethylmercury silicate, 3-(4-chlorophenyl)-5-methylrhodanine, 4-(2-nitroprop-1- enyl)phenyl thiocyanateme, ampropylfos, anilazine, azithiram, barium polysulfide, Bayer 32394, benodanil, benquinox, bentaluron, benzamacril, benzamacril-isobutyl, benzamorf, binapacryl, bis(methylmercury) sulfate, bis(tributyltin) oxide, buthiobate, cadmium calcium copper zinc chromate sulfate, carbamorph, CECA, chlobenthiazone, chloraniformethan, chlorfenazole, chlorquinox, climbazole, copper bis(3-phenylsalicylate), copper zinc chromate, coumoxystrobin, cufraneb, cupric hydrazinium sulfate, cuprobam, cyclafuramid, cypendazole, cyprofuram, decafentin, dichlobentiazox, dichlone, dichlozoline, diclobutrazol, dimethirimol, dinocton, dinosulfon, dinoterbon, dipymetitrone, dipyrithione, ditalimfos, dodicin, drazoxolon, EBP, enoxastrobin, ESBP, etaconazole, etem, ethirim, fenaminstrobin, fenaminosulf, fenapanil, fenitropan, fenpicoxamid, florylpicoxamid, flubeneteram, flufenoxystrobin, fluopimomide, fluotrimazole, furcarbanil, furconazole, furconazole-cis, furmecyclox, furophanate, glyodine, griseofulvin, halacrinate, Hercules 3944, hexylthiofos, ICIA0858, ipfentrifluconazole, ipflufenoquin, isopamphos, isovaledione, mandestrobin, mebenil, mecarbinzid, mefentrifluconazole, metazoxolon, methfuroxam, methylmercury dicyandiamide, metsulfovax, metyltetraprole, milneb, mucochloric anhydride, myclozolin, N-3,5-dichlorophenyl-succinimide, N-3-nitrophenylitaconimide, natamycin, N-ethylmercurio-4-toluenesulfonanilide, nickel bis(dimethyldithiocarbamate), OCH, phenylmercury dimethyldithiocarbamate, phenylmercury nitrate, phosdiphen, prothiocarb; prothiocarb hydrochloride, pyracarbolid, pyrapropoyne, pyridachlometyl, pyridinitril, pyrisoxazole, pyroxychlor, pyroxyfur, quinacetol; quinacetol sulfate, quinazamid, quinconazole, quinofumelin, rabenzazole, salicylanilide, SSF-109, sultropen, tecoram, thiadifluor, thicyofen, thiochlorfenphim, thiophanate, thioquinox, tioxymid, triamiphos, triarimol, triazbutil, trichlamide, triclopyricarb, triflumezopyrim, urbacid, zarilamid, (2S,3S)-3-(o-tolyl)butan-2-yl (4-methoxy-3-(propionyloxy)picolinoyl)-L-alaninate, and any combinations thereof.Docket # 118737-WO-SEC-1

[0126] Accordingly, provided is a method for preventing ASR associated damage to a legume crop species comprising planting a field with seed comprising at least one NLR polynucleotide encoding at least one NLR polypeptide described herein. In certain embodiments, the method further comprises treating the field with a fungicide.

[0127] Also provided herein is a method of identifying germplasm or plants comprising an NLR gene of the disclosure. The method comprises obtaining a nucleic acid sample from one or more plants, and contacting said nucleic acid sample with a nucleic acid sequence that specifically binds to a NLR polynucleotide of the disclosure and detecting the specific binding of the nucleic acid to its target sequence. For example, the method can detect the target sequence through the use of a labeled probe or by conducting a PCR reaction with suitable PCR primers that only produce an amplicon in the presence of the target sequence. In one embodiment the method comprises obtaining a nucleic acid sample from one or more plants, and contacting the nucleic acid sample with either:

[0128] i) a polynucleotide that comprises a sequence of at least 8 nucleotides that are identical or have at least 90-95% sequence identity to a contiguous sequence selected from the group consisting of SEQ ID NOs: 7-14, or complements thereof; wherein said method further comprises subjecting said sample and said polynucleotide to stringent hybridization conditions; and assaying said sample for hybridization of said polynucleotide to said DNA; or

[0129] ii) a pair of PCR primers, wherein a first and second PCR primer each specifically bind to a sequence selected from the group consisting of SEQ ID NOs: 7-14, wherein said first and second PCR primers are capable of producing an amplicon when bound to their target complementary sequences and subjected to standard PCR reaction conditions; subjecting said sample to polymerase chain reaction conditions; and assaying for an amplicon generated between said first and second primers.

[0130] As used herein, “stringent conditions” encompass conditions under which hybridization will only occur if there is less than 20% mismatch between the hybridization molecule and a sequence within the target nucleic acid molecule. “Stringent conditions” include further particular levels of stringency. Thus, as used herein, “moderate stringency” conditions are those under which molecules with more than 20% sequence mismatch will not hybridize; conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize; and conditions of “very high stringency” are those under which sequences with moreDocket # 118737-WO-SEC-1 than 5% mismatch will not hybridize. The following are representative, non-limiting hybridization conditions.

[0131] High Stringency condition (detects sequences that share at least 90% sequence identity): Hybridization in 5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 16 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 15 minutes each; and wash twice in 0.5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 20 minutes each.

[0132] Moderate Stringency condition (detects sequences that share at least 80% sequence identity): Hybridization in 5x-6x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65-70 °C for 16-20 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 5-20 minutes each; and wash twice in 1x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 55-70 °C for 30 minutes each.

[0133] Non-stringent control condition (sequences that share at least 50% sequence identity will hybridize): Hybridization in 6x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature to 55 °C for 16-20 hours; wash at least twice in 2x-3x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature to 55 °C for 20-30 minutes each.

[0134] As used herein, the term "germplasm" refers to genetic material of, or from, an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety, or family), or a clone derived from a line, variety, species, or culture. The germplasm can be part of an organism or cell or can be separate from the organism or cell. The germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. Germplasm in the context of the present disclosure includes cells, seed or tissues from which new plants can be grown, or plant parts, such as leaves, stems, pollen, or cells, that can be cultured into a whole plant.Docket # 118737-WO-SEC-1

[0135] In certain embodiments, the germplasm for use in the method of identifying germplasm comprising an NLR of the disclosure may be from any plant. In certain embodiments the germplasm is from a nonhost resistant plant or a legume crop species from a genus selected from the group consisting of Glycine, Vigna, and Lablab.

[0136] In certain embodiments, the legume crop species or legume-derived gene is derived from the genus Glycine. Examples of Glycine species include, but are not limited to, Glycine arenaria, Glycine argyrea, Glycine cyrtoloba, Glycine canescens, Glycine clandestine, Glycine curvata, Glycine falcata, Glycine latifolia, Glycine microphylla, Glycine pescadrensis, Glycine stenophita, Glycine syndetica, Glycine soja, Glycine tabacina and Glycine tomentella.

[0137] In certain embodiments, the legume crop species or legume-derived gene is derived from the genus Vigna. Vigna is a pantropic genus that comprises approximately 100 species. It is a taxonomic group subdivided into the subgenera Vigna, Haydonia, Plectotropis (African), Ceratotropis (Asian), Sigmoidotropis , and Lasiopron. The genus includes economically relevant species such as Vigna unguiculata (L.) Walp (cowpea), Vigna radiata (L.) Wilczek (mung bean), Vigna angularis (Willd.) Ohwi and Ohashi (azuki bean), Vigna mungo (L.) Hepper (black gram), and Vigna umbellata (Thunb.) Ohwi and Ohashi (rice bean). Four subspecies are recognized within Vigna unguiculata: dendtiana, a wild relative of cultivated subspecies; cylindrica, cultivated catjang; sesquipedalis, cultivated yardlong bean; and unguiculata, cultivated black- eyed pea. Vigna unguiculata ssp. unguiculata is further divided into cultivar groups Unguiculata, grown as a pulse; Biflora or Cilindrica (catjang), mainly used as a forage; Sesquipedalis (yardlong or asparagus bean), grown as a vegetable; Textilis, cultivated for the fibres of its long floral peduncles; and Melanophthalmus (black-eyed pea). Susceptibility of several Vigna species, including Vigna radiata, Vigna mungo and Vigna unguiculata to Phakopsora pachyrhizi has been reported under field and greenhouse conditions.

[0138] In certain embodiments, the legume crop species or legume-derived gene is derived from the genus Lablab. Lablab purpureus (L.) Sweet is a leguminous species (Verdcourt (1971) Flora of Tropical East Africa, pp.696-699, Crown Agents, London, UK; and Duke et al. (1981) Handbook of Legumes of World Economic Importance, pp.102-106, Plenum Press, New York, USA and London, UK) native to Asia and Africa (Pengelly and Maass, (2001) Gen. resour. crop ev.48: 261-272). It is commonly known as lablab bean, hyacinth bean, bonavist bean, field bean, Egyptian bean, poor man's bean, Tonga bean (English) and by at least 20 additional vernacularDocket # 118737-WO-SEC-1 names. It is grown in Africa, Asia, and the Caribbean as either a pulse crop or as a green vegetable (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. resour. crop ev.48: 261-272).

[0139] Lablab purpureas has been reported as an alternative host for Phakopsora pachyrhizi (Perez-Hernandez, (2007) Alternative hosts of Phakopsora pachyrhizi in the Americas: An analysis of their role in the epidemiology of Asian soybean rust in the continental U.S. M.Sc. thesis. Iowa State University. Ames, Iowa. U.S.A.; Vakili (1981) Plant Dis.65: 817-819; and Poonpolgul and Surin, (1980) Soybean Rust Newsletter, 3: 30-31).

[0140] In certain embodiments, the legume crop species or legume-derived gene is derived from the genus Cicer, Cajanus, Medicago, Phaseolus, Pisum, Pueraria, or Trifolium. Examples of Cicer species include, but are not limited to, Cicer arietinum, Cicer echinospermum, Cicer reticulatum and Cicer pinnatifldum. An example of the Cajanus species include, but is not limited to, Cajanus cajan. Examples of the Medicago species include, but are not limited to, Medicago truncatula and Medicago sativa. Examples of the Phaseolus species include, but are not limited to, Phaseolus vulgaris, Phaseolus lunatus, Phaseolus acutifolius and Phaseolus coccineus. Examples of the Pisum species include, but are not limited to, Pisum abyssinicum, Pisum sativum, Pisum elatius, Pisum fulvum, Pisum transcaucasium and Pisum humile. An example of the Pueraria species includes, but is not limited to, Pueraria lobata. Examples of the Trifolium species include, but are not limited to, Trifolium aureum and Trifolium occidentale.

[0141] In certain embodiments, the method comprises identifying a legume crop species comprising the desired NLR gene of the present disclosure and introgressing the NLR gene into a different plant. For example, a different plant of the same legume crop species or a plant of a different legume crop species.

[0142] The present disclosure also includes kits for the assays described herein. The polypeptide sequences and polynucleotides can be packaged as a component of a kit with instructions for completing the assay disclosed herein. The kits of the present disclosure can include any combination of the polypeptides and / or polynucleotides described herein and suitable instructions (written and / or provided as audio-, visual-, or audiovisual material). In one embodiment, the kit relates to a DNA detection kit for identifying NLR genes against ASR. Kits utilizing any of the sequences disclosed herein for the identification of a transgenic event in aDocket # 118737-WO-SEC-1 plant for efficacy against ASR are provided. For example, the kits can comprise a specific probe having a sequence corresponding to, or complementary to, a sequence having between 80% and 100% sequence identity with a specific region of the transgenic event. The kits can include any reagents and materials required to carry out the assay or detection method.

[0143] The following are examples of specific embodiments of some aspects of the invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way.Docket # 118737-WO-SEC-1 EXAMPLE 1

[0144] This example demonstrates the identification of an ASR resistant plant.

[0145] Tepary bean (Phaseolus acutifolius) accessions were screened against Phakopsora pachyrhizi for resistance in a controlled environments bioassay. Plants were inoculated at the seedling stage and phenotyped for disease reaction 14 days post-inoculation. PI 535221 was identified as resistant, and PI 321637 was identified as susceptible. EXAMPLE 2

[0146] This example demonstrates map-based cloning to identify ASR resistance QTLs.

[0147] The resistant line, PI 535221, and the susceptible line, PI 321637, were used to generate an F2 population for map-based cloning using phenotypes assessed in the controlled environments assay described in Example 1.187 markers spanning the whole genome were selected for the initial QTL mapping. Markers were developed by doing RNA-seq on both PI 535221 and PI 321637, aligning the reads to a publicly available Tepary bean genome to get an approximate genomic location, and looking for SNPs between the two lines.96 F2 individuals were used for the initial mapping. Of the 96 individuals, 71 individuals had a resistant phenotype, and 25 individuals had a susceptible phenotype, matching the segregation ratios expected based on the segregation of a single, dominant locus (Table 2). Table 2: Segregation Ratios of the Generated F2 Population GE Name Note # of Resistant # of Susceptible Pl Pl

[0148] After genotyping with KASP SNP markers, a single, strong effect QTL was identified at the beginning of chromosome 8, to the left of the marker, TB_A. An additional 205 F2 individuals were grown, for a total of 301 F2s, and additional markers were added within the QTL interval. The QTL interval was narrowed to a region between the markers TB_B and TB_C. The genomes of the resistant and susceptible parents were sequenced. Based on genome comparison, additional SNPs were identified. Running these additional SNPs on the F2Docket # 118737-WO-SEC-1 individuals delimited the interval to between the markers TB_D and TB_E. This represents an interval size of 73,121bp in the resistant parent, PI 535221 and 27,122bp in the susceptible parent, PI 321637. An additional marker, TB_F, was run, however, it was co-segregating with the phenotype and did not help to narrow the interval.

[0149] To try to narrow the interval further, 513 F3 individuals were grown. Multiple individuals were found supporting a new left flanking marker of TB_F. No new individuals were found supporting the right flanking marker of TB_E and the region was not narrowed from the right side. The final fine-mapped interval between TB_F and TB_E is 64,657bp in the resistant parent and 18,660bp in the susceptible parent. EXAMPLE 3

[0150] This example demonstrates the identification of candidate ASR resistance genes.

[0151] Paired-end RNA-seq and PacBio Iso-seq libraries were constructed from RNA extracted from PI535221 and then aligned to the PI535221 genome using HISAT2 and MiniMap2, respectively. StringTie2 was then employed to assemble gene models based on aligned reads. Augustus was also used to predict gene models and its results were merged with alignment-based assemblies using StringTie2 to create the final gene annotation set.

[0152] The interval was found to contain a total of six genes, four of which are canonical resistance genes (NLRs). One NLR was significantly expressed in the RNA-seq data and produces multiple isoforms, one isoform of which was predicted to encode the fully functional NLR with all associated domains (coiled-coil, NB-ARC and LRRs). Stable expression of the isoform was driven by the constitutive H2B promoter in soy transgenic plants, along with all other transcripts predicted to encode full or partial NLR proteins. EXAMPLE 4

[0153] This example demonstrates expression of the identified tepary bean ASR resistance gene in soybean.

[0154] T0 plants expressing the isoform encoding SEQ ID NO: 1 were generated and selfed to generate T1 seed for bioassay screening.32 segregating T1 seeds were planted per event for bioassay testing. Plants were sampled for copy number and expression analysis prior to inoculation. Plants were then inoculated in controlled environments with P. pachyrhizi andDocket # 118737-WO-SEC-1 scored 14 days post inoculation for lesion type and sporulation level. Three events encoding SEQ ID NO: 1 (i.e., PaRpp1), referred to as, GMER6Z.001.5A, GMER6Z.001.20A, GMER6Z.001.28A, were positively expressing and exhibited resistant phenotypes (Table 3). Table 3: Phenotype of Soybean Plants Expressing a Tepary Bean ASR Resistance Gene Number Numbe Event / Copy Number of total Lesion r of plants by T Sporulation LevelEXAMPLE 5

[0155] This example demonstrates the identification of an ASR resistant plant.

[0156] Tepary bean (Phaseolus acutifolius) accessions were screened against Phakopsora pachyrhizi for resistance in a controlled environments bioassay. Plants were inoculated at the seedling stage and phenotyped for disease reaction 14 days post-inoculation. W624877 was identified as resistant, and PI 440789 was identified as susceptible. EXAMPLE 6

[0157] This example demonstrates map-based cloning to identify ASR resistance QTLs.

[0158] The resistant line, W624877, and the susceptible line, PI 440789, were used to generate an F2 population for map-based cloning using phenotypes assessed in the controlled environments assay described in Example 5.186 markers spanning the whole genome were selected for the initial QTL mapping. Markers were developed by doing RNA-seq on both W624877 and PI 440789, aligning the reads to a publicly available Tepary bean genome to get an approximate genomic location, and looking for SNPs between the two lines. 92 F2Docket # 118737-WO-SEC-1 individuals were used for the initial mapping. Of the 92 individuals, 70 had a resistant phenotype, and 22 individuals had a susceptible phenotype, matching the segregation ratios expected based on the segregation of a single, dominant locus (Table 4). Table 4: Segregation Ratios of the Generated F2 Population GE Name Note # of Resistant # of Susceptible Plants Plants[ ] er genoyp ng w mar ers, a s ng e, s rong e ec Q was identified at the beginning of chromosome 8, to the left of the marker, 08:03059935, and to the right of the marker, 08:00911298. An additional 193 F2 individuals were grown, for a total of 285 F2s, and additional markers were added within the QTL interval. The QTL interval was narrowed to a region between the markers Chr08:01807772 and Chr08:01962953, which were markers used for another Tepary bean QTL mapping project and were identified by an RNA- seq comparison of PI 535221 and PI 321637. A lack of useful recombination events prevented narrowing the interval further.

[0160] To try to narrow the interval further, 438 additional F2 individuals were grown. Using these individuals, the QTL region was narrowed to the interval between the markers, Chr08:01807772 and Chr08:01908870. The genomes of the resistant parent, W624877, and susceptible parent, PI 440789, were sequenced. Based on genome comparison, additional SNPs were identified. Running these additional SNPs on the useful recombinants from all the individuals used for mapping delimited the interval to between TB2_02 and TB2_12. This represents an interval size of 98,227bp in the resistant parent, W624877, and 53,264bp in the susceptible parent, PI440789. Several additional markers within the interval were run, however they were all co-segregating with the phenotype and did not help to narrow the interval. EXAMPLE 7

[0161] This example demonstrates the identification of a candidate ASR resistance gene.

[0162] Paired-end RNA-seq and PacBio libraries were constructed and aligned to the TeparyDocket # 118737-WO-SEC-1 bean donor genome using HISAT2 and MiniMap2, respectively. StringTie2 was then employed to assemble gene models based on aligned reads. Augustus was used to predict gene models and its results were merged with alignment-based assemblies using StringTie2 to create the final gene annotation set.

[0163] The interval was found to contain a total of nine genes, four of which are canonical resistance genes (NLRs). One NLR encoding SEQ ID NO: 6 was found to be significantly expressed in the RNA-seq data. EXAMPLE 8

[0164] This example demonstrates expression of the identified tepary bean ASR resistance gene in soybean.

[0165] T0 plants expressing the NLR found to be significantly expressed in Example 7 were generated and selfed to generate T1 seed for bioassay screening.32 segregating T1 seeds were planted per event for bioassay testing. Plants were sampled for copy number and expression analysis prior to inoculation. Plants were then inoculated in controlled environments with P. pachyrhizi and scored 14 days post inoculation for lesion type and sporulation level.

[0166] Four events encoding SEQ ID NO: 6 (i.e., PaRpp2), referred to as, GMKN7V.001.1A, GMKN7V.001.8A, GMKN7V.001.16A, and GMKN7V.001.21A were positively expressing and exhibited resistant phenotypes (Table 5). Table 5: Phenotype of Soybean Plants Expressing a Tepary Bean ASR Resistance Gene Event / Copy Number Number Lesion Number of plants byDocket # 118737-WO-SEC-1 GMKN7V.001.21A – 1 copy 14 RB 14 0 0 GMKN7V.001.21A - Null 10 T 0 1 9

[0167] All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.

[0168] Unless defined otherwise, 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 belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.

[0169] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0170] Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

Claims

Docket # 118737-WO-SEC-1 We claim:

1. A polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-6, wherein said polypeptide when expressed in the cells of a plant confers resistance to Asian soybean rust (ASR) disease.

2. The polynucleotide of claim 1, wherein the encoded polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1 or 6.

3. The polynucleotide of claim 1, wherein the polynucleotide comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 7-14.

4. The polynucleotide of any one of claims 1-3, further comprising a heterologous regulatory sequence operably linked to the polynucleotide.

5. The polynucleotide of claim 4, wherein the heterologous regulatory sequence is a promoter operable in a plant.

6. A polypeptide encoded by the polynucleotide of any one of claims 1-5.

7. The polypeptide of claim 6, wherein the polypeptide is tagged with a detectable marker.

8. A nucleic acid construct comprising the polynucleotide of any one of claims 1-5.

9. A host cell comprising the polynucleotide of any one of claims 1-5 or the nucleic acid construct of claim 8.

10. A transgenic plant cell comprising the polynucleotide of any one of claims 1-5 or the nucleic acid construct of claim 8.

11. A plant or plant part comprising the transgenic plant cell of claim 10.

12. The plant or plant part of claim 11, wherein the plant part is a seed.

13. The plant or plant part of claim 11 or 12, wherein the plant is a legume crop plant.

14. The plant or plant part of claim 13, wherein the legume crop plant is selected from alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut and tamarind.

15. The plant or plant part of claim 13, wherein the legume crop plant is soybean.Docket # 118737-WO-SEC-1 16. A plant cell comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-6 operably linked to a heterologous regulatory element.

17. The plant cell of claim 16, wherein the encoded polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1 or 6.

18. The plant cell of claim 16, wherein the plant cell comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 operably linked to a first regulatory element and a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5 operably linked to a second regulatory element, wherein the first regulatory element, the second regulatory element, or both is a heterologous regulatory element.

19. The plant cell of claim 18, wherein the heterologous regulatory element is a heterologous promoter.

20. A plant or plant part comprising the plant cell of any one of claims 16-19, wherein the plant has increased resistance to Asian soybean rust (ASR) as compared to a control plant not comprising the polynucleotide.

21. The plant or plant part of claim 20, wherein the plant is a legume crop plant.

22. The plant or plant part of claim 21, wherein the legume crop plant is selected from the group consisting of alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut and tamarind.

23. The plant or plant part of claim 21, wherein the legume crop plant is soybean.

24. A seed comprising the plant cell of any one of claims 16-19.

25. A method of detecting an ASR resistance polypeptide encoding nucleic acid present in plant tissues, said method comprising obtaining a nucleic acid sample from said plant tissues; and i) contacting said nucleic acid sample with a polynucleotide that comprises a sequence of at least 8 nucleotides that are identical to a contiguous sequence of any one of SEQ ID NOs: 7-14, or complements thereof;Docket # 118737-WO-SEC-1 subjecting said sample and said polynucleotide to stringent hybridization conditions; and assaying said sample for hybridization of said polynucleotide to said DNA; or ii) contacting said nucleic acid sample with a first and second PCR primer, wherein said first and second PCR primer each specifically bind to any one of SEQ ID NOs: 7-14; subjecting said sample to polymerase chain reaction; and assaying for an amplicon generated between said first and second primers.

26. A method for conferring resistance to Asian soybean rust (ASR) in a legume crop species, the method comprising introducing into a regenerable plant cell of a legume crop species a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-6; and generating a plant from the plant cell, wherein the plant comprises the polynucleotide and has increased resistance to ASR as compared to a control plant not comprising the polynucleotide.

27. The method of claim 26, wherein the polynucleotide is introduced into the regenerable plant cell using a nucleic acid construct comprising the polynucleotide operably linked to a regulatory element.

28. The method of claim 27, wherein the regulatory element is a heterologous regulatory element.

29. The method of claim 28, wherein the heterologous regulatory element is a promoter.

30. The method of claim 26, wherein the polynucleotide is introduced into the regenerable plant cell using genome editing technologies.

31. The method of claim 30, wherein the genome editing technology uses an enzyme selected from the group consisting of a polynucleotide-guided endonuclease, CRISPR-Cas endonucleases, base editing deaminases, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) and engineered site-specific endonucleases.

32. The method of any one of claims 26-31, wherein the legume crop species is selected from the group consisting of alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut and tamarind.

33. The method of claim 32, wherein the legume crop species is soybean.Docket # 118737-WO-SEC-1 34. The method of any one of claims 26-33, wherein the ASR is caused by a plant pathogen selected from the group consisting of Phakopsora pachyrhizi or Phakopsora meibomiae, or a combination thereof.

35. A method for producing Asian soybean rust (ASR) resistant plants, the method comprising crossing a first plant comprising a polynucleotide of any one of claims 1-6 with a second plant, harvesting the seed, isolating harvested seeds comprising the polynucleotide and growing the isolated seeds to produce the resistant plant, wherein the resistant plant comprises the polynucleotide.

36. A method of preventing ASR associated damage to a legume crop species, the method comprising planting a field with the seed of claim 24.

37. The method of claim 36, wherein the field is treated with a fungicide.