CcRpp1 homologous effectors and methods of use

Abstract

Disclosed herein are compositions and methods for identifying genes that improve or enhance pathogen resistance. Compositions comprising effector polypeptides encoded by polynucleotides disclosed herein can be used to identify genes for improving resistance to Asian soybean rust (ASR). Methods for selecting ASR resistant plants for planting in a cultivation area are also disclosed.

Description

[0001] References to electronically submitted sequence listings

[0002] An official copy of the sequence listing is submitted electronically via the Patent Center as an XML-formatted sequence listing, with the file name 108283_SequenceListing, created on August 7, 2023, and with a size of 763,782 bytes, and is submitted simultaneously with this specification. The sequence listing contained in this XML-formatted file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to the field of molecular biology, and in particular to compositions and methods that can be used to identify genes that enhance resistance to the causative agent of Asian soybean rust (ASR) in leguminous plants. The present disclosure further relates to effector polypeptides that can interact with polypeptides encoded by genes that enhance ASR resistance, methods for isolating polypeptides encoded by genes that enhance ASR resistance using these polypeptide sequences, and methods for selecting plants for planting in a cultivation area. Background Art

[0004] Soybean diseases are a major threat to soybean production, resulting in yield losses and reduced kernel 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%.

[0005] Therefore, there is a need to develop compositions and methods for identifying ASR resistance genes. The present disclosure provides such compositions and methods. Summary of the invention

[0006] Nucleic acid constructs and expression cassettes are provided, comprising a polynucleotide encoding an effector polypeptide or a functional fragment thereof operably linked to a regulatory element, the 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 NO: 1-8 or 10-309. In certain embodiments, at least one regulatory sequence is a promoter (e.g., a heterologous promoter).

[0007] Also provided are chimeric polypeptides comprising an effector polypeptide or a functional fragment thereof operably linked to a heterologous peptide (e.g., a protein tag), the 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. Further provided are polynucleotides encoding the chimeric polypeptides and nucleic acid constructs and expression cassettes comprising the polynucleotides encoding the chimeric polypeptides.

[0008] Also provided are host cells (e.g., bacterial cells, fungal cells, plant cells) comprising any of the polynucleotides (e.g., isolated polynucleotides, recombinant polynucleotides, nucleic acid constructs, expression cassettes) encoding the effector polypeptides, functional fragments thereof, or chimeric polypeptides described herein.

[0009] Methods for identifying CcRpp1-like proteins are provided, the methods comprising identifying one or more polypeptides that bind to a chimeric polypeptide comprising an effector polypeptide or a functional fragment thereof operably linked to a heterologous peptide (e.g., a protein tag), the 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. In certain embodiments, the method further comprises expressing the chimeric polypeptide and the one or more identified interacting polypeptides in a host cell, and determining the HR cell death, H 2 O 2 accumulation, presence of marker genes or marker metabolite expression, or any combination thereof.

[0010] Also provided are methods for identifying CcRpp1-like proteins, the methods comprising identifying one or more polypeptides that bind to an effector polypeptide or a functional fragment thereof, the 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 higher sequence identity to any one of SEQ ID NO: 1-8. In certain embodiments, the method further comprises expressing the effector polypeptide or a functional fragment thereof and the one or more identified interacting polypeptides in a host cell, and determining the HR cell death, H 2 O 2 accumulation, presence of marker genes or marker metabolite expression, or any combination thereof.

[0011] Further provided are methods for selecting plants for planting in a cultivation area, the methods comprising providing a sample comprising a plurality of Psora pachyrhizi isolates from the cultivation area, determining 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 plants expressing a CcRpp1 polypeptide, a CcRpp1-like polypeptide, or a combination thereof for planting in the cultivation area when the polynucleotide is present.

[0012] Also provided are methods for identifying ASR26 alleles, the methods comprising providing a population comprising a plurality of Psora pachyrhizi isolates, and detecting the ASR26 allele in the population. In certain embodiments, the method further comprises expressing the identified ASR26 allele and the CcRPP1 polypeptide, the CcRPP1-like polypeptide, or a combination thereof in a host cell, and determining the HR cell death, H 2 O 2 accumulation, presence of marker genes or marker metabolite expression, or any combination thereof.

[0013] Methods for identifying ASR resistance polypeptides that do not compete with CcRpp1 are provided, the methods comprising identifying one or more polypeptides that bind to an effector polypeptide or a functional fragment thereof, the 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, determining the identity of the one or more identified polypeptides to any one of SEQ ID NOs: 10-309, NO:1-8 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, and selecting not to interact 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 Any of NO:1-8 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 one or more polypeptides that interact with the polypeptide.

[0014] Further provided are methods for detecting soluble CcRpp1 protein in a sample, the methods comprising incubating a 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 NO: 1-8 and a sample containing CcRpp1 protein, separating the ASR26 polypeptide and polypeptides bound thereto from the mixture, and detecting the presence of CcRpp1 protein in the separated ASR26 polypeptide and polypeptides bound thereto, thereby detecting soluble CcRpp1 protein.

[0015] Brief description of sequence listing

[0016] The present disclosure may be more fully understood from the following detailed description and the accompanying sequence listing, which form a part of this application.The sequence description and sequence listing attached hereto comply with the rules regarding nucleotide and amino acid sequence disclosure in patent applications as set forth in 37 CFR §§1.831-1.835. DETAILED DESCRIPTION

[0017] The present disclosure provides compositions and methods for identifying and selecting Asian soybean rust (ASR) resistance genes using effector proteins. These methods involve expressing nucleic acid sequences encoding effector proteins from Psora pachyrhizi and Psora dasyphylla in host cells, and identifying interacting polypeptides or incubating isolated and / or chimeric effector proteins and identifying interacting polypeptides. These methods also involve selecting plants for planting in a cultivation area by determining the presence of specific ASR effectors in the cultivation area.

[0018] Pathogens that cause ASR, such as Psora pachyrhizium and Psora tataricola, can break through the plant's first layer of immunity by secreting molecules called "effector proteins" or "effectors." In some cases, plants have evolved a second layer of immunity in which R gene products (e.g., NLR proteins) recognize specific effectors, leading to effector-triggered immunity. One such NLR protein, CcRpp1 (U.S. Patent 10,842,097), has been shown to provide robust resistance to ASR when expressed in plants. The identification of effector proteins recognized by CcRpp1 allows the identification of CcRpp1-like proteins that may have similar or identical modes of action, as well as the identity of non-competitive ASR resistance genes that have different modes of action than CcRpp1 (e.g., recognizing different effectors).

[0019] Thus, one aspect of the present disclosure provides effector encoding polynucleotides (e.g., isolated polynucleotides and recombinant polynucleotides) encoding effector polypeptides (e.g., isolated polypeptides and recombinant polypeptides) or functional fragments thereof, which effector polypeptides comprise, consist essentially of, or consist 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. 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.

[0020] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0021] In the present disclosure, "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in single-stranded or double-stranded form, and unless otherwise limited, encompasses known analogs (e.g., peptide nucleic acids) that have the essential properties of natural nucleotides because they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

[0022] "Isolated" polynucleotides (or DNA) are used herein to refer to nucleic acid sequences (or DNA) that are no longer in their natural environment, such as in vitro or in heterologous recombinant bacteria or plant host cells. Isolated polynucleotides or their biologically active parts are substantially free of other cell materials or culture media when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when synthesized by chemical methods. Isolated polynucleotides do not contain sequences that are naturally located at the flanks of nucleic acids in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences at the 5' and 3' ends of the nucleic acid) (optimally protein coding sequences). "Recombinant" polynucleotides (or DNA) are used herein to refer to nucleic acid sequences (or DNA) in recombinant bacteria or plant host cells. In some embodiments, "isolated" or "recombinant" nucleic acids do not contain sequences that are naturally located at the flanks of nucleic acids in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences at the 5' and 3' ends of the nucleic acid) (preferably protein coding sequences). For the purposes of this disclosure, "isolated" or "recombinant" excludes isolated chromosomes when used to refer to nucleic acid molecules. For example, in various embodiments, a recombinant nucleic acid molecule encoding an effector polypeptide of the present disclosure may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleic acid sequence that naturally flanks the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.

[0023] As used herein, "percentage (%) of sequence identity" relative to a reference sequence (subject sequence) is determined as the percentage of amino acid residues or nucleotides identical to the corresponding amino acid residues or nucleotides in the reference sequence in the candidate sequence (query sequence) after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions of amino acids as part of the sequence identity. The comparison for the purpose of determining the percentage of sequence identity can be achieved in various ways within the technical scope of the art, such as using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the sequence being compared. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (e.g., percentage of identity of the query sequence = number of identical positions between the query sequence and the subject sequence / total number of positions of the query sequence × 100).

[0024] Unless otherwise indicated, sequence identity / similarity values ​​provided herein refer to the values ​​obtained using the BLAST 2.0 program package with default parameters (Altschul et al., (1997) Nucleic Acids Res. 25:3389-402).

[0025] As used herein, "functional fragment", "fragment that is functionally equivalent", "functionally equivalent fragment", etc. refers to a portion or subsequence of a polypeptide sequence of the present disclosure that retains its native ability. For example, a functional fragment of an effector polypeptide (such as those described herein) retains the ability to be recognized by and bind to an NLR protein. Thus, in certain embodiments, a functional fragment of an ASR26 effector polypeptide (e.g., SEQ ID NO: 1-8) binds to the CcRpp1 polypeptide sequence of SEQ ID NO: 9. In certain embodiments, a functional fragment of a polypeptide described herein comprises an N-terminal and / or 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.

[0026] Functional fragments of the specific polypeptides of the embodiments (ie, reference polypeptides) can also be evaluated by comparing the percentage of sequence identity between the polypeptides and the reference polypeptides. The percentage of sequence identity between any two polypeptides can be calculated using sequence alignment programs known in the art.

[0027] The functional fragment of the native protein of the embodiment 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 with the amino acid sequence of the native protein, as determined by sequence alignment programs known in the art. The functional fragment of the protein of the present disclosure can differ from the protein by as little as about 1-15 amino acid residues, as little as about 1-10 (such as about 6-10), as little as about 5, as little as 4, 3, 2 or even 1 amino acid residue.

[0028] Polynucleotides as described herein can be used to separate corresponding sequences from other organisms, particularly other pachyrhizome rust fungi and schizophyllum rust fungi. In this way, methods such as PCR or hybridization can be used to identify these sequences based on the sequence identity of such sequences and sequences shown herein. The disclosure encompasses sequences separated based on the sequence identity of the complete sequence shown herein or its variants and fragments. Such sequences include sequences that are orthologs or allelomorphs of the disclosed sequences. The term "orthologs" refers to genes derived from common ancestral genes and found in different species due to speciation. The term "allele" refers to the variation (for example, SNP) of the nucleotide sequence synthesized by the same position encoding gene product on a DNA molecule. When the nucleotide sequences of genes found in different species and / or the protein sequences encoded by them have 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 more sequence identity, these genes are considered to be orthologs. The functions of orthologs are generally highly conserved between species. Therefore, the present disclosure encompasses isolated polynucleotides encoding effector proteins that hybridize to the sequences disclosed herein, or functional fragments thereof.

[0029] As used herein, "encoding", "encoded", etc., with respect to a given nucleic acid means that it contains information for translation into a given protein. A nucleic acid encoding a protein may contain non-translated sequences (e.g., introns) within the translated region of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in a cDNA). Information used to encode a protein is specified by codon usage. Typically, amino acid sequences are encoded by nucleic acids using a "universal" genetic code. However, when these organisms are used to express nucleic acids, variants of the universal code may be used, such as those present in some plants, animals, and fungal mitochondria, the bacterium Mycoplasma capricolum (Yamao et al., (1985) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 82:2306-9), or ciliate macronuclei.

[0030] When nucleic acids are synthetically prepared or altered, known codon biases of the intended host for the nucleic acid to be expressed can be utilized. For example, although nucleic acid sequences of the invention can be expressed in both monocotyledonous and dicotyledonous plant species, the sequences can be modified to account for specific codon biases and GC content biases of monocotyledonous or dicotyledonous plants, as these biases have been shown to differ (Murray et al., (1989) Nucleic Acids Res. 17:477-98 and incorporated herein by reference).

[0031] In certain embodiments, the effector encoding polynucleotide encodes a CcRpp1 effector polypeptide (also referred to herein as ASR26) or a functional fragment thereof, which effector 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. In certain embodiments, the CcRpp1 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.

[0032] 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., a 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.

[0033] As used herein, "heterologous" with respect to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified relative to its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide is derived, or, if from the same / similar species, one or both are substantially modified relative to their native form and / or genomic locus, or the promoter is not the native promoter of the operably linked polynucleotide.

[0034] As used herein, "operably linked" is intended to mean a functional connection between two or more elements. For example, an operable connection between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional connection that allows the expression of the polynucleotide of interest. Operably linked elements can be continuous or non-continuous. When used to refer to the connection of two protein coding regions, operably linked means that the coding regions are in the same reading frame.

[0035] As used herein, "regulatory element" generally refers to a transcriptional regulatory element that participates in regulating the transcription of a nucleic acid molecule (such as a gene or a target gene). A regulatory element is a nucleic acid and may include a promoter, an enhancer, an intron, an expression control element (EME), a 5'-untranslated region (5'-UTR, also referred to 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", that is, it activates the expression of a gene on the same nucleic acid molecule (e.g., chromosome) where the regulatory element is located.

[0036] "Enhancer" element is any nucleic acid molecule that increases the transcription of a nucleic acid molecule when functionally connected to a promoter (regardless of its relative position). Various enhancers are known in the art, including, for example, introns with gene expression enhancement properties in plants, ubiquitin introns (i.e., maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), ω enhancers or ω' enhancers (Gallie et al., (1989) Molecular Biology of RNA [RNA molecular biology] editor Cech (Liss [Lees Company], New York) 237-256 and Gallie et al., (1987) Gene [gene] 60: 217-25), CaMV35S enhancer (see, for example, Benfey et al., (1990) EMBO J. [European Molecular Biology Association Journal] 9: 1685-96), and the enhancer of U.S. Patent No. 7,803,992 can also be used. The list of the above transcription enhancers is not intended to be restrictive. Any suitable transcription enhancer may be used in the embodiments described herein.

[0037] "Repressors" (also sometimes referred to herein as silencers) are defined as any nucleic acid molecule that inhibits transcription when functionally linked to a promoter (regardless of relative position). The term "cis-element" generally refers to a transcriptional regulatory element that affects or regulates the expression of an operably linked transcribable polynucleotide, wherein the transcribable polynucleotides are present in the same DNA sequence. Cis-elements can function to bind transcription factors, which are trans-acting polypeptides that regulate transcription.

[0038] "Intron" is an intervening sequence in a gene that is transcribed into RNA, but then excised in the process of producing mature mRNA. The term is also used for excised RNA sequences. "Exon" is a part of a gene's sequence that is transcribed and found in the mature mRNA derived from the gene, but is not necessarily a part of the sequence encoding the final gene product. The 5' untranslated region (5'UTR) (also referred to as the translation leader sequence or leader RNA) is the mRNA region directly upstream of the start codon. This region participates in regulating the translation of transcripts by different mechanisms in viruses, prokaryotes and eukaryotes. "3' non-coding sequence" refers to a DNA sequence located downstream of the coding sequence, and includes polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are usually characterized as affecting the 3' end of the polyadenylic acid segment added to the mRNA precursor.

[0039] As used herein, "promoter" refers to a DNA region upstream of the start of transcription and involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. "Plant promoter" is a promoter that can initiate transcription in plant cells. In certain embodiments, the polynucleotides described herein are operably linked to a promoter that drives expression in plant cells. Any promoter known in the art can be used in the method of the present disclosure, including but not limited to constitutive promoters, pathogen-inducible promoters, wound-inducible promoters, tissue-preferred promoters, and chemically regulated promoters. The selection of promoters can depend on the desired expression time and position in the transformed plant and other factors known to those skilled 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; core CaMV 35S promoter; rice actin; ubiquitin; pEMU; MAS; ALS; etc. Other constitutive promoters include, for example, those disclosed in the following 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 are contemplated for use in the present disclosure.

[0040] Generally, it is beneficial to express genes from inducible promoters, particularly from pathogen-inducible promoters. Such promoters include those from pathogenesis-related proteins (PR proteins) induced after infection by pathogens (e.g., PR proteins, SAR proteins, β-1,3-glucanase, chitinase, etc.).

[0041] Of interest are promoters that are locally expressed at or near the site of pathogen infection. In addition, since pathogens find entry into plants through wounds or insect damage, wound-inducible promoters can be used in the construction of the present disclosure. Such wound-inducible promoters include the potato proteinase inhibitor (pin II) gene, wun1 and wun2, win1 and win2, systemin, WIP1, MPI genes, etc.

[0042] Chemically regulated promoters can be used to regulate the expression of genes in plants by using exogenous chemical regulators.Depending on target, promoters can be chemically induced promoters when chemical substances are used to induce gene expression, or promoters can be chemically repressed promoters when chemical substances are used to suppress gene expression.Chemically induced promoters are known in the art, and include but are not limited to the maize In2-2 promoter activated by benzsulfamide herbicide safeners, the maize GST promoter activated by the hydrophobic electrophilic compound used as pre-emergence herbicide, and the tobacco PR-1a promoter activated by salicylic acid.Other purpose chemically regulated promoters include steroid responsive promoters (for example, glucocorticoid inducible promoters and tetracycline inducible and tetracycline repressible promoters).

[0043] Tissue-preferred promoters can be used to target the enhanced expression of target genes or proteins in specific plant tissues. 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. 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 may be modified.

[0044] 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.

[0045] "Seed-preferred" promoters include both "seed-specific" promoters (those active during seed development, such as promoters of seed storage proteins) and "seed germination" promoters (those active during seed germination). Such seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced signal), cZ19Bl (maize 19kDa zein), milps (inositol-1-phosphate synthase), and celA (cellulose synthase) (see WO 00 / 11177, incorporated herein by reference). γ-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicotyledons, seed-specific promoters include, but are not limited to, bean β-phaseolin, rapeseed protein, 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 protein, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00 / 12733, which discloses seed-preferred promoters from end1 and end2 genes; incorporated herein by reference.

[0046] Also provided is a nucleic acid (e.g., DNA) construct comprising any of the effector encoding polynucleotides described herein. The use of the terms "nucleic acid construct", "nucleotide construct", etc. herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. One of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides consisting of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, can also be used 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. In addition, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences that can be used in the plant transformation methods of the embodiments, including but not limited to those consisting of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. 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-loop structures, and the like.

[0047] In certain embodiments, the effector encoding polynucleotides described herein are provided in expression cassettes (e.g., plasmids, cosmids, viruses, autonomously replicating sequences, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleotide sequences) for expression in a target host or any target plant. The cassette may comprise 5' and 3' regulatory sequences operably linked to the effector encoding polynucleotides. The cassette may additionally contain at least one additional gene to be co-transformed into an organism. Alternatively, one or more additional genes may be provided on a plurality of expression cassettes. Such expression cassettes are provided with a plurality of restriction sites and / or recombination sites for inserting the effector encoding polynucleotides so as to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain a selective marker gene.

[0048] The expression cassette may comprise a transcriptional and translational initiation region (e.g., a promoter), an effector encoding polynucleotide, and a transcriptional and translational termination region (e.g., a termination region) that functions in a plant in a 5'-3' transcriptional direction. The regulatory region (e.g., a promoter, a transcriptional regulatory region, and a translational termination region) and / or the effector encoding polynucleotide may be native / similar to the host cell or to each other. Alternatively, the regulatory region and / or the effector encoding polynucleotide may be heterologous to the host cell or to each other.

[0049] The termination region may be native to the transcription initiation region, to the plant host, or may be derived from another source (ie, foreign or heterologous) to the promoter, the effector encoding polynucleotide, the plant host, or any combination thereof.

[0050] The expression cassette may additionally contain a 5' leader sequence. Such leader sequences may serve to enhance translation. Translation leaders are known in the art and include viral translation leader sequences.

[0051] Typically, the expression cassette may include a selective marker gene for selecting transformed cells. Selective marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes that confer resistance to herbicidal compounds such as glyphosate, glufosinate, bromoxynil, imidazolinone and 2,4-dichlorophenoxyacetic acid (2,4-D). The list of the above selective marker genes is not intended to be restrictive. Any selective marker gene may be used in the present disclosure.

[0052] In preparing the expression cassette, various DNA fragments may be manipulated to provide DNA sequences in the proper orientation and, where appropriate, in the proper reading frame. To this end, adapters or joints may be employed to connect the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction enzyme cutting (restriction), annealing, re-substitution (e.g., conversion and transversion) may be involved.

[0053] Further provided is a host cell 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 one 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, a tobacco protoplast, an Arabidopsis (Arabidopsis) cell, an Arabidopsis protoplast, a soybean cell or a soybean protoplast. In certain embodiments, a nucleic acid construct or expression cassette as described herein is expressed in a 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, a candidate ASR resistance gene is introduced into a host cell using a nucleic acid construct comprising a candidate ASR resistance gene operably connected to a regulatory element (e.g., such as a heterologous promoter).

[0054] As used herein, "introduced", "introducing", etc. are intended to mean presenting the polynucleotides or resulting polypeptides of the present invention to a host cell in such a manner that the sequence is able to enter the interior of the cell. The methods of the present disclosure do not depend on a specific method for introducing a sequence into a host cell, as long as the polynucleotide or polypeptide is able to enter the interior of the cell.

[0055] In certain embodiments, transient transformation techniques are used to transiently express polynucleotides described herein in host cells. In certain embodiments, stable transformation techniques are used to stably express polynucleotides in host cells. In certain embodiments, nucleic acid constructs or expression cassettes described herein are used to introduce polynucleotides into plants, plant cells, plant parts, seeds or grains. In certain embodiments, genome editing techniques are used to introduce polynucleotides into plants, plant cells, plant parts, seeds or grains.

[0056] "Stable transformation" is intended to mean that a polynucleotide introduced into a plant is integrated into the genome of the plant of interest and is heritable by its progeny. "Transient transformation" is intended to mean that a polynucleotide is introduced into a plant of interest and is not integrated into the genome of the plant or organism, or that a polypeptide is introduced into a plant or organism.

[0057] Transformation protocols and 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, virus-mediated, ballistic particle acceleration, heat shock, and lipid-mediated transfection. The type of transformation or transfection method used for use with the compositions and methods described herein may vary depending on the type of host cell (e.g., bacterial cells, fungal cells, or plant cells) used for transformation.

[0058] 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 higher sequence identity to any one of SEQ ID NOs: 1-8 and 10-309, or a functional fragment thereof, operably linked to a heterologous peptide. In certain embodiments, the polynucleotide encoding the chimeric polypeptide is in a nucleic acid construct comprising a polynucleotide operably linked to a regulatory element, such as, for example, a heterologous promoter. In certain embodiments, the chimeric polypeptide binds to a CcRpp1 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 a functional fragment thereof.

[0059] In certain embodiments, the heterologous peptide includes a protein tag. As used herein, "protein tag" includes, but is not limited to, affinity tags, solubility tags, chromatographic tags, epitope tags, and fluorescent tags. The position of the protein tag in the chimeric polypeptide is not particularly limited, as long as the chimeric polypeptide maintains binding to the CcRpp1 polypeptide or the corresponding resistance polypeptide, so that in certain embodiments, the protein tag can be operably connected to the N-terminus, C-terminus, or both the N-terminus and the 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.

[0060] Also provided herein are methods for identifying a CcRpp1-like protein by identifying one or more polypeptides that bind to a chimeric effector polypeptide or an effector polypeptide described herein, or a functional fragment thereof, 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.

[0061] As used herein, a "CcRpp1-like" protein refers to a protein that has the same or similar biological activity as CcRpp1, but does not have 100% sequence identity with CcRpp1 (i.e., SEQ ID NO: 9) over the full length of the protein. In certain embodiments, the CcRpp1-like protein binds to at least one of the ASR26 effector protein alleles described herein (e.g., SEQ ID NOs: 1-8).

[0062] 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 (e.g., SEQ ID NOs: 1-8) described herein or a 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 at least one effector polypeptide and / or at least one chimeric polypeptide and polypeptides that interact therewith from the population, and identifying polypeptides that interact with the effector polypeptide or the chimeric polypeptide.

[0063] 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 polypeptides (e.g., SEQ ID NOs: 1-8) described herein or functional fragments thereof, or at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more) chimeric polypeptides described herein, or a combination of at least one effector polypeptide and at least one chimeric polypeptide, isolating at least one effector polypeptide and / or at least one chimeric polypeptide and an R gene polypeptide interacting therewith from the population, and identifying an R gene polypeptide interacting with the effector polypeptide or the chimeric polypeptide.

[0064] In certain embodiments of the methods described herein, incubation is performed under conditions that allow at least one chimeric polypeptide or at least one effector polypeptide or functional fragment thereof to bind to one or more polypeptides of the population. One of ordinary skill in the art will recognize conditions that can be manipulated to allow or improve capture of protein-protein interactions. For example, conditions that can be varied include, but are not limited to, buffer composition, pH, incubation time, and temperature. In certain embodiments, incubation is performed in the presence of a cross-linking agent. The type of cross-linking agent used is not particularly limited, as long as the cross-linking agent enhances the binding of the effector and the bound polypeptide. The types of cross-linking agents used in the methods described herein include, but are not limited to, homobifunctional cross-linking agents and photoreactive cross-linking agents.

[0065] As used in the methods described herein, "separation" encompasses the removal of polypeptides and bound polypeptides from a biological environment (e.g., an incubated culture). The method for separating the effector polypeptide or chimeric polypeptide and bound polypeptide (e.g., R gene candidate) described herein is not particularly limited, and may be any method known in the art that can separate effector polypeptides or chimeric polypeptides and allow subsequent identification of bound polypeptides. In certain embodiments, the method includes immunoprecipitation using antibodies specific for effector proteins and / or chimeric proteins. In certain embodiments, the antibody is specific for a protein tag of a chimeric polypeptide (e.g., an anti-HA antibody). In certain embodiments, the method includes a pull-down assay, such as a pull-down using a peptide that recognizes a protein tag of a chimeric polypeptide. In certain embodiments, the method includes column chromatography, such as affinity chromatography. In certain embodiments, column chromatography uses antibodies that bind to effector polypeptides or functional fragments. In certain embodiments, column chromatography uses a ligand (e.g., antibody, protein A and / or G, peptide, glutathione) that binds to a protein tag of a chimeric polypeptide. In certain embodiments, the method includes an enzyme-linked immunosorbent assay (ELISA).

[0066] The method for detecting a polypeptide that interacts or binds to an effector polypeptide or chimeric polypeptide (e.g., an R gene protein or polypeptide from an ASR-resistant plant cell) is not particularly limited, so that any method known in the art can be used to identify the polypeptide. For example, in certain embodiments, the method includes mass spectrometry.

[0067] In certain embodiments, the plant cells providing the population are from an ASR-resistant plant or from a Leguminosae crop species of a genus selected from the group consisting of Glycine, Vigna, and Lablab.

[0068] In certain embodiments, the leguminous crop species or leguminous source 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 neclandestine, Glycine curvata, Glycine falcata, Glycine latifolia, Glycine microphylla, Glycine pescadrensis, Glycine stenophita, Glycine syndetica, Glycine soja, Glycine tabacina, and Glycine tomentella.

[0069] In another embodiment, the gene of the leguminous crop species or leguminous source is derived from Vigna. Vigna is a pantropic genus including about 100 species. It is a taxon subdivided into the following subgenera: Vigna, Haydonia, Plectotropis (Africa), Ceratotropis (Asia), Sigmoidotropis and Lasiopron. The genus includes economically relevant species such as cowpea (Vigna unguiculata (L.) Walp, cowpea), mung bean (Vigna radiata (L.) Wilczek, mung bean), red bean (Vigna angularis (Willd.) Ohwi and Ohashi, azuki bean), black bean (Vigna mungo (L.) Hepper, black gram) and adzuki bean (Vigna umbellata (Thunb.) Ohwi and Ohashi, rice bean). The following four subspecies are recognized within cowpea: dendtiana, a wild relative of the cultivated subspecies; cylindrica, the cultivated black cowpea; sesquipedalis, the cultivated long bean; and unguiculata, the cultivated black-eyed pea. The Vigna species cowpea is further divided into the following cultivar groups: Unguiculata, cultivated as a legume; Biflora or Cilindrica (black cowpea), grown primarily as fodder; Sesquipedalis (long bean or cowpea), cultivated as a vegetable; Textilis, cultivated for its long pedunculated fiber; and Melanophthalmus (black-eyed pea). Susceptibility of several Vigna species, including mung bean (Vigna radiata), black bean (Vigna mungo), and cowpea (Vigna unguiculata) to Psora pachyrhizi has been reported under field and greenhouse conditions.

[0070] In another embodiment, the legume crop species or legume-derived gene is derived from Lablab purpureus (L.) Sweet is a species of the Leguminosae family (Verdcourt (1971) Flora of Tropical East Africa, pp. 696-699, Crown Agents, London, England; and Duke et al. (1981) Handbook of Legumes of World Economic Importance, pp. 102-106, Plenum Press, New York, USA and London, England), 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 (UK), and at least 20 other common names. It is grown as a legume crop or as a green vegetable in Africa, Asia, and the Caribbean (Duke et al. (1981) Handbook of Legumes of World Economic Importance, pp. 102-106, Plenum Press, New York, USA and London, UK; and Pengelly and Maass, (2001) Gen. resour. crop ev. 48:261-272).

[0071] 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 USMSc. thesis. Iowa State University. Ames, Iowa, USA; Vakili (1981) Plant Dis. 65:817-819; and Poonpolgul and Surin, (1980) Soybean Rust Newsletter, 3:30-31).

[0072] In one aspect, the leguminous crop species or leguminous source gene is derived from 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. Examples of Cajanus species include, but are not limited to, Cajanus cajan. Examples of Medicago species include, but are not limited to, Medicago truncatula and Medicago sativa. Examples of Phaseolus species include, but are not limited to, Phaseolus vulgaris, Phaseolus lunatus, Phaseolus acutifolius, or Phaseolus coccineus. Examples of Pisum species include, but are not limited to, Pisum abyssinicum, Pisum sativum, Pisum elatius, Pisum fulvum, Pisum transcaucasium, and Pisum humile. Examples of Pueraria species include, but are not limited to, Pueraria lobata. Examples of Trifolium species include, but are not limited to, Trifolium aureum and Trifolium occidentale.

[0073] R gene polypeptide is the protein that recognizes the activity of specific effector and causes the immunity triggered by effector. R gene typically encodes the protein characterized by C-terminal leucine-rich repeat sequence (LRR) and nucleotide binding site (NBS) domain. Such nucleic acid binding LRR is called nucleotide binding LRR (NLR) protein. NBS domain acts as a molecular switch according to the nucleotide bound: ADP-binds in the static state, and ATP-binds in the active state. It is generally believed that LRR domain participates in effector recognition and autoinhibition (Ting et al., Immunity [immunity], 28 (2008), pp. 285-287). Typical plant NLR is almost universally characterized by other coiled coil (CC) or Toll / interleukin-1 receptor (TIR) ​​N-terminal domain. These N-terminal domains are used to classify plant NLR into two main groups, called CNL (CC-NLR) and TNL (TIR-NLR). It has been shown that both CC and TIR domains play a key role in the formation of dimers and oligomers.

[0074] Any method known in the art can be used to produce a population comprising multiple R gene polypeptides. In certain embodiments, a population is produced by isolating R gene polypeptides from ASR-resistant plant species (such as leguminous crop species disclosed herein). In certain embodiments, a population is produced by isolating R gene polypeptides from one or more leguminous crop species. In certain embodiments, a population is produced by producing variants of known ASR R gene polypeptides. As used herein, a variant refers to a protein derived from a natural protein by deleting or adding one or more amino acids at one or more sites in a natural protein and / or replacing one or more amino acids at one or more sites in a natural protein. Methods for such operations are known in the art. For example, amino acid sequence variants and fragments of resistance proteins can be prepared by mutations in DNA. Methods for mutagenesis and polynucleotide changes 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 will be understood by those of ordinary skill in the art, R gene polypeptide populations can be obtained from a variety of sources or methods, such that, for example, a population can contain R genes from resistant plant species as well as R gene variants.

[0075] In certain embodiments, the method for identifying a CcRpp1-like polypeptide comprises expressing at least one of the polynucleotides described herein (e.g., a polynucleotide encoding an effector polypeptide or a functional fragment thereof or a chimeric polypeptide) in a host cell, separating the expressed effector polypeptide and / or chimeric polypeptide and the protein bound thereto, and identifying one or more polypeptides that interact with the expressed effector polypeptide and / or chimeric polypeptide. The method for separating effector polypeptides may be any method described herein or known in the art. Similarly, the method for identifying 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). One or more candidate ASR resistance genes may be introduced into the host cell via a nucleic acid construct, may be a natural ASR resistance gene, or may be a combination of an introduced gene and a natural gene. The host cell may be any host cell provided herein. In certain embodiments, the host cell is a plant cell, such as Nicotiana benthamiana or maize (optionally using a method for detecting interactions as described in International Patent Application Publication No. WO 2021 / 211227).

[0076] In certain embodiments, the method for identifying CcRpp1-like polypeptides further comprises verifying whether one or more identified polypeptides are polypeptides that increase resistance to ASR. In certain embodiments, the method comprises expressing at least one of the effector proteins or chimeric proteins described herein and one or more of the identified polypeptides in a host cell, and determining the host cell's hypersensitive response (HR) cell death, H 2 O 2 In certain embodiments, the effector protein or chimeric protein comprises an ASR26 allele (e.g., SEQ ID NO: 1-8) or a functional fragment thereof. 2 O 2 In some embodiments, the effector protein or chimeric protein comprises an ASR26 allele or a functional fragment thereof encoding a polypeptide that produces HR cell death, H cell death, or a combination thereof. 2 O 2 accumulation, marker gene or marker metabolite expression or any combination thereof. The host cell can be any host cell provided herein. In certain embodiments, the host cell is a plant cell, such as, for example, Nicotiana benthamiana. For determining HR cell death and H 2 O 2Methods for accumulation are known in the art and include, but are not limited to, visual identification of HR cell death and H 2 O 2 DAB staining of accumulation. Examples of changes in metabolite expression or gene expression in response to pathogen infection can be found, for example, in 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):e1005827(2016)).

[0077] In certain embodiments, methods for verifying the identified polypeptides include expressing the polypeptides in leguminous plants, plant cells, tissues or organs (e.g., soybeans), and determining the resistance, immunity or susceptibility of the plants to ASR. General methods for determining the resistance, immunity or susceptibility of plants to specific pathogens are known to those skilled in the art. For example, methods for screening or determining the resistance, immunity or susceptibility of leguminous plants to plant diseases may include exposing plant cells, tissues or organs (e.g., leaves) to pathogens (e.g., Puccinia pachyrhizi), and then determining and / or measuring the degree of resistance, immunity and / or susceptibility of the exposed plants to plant diseases (e.g., ASR) caused by the pathogen. The method may further include measuring any observable plant disease symptoms on plants exposed to plant pathogens, and then comparing the plant disease symptoms to a reference standard to determine the degree or range of disease resistance. Methods for exposing plant cells, tissues or organs to pathogens are known in the art. Methods for measuring, comparing and determining the level of resistance, immunity and / or susceptibility (eg, plant disease symptoms) to diseases caused by pathogens (such as, for example, ASR) are also known in the art.

[0078] In certain embodiments, the method for identifying CcRpp1-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 determining HR cell death, H 2 O 2 In some embodiments, the effector polypeptide, chimeric polypeptide or functional fragment comprises an ASR26 allele. In some embodiments, the host cell is a cell isolated from an ASR-resistant plant, such that the cell comprises a candidate ASR resistance gene. In some embodiments, the candidate ASR resistance gene is introduced into the host cell.

[0079] Also provided herein is a method for selecting plants for planting in a cultivation area, the method comprising providing a sample comprising a plurality of isolates of Psora pachyrhizi, Psora dasyphylla, or a combination thereof from the cultivation area, determining the presence of an ASR26 allele or an ortholog thereof of the isolates, and selecting plants expressing a CcRpp1 polypeptide, a CcRpp1-like polypeptide, or a combination thereof for planting in the cultivation area 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 CcRpp1 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, plants expressing a CcRpp1 polypeptide, a CcRpp1-like polypeptide, or a combination thereof are 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 (eg, a P. pachyrhizium, P. dasyphylla isolate) are well known in the art and include, for example, PCR.

[0080] As used herein, a cultivation area includes any area where people wish to grow plants. Such cultivation areas include, but are not limited to, fields (such as crop fields, turf fields, wood fields, managed forests, fields for growing fruits and vegetables, etc.), greenhouses or growth chambers where plants are grown.

[0081] In certain embodiments, the plant expressing the CcRpp1 polypeptide, CcRpp1-like polypeptide or a combination thereof described herein is an elite plant strain (e.g., an elite soybean strain). In certain embodiments, plant cells, plant parts, seeds and grains are isolated from or produced by an elite plant strain. As used herein, "elite strain" refers to any strain obtained by breeding and selection for excellent agronomic traits that allow producers to harvest commercially significant products. Many superior strains are available and are known to those skilled in the art of plant breeding (e.g., soybean, canola and sunflower breeding). "Excellent population" is a mixture of state-of-the-art individuals or strains that can be used to represent a given crop species (e.g., soybean) with an agronomically excellent genotype.

[0082] The plant species of the disclosed compositions and methods (eg, the plant from which the host cell is isolated and the plant expressing the CcRpp1 polypeptide, CcRpp1-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 species (e.g., Brassica napus, Brassica rapa, Brassica juncea), particularly those Brassica species that can be used as a source of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), millet (Panicum miliaceum), foxtail millet (Setaria italica), millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffea species), coconut (Cocos nucifera), pineapple (Ananascomosus), citrus (Citrus species), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa species), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango,Mangiferaindica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugar cane (Saccharum species), oats, barley, vegetables, ornamentals and conifers.

[0083] In certain embodiments, the plant of the compositions and methods described herein is a leguminous crop species, including but not limited to alfalfa (Medicago sativa); clover or clover (Trifolium species); pea, including pea (Pisum satinum), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata), and Lathyrus species; bean (Fabaceae or Leguminosae); lentil (Lens culinaris); lupine (Lupinus species); mesquite (Prosopis species); carob (Ceratonia siliqua), soybean (Glycine max), peanut (Arachis hypogaea), or tamarind (Tamarindus indica). The terms "leguminous species" and "leguminous crop species" are used herein to refer to plants, and can be, for example, a plant of interest. In certain embodiments, the leguminous species or leguminous crop species is a plant, a plant part, or a plant cell.

[0084] Also provided herein is a method for identifying an ASR26 allele, the method comprising providing a population comprising a plurality of Puccinia pachyrhizi isolates, Puccinia oleracea isolates, or a combination thereof, and detecting the allele of ASR26 in the population. In certain embodiments, the plurality of isolates are taken from a cultivation area. The method for identifying the allele is not particularly limited and may be any method known in the art. In certain embodiments, the detection comprises genome sequencing or RNA sequencing.

[0085] In certain embodiments, the method further comprises assaying the identified ASR26 allele to determine whether the identified allele is recognized by a CcRpp1 polypeptide, a chimeric CcRpp1 polypeptide, or a CcRpp1-like polypeptide, or any combination thereof. The assay can be any of the assays described herein or known in the art that determine the interaction between the ASR26 allele and a CcRpp1, CcRpp1-like, or chimeric CcRpp1 polypeptide. In certain embodiments, the assay is a pull-down assay or an immunoprecipitation assay for determining binding between the allele and a CcRpp1, CcRpp1-like, or chimeric CcRpp1 polypeptide. In certain embodiments, the ASR26 allele and a CcRpp1, CcRpp1-like, or chimeric CcRpp1 polypeptide are expressed in a host cell, and HR cell death, H cell death, or H cell death are measured. 2 O 2 accumulation, presence of marker gene expression and / or marker metabolite expression.

[0086] As will be appreciated by one of ordinary skill in the art, the methods described herein for identifying CcRpp1-like polypeptides using ASR26 alleles or functional fragments thereof can be adapted to identify candidate ASR resistance polypeptides using effector polypeptides (e.g., non-ASR26 polypeptides) described herein. Thus, also provided herein are methods for identifying ASR resistance proteins by identifying one or more polypeptides that bind to an effector polypeptide or a functional fragment thereof or a chimeric polypeptide produced therefrom, the 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. The method for identifying one or more polypeptides can be any method described herein or known in the art that allows identification of interacting proteins. The methods may further comprise validating the identified interacting proteins using any of the validation methods described herein, including but not limited to expression in host cells to assay for HR cell death, H 2 O 2 Accumulation, marker gene or marker metabolite expression or any combination thereof, and expression in plants to determine resistance to ASR.

[0087] In certain embodiments, the binding of a candidate resistance gene to an ASR26 allele is determined. In certain embodiments, a candidate resistance gene that does not bind to an ASR26 allele is selected. In certain embodiments, a population of R gene polypeptides used in identifying candidates that bind or interact with an effector polypeptide (e.g., SEQ ID NO: 10-309) is prescreened to remove R gene polypeptides that bind to an ASR26 allele. Methods for removing polypeptides from a sample are known in the art and include, but are not limited to, pull-down assays, immunoprecipitation, and column chromatography.

[0088] Also provided herein are methods for identifying ASR resistance polypeptides that do not compete with CcRpp1 (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 or a functional fragment thereof, the effector polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 10-309, determining the interaction of one or more identified polypeptides with a polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 1-8, and selecting one or more polypeptides that do not interact with a polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 1-8. One or more identified ASR resistance polypeptides that do not compete with CcRpp1 can be expressed in plants alone or in combination with another ASR resistance polynucleotide (e.g., such as CcRpp1) using methods described herein or known in the art to produce plants with ASR resistance. The method for 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 can be any method described herein (e.g., in combination with assaying and / or measuring HR cell death, H ... 2 O 2Accumulation, marker gene or marker metabolite expression or any combination thereof). In certain embodiments, the method comprises providing a polypeptide population from ARS-resistant plant cells; incubating the polypeptide population with an effector polypeptide, a functional fragment thereof, or a chimeric polypeptide comprising an effector polypeptide or a functional fragment thereof under conditions that allow at least one effector polypeptide, a functional fragment thereof, or a chimeric polypeptide to bind to one or more polypeptides of the population; separating the effector polypeptide or chimeric polypeptide and the polypeptide interacting therewith from the population; and identifying one or more polypeptides. In certain embodiments, one or more polypeptides are identified by providing a population comprising a plurality of R gene candidates, optionally prescreened to remove candidates that bind to ASR26 alleles; incubating the population with an effector polypeptide or a chimeric polypeptide comprising an effector polypeptide or a functional fragment thereof under conditions that allow the effector polypeptide or chimeric polypeptide to bind to one or more R gene candidates in the population; separating the effector polypeptide or chimeric polypeptide and the interacting R gene candidates from the population; and identifying the R gene candidates. In certain embodiments, one or more polypeptides are identified by: introducing a nucleic acid construct comprising a polynucleotide encoding an effector polypeptide or a functional fragment thereof or a chimeric polypeptide comprising an effector polypeptide or a functional fragment thereof into a host cell comprising one or more candidate ASR resistance genes; isolating the expressed effector polypeptide or chimeric polypeptide; and identifying one or more polypeptides that interact with the expressed effector polypeptide or chimeric polypeptide. The method for determining the interaction of one or more identified polypeptides with a polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 1-8 can be any method described herein (e.g., in combination with determining and / or measuring HR cell death, H ... 2 O 2 accumulation, marker gene or marker metabolite expression, or any combination thereof).

[0089] In certain embodiments, the method for identifying an ASR resistance polypeptide that does not compete with CcRpp1 comprises expressing 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 in a host cell comprising one or more R gene candidate polypeptides; and determining HR cell death, H 2 O 2 accumulation, expression of marker genes or marker metabolites, or any combination thereof; identifying the effector peptides that cause HR cell death, H 2 O 2The invention relates to a method for expressing a candidate R gene polypeptide that accumulates, marks a gene or marks a metabolite; expressing one or more identified candidate R gene polypeptides and an ASR26 polypeptide having at least 90% sequence identity with any one of SEQ ID NOs: 1-8 in a second host cell; and determining HR cell death, H 2 O 2 accumulation, marker gene or marker metabolite expression, or any combination thereof; and selecting for HR cell death, H ... 2 O 2 accumulation, marker gene or marker metabolite expression, or any combination thereof, of one or more identified R gene candidate polypeptides.

[0090] Further provided are methods for detecting and / or isolating a soluble form (e.g., an active form) of a CcRpp1 protein in a sample. In certain embodiments, the method comprises incubating a mixture comprising an ASR26 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 1-8 and a sample containing a CcRpp1 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; separating the ASR26 polypeptide and polypeptides bound thereto from the mixture; and detecting the presence of the CcRpp1 protein in the separated 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 CcRpp1 polypeptide is operably linked to a protein tag, such as those described herein. In certain embodiments, the protein tag operably linked to the CcRpp1 polypeptide is a protein tag that is different from the protein tag operably linked to ASR26. The method for isolating the ASR26 polypeptide is not particularly limited and can be any method described herein or known in the art that can separate proteins from a mixture while maintaining protein-protein interactions, such as, for example, immunoprecipitation and pull-down assays. The method for detecting the CcRpp1 polypeptide can 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.

[0091] The following are examples of specific embodiments of some aspects of the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0092] Example 1

[0093] Identification of predicted effectors of Phakopsora pachyrhizi.

[0094] The transcriptome of P. pachyrhizi (the causal agent of ASR) was generated by aligning paired-end RNA-seq reads from infected soybean plants to the P. pachyrhizi genome. Transcripts that were significantly expressed in the dataset were then used to generate a predicted P. pachyrhizi proteome.

[0095] The resulting proteins were then evaluated for effector features, including the presence of signal peptides (SignalP 3.0 and Phobius), sequence-based machine learning (EffectorP 3.0), conserved domains (HMMer), and the absence of transmembrane domains (TMHMM).

[0096] Example 2

[0097] Identification of candidate avirulence (Avr) genes for CcRpp1.

[0098] CcRpp1 is an NLR gene from pigeon pea and confers resistance to Psora pachyrhizi. A biochemical approach was used to identify CcRpp1 homologous effectors. FLAG-tagged CcRpp1 protein was expressed in E. coli cells and then incubated with extracts of soybean leaf tissue infected with ASR. CcRpp1 interacting proteins were pulled down with anti-FLAG agarose beads and subsequently analyzed by mass spectrometry. When searched against a list of predicted Psora pachyrhizi effectors, eight putative ASR effectors were identified.

[0099] Example 3

[0100] Identification of ASR26 as a cognate effector recognized by CcRpp1.

[0101] Recognition of pathogen effectors by NLR proteins leads to effector-triggered immunity, often ultimately leading to HR cell death accompanied by reactive oxygen species production. The recognition between CcRpp1 and putative effectors was confirmed using an Agrobacterium-mediated transient expression system in Nicotiana benthamiana. All eight ASR candidate effectors and CcRpp1 identified in Example 2 were cloned individually into binary vectors and introduced into Agrobacterium tumefaciens AGL1. When co-expressed with CcRpp1 in Nicotiana benthamiana, only one effector, ASR26, caused HR cell death (visual observation) and accumulation of H 2 O 2 (DAB staining), indicating that CcRpp1 can recognize ASR26 and trigger an immune response.

[0102] Co-immunoprecipitation was performed to examine the physical interaction between CcRpp1 and ASR26. The results of these studies confirmed that CcRpp1 interacts with ASR26 in plants.

[0103] Example 4

[0104] CcRpp1 recognizes four known ASR26 alleles.

[0105] Surveying ASR26 alleles using genome sequences and / or RNA-seq datasets from thirteen P. pachyrhizi isolates resulted in the identification of a total of four ASR26 alleles. All four ASR26 alleles caused HR cell death and accumulation of H when co-expressed with CcRpp1 in N. benthamiana. 2 O 2 , indicating that CcRpp1 can recognize all four ASR26 alleles and is expected to provide resistance to all thirteen isolates.

[0106] Example 5

[0107] Soluble CcRpp1 protein, but not insoluble CcRpp1 protein, physically interacts with ASR26.

[0108] CcRpp1 protein with His tag (CcRpp1-His) was produced using the Bl21Gold E. coli expression system or the Sf9 insect cell expression system. The CcRpp1-His protein produced from E. coli cells is insoluble, while the CcRpp1-His protein from insect cells is soluble. ASR26 protein fused to the MBP tag (MBP-ASR26) was produced in E. coli cells and determined to be soluble. In order to determine whether ASR26 can interact with the soluble CcRpp1 protein predicted to be in active form, in vitro pull-down assays were performed using both soluble and insoluble forms of the expressed CcRpp1 protein as capture and labeled ASR26 protein as bait.

[0109] For in vitro pull-down assays, purified CcRpp1-His (capture) and MBP-ASR26 (bait) proteins were obtained. The MBP-ASR26 (bait) protein was fixed to anti-MBP magnetic beads and any unattached protein was removed by washing with MBP column binding / washing buffer. Thereafter, the CcRpp1-His (capture) protein was introduced into the beads bound to MBP-ASR26 and allowed to incubate for a minimum of 30 minutes. After washing away the unbound proteins, the formed protein complex was released from the beads. The eluted proteins were analyzed using SDS-PAGE and Western blotting with anti-MBP antibodies and / or anti-His antibodies to visualize and detect protein interactions.

[0110] These results suggest that ASR26 can be used to determine whether purified CcRpp1 protein is in a soluble form.

[0111] All publications and patent applications in this specification are indicative of the levels of ordinary skill in the art to which the 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 to be incorporated by reference.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. Unless otherwise mentioned, the techniques employed or contemplated herein are standard methods well known to those of ordinary skill in the art. The materials, methods and examples are illustrative only and are not limiting.

[0113] Many modifications and other embodiments of the invention set forth herein will occur to those skilled in the art with the aid of the teachings given in the foregoing description and the accompanying drawings. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and 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 only in a general and descriptive sense and not for purposes of limitation.

[0114] Units, prefixes and symbols may be represented in their SI accepted form. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino to carboxyl direction. Numerical ranges include the values ​​of the defined ranges. Amino acids may be referred to herein by their commonly known three letter symbols or by the single letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single letter codes.

Claims

1. A nucleic acid construct comprising a polynucleotide encoding an effector polypeptide or a functional fragment thereof having at least 90% sequence identity with any one of SEQ ID NOs: 1-8, which is operably linked to a regulatory element.

2. The nucleic acid construct of claim 1, wherein the regulatory element is a heterologous regulatory element.

3. The nucleic acid construct of claim 2, wherein the heterologous regulatory element is a promoter that functions in plant cells.

4. A chimeric polypeptide comprising an effector polypeptide or a functional fragment thereof operably linked to a heterologous peptide, wherein the effector polypeptide comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 1-8.

5. The chimeric polypeptide of claim 4, wherein the heterologous peptide comprises a protein tag.

6. The chimeric polypeptide of claim 5, wherein the protein tag is selected from the group consisting of a FLAG tag, an HA tag, a His tag, a myc tag, a glutathione-s-transferase (GST) tag, and an Fc tag.

7. A polynucleotide encoding the chimeric polypeptide according to any one of claims 4 to 6.

8. A nucleic acid construct comprising the polynucleotide according to claim 7.

9. A host cell comprising the nucleic acid construct of any one of claims 1-3 or 8.

10. The host cell of claim 9, wherein the host cell is a bacterial cell, a fungal cell or a plant cell.

11. The host cell of claim 10, wherein the host cell is a plant cell.

12. The host cell of claim 11, wherein the plant cell is a plant protoplast.

13. The host cell of any one of claims 10-12, wherein the plant cell further comprises a candidate resistance gene.

14. The host cell of claim 13, wherein the candidate resistance gene is introduced into the host cell using a nucleic acid construct comprising the candidate resistance gene operably linked to a regulatory element.

15. A method for identifying a CcRpp1-like protein, the method comprising identifying one or more polypeptides that bind to the chimeric polypeptide of any one of claims 4-6.

16. The method of claim 15, wherein the method comprises: a. Providing a polypeptide population from an ASR-resistant plant cell; b. incubating the polypeptide population with the at least one chimeric polypeptide under conditions that allow the at least one chimeric polypeptide to bind to one or more polypeptides of the population; c. separating said at least one chimeric polypeptide and its interacting polypeptide from said population; as well as d. identifying one or more polypeptides that interact with the chimeric polypeptide.

17. The method of claim 15, wherein the method comprises: a. providing a population comprising a plurality of R gene candidates; b. incubating the population with the at least one chimeric polypeptide under conditions that allow the at least one chimeric polypeptide to bind to one or more R gene candidates in the population; c. isolating said at least one chimeric polypeptide and interacting R gene candidate from said population; and d. Identifying R gene candidates that interact with the at least one chimeric polypeptide.

18. The method of claim 16 or 17, wherein the incubation is performed in the presence of a cross-linking agent.

19. The method of claim 18, wherein the crosslinker is a homobifunctional crosslinker or a photoreactive crosslinker.

20. The method of claim 15, wherein the method comprises: a. introducing the nucleic acid DNA construct as claimed in claim 8 into a host cell comprising one or more candidate ASR resistance genes; b. isolating the expressed chimeric polypeptide; and c. Identifying one or more polypeptides that interact with the expressed chimeric polypeptide.

21. A method for identifying a CcRpp1-like protein, the method comprising identifying one or more polypeptides that bind to an effector polypeptide or a functional fragment thereof, wherein the effector polypeptide has at least 90% sequence identity with any one of SEQ ID NOs: 1-8.

22. The method of claim 21, wherein the method comprises: a. Providing a polypeptide population from ARS-resistant plant cells; b. incubating the polypeptide population with the at least one effector polypeptide under conditions that allow binding of the at least one effector polypeptide to one or more polypeptides of the population; c. separating the at least one effector polypeptide and the polypeptide interacting therewith from the population; as well as d. Identifying a polypeptide that interacts with the at least one effector polypeptide.

23. The method of claim 21, wherein the method comprises: a. providing a population comprising a plurality of R gene candidates; b. incubating the population with the at least one effector polypeptide under conditions that allow the at least one effector polypeptide to bind to one or more R gene candidates in the population; c. isolating the at least one effector polypeptide and interacting R gene candidate from the population; and d. Identifying R gene candidates that interact with the at least one effector polypeptide.

24. The method of claim 22 or 23, wherein the incubation is performed in the presence of a cross-linking agent.

25. The method of claim 24, wherein the crosslinker is a homobifunctional crosslinker or a photoreactive crosslinker.

26. A method for identifying a CcRpp1-like protein, the method comprising: a. introducing into a host cell comprising one or more candidate ASR resistance genes a nucleic acid construct as described in any one of claims 1 to 3; b. separating the expressed effector polypeptide; as well as c. Identifying one or more polypeptides that interact with the expressed effector polypeptide.

27. The method of any one of claims 15-26, wherein the method further comprises expressing the at least one effector polypeptide and the one or more identified interacting polypeptides 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.

28. The method of claim 27, wherein the one or more identified interacting polypeptides are expressed individually in the host cell.

29. The method of any one of claims 27-29, wherein the host cell is a plant cell.

30. A method for identifying a CcRpp1-like protein, the method comprising measuring HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof, of the host cell of claim 13 or 14.

31. A polypeptide comprising a functional fragment of a polypeptide comprising an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 1-8, wherein the functional fragment interacts with the CcRpp1 polypeptide of SEQ ID NO: 9 or a functional fragment thereof.

32. A method for selecting plants for planting in an area of ​​cultivation, the method comprising: a. providing a sample comprising a plurality of isolates of Phakopsora pachyrhizi from a cultivation area; b. determining the presence of a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-8; and c. When the polynucleotide is present, selecting plants expressing a CcRpp1 polypeptide, a CcRpp1-like polypeptide or a combination thereof for planting in the cultivation area.

33. The method of claim 32, wherein the CcRpp1 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

9.

34. The method of claim 32 or 33, wherein when the polynucleotide is present in at least 30% of the isolates tested, the plants expressing the CcRPP1 polypeptide, the CcRPP1 -like polypeptide or a combination thereof are selected for planting in the area of ​​cultivation.

35. A method for identifying an ASR26 allele, the method comprising: a. providing a population comprising a plurality of isolates of Phakopsora pachyrhizi, and b. Detecting ASR26 alleles in said population.

36. The method of claim 35, wherein the population comprising a plurality of P. pachyrhizi isolates is taken from an area under cultivation.

37. The method of claim 35 or 36, wherein the detecting comprises genome sequencing, RNA sequencing, or a combination thereof.

38. The method of any one of claims 35-37, wherein the method further comprises expressing the identified ASR26 allele and CcRPP1 polypeptide, CcRPP1-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.

39. A method for identifying an ASR resistance polypeptide that does not compete with CcRpp1, the method comprising: a. Identifying one or more polypeptides that bind to an effector polypeptide or a functional fragment thereof, wherein the effector polypeptide has at least 90% sequence identity with any one of SEQ ID NOs: 10-309; b. determining the interaction of the one or more identified polypeptides with a polypeptide having at least 90% sequence identity with any one of SEQ ID NOs: 1-8; and c. Selecting 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.

40. The method of claim 39, wherein the one or more polypeptides are identified by: a. Providing a polypeptide population from ARS-resistant plant cells; b. incubating the polypeptide population with the effector polypeptide or a functional fragment thereof or a chimeric polypeptide comprising the effector polypeptide or a functional fragment thereof under conditions that allow at least one chimeric polypeptide to bind to one or more polypeptides of the population; c. separating the effector polypeptide or the chimeric polypeptide and the polypeptide interacting therewith from the population; and d. identifying the one or more polypeptides.

41. The method of claim 39, wherein the one or more polypeptides are identified by: a. providing a population comprising a plurality of R gene candidates; b. incubating the population with the effector polypeptide, a functional fragment thereof, or a chimeric polypeptide comprising the effector polypeptide or a functional fragment thereof under conditions that allow the effector polypeptide or the chimeric polypeptide to bind to one or more R gene candidates in the population; c. isolating the effector polypeptide or the chimeric polypeptide and interacting R gene candidate from the population; and d. Identify the R gene candidates.

42. The method of claim 41, wherein the population comprising a plurality of R gene candidates is pre-screened to remove candidates that bind to the ASR26 allele.

43. The method of claim 39, wherein the method comprises: a. introducing into a host cell comprising one or more candidate ASR resistance genes a nucleic acid construct comprising a polynucleotide encoding the effector polypeptide, a functional fragment thereof, or a chimeric polypeptide comprising the effector polypeptide or a functional fragment thereof; b. isolating the expressed effector polypeptide or chimeric polypeptide; and c. Identifying one or more polypeptides that interact with the expressed effector polypeptide or chimeric polypeptide.

44. A method for identifying an ASR resistance polypeptide that does not compete with CcRpp1, the method comprising: a. expressing a polynucleotide in a host cell comprising one or more R gene candidate polypeptides, wherein the polynucleotide encodes an effector polypeptide or a functional fragment thereof having at least 90% sequence identity with any one of SEQ ID NOs: 10-309; b. determining the presence of HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof, of the host cell; c. identifying R gene candidate polypeptides that cause HR cell death, H2O2 accumulation, marker gene or marker metabolite expression in the presence of the effector polypeptide; d. expressing the one or more identified R gene candidate polypeptides and an ASR26 polypeptide having at least 90% sequence identity with any one of SEQ ID NOs: 1-8 in a second host cell; e. determining the presence of HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof, of the host cell; and f. selecting one or more identified R gene candidate polypeptides that do not cause HR cell death, H2O2 accumulation, marker gene or marker metabolite expression, or any combination thereof, in the presence of the ASR26 effector polypeptide.

45. A method for detecting soluble CcRpp1 protein in a sample, the method comprising a. incubating a mixture comprising an ASR26 polypeptide having at least 90% sequence identity with any one of SEQ ID NOs: 1-8 and a sample containing a CcRpp1 protein; b. separating the ASR26 polypeptide and the polypeptide bound thereto from the mixture; and c. Detecting the presence of CcRpp1 protein in the isolated ASR26 polypeptide and the polypeptide bound thereto, thereby detecting soluble CcRpp1 protein.

46. ​​The method of claim 45, wherein the CcRpp1 protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9.