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 108283_SequenceListing created on August 7, 2023 and having a size of 763,782 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 identifying genes that enhance resistance to the causal agent of Asian soybean rust (ASR) in legume plants. The disclosure further relates to effector polypeptides capable of interacting with the polypeptides encoded by the genes enhancing ASR resistance, methods of using these polypeptide sequences to isolate the polypeptides encoded by the genes enhancing ASR resistance, and methods for selecting plants to grow in an area of cultivation.

BACKGROUND

[0003] Soybean diseases are a major threat for soybean production, resulting in yield losses and decrease 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 identifying ASR resistance genes. This disclosure provides such compositions and methods.

SUMMARY

[0005] Provided are nucleic acid constructs and expression cassettes comprising a polynucleotide encoding an effector polypeptide 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% or more sequence identity to any one of SEQ ID NOs: 1-8 or 10-309, or a functional fragment thereof, operably linked to a regulatory element. In certain embodiments, the at least one regulatory sequence is a promoter (e.g., heterologous promoter).

[0006] Also provided are chimeric polypeptides comprising an effector 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% or more sequence identity to any one of SEQ ID NOs: 1-8 or 10-309, or a functional fragment thereof, operably linked to a heterologous peptide (e.g., protein tag). Further provided are polynucleotides encoding the chimeric polypeptides and nucleic acid constructs and expression cassettes comprising the polynucleotides encoding the chimeric polypeptides.

[0007] Also provided are host cells (e.g., bacterial cell, fungal cell, plant cell) comprising any of the polynucleotides (e.g., isolated polynucleotides, recombinant polynucleotides, nucleic acid constructs, expression cassettes) encoding effector polypeptides, functional fragments thereof, or chimeric polypeptides described herein.

[0008] Provided are methods for identifying CcRppl-like proteins comprising identifying one or more polypeptides that bind to a chimeric polypeptide comprising an effector 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% or more sequence identity to any one of SEQ ID NOs: 1-8, or a functional fragment thereof, operably linked to a heterologous peptide (e.g., protein tag). In certain embodiments, the method further comprises expressing in a host cell the chimeric polypeptide and the one or more identified interacting polypeptides and assaying the host cell for the presence of HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof. [0009] Also provided are methods for identifying CcRppl-like proteins comprising identifying one or more polypeptides that bind to an effector polypeptide 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% or more sequence identity to any one of SEQ ID NOs: 1-8, or a functional fragment thereof. In certain embodiments, the method further comprises expressing in a host cell the effector polypeptide or functional fragment thereof and the one or more identified interacting polypeptides and assaying the host cell for the presence of HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof. [0010] Further provided are methods for selecting a plant for growing in an area of cultivation comprising providing a sample comprising a plurality of Phakopsora pachyrhizi isolates from an area of cultivation, assaying for the presence of a polynucleotide comprising a nucleotide 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% or more sequence identity to any one of SEQ ID NOs: 1-8, and selecting a plant expressing a CcRppl polypeptide, a CcRppl -like polypeptide or a combination thereof for growing in the area of cultivation when the polynucleotide is present.

[0011] Also provide are methods for identifying ASR26 alleles comprising providing a population comprising a plurality of Phakopsora pachyrhizi isolates and detecting in the population ASR26 alleles. In certain embodiments, the method further comprises expressing the identified ASR26 allele and a CcRPPl polypeptide, a CcRPPl -like polypeptide or a combination thereof in a host cell and assaying the host cell for the presence of HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof.

[0012] Provided are methods for identifying ASR resistance polypeptides that do not compete with CcRppl comprising identifying one or more polypeptides that bind to an effector polypeptide 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% or more sequence identity to any one of SEQ ID NOs: 10-309, or a functional fragment thereof, assaying the one or more identified polypeptides for interaction with a polypeptide 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% or more sequence identity to any one of SEQ ID NOs: 1-8, and selecting the one or more polypeptides that do not interact with the polypeptide 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% or more sequence identity to any one of SEQ ID NOs: 1-8.

[0013] Further provided are methods for detecting a soluble CcRppl protein in a sample comprising incubating a mixture, the mixture comprising an ASR26 polypeptide 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% sequence identity to any one of SEQ ID NOs: 1-8 and a sample comprising a CcRppl protein, isolating the ASR26 polypeptide and polypeptides bound thereto from the mixture, and detecting in the isolated ASR26 polypeptide and polypeptides bound thereto the presence of a CcRppl protein, thereby detecting a soluble CcRppl protein.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0014] 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

[0015] The present disclosure provides compositions and methods for using effector proteins for identifying and selecting Asian soybean rust (ASR) resistance genes. The methods involve expressing in host cells nucleic acid sequences encoding effector proteins from Phcikopsora pachyrhizi and Phakopsora meibomiae and identifying interacting polypeptides or incubating isolated and/or chimeric effector proteins and identifying interacting polypeptides. The methods also involve selecting plants for growing in an area of cultivation by determining the presence of specific ASR effectors in the area of cultivation.

[0016] 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. One such NLR protein, CcRppl (U.S. Patent 10,842,097), has been shown to provide robust resistance to ASR when expressed in a plant. The identification of the effector protein recognized by CcRppl allows for the identification of CcRppl -like proteins that may share a similar or the same mode of action, as well as the identity of non-competing ASR resistance genes that have a different mode of action (e.g., recognizes a different effector) than CcRppl.

[0017] Accordingly, one aspect of the disclosure provides effector-encoding polynucleotides (e.g., isolated polynucleotides and recombinant polynucleotides) encoding effector polypeptides (e.g., isolated polypeptides and recombinant polypeptides) comprising, consisting essentially of, or consisting of 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% or more sequence identity to any one of SEQ ID NOs: 1-8 and 10-309, or functional fragments thereof In certain embodiments, the effector-encoding polynucleotide comprises a nucleotide 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% or more sequence identity to any one of SEQ ID NOs: 310-317 and 319-618. [0018] 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.

[0019] 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.

[0020] 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 (optimally protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. 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 (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, “isolated” or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the recombinant nucleic acid molecules encoding the effector 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.

[0021] 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 achieve 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 x lOO).

[0022] 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).

[0023] As used herein, "functional fragment," "fragment that is functionally equivalent," "functionally equivalent fragment" and the like refer to a portion or subsequence of a polypeptide sequence of the present disclosure in which its native ability is retained. For example, a functional fragment of an effector polypeptide, such as those described herein, maintains the ability to be recognized by and bind to an NLR protein. Accordingly, in certain embodiments, a functional fragment of an ASR26 effector polypeptide (e.g., SEQ ID NOs: 1-8) binds to the CcRppl polypeptide sequence of SEQ ID NO: 9. In certain embodiments, the functional fragments of the polypeptides described herein comprises an N-terminal and/or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more amino acids. [0024] Functional fragments of a particular polypeptide of the embodiments (i.e., the reference polypeptide) can also be evaluated by comparison of the percent sequence identity between the polypeptide and the reference polypeptide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs known in the art.

[0025] Functional fragments of a native protein of the embodiments can have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs known in the art. A functional fragment 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.

[0026] The polynucleotides described herewith can be used to isolate corresponding sequences from other organisms, particularly other Phakopsora pachyrhizi and Phakopsora meibomiae. In this manner, methods such as PCR or hybridization can be used to identify such sequences based on their sequence identity to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire sequences set forth herein or to variants and fragments thereof are encompassed by the present disclosure. Such sequences include sequences that are orthologs or alleles 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. The term “allele” refers to a variation of a nucleotide sequence (e.g., SNP) that encodes the synthesis of a gene product at the same place on a DNA molecule. 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 an effector protein that hybridize to the sequences disclosed herein, or functional fragments thereof, are encompassed by the present disclosure.

[0027] As used herein “encoding,” “encoded,” or the like, with respect to a specified nucleic acid, is meant 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 in 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 a!.. (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.

[0028] 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, etal., (1989) Nucleic Acids Res. 17:477-98 and herein incorporated by reference).

[0029] In certain embodiments the effector-encoding polynucleotide encodes a CcRppl effector polypeptide, also referred to herein as ASR26, 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% or more sequence identity to any one of SEQ ID NOs: 1-8, or a functional fragment thereof. In certain embodiments, the CcRppl effector-encoding polynucleotide comprises a nucleotide 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% or more sequence identity to any one of SEQ ID NOs: 310-317.

[0030] In certain embodiments, the polynucleotide encoding the 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 one or more of the effector polypeptides disclosed herein. In certain embodiments, the heterologous regulatory element operably linked to the polynucleotide is heterologous to the host cell. [0031] 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, 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.

[0032] 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 of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest. 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.

[0033] 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 it 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. [0034] 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 ofRNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, etal., (\99G)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. [0035] 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 may function to bind transcription factors, which are trans-acting polypeptides that regulate transcription.

[0036] 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 ¹ 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.

[0037] As used herein “promoter” refers to a region of DNA upstream from the start of transcription and 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, pathogeninducible 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 WO 99/43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter; rice actin; ubiquitin; pEMU; MAS; ALS; and the like. Other constitutive promoters include, for example, those disclosed in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6, 177,611, which are known in the art, and can be contemplated for use in the present disclosure. [0038] 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-1, 3-glucanase, chitinase, etc.

[0039] 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 woundinducible promoters include potato proteinase inhibitor (pin II) gene, wunl and wun2, winl and win2, systemin, WIP1, MPI gene, and the like.

[0040] 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).

[0041] 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. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2): 255 -265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803 ; Hansen et al. (1997) Mol. Gen Genet. 254(3): 337-343 ; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol.

112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (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.

[0042] Leaf-specific promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2)255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0043] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seedgerminating" 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, herein incorporated by reference). 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 P-phaseolin, napin, P-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.

[0044] Also provided is a nucleic acid (e.g., DNA) construct comprising any of the effectorencoding polynucleotides described herein. The use of the term “nucleic acid construct”, "nucleotide constructs" or the like 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 Such 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.

[0045] In certain embodiments, the effector-encoding 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 host of interest or any plant of interest. The cassette can include 5' and 3' regulatory sequences operably linked to an effector-encoding 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 effector-encoding polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0046] The expression cassette can include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (e.g., a promoter), an effector-encoding polynucleotide, 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 effector-encoding polynucleotide may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the effector-encoding polynucleotide may be heterologous to the host cell or to each other.

[0047] 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 effector-encoding polynucleotide, the plant host, or any combination thereof. [0048] 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. [0049] 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 as 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.

[0050] 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.

[0051] Further provided are host cells comprising at least one of the effector-encoding polynucleotide sequences, nucleic acid constructs, or expression cassettes described herein, so that the host cell expresses any of the effector polypeptides described herein. In certain embodiments, the host cell is a bacterial cell, a fungal cell, or a plant cell. In certain embodiments, the plant cell is a plant protoplast. In certain embodiments, the plant cell or plant protoplast is a tobacco cell, tobacco protoplast, Arabidopsis cell, Arabidopsis protoplast, soybean cell or soybean protoplast. In certain embodiments, the nucleic acid construct or expression cassette, described herein, is expressed in the host cell. In certain embodiments, the host cell further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50, or more) candidate ASR resistance genes. In certain embodiments, the candidate ASR resistance gene is introduced into the host cell using a nucleic acid construct comprising the candidate ASR resistance gene operably linked to a regulatory element, such as, for example a heterologous promoter.

[0052] As used herein, “introduced”, “introducing” or the like is intended to mean presenting to the host cell the inventive polynucleotide or resulting polypeptide in such a manner that the sequence gains access to the interior of the cell. The methods of the disclosure do not depend on a particular method for introducing a sequence into a host cell, only that the polynucleotide or polypeptide gains access to the interior of the cell.

[0053] In certain embodiments, the polynucleotides described herein are transiently expressed in the host cell using a transient transformation technique. In certain embodiments, the polynucleotides are stably expressed in the host cell 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 are introduced into the plant, plant cell, plant part, seed or grain using a genome editing technique.

[0054] "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.

[0055] Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into host cells may include but are not limited to microinjection, electroporation, Agrobacterium-mediated transformation, Ochrobacterium-mediated transformation, direct gene transfer, viral mediated, ballistic particle acceleration, heat shock, and lipid-mediated transfection. The type of transformation or transfection method for use with the compositions and method described herein may vary depending on the type of host cell, e.g., bacterial cell, fungal cell, or plant cell, targeted for transformation.

[0056] Also provided herein are chimeric polypeptides and polynucleotides encoding chimeric polypeptides 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% or more sequence identity to any one of SEQ ID NOs: 1-8 and 10-309, or functional fragments thereof, operably linked to a heterologous peptide. In certain embodiments, the polynucleotide encoding the chimeric polypeptide is in a nucleic acid construct comprising the polynucleotide operably linked to a regulatory element, such as, for example, a heterologous promoter. In certain embodiments, the chimeric polypeptide binds to a CcRppl polypeptide sequence comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 9. In certain embodiments, the chimeric polypeptide comprises 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% or more sequence identity to any one of SEQ ID NOs: 1-8, or functional fragments thereof

[0057] In certain embodiments, the heterologous peptide comprises a protein tag. As used herein, a “protein tag” includes, but is not limited to, affinity tags, solubilization tags, chromatography tags, epitope tags, and fluorescent tags. The location of the protein tag in the chimeric polypeptide is not particularly limited so long as the chimeric polypeptide maintains binding to the CcRppl polypeptide or corresponding resistance polypeptide, such that in certain embodiments the protein tag may be operably linked to the N-terminus, C-terminus, or both the N- and C-terminus of the amino acid sequence. In certain embodiments, the protein tag is a FLAG tag (e.g., 3X-FLAG), a myc tag, a His tag, an MBP tag, a glutathione-s-transferase tag, an HA tag, or an Fc tag.

[0058] Also provided herein are methods for identifying CcRppl-like proteins by identifying one or more polypeptides that bind to a chimeric effector polypeptide or an effector polypeptide or functional fragment thereof described herein such as those 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% or more sequence identity to any one of SEQ ID NOs: 1-8.

[0059] As used herein a “CcRppl -like” protein refers to a protein that has the same or similar biological activity to CcRppl but does not share 100% sequence identity with CcRppl (i.e., SEQ ID NO: 9) over the full length of the protein. In certain embodiments, the CcRppl -like protein binds to at least one of the ASR26 effector protein alleles described herein (e.g., SEQ ID NOs: 1- 8).

[0060] In certain embodiments, the method comprises providing a population of polypeptides from a plant cell, optionally an ASR resistant plant cell, incubating the population of polypeptides with at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more) effector polypeptide, or functional fragment thereof, described herein (e.g., SEQ ID NOs: 1-8) or at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more) chimeric polypeptide described herein or a combination of at least one effector polypeptide and at least one chimeric polypeptide, isolating the at least one effector polypeptide and/or the at least one chimeric polypeptide along with polypeptides interacting therewith from the population, and identifying polypeptides interacting with the effector polypeptide or chimeric polypeptide.

[0061] In certain embodiments, the method comprises providing a population comprising a plurality of R-gene polypeptides, incubating the population of R-gene polypeptides with at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more) effector polypeptide described herein (e.g., SEQ ID NOs: 1-8), or functional fragment thereof, or at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more) chimeric polypeptide described herein or a combination of at least one effector polypeptide and at least one chimeric polypeptide, isolating the at least one effector polypeptide and/or the at least one chimeric polypeptide along with R-gene polypeptides interacting therewith from the population, and identifying the R-gene polypeptides interacting with the effector polypeptide or chimeric polypeptide.

[0062] In certain embodiments of the methods described herein, the incubation is performed under conditions that allow for binding of the at least one chimeric polypeptide or the at least one effector polypeptide, or functional fragment thereof, to one or more polypeptides of the population. Those of ordinary skill in the art will recognize the conditions that can be manipulated to allow for, or improve, the capture of protein-protein interactions. For example, conditions that can be altered include, but are not limited to, buffer composition, pH, incubation time, and temperature. In certain embodiments, the incubation is performed in the presence of a crosslinker. The type of crosslinker used is not particularly limited so long as the crosslinker enhances the binding of the effector and bound polypeptide. Types of crosslinkers for use in the methods described herein include, but are not limited to, homobifunctional crosslinkers and photoreactive crosslinkers.

[0063] “Isolating” as used in the methods described herein encompasses removing a polypeptide and bound polypeptides from a biological environment (e.g., incubation culture). The method for isolating the effector or chimeric polypeptides and bound polypeptides (e.g., R-gene candidates) of the methods described herein is not particularly limited and may be any method known in the art that can isolate the effector or chimeric polypeptide and allow for subsequent identification of the bound polypeptides. In certain embodiments, the method comprises immunoprecipitation using an antibody specific for the effector protein and/or chimeric protein. In certain embodiments, the antibody is specific for the protein tag of the chimeric polypeptide (e.g., anti- HA antibody). In certain embodiments, the method comprises a pulldown assay, such as, for example pulldown using a peptide that recognizes the protein tag of the chimeric polypeptide. In certain embodiments, the method comprises column chromatography, e.g., affinity chromatography. In certain embodiments, the column chromatography uses an antibody that binds to the effector polypeptide or functional fragment. In certain embodiments, the column chromatography uses a ligand (e.g., antibody, Protein A and/or G, peptide, glutathione) that binds to the protein tag of the chimeric polypeptide. In certain embodiments, the method comprises an enzyme linked immunosorbent assay (ELISA).

[0064] The method for detecting a polypeptide (e.g., R-gene proteins or polypeptides from the ASR resistant plant cell) interacting with or bound to the effector polypeptide or chimeric polypeptide is not particularly limited such that the polypeptide may be identified using any method known in the art. For example, in certain embodiments, the method comprises mass spectrometry.

[0065] In certain embodiments the plant cell from which the population is provided is from an ASR resistant plant or a legume crop species from a genus selected from the group consisting of Glycine, Vigna, and Lablab.

[0066] 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.

[0067] In another embodiment, 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.

[0068] In another embodiment, 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 vernacular 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).

[0069] 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).

[0070] In an aspect, 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. [0071] R gene polypeptides are proteins that recognize the activity of specific effectors resulting in an effector-triggered immunity. R genes typically encode proteins that feature C-terminal leucine-rich repeats (LRRs) and nucleotide-binding site (NBS) domains. Such nucleic acid binding LRRs are designated nucleotide-binding LRR (NLR) proteins. The NBS domain functions as a molecular switch depending on the bound nucleotide: ADP -bound in the resting state and ATP-bound in the active state. The LRR domain is generally thought to be involved in effector recognition and autoinhibition (Ting et al., Immunity, 28 (2008), pp. 285-287). Typical plant NLRs almost universally feature the additional coiled-coil (CC) or Toll/interleukin-1 receptor (TIR) N-terminal domain. These N-terminal domains are used to sort plant NLRs into two main groups termed CNLs (CC-NLRs) and TNLs (TIR-NLRs). Both CC and TIR domains have been demonstrated to play key roles in the formation of dimers and oligomers.

[0072] The population comprising a plurality of R-gene polypeptides may be produced using any method known in the art. In certain embodiments, the population is generated by isolating R- gene polypeptides from ASR resistant plant species, such as legume crop species disclosed herein. In certain embodiments, the population is generated by isolating R-gene polypeptides from one or more legume crop species. In certain embodiments, the population is generated by producing variants of known ASR R-gene polypeptides. As used herein, a variant refers to a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. 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, including, but not limited to, procedures such as DNA shuffling. Strategies for such DNA shuffling are known in the art. As would be understood by a person of ordinary skill in the art, the population of R gene polypeptides may be obtained from multiple sources or methods, such that, for example, the population may contain R-genes from a resistant plant species along with R gene variants.

[0073] In certain embodiments, the method for identifying CcRppl-like polypeptides comprises expressing in a host cell at least one of the polynucleotides described herein (e.g., polynucleotide encoding effector polypeptide, or functional fragment thereof, or chimeric polypeptide), isolating the expressed effector polypeptide and/or chimeric polypeptide and proteins bound thereto, and identifying one or more polypeptides interacting with the expressed effector polypeptide and/or chimeric polypeptide. The methods for isolating the effector polypeptide may be any method described herein or known in the art. Similarly, the method for identifying the bound (e.g., interacting) polypeptides may be any method described herein or known in the art. In certain embodiments, the host cell comprises one or more candidate ASR resistance genes (e.g., R genes). The one or more candidate ASR resistance genes may be introduced into the host cells by a nucleic acid construct, may be native ASR resistance genes or may be a combination of introduced and native genes. The host cell may be any host cell provided herein. In certain embodiments, the host cell is a plant cell such as, for example, Nicotiana benthamiana or maize (optionally using the methods for detecting interaction as described in International Patent Application Publication Number WO 2021/211227).

[0074] In certain embodiments, the method for identifying CcRppl-like polypeptides further comprises validating the one or more identified polypeptides as a polypeptide increasing resistance to ASR. In certain embodiments, the method comprises expressing in a host cell at least one of the effector proteins or chimeric proteins described herein and one or more of the identified polypeptides and assaying the host cells for hypersensitive response (HR) cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof. In certain embodiments, the effector protein or chimeric protein comprises an ASR26 allele (e.g., SEQ ID NOs: 1-8) or a functional fragment thereof. Host cells showing either HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof would indicate that the identified polypeptide is expected to increase resistance to ASR, such that embodiments in which the effector protein or chimeric protein comprises an ASR26 allele or a functional fragment thereof encoding a polypeptide producing HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof would indicate that the identified polypeptide is a CcRppl-like polypeptide. The host cell may be any host cell provided herein. In certain embodiments, the host cell is a plant cell such as, for example, Nicotiana benthamiana. Methods for assaying HR cell death and H2O2 accumulation are known in the art and include, but not limited to, visual identification of HR cell death and DAB staining for H2O2 accumulation. Examples of changes in metabolite expression or gene expression in response to pathogen infection can be found in, for example, Silva et al. (Metabolites, 11(3): 179 (2021)), Castro-Moretti et al. (Metabolites, 10(2): 52 (2020)), and Qi et al (PLoS Pathog, 12(9): el005827 (2016)).

[0075] In certain embodiments, the method for validating the identified polypeptide comprises expressing the polypeptide in a legume plant, plant cell, tissue or organ (e.g., soybean) and assaying the plant for resistance, immunity, or susceptibility to ASR. 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. 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.

[0076] In certain embodiments, the method for identifying CcRppl-like polypeptides comprises expressing one or more effector polypeptides, or functional fragments thereof, or chimeric polypeptides or any combination thereof in a host cell comprising one or more candidate ASR resistance genes and assaying the host cell for HR cell death, H2O2 accumulation, marker gene expression, marker metabolite expression, or any combination thereof. In certain embodiments, the effector polypeptide, chimeric polypeptide, or functional fragment comprises an ASR26 allele. In certain embodiments, the host cell is a cell isolated from an ASR resistant plant such that the cell comprises candidate ASR resistance genes. In certain embodiments, the candidate ASR resistance genes are introduced into the host cell.

[0077] Also provided herein is a method for selecting a plant for growing in an area of cultivation comprising providing a sample comprising a plurality of Phakopsora pachyrhizi isolates, Phakopsora meibomiae isolates or a combination thereof from an area of cultivation, assaying the isolates for the presence of an ASR26 allele or ortholog thereof, and selecting a plant expressing a CcRppl polypeptide, a CcRppl-like polypeptide, or a combination thereof for growing in the area of cultivation when the polynucleotide is present. In certain embodiments, the ASR26 allele comprises a nucleotide 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% or more sequence identity to any one of SEQ ID NOs: 310-317. In certain embodiments, the ASR26 allele comprises a polypeptide 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% or more sequence identity to any one of SEQ ID NOs: 1-8. In certain embodiments, the CcRppl polypeptide comprises 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% or more sequence identity to SEQ ID NO: 9. In certain embodiments, the plant expressing a CcRppl polypeptide, a CcRppl -like polypeptide, or a combination thereof is selected when the polynucleotide is present in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the isolates tested. Methods for detecting the presence of a nucleotide sequence in a sample (e.g., Phakopsora pachyrhizi, Phakopsora meibomicie isolates) are well known in the art and include, for example, PCR.

[0078] As used herein, an “area of cultivation” comprises any region in which one desires to grow a plant. Such areas of cultivations include, but are not limited to, a field in which a plant is cultivated (such as a crop field, a sod field, a tree field, a managed forest, a field for culturing fruits and vegetables, etc), a greenhouse, or a growth chamber.

[0079] In certain embodiments, the plant expressing the CcRppl polypeptide, the CcRppl -like polypeptide, or a combination thereof described herein are elite plant lines (e.g., elite soybean line). In certain embodiments, 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, canola, and sunflower 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. [0080] The plant species of the compositions and methods of the present disclosure (e.g., plants from which host cells are isolated and plants expressing the CcRppl polypeptide, the CcRppl- like polypeptide, or a combination thereof) can be any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica spp. (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), 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.

[0081] 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 legume species or legume crop species is a plant, plant part or plant cell.

[0082] Also provided herein are methods for identifying ASR26 alleles comprising providing a population comprising a plurality of Phakopsora pachyrhizi isolates, Phakopsora meibomiae isolates or a combination thereof and detecting in the population alleles of ASR26. In certain embodiments, the plurality of isolates is taken from an area of cultivation. The method for identifying the allele is not particularly limited and may be any method known in the art. In certain embodiments, the detecting comprises genomic sequencing or RNA sequencing.

[0083] In certain embodiments, the method further comprises assaying the identified ASR26 alleles to determine if the identified allele is recognized by the CcRppl polypeptide, a chimeric CcRppl polypeptide, or the CcRppl -like polypeptide, or any combination thereof. The assay can be any of the assays described herein, or known in the art, that determines an interaction between the ASR26 allele and CcRppl, CcRppl -like, or chimeric CcRppl polypeptide. In certain embodiments, the assay is a pulldown assay or immunoprecipitation assay to determine binding between the allele and CcRppl, CcRppl -like, or chimeric CcRppl polypeptide. In certain embodiments, the ASR26 allele and CcRppl, CcRppl -like, or chimeric CcRppl polypeptide are expressed in a host cell and the presence of HR cell death, H2O2 accumulation, marker gene expression and/or marker metabolite expression is measured.

[0084] As would be understood by a person of ordinary skill in the art, the methods described herein to identify CcRppl -like polypeptides using an ASR26 allele or functional fragment thereof, could be adapted to identify candidate ASR resistance polypeptides using the effector polypeptides (e.g., non-ASR26 polypeptides) described herein. Accordingly, also provided herein are methods for identifying ASR resistance proteins by identifying one or more polypeptides that bind to a effector 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% or more sequence identity to any one of SEQ ID NOs: 10-309, a functional fragment thereof or a chimeric polypeptide produced therefrom. The methods for identifying the one or more polypeptides may be any method described herein, or known in the art, that allows for the identification of interacting proteins. The methods may further include validating the identified interacting protein using any of the validation methods described herein including, but not limited to, expression in host cells to assay HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof and expressing in plants to determine resistance to ASR.

[0085] In certain embodiments, the candidate resistance genes are assayed for binding to an ASR26 allele. In certain embodiments, candidate resistance genes are selected that do not bind an ASR26 allele. In certain embodiments, the population of R-gene polypeptides for use in identifying candidates binding to or interacting with the effector polypeptides (e.g., SEQ ID NOs: 10-309) is pre-screened to remove R-gene polypeptides that bind to an ASR26 allele. Methods to remove polypeptides from a sample are known in the art and include, but are not limited to, pulldown assays, immunoprecipitation, and column chromatography.

[0086] Also provided herein are methods for identifying ASR resistance polypeptides that do not compete with CcRppl (e.g., have a different mode of action). In certain embodiments, the method comprises identifying one or more polypeptides that bind to an effector polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 10-309, or a functional fragment thereof, assaying the one or more identified polypeptides for interaction with a polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 1-8, and selecting the one or more polypeptides that do not interact with the polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 1-8. The one or more identified ASR resistance polypeptides that do not compete with CcRppl can be expressed in a plant either alone or in combination with another ASR resistance polynucleotide, such as, for example, CcRppl using the methods described herein or known in the art, to generate a plant having ASR resistance. The method for identifying the one or more polypeptides that bind to an effector polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 10-309 may be any method described herein (e.g., binding assays and/or measuring HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof in a host cell). In certain embodiments, the method comprises providing a population of polypeptides from an ARS resistant plant cell, incubating the population of polypeptides with the effector polypeptide, a functional fragment thereof or a chimeric polypeptide comprising the effector polypeptide or the functional fragment thereof under conditions that allow for binding of the at least one effector polypeptide, functional fragment thereof or chimeric polypeptide to one or more polypeptides of the population, isolating the effector polypeptide or the chimeric polypeptide and polypeptides interacting therewith from the population, and identifying the one or more polypeptides. In certain embodiments, the one or more polypeptides are identified by providing a population comprising a plurality of R-gene candidates, optionally pre-screened to remove candidates that bind to an ASR26 allele, incubating the population with the effector polypeptide or a chimeric polypeptide comprising the effector polypeptide or the functional fragment thereof under conditions that allow for binding of the effector polypeptide or the chimeric polypeptide to one or more R-gene candidates in the population, isolating the effector polypeptide or the chimeric polypeptide and interacting R-gene candidates from the population, and identifying the R-gene candidates. In certain embodiments, the one or more polypeptides are identified by introducing into a host cell comprising one or more candidate ASR resistance genes a nucleic acid construct comprising a polynucleotide encoding the effector polypeptide, or a functional fragment thereof, or a chimeric polypeptide comprising the effector polypeptide or the functional fragment thereof, isolating the expressed effector polypeptide or chimeric polypeptide, and identifying one or more polypeptides interacting with the expressed effector polypeptide or chimeric polypeptide. The method for assaying the one or more identified polypeptides for interaction with a polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 1-8 may be any method described herein (e.g., binding assays and/or measuring HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof in a host cell).

[0087] In certain embodiments, the method for identifying ASR resistance polypeptides that do not compete with CcRppl comprises expressing in a host cell comprising one or more R-gene candidate polypeptides a polynucleotide encoding an effector polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 10-309, or a functional fragment thereof or a chimeric polypeptide comprising an effector polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 10-309, or a functional fragment thereof, assaying the host cell for the presence of HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof, identifying R-gene candidate polypeptides that result in HR cell death, H2O2 accumulation, marker gene or marker metabolite expression in the presence of the effector polypeptide, expressing in a second host cell the one or more identified R-gene candidate polypeptides and an ASR26 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 1-8, assaying the host cell for the presence of HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof, and selecting the one or more identified R-gene candidate polypeptides that do not result in HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof in the presence of the ASR26 effector polypeptide.

[0088] Further provided are methods for detecting and/or isolating the soluble form (e.g., active form) of CcRppl protein in a sample. In certain embodiments, the method comprises incubating a mixture, the mixture comprising an ASR26 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 1-8 and a sample comprising a CcRppl protein comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 9, isolating the ASR26 polypeptide and polypeptides bound thereto from the mixture, and detecting the presence of a CcRppl protein in the isolated ASR26 polypeptide and polypeptides bound thereto. In certain embodiments, the ASR26 polypeptide is operably linked to a protein tag, such as those described herein. In certain embodiments, the ASR26 polypeptide is operably linked to an MBP tag. In certain embodiments, the CcRppl polypeptide is operably linked to a protein tag, such as those described herein. In certain embodiments, the protein tag operably linked to the CcRppl polypeptide is a different protein tag from the protein tag operably linked to ASR26. The method for isolating the ASR26 polypeptide is not particularly limited and may be any method described herein or known in the art that can isolate a protein from a mixture while maintaining the protein-protein interactions, such as, for example, immunoprecipitation and pull-down assays. The method for detecting the CcRppl polypeptide may be any method described herein or known in the art for detecting the presence of a protein such as, for example, Western blotting, mass spectrometry and ELISA. [0089] 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.

EXAMPLE 1

[0090] Identification of predicted Phakopsora pachyrhizi effectors.

[0091] The Phakopsora pachyrhizi (causal agent of ASR) transcriptome was generated by aligning paired-end RNA-seq reads from infected soybean plants to the Phakopsora pachyrhizi genome. Transcripts which were significantly expressed in the dataset were then utilized to generate a predicted Phakopsora pachyrhizi proteome.

[0092] The resulting proteins were then assessed for effector characteristics, including presence of a signal peptide (SignalP 3.0 and Phobius), sequence-based machine learning (EffectorP 3.0), conserved domains (HMMer) and lack of a transmembrane domain (TMHMM).

EXAMPLE 2

[0093] Identification of candidate avirulence (Avr) genes for CcRppl.

[0094] CcRppl is an NLR gene from pigeon pea and confers resistance to Phakopsora pachyrhizi. A biochemical approach was used to identify a CcRppl cognate effector. FLAG- tagged CcRppl protein was expressed in E. coli cells and then incubated with extracts of ASR- infected soybean leaf tissues. CcRppl -interacting proteins were pulled down with anti-FLAG agarose beads, followed by mass spectrometry analysis. Eight putative ASR effectors were identified when searched against the predicted Phakopsora pachyrhizi effector list.

EXAMPLE 3

[0095] Identification of ASR26 as the cognate effector recognized by CcRppl.

[0096] Recognition of pathogen effectors by NLR proteins leads to effector-triggered immunity, often culminating in a HR cell death accompanied with reactive oxygen species production. An Agrobacterium -mediated transient expression system in Nicotiana benthamiana was used to confirm the recognition between the CcRppl and the putative effectors. All eight ASR candidate effectors identified in Example 2 and CcRppl were individually cloned into a binary vector and introduced into Agrobacterium tumefaciens AGL1. Only one of the effectors, ASR26, caused HR cell death (visual) and accumulated H2O2 (DAB staining) when co-expressed with CcRppl in N. benthamiana, indicating that CcRppl can recognize ASR26 and trigger an immune response. [0097] Co-immunoprecipitation was performed to examine the physical interaction between CcRppl and ASR26. The result from these studies confirmed that CcRppl interacts with ASR26 in planta.

EXAMPLE 4

[0098] CcRppl recognizes four known ASR26 alleles.

[0099] Surveying ASR26 alleles with genomic sequences and/or RNA-seq data sets from thirteen P. pachyrhizi isolates resulted in the identification of a total of four ASR26 alleles. When co-expressed with CcRppl in N. benthamiana, all four ASR26 alleles caused HR cell death and accumulated H2O2, indicating that CcRppl can recognize all four ASR26 alleles and is expected to provide resistance against all the thirteen isolates.

EXAMPLE 5

[0100] Soluble CcRppl protein, but not insoluble CcRppl protein, physically interacts with ASR26.

[0101] CcRppl protein with a His tag (CcRppl-His) was produced using either the B121Gold E. coli expression system or the Sf9 insect cell expression system. CcRppl -His protein produced from E. coli cells was insoluble, whereas the CcRppl-His protein from insect cells was soluble. ASR26 protein fused with an MBP tag (MBP-ASR26) was produced in E. coli cells and determined to be soluble. To determine if ASR26 can interact with the soluble CcRppl protein, which is predicted to be the active form, in vitro pulldown assays were performed using both the soluble and insoluble versions of the expressed CcRppl protein as prey and the tagged ASR26 protein as bait.

[0102] For the in vitro pull-down assays, purified CcRppl-His (prey) and MBP-ASR26 (bait) proteins were obtained. The MBP-ASR26 (bait) protein was immobilized onto anti-MBP magnetic beads, and any unattached proteins were removed through washes using MBP Column Binding/Washing Buffer. Following this, the CcRppl-His (prey) protein was introduced to the beads bound with MBP-ASR26 and allowed to incubate for a minimum of 30 minutes. The protein complex formed was released from the beads after washing away unbound proteins. The eluted proteins were subjected to analysis using SDS-PAGE and Western blotting with anti-MBP antibodies and/or anti-His antibodies to visualize and detect the protein interactions. [0103] These results indicate that ASR26 could be used to determine if a purified CcRppl protein is in a soluble form.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.