Methods for modulating immune function in plants

Abstract

The present invention relates to genetically modified plants, parts thereof, and plant cells that contain one or more mutations in one or more NLR proteins, such as the helper NLR proteins NRC2 and / or NRC3, and to methods for conferring or improving plant immunity against pathogens or pests by introducing one or more mutations into one or more NLR genes.

Description

[Technical Field]

[0001] The present invention relates to genetically modified plants, plant parts thereof, and plant cells containing one or more mutations in one or more NLR proteins, such as the helper NLR proteins NRC2 and / or NRC3, and methods for conferring or improving immunity of plants against pathogens or pests by introducing one or more mutations in one or more NLR genes. [Background technology]

[0002] Plant diseases and pests threaten agricultural productivity and food quality and pose a clear and present crisis to humanity's food systems. Global crop yield losses due to pathogens and pests have reached 30%, costing hundreds of billions of dollars in lost food production—enough to feed billions of people. It is estimated that by 2050, global food production will need to increase by approximately 70% to feed the projected world population.

[0003] Global trade, climate change, and the tendency of plant pathogens to destroy disease resistances painstakingly engineered into crop plants are increasing the frequency of plant disease outbreaks. Current strategies for controlling crop diseases rely on chemical control, but common fungicides / pesticides face increasingly strict regulation due to environmental and health concerns. The most sustainable strategy for dealing with plant pathogens and pests is to engineer crops with broad-spectrum disease resistance. However, current disease resistance breeding approaches are slow, inefficient, and do not fully utilize our current fundamental understanding of plant immunity.

[0004] A sustainable way to combat pathogens is through the genetic improvement of crops. Plants possess a genetic toolkit to fight disease, but resistance breeding has been limited by pathogens' ability to adapt and evade the plant's immune system. Plants defend themselves against parasites by encoding specialized disease resistance proteins, which act as immune sensors and activate plant immunity upon detecting pathogens. However, some pathogens evade or suppress plant disease resistance, limiting the utility of these immune sensors in agriculture. Manipulation of disease resistance functions is another strategy, but historically has been limited by a limited understanding of the underlying mechanisms.

[0005] Thus, there is a need to develop new genetic mechanisms for conferring disease resistance to plants, particularly commercially important crops.

[0006] The nucleotide-binding and leucine-rich repeat (NLR) class of intracellular immune receptors is a key component of innate immunity in plants and animals. It mediates intracellular recognition of pathogens and subsequently initiates a series of immune responses to prevent infection. NLRs can be activated by pathogen-secreted virulence proteins called effectors, which are released by pathogens into host cells and modulate host physiology. NLR activation in plants and animals is characterized by oligomerization to form higher-order immune complexes called resistosomes and inflammasomes, respectively. These immune complexes initiate immune signaling by diverse mechanisms, which often trigger a form of programmed cell death called the hypersensitive response (HR) in plants and pyroptosis in animals. Recent studies have reported that NLR-like proteins in prokaryotes mediate antiviral immunity and programmed cell death through mechanisms similar to those observed in eukaryotic NLRs, suggesting that this is a conserved defense mechanism across all three domains of life. Notably, pathogen effectors can act as both triggers and suppressors of NLR-mediated immunity. In some cases, adapted pathogens utilize effectors that directly or indirectly inhibit NLR signaling and suppress immune activation through diverse strategies. The precise mechanisms by which pathogen effectors attenuate NLR-mediated immunity and allow disease progression remain largely unknown. Furthermore, although various strategies have been proposed in recent years to engineer novel effector recognition specificities in NLRs, approaches to mitigate the immunosuppressive effects of effector-mediated NLRs remain lacking. Summary of the Invention

[0007] The techniques described herein allow for the engineering of disease-resistant crops with minimal changes to the genome, thus enabling increased food production while minimizing the use of chemicals. Until now, it has been difficult to determine how to utilize modern breeding techniques to improve disease resistance. Thus, the inventions described herein represent a valuable tool to support farmers in their fight against plant diseases and aid in their efforts to feed the world.

[0008] The following embodiments apply to all aspects of the present invention.

[0009] In one embodiment of the present invention, there is provided a genetically modified plant, plant part, or plant cell, comprising at least one mutation in at least one nucleic acid sequence encoding an NLR (nucleotide-binding domain and leucine-rich repeat-containing) protein; the NLR protein comprises an HD1 domain; and the at least one mutation is a mutation of one or more amino acids at the N-terminal portion of the HD1 domain.

[0010] In one embodiment, the at least one mutation reduces or prevents inhibition by pathogen effectors of the initiation of an immune response by the NLR protein.

[0011] In another aspect of the present invention, there is provided a genetically modified plant, plant part, or plant cell, comprising at least one mutation in at least one nucleic acid sequence encoding an NLR (nucleotide-binding domain and leucine-rich repeat-containing) protein; said NLR protein initiates an immune response pathway; and said at least one mutation reduces or prevents inhibition by pathogen effectors of the initiation of said immune response pathway by said NLR protein.

[0012] In another aspect of the present invention, there is provided a method for conferring or improving pathogen resistance in a plant, the method comprising introducing at least one mutation into at least one nucleic acid sequence encoding an NLR (nucleotide-binding domain and leucine-rich repeat-containing) protein; the NLR protein comprises an HD1 domain; and the at least one mutation is a mutation of one or more amino acids at the N-terminal portion of the HD1 domain.

[0013] In another aspect of the present invention, there is provided a method for conferring or improving pathogen resistance in a plant, the method comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein; the NLR protein comprises an HD1 domain; and the NLR protein comprises one or more amino acid mutations at the N-terminal portion of the HD1 domain.

[0014] In another aspect of the present invention, there is provided a method for producing a plant with improved pathogen resistance, the method comprising introducing at least one mutation into at least one nucleic acid sequence encoding an NLR (nucleotide-binding domain and leucine-rich repeat-containing) protein; the NLR protein comprises an HD1 domain; and the at least one mutation is a mutation of one or more amino acids at the N-terminal portion of the HD1 domain.

[0015] In another aspect of the present invention, there is provided a method for producing a plant with improved pathogen resistance, comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein; the NLR protein comprises an HD1 domain; and the NLR protein comprises one or more amino acid mutations at the N-terminal portion of the HD1 domain.

[0016] In another aspect of the present invention, there is provided a method for generating a modified NLR protein (wherein a pathogen effector is unable to bind to said NLR protein or the pathogen effector has a reduced ability to bind to said NLR protein), the method comprising introducing at least one mutation into at least one nucleic acid sequence encoding an NLR protein; said NLR protein comprises an HD1 domain; and said at least one mutation is a mutation of one or more amino acids at the N-terminal portion of said HD1 domain.

[0017] In one embodiment, the NLR protein initiates an immune response by interacting with one or more downstream corresponding signaling factors, and the at least one mutation preserves the interaction(s).

[0018] In another aspect of the present invention, there is provided a genetically modified plant, plant part, or plant cell, which expresses a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein; the NLR protein comprises an HD1 domain; and the NLR protein comprises one or more amino acid mutations at the N-terminal portion of the HD1 domain.

[0019] In one embodiment, the NLR protein is an NRC (nucleotide-binding domain and leucine-rich repeat-containing required for cell death) protein (a protein containing a nucleotide-binding domain and leucine-rich repeat required for cell death).

[0020] In one embodiment, the at least one mutation reduces or prevents inhibition by pathogen effectors of NRC protein oligomerization to form complexes to initiate an immune response.

[0021] In one embodiment, the mutation reduces or prevents binding between the NLR protein and the pathogen effector.

[0022] In one embodiment, the N-terminal portion of the HD1 domain comprises 42 or fewer amino acids at the N-terminus of the HD1 domain, more preferably 38 or fewer amino acids at the N-terminus of the HD1 domain.

[0023] In one embodiment, the N-terminal portion of the HD1 domain comprises the sequence set forth in SEQ ID NO: 2, 5, 8, 14, 17, 23, 26, or 29, or a variant or fragment thereof. Preferably, the variant has at least 60% overall sequence identity to SEQ ID NO: 2, 5, 8, 14, 17, 23, 26, or 29.

[0024] In one embodiment, the at least one mutation is in the RNBS-C motif of the HD1 domain. Preferably, the RNBS-C motif comprises the sequence set forth in SEQ ID NO: 3, 6, 9, 15, 18, 24, 27, or 30, or a variant or fragment thereof. More preferably, the variant has at least 60% overall sequence identity to the sequence set forth in SEQ ID NO: 3, 6, 9, 15, 18, 24, 27, or 30.

[0025] In one embodiment, the mutation is a substitution.

[0026] Preferably, the mutations are substitutions at one or more positions in the amino acid sequence, wherein the positions are selected from positions 315, 316, or 317 of SEQ ID NO: 32, 34, 36, 41, 43, 47, 79, or 51, or any of their homologs or functional variants. Alternatively, the substitutions are at homologous positions in homologous sequences.

[0027] In one embodiment, the substitution is with a hydrophilic amino acid and / or a positively charged amino acid.

[0028] In one embodiment, the mutation is a substitution of a D residue. Alternatively, the mutation is a substitution of an E residue. Alternatively, the mutation is a substitution of an N residue. Alternatively, the mutation is a substitution of an S residue.

[0029] In one embodiment, the substitution is a D317K substitution in SEQ ID NO: 32 or 34, or a homologue or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0030] In one embodiment, the substitution is an N317K substitution in SEQ ID NO: 36, or a homologue or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0031] In one embodiment, the substitution is a D315K substitution in SEQ ID NO: 41, 43, 49, or 51, or a homolog or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0032] In one embodiment, the substitution is a D316K substitution in SEQ ID NO: 47, or a homologue or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0033] In one embodiment, the substitution is a S317K substitution in SEQ ID NO: 41, 43, 49, or 51, or a homolog or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0034] In one embodiment, the substitution is an E317K substitution in SEQ ID NO: 47, or a homologue or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0035] Preferably, as shown in Figure 5, said substitution is preferably at position D317 of SEQ ID NO: 32 or 34, or at a homologous position in a homologous sequence, wherein said substitution is preferably D317K.

[0036] In one embodiment, the mutation is a substitution of all or part of the N-terminal portion of the HD1 domain with a corresponding portion in a second NLR protein, wherein the second NLR protein is not inhibited by the pathogen effector.

[0037] In one embodiment, the mutation is a replacement of all or most of the RNBS-C motif with a corresponding RNBS-C motif in a second NLR protein, wherein the second NLR protein is not inhibited by the pathogen effector.

[0038] In one embodiment, the nucleic acid construct expresses a nucleic acid sequence encoding an NLR protein, wherein the NLR protein is selected from SEQ ID NO: 37, 38, 39, 44, 45, 52, 53, 54, 55, 56, 57, 58, or 59, and preferably, the regulatory sequence is operably linked to a regulatory sequence.

[0039] In one embodiment, the pathogen is Potato Cyst Nematode. Preferably, the pathogen effector is SPRYSEC15.

[0040] In one embodiment, the plant is a monocotyledonous or dicotyledonous plant. Preferably, the plant is a crop plant. More preferably, the plant is a Solanaceae plant.

[0041] In one embodiment, the NLR protein is NRC1 and / or NRC2 and / or NRC3.

[0042] In one embodiment, the plant part is a seed or grain.

[0043] In another aspect of the present invention, there is provided a plant obtained or obtainable by the method of the present invention.

[0044] In another aspect of the present invention, there is provided a method for screening a population of plants to identify and / or select plants that exhibit pathogen resistance or that will exhibit improved pathogen resistance, the method comprising detecting in said plants or plant reproductive cells at least one polymorphism in the NRC2 and / or NRC3 gene (preferably, said polymorphism is a polymorphism in the N-terminal portion of the HD1 domain of said NRC2 and / or NRC3 gene).

[0045] In one embodiment, the N-terminal portion of the HD1 domain comprises 42 or fewer amino acids at the N-terminus of the HD1 domain, more preferably 38 or fewer amino acids at the N-terminus of the HD1 domain.

[0046] In another aspect of the present invention, there is provided an isolated NLR (nucleotide-binding domain and leucine-rich repeat-containing) protein, wherein said NLR protein comprises a sequence selected from SEQ ID NOs: 37, 38, 39, 44, 45, 52, 53 54, 55, 56, 57, 58, or 59, or a functional variant or homolog thereof.

[0047] The invention is further illustrated in the following non-limiting drawings. [Brief explanation of the drawings]

[0048]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0049] The present invention will now be further described. In the following description, different aspects of the present invention will be defined in more detail. Each aspect so defined may be combined with any other aspect(s), unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature(s) indicated as being preferred or advantageous.

[0050] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, bacteriology, tissue culture, molecular biology, chemistry, biochemistry, recombinant DNA technology, and bioinformatics, which are within the skill of the art, and such techniques are fully explained in the literature.

[0051] As used herein, the terms "nucleic acid," "nucleic acid sequence," "nucleotide," "nucleic acid molecule," or "polynucleotide" are intended to encompass DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural, mutated, or synthetic DNA or RNA molecules, and analogs of DNA or RNA produced using nucleotide analogs. They may be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, antisense sequences, and non-coding regulatory sequences that do not encode an mRNA or protein product. These terms also encompass genes. The terms "gene" or "gene sequence" are used broadly to refer to DNA nucleic acids associated with a biological function. Thus, a gene may include introns and exons, as in a genomic sequence, or may include only the coding sequence, as in a cDNA, and / or may include cDNA in combination with regulatory sequences.

[0052] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length joined by peptide bonds.

[0053] For purposes of the present invention, a "genetically modified plant" is a plant that has been genetically modified relative to a native wild-type (WT) plant. In one embodiment, a genetically modified plant is a plant that has been modified relative to a native wild-type (WT) plant using a mutagenesis method, such as targeted genome modification or genome editing. In one embodiment, the plant genome has been modified relative to the wild-type using a mutagenesis method. Such plants have modified phenotypes, such as those described herein, such as improved immunity to pathogens. Thus, in this example, these phenotypes are conferred by the presence of a modified plant genome, e.g., the presence of a mutation in at least one gene encoding an NLR gene. In particular, aspects of the present invention encompass recombinant DNA technology; embodiments based solely on the production of plants by conventional breeding techniques are excluded from these aspects of the present invention.

[0054] In accordance with all aspects of the present invention, including the methods described below, as well as the plants, methods, and uses described below, the term "control sequence" is used interchangeably herein with "promoter," and both of these terms should be interpreted in a broad context to refer to regulatory nucleic acid sequences capable of influencing the expression of sequences to which they are linked. The term "control sequence" also encompasses synthetic fusion molecules or derivatives thereof that cause, activate, or enhance expression of a nucleic acid molecule in a cell, tissue, or organ.

[0055] In one embodiment, the promoter may be a constitutive or strong promoter. Alternatively, the promoter may be a tissue-specific promoter.

[0056] A "constitutive promoter" refers to a promoter that is transcriptionally active in at least one cell, tissue, or organ during most (but not necessarily all) phases of growth and development under most environmental conditions. Examples of constitutive promoters include the cauliflower mosaic virus promoter (CaMV35S or 19S), rice actin promoter, maize ubiquitin promoter, rubisco small subunit promoter, maize H3 histone or alfalfa H3 histone promoter, OCS promoter, SAD1 or 2 promoter, GOS2 promoter, or any promoter that enhances transcription.

[0057] A "strong promoter" refers to a promoter that increases gene expression or causes overexpression of a gene. Examples of strong promoters include, but are not limited to, the CaMV-35S promoter, the CaMV-35Somega promoter, the Arabidopsis ubiquitin UBQ1 promoter, the rice ubiquitin promoter, the actin promoter, or the maize Adh-1 (alcohol dehydrogenase 1) promoter.

[0058] The term "operably linked," as used herein, refers to a functional linkage between a promoter sequence and a genome of interest such that the promoter sequence is capable of initiating transcription of the genome of interest.

[0059] In one embodiment, the progeny plant is stably transformed with a nucleic acid construct described herein and contains an exogenous polynucleotide, which is genetically maintained in the plant cell. The method may include a step of confirming stable integration of the construct. The method may also include the additional step of collecting seeds from the selected progeny plant.

[0060] In one aspect of the present disclosure, there is provided a genetically modified plant, plant part, or plant cell, comprising at least one mutation in at least one nucleic acid sequence encoding a nucleotide-binding domain and leucine-rich repeat-containing (NLR) protein.

[0061] In another aspect of the present invention, there is provided a genetically modified plant, plant part, or plant cell, which expresses a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein; said NLR protein comprises an HD1 domain; and said NLR protein comprises one or more amino acid mutations at the N-terminal portion of said HD1 domain, as described herein.

[0062] In one embodiment, the nucleic acid construct expresses a nucleic acid sequence encoding an NLR protein, wherein the NLR protein is selected from SEQ ID NO: 37, 38, 39, 44, 45, 52, 53, or 54, or a functional variant or homolog thereof, and wherein preferably the regulatory sequence is operably linked to a regulatory sequence.

[0063] NLRs belong to the STAND (signal ATPases with numerous domains) superfamily. NLRs typically have a three-domain structure consisting of an N-terminal signaling domain, a central nucleotide-binding domain, and a repeat-forming C-terminal superstructure. The central domain (called NB-ARC (nucleotide-binding adaptor shared by APAF-1, plant R proteins, and CED-4) in plant NLRs and NACHT (shared by NAIP2, C2TA, HET-E, and TP1) in animal NLRs) is a hallmark of this protein family and plays an important role as a molecular switch, mediating the conformational changes required for activation. In NB-ARC, this central domain consists of a nucleotide-binding domain (NB), a helical domain (HD1), and a winged-helix domain (WHD), while in NACHT domains, a second helical domain (HD2) is also present. Diverse NLR activation and signaling strategies are known in nature. In some cases, a single NLR protein (called a singleton) can mediate both elicitor recognition and subsequent immune signaling (19). However, some NLRs can function as receptor pairs or in higher-dimensional structures called immune receptor networks (13, 20). In these cases, one NLR acts as a pathogen sensor and requires a second, "helper" NLR to initiate immune signaling. This is also true for the NRC immune receptor network in Solanaceae, which is composed of numerous sensor NLRs that require an array of downstream helper NLRs, called NRCs (NLR required for cell death), to successfully initiate immune signaling. This NRC network can encompass up to half of the NLRome in some Solanaceae species and plays an important role in mediating immunity against diverse plant pathogens, including oomycetes, bacteria, viruses, nematodes, and insects.

[0064] Engaging in a coevolutionary arms race with their hosts, diverse plant and metazoan pathogens have evolved effectors that can disrupt host NLR signaling and promote disease through different strategies. In some cases, effectors can indirectly suppress NLR-mediated immunity by interfering with host proteins that regulate or activate downstream NLR signaling (15, 17, 21, 22). In other cases, several effectors have evolved that directly interact with NLRs and inhibit their function (15, 16, 23). One example is the potato cyst nematode effector SS15, which can inhibit signaling mediated by the helper NLRs NRC1, NRC2, and NRC3 by directly binding to their central NB-ARC domain (15) (Figure 1A).

[0065] Thus, a genetically modified plant, plant part, or plant cell can contain at least one mutation in at least one nucleic acid sequence encoding a sensor NLR, a singleton NLR, or a helper NLR, such as an NRC protein (e.g., NRC2 or NRC3). Singleton and helper NLRs are sometimes collectively referred to as executer NLRs.

[0066] "At least one mutation in at least one nucleotide sequence encoding an NLR" means that when an NLR gene exists as two or more copies or homeologs (having the same sequence or slightly different sequences), at least one mutation is present in at least one (endogenous) gene. In one embodiment, the entire gene is mutated. Additionally or alternatively, "at least one mutation in at least one nucleotide sequence encoding an NLR" means that the nucleic acid sequences encoding one or more NLR proteins are mutated. For example, only NRC2 (and at least one or all of its homeologs) is mutated. Alternatively, only NRC3 (and at least one or all of its homeologs) is mutated. Alternatively, both NRC2 and NRC3 (and at least one or all of their homeologs) are mutated. Preferably, the NLR is not NRC4.

[0067] An "endogenous" nucleic acid or gene can refer to the native or naturally occurring sequence in the plant genome.

[0068] In one embodiment, the NLR can be at least one of NRC1, NRC2, or NRC3.

[0069] The nucleic acid sequence of NRC1 may encode the NRC1 protein set forth in SEQ ID NO: 47, or a functional variant or homologue thereof. Preferably, the nucleic acid sequence of NRC2 comprises or consists of SEQ ID NO: 46, or a functional variant or homologue thereof.

[0070] The nucleic acid sequence of NRC2 may encode the NRC2 protein set forth in SEQ ID NO: 32, 34, 41, or 49, or a functional variant or homologue thereof. Preferably, the nucleic acid sequence of NRC2 comprises or consists of SEQ ID NO: 31, 33, 40, or 48, or a functional variant or homologue thereof.

[0071] The nucleic acid sequence of NRC3 may encode the NRC3 protein set forth in SEQ ID NO: 36, 43, or 51. Preferably, the nucleic acid sequence of NRC3 comprises or consists of SEQ ID NO: 35, 42, or 50, or a functional variant or homologue thereof.

[0072] In one embodiment, the NLR protein comprises an HD1 domain, and the at least one mutation is a mutation of one or more nucleotides in the HD1 domain. The HD1 domain may also be referred to as a helical domain or an ARC1 domain. These terms may be used interchangeably herein.

[0073] The HD1 domain is a conserved domain containing one or more conserved motifs. Preferably, the HD1 domain contains the RNBS-C and / or GLPL motifs. However, in particular, the HD1 domain can be considered to be a domain containing the RNBS-C motif, having 50 to 100 amino acids, more preferably about 60 to 90 amino acids, and particularly about 80 amino acids.

[0074] The RNBS (resistance nucleotide binding site)-C motif may comprise the consensus sequence LxxxExWxLF (wherein the leucines at positions 0 and 8 are highly conserved). In further embodiments, the RNBS-C motif may comprise the amino acid sequence set forth in SEQ ID NO: 3, 6, 9, 15, 18, 24, 27, or 30, or a functional fragment or variant thereof.

[0075] As described in connection with Figure 2, we found that only the N-terminal half of the HD1 domain is important for binding pathogen effectors, and that mutations in this region in particular result in NLR proteins that can evade repression by pathogen effectors while retaining full functionality (e.g., being able to oligomerize and mediate cell death when activated by effector or sensor NLRs).

[0076] Therefore, the at least one mutation in the NLR protein is preferably at least one mutation in the N-terminal portion of the HD1 domain. In one example, the N-terminal portion of the HD1 domain containing one or more mutations may comprise 42 or fewer amino acids (e.g., 1 to 42) from the N-terminus of the HD1 domain. Preferably, the N-terminal portion of the HD1 domain may comprise 38 or fewer amino acids (e.g., amino acids 1 to 38) from the N-terminus of the HD1 domain. Preferably, the N-terminal portion of the HD1 domain may comprise or consist of the RNBS-C motif described above. The N-terminal portion of HD1 may also be referred to herein as HD1-1.

[0077] The HD1-1 region may comprise or consist of a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 14, 17, 23, 26, or 29, or a functional variant or fragment thereof. Preferably, the functional variant or fragment comprises at least the RNBC-C motif described above. Preferably, the at least one mutation is in the RNBC-C motif.

[0078] The term "variant" or "functional variant," as used herein with respect to any sequence defined herein, refers to a variant gene sequence, or a portion of that gene sequence, that retains the biological function of the full-length, non-variant sequence. Thus, in the context of an NLR protein, a functional variant may be one that can mediate an immune response, such as a hypersensitive response (HR). For an NRC protein, a functional variant may be one that can oligomerize and initiate an immune response. In the context of HD1 or HD1-1, a functional variant may be one that promotes oligomerization of the NRC protein and initiates an immune response.

[0079] Functional variants also include genomic variants of interest that have sequence changes that do not affect function, such as sequence changes at non-conserved residues. Also included are variants that are substantially identical, i.e., have only some sequence variations (e.g., at non-conserved residues), compared to the wild-type sequences described herein, and are biologically active (e.g., can oligomerize and cause cell death). Mutations in nucleic acid sequences that result in different amino acids at specific sites without affecting the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine (a hydrophobic amino acid) can be substituted with a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, mutations that result in the substitution of one negatively charged residue with another, such as aspartic acid with glutamic acid, or one positively charged residue with another, such as lysine with arginine, can also be expected to result in functionally equivalent products. Nucleotide mutations resulting in changes to the N- and C-terminal portions of a polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the modifications listed above is well within the routine skill of those in the art, as is determining retention of biological activity of the encoded product.

[0080] As used in any aspect of the invention described herein, a "variant" or "functional variant" refers to a variant that is at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, or at least 99% overall sequence identity.

[0081] The term "homolog" as used herein also refers to the orthologs of NLR genes from other plant species.Suitable homologs can be identified by sequence comparison and identifying conserved domains as described above.In the art, there are predictors that can be used to identify such sequences.The function of homologs can be identified as described herein, and therefore, those skilled in the art can confirm their function, for example, when overexpressed in plants.

[0082] Homologues may also have at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity (higher orders being more preferred) to the amino acid sequences referred to herein or the nucleic acid sequences referred to herein. Functional variants of the NLR gene homologs defined above are also within the scope of the present invention.

[0083] Two nucleic acid sequences or polypeptides can be referred to as "identical" if the sequence of nucleotides or the sequence of amino acid residues in the two sequences, respectively, are identical when aligned for maximum identity as described below. The term "identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical, or that have a specified percentage (%) of identical amino acid residues or nucleotides, when aligned for maximum identity over a comparison window, as measured using one of the sequence comparison algorithms described below, or by manual alignment and visual inspection. When percent sequence identity is used with respect to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions (in which an amino acid sequence is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), thereby not altering the functional properties of the molecule). When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. In sequence comparison, typically, one sequence is used as a reference sequence, and this sequence is compared with the test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the sequence identity (%) of the test sequence to the reference sequence based on the program parameters. Non-limiting examples of algorithms suitable for determining sequence identity (%) and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm. The overall sequence identity of variants can be determined using any number of sequence alignment programs known in the art.As an example, one can use the Emboss Stretcher provided by EMBL-EBI: https: / / www.ebi.ac.uk / Tools / psa / emboss_stretcher / (using default parameters, i.e., for proteins, pair output format, Matrix = BLOSUM62, Gap open = 1, Gap extend = 1; for nucleotides, pair output format, Matrix = DNAfull, Gap open = 16, Gap extend = 4). Do).

[0084] Those skilled in the art will understand that suitable homologs and homologous positions in the sequences can be identified by sequence comparison (e.g., BLAST, alignment) and identification of conserved domains. Phylogenetic tree analysis using nucleotide or amino acid sequences can be used to confirm orthology to NLR genes. Predictors exist in the art that can be used to identify such sequences. The function of a homolog can be identified as described herein. Thus, one skilled in the art can confirm the function of a homolog using, for example, a programmed cell death assay. Thus, a homologous position, or "corresponding position in a homologous sequence," as used herein, can be determined by sequence alignment once a homologous sequence has been identified. For example, a homolog can be identified by a BLAST search of the plant genome of interest using the NRC2 or NRC3 sequence of S. tuberosum or N. benthamiana (i.e., one of the sequences set forth in SEQ ID NOS: 31-36 or 40-43) as a query.

[0085] Thus, the nucleotide sequences of the present invention described herein can also be used to isolate corresponding sequences from other organisms, particularly other plants, such as crop plants. In this manner, methods such as PCR and hybridization can be used to identify such sequences based on sequence homology to the sequences described herein. Sequence topology and characteristic domain structure (e.g., the presence of the RNBS-C motif) can also be considered when identifying and isolating homologs. Sequences can be isolated based on sequence identity to the entire sequence or fragments thereof. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA or cDNA fragments (i.e., a genomic DNA or cDNA library) from a selected plant. Hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and can be labeled with a detectable group or any other detectable marker. Methods for preparing probes for hybridization and for constructing cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0086] Hybridization of such sequences may be performed under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" refer to conditions under which a probe will hybridize detectably (e.g., at least twice as strongly as background) to its target sequence than to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. By adjusting the stringency of hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatch in the sequence to detect sequences with lower similarity (heterologous probing). Generally, probes are less than about 1000 nucleotides in length, and preferably less than 500 nucleotides in length.

[0087] Typically, stringent conditions would be conditions in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salt), the pH is 7.0 to 8.3, and the temperature is about 30°C or higher for short probes (e.g., 10 to 50 nucleotides) and about 60°C or higher for long probes (e.g., more than 50 nucleotides). Hybridization times are generally less than 24 hours, usually about 4 to 12 hours. Stringent conditions can also be achieved by adding destabilizing agents such as formamide.

[0088] In a further embodiment, a variant as used herein may comprise a nucleic acid sequence encoding an NLR polypeptide as defined herein that is capable of hybridizing to a nucleic acid sequence as defined herein under stringent conditions as defined herein.

[0089] In one embodiment, a genetically modified plant, plant part, or plant cell is provided, comprising at least one mutation in at least one nucleic acid sequence encoding at least one NLR protein, preferably encoding NRC1, NRC2, and / or NRC3, wherein the NRC1 gene is a. a nucleic acid sequence encoding a polypeptide set forth in one of SEQ ID NOs: 47; b. a nucleic acid sequence set forth in one of SEQ ID NOs: 46; c. a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to either (a) or (b); or d. A nucleic acid sequence encoding an NRC2 polypeptide as defined herein, which is capable of hybridizing to any of the nucleic acid sequences (a) to (c) under stringent conditions as defined herein. comprising or consisting of a nucleic acid sequence such as: In addition, the NRC2 gene is e. a nucleic acid sequence encoding a polypeptide set forth in one of SEQ ID NOs: 32, 34, 41, or 49; f. a nucleic acid sequence set forth in one of SEQ ID NOs: 31, 33, 40, or 48; g. a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to either (e) or (f); or h. A nucleic acid sequence encoding an NRC2 polypeptide as defined herein, which is capable of hybridizing to any of the nucleic acid sequences (e) to (g) under stringent conditions as defined herein. comprising or consisting of a nucleic acid sequence such as: In addition, the NRC3 gene is i. a nucleic acid sequence encoding a polypeptide set forth in one of SEQ ID NOs: 36, 43, or 51; j. a nucleic acid sequence set forth in one of SEQ ID NOs: 35, 42, or 50; k. a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to either (a) or (b); or l. A nucleic acid sequence encoding an NRC3 polypeptide as defined herein, which is capable of hybridizing to any of the nucleic acid sequences (a) to (c) under stringent conditions as defined herein. or consisting of such a nucleic acid sequence.

[0090] Preferably, the at least one mutation introduced into at least one NLR protein, specifically into the HD1-1 region of the NLR protein, causes a change or mutation in the corresponding amino acid sequence of the NLR protein. As shown in Figure 5, when the NLR protein is an NRC protein, the at least one mutation preferably reduces or prevents pathogen effectors from inhibiting the oligomerization of the NRC protein to form a complex for initiating an immune response. That is, the pathogen effectors can no longer suppress the function (immune activation) of the NRC protein. NRC protein oligomerization can be measured, for example, by BN-PAGE.

[0091] More preferably, the at least one mutation reduces or prevents binding between the NLR protein and the pathogen effector. Binding between the NLR protein and the pathogen effector can be determined by any method routine in the art, such as co-immunoprecipitation, as described in the Examples.

[0092] Importantly, the mutation does not adversely affect the function of the NLR protein. In one example, the at least one mutation reduces or prevents pathogen effectors from inhibiting the initiation of an immune response pathway by the NLR protein. In the example of an NRC protein, the NRC protein can (still) oligomerize and / or mediate an immune response, such as a cell death response. In one example, the mutation preserves (or does not change) the downstream interaction / signaling pathway of the native NLR protein. For example, if the native NLR protein initiates an immune response through one or more interactions with one or more downstream molecules, the mutant NLR protein initiates an immune response through the same interaction.

[0093] As shown in Figures 3 and 5, we demonstrate that mutations in the HD1-1 region, in particular, maintain upstream signaling with the sensor NLR, evade inhibition by pathogen effectors, and prevent pathogen effector binding to the NLR (i.e., reduced or no binding to the NLR).

[0094] The at least one mutation introduced into the at least one nucleic acid sequence encoding at least one NRC protein is one of the following types of mutations: 1. "missense mutations," which are mutations in a nucleic acid sequence that result in the substitution of one amino acid for another; 2. "Nonsense mutations" or "stop codon mutations", which are mutations in nucleic acid sequences that introduce premature stop codons, thereby causing translation termination (resulting in truncated proteins); in plants, translation stop codons are selected from "TGA" (UGA in RNA), "TAA" (UAA in RNA), and "TAG" (UAG in RNA); therefore, any nucleotide substitution, insertion, or deletion that results in one of these codons in (the reading frame of) the mature mRNA being translated will stop translation; 3. "insertion mutations" of one or more nucleotides or one or more amino acids (due to the addition of one or more codons in the coding sequence of the nucleic acid); 4. "Deletion mutations" of one or more nucleotides or one or more amino acids (due to the deletion of one or more codons in the coding sequence of the nucleic acid); 5. "Frameshift mutations" in which the nucleic acid sequence downstream of the mutation is translated in a different frame. Frameshift mutations can arise from a variety of causes, for example, the insertion, deletion, or duplication of one or more nucleotides; 6. "Splice site" mutations, which are mutations that result in the insertion, deletion, or substitution of nucleotides at the splice site (i.e., either splice acceptor mutations or splice donor mutations). (wherein, preferably, any one or more of the above mutations reduce or eliminate binding between the NLR protein and a pathogen effector) As described above, "binding" refers to direct or indirect mutual association. That is, when expressed in a plant, the NLR and the pathogen effector co-immunoprecipitate. The reduction in binding between the mutant NLR and the pathogen effector can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the degree of binding between the wild-type NLR and the pathogen effector (e.g., as measured by co-ip). Alternatively, no interaction between the NLR and the pathogen effector can be detected, i.e., binding can be abolished.

[0095] Preferably, the one or more mutations are substitution mutations, for example, any substitution in HD1-1, more preferably any substitution in the RNBS-C motif of HD1-1.

[0096] As shown in Figure 3, the substitutions are at one or more positions in the amino acid sequence, and the substitution positions are selected from positions 315, 316, or 317 of SEQ ID NO: 32, 34, 36, 41, 47, 49, or 51, or any of their homologs or functional variants. Alternatively, the substitutions are at homologous positions in homologous sequences.

[0097] Preferably, the substitution is for a hydrophilic and / or positively charged amino acid, hi one embodiment, the substitution is for an amino acid with the opposite biochemical properties, such as, but not limited to, lysine for aspartic acid (positive to negative charge) or serine for alanine (polar to non-polar).

[0098] In one embodiment, the mutation is a substitution of a D residue. Alternatively, the mutation is a substitution of an E residue. Alternatively, the mutation is a substitution of an N residue. Alternatively, the mutation is a substitution of an S residue.

[0099] In one embodiment, the substitution is a D317K substitution in SEQ ID NO: 32 or 34, or a homologue or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0100] In one embodiment, the substitution is an N317K substitution in SEQ ID NO: 36, or a homologue or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0101] In one embodiment, the substitution is a D315K substitution in SEQ ID NO: 41, 43, 49, or 51, or a homolog or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0102] In one embodiment, the substitution is a D316K substitution in SEQ ID NO: 47, or a homologue or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0103] In one embodiment, the substitution is a S317K substitution in SEQ ID NO: 41, 43, 49, or 51, or a homolog or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0104] In one embodiment, the substitution is an E317K substitution in SEQ ID NO: 47, or a homologue or functional variant thereof, or a substitution at a homologous position in a homologous sequence.

[0105] Preferably, as shown in Figure 5, the substitution is preferably at position D317 of SEQ ID NO: 32 or 34, or at a homologous position of a homologous sequence, and among these, preferably, the substitution is a D317K substitution.

[0106] Homologous positions, or as used herein, "corresponding positions in homologous sequences," can be determined by performing sequence alignments once homologous sequences have been identified. For example, homologs can be identified by BLAST searching the plant genome of interest using the S. tuberosum or N. benthamiana NRC1, NRC2, or NRC3 sequence (i.e., one of the sequences set forth in SEQ ID NOs: 32, 34, 36, 41, 43, 47, 49, or 51) as a query.

[0107] Alternatively, the mutation is a substitution of all or most of the HD1-1 region with a corresponding HD1-1 region in a second NLR protein, wherein said second NLR protein is not inhibited by pathogen effectors.

[0108] The mutation may also be a substitution of all or most of the RNBS-C motif with a corresponding RNBS-C motif in a second NLR protein, wherein said second NLR protein is not inhibited by the pathogen effector.

[0109] "Not inhibited" means that the pathogen effector either cannot bind or cannot sufficiently bind to the second NLR protein, or that the pathogen effector does not suppress or inhibit the induction of an immune response, such as induction of cell death, by the second NLR protein. In one example, the second NLR protein may be NRC4.

[0110] The nucleic acid sequence of the RNBS-C motif of NRC4 may encode the RNBS-C motif set forth in SEQ ID NO: 12, or a homologue or functional variant thereof.

[0111] In one embodiment, the mutation is introduced using targeted genome editing. Thus, in one embodiment, the present invention relates to methods and plants produced by the genetic engineering methods described above, and does not include natural plant species or the production of plants by conventional breeding methods.

[0112] Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to promote genome editing through recombination events that occur via homologous recombination (HR).

[0113] In a preferred embodiment, the genome editing method used in accordance with various aspects of the present invention is CRISPR. Alternatively, targeted genome editing can be performed using TALEN.

[0114] In a preferred embodiment of any aspect of the invention, sgRNAs can be used in conjunction with a modified Cas9 protein (e.g., nickase Cas9 or nCas9, or inactive Cas9 ("dead" Cas9, or dCas9)) conjugated with a "base editor" (e.g., an enzyme such as a deaminase (e.g., a cytidine deaminase, or TadA (tRNA adenosine deaminase) or ADAR, or APOBEC, etc.). These enzymes can replace one base with another, resulting in a single-base substitution without removing DNA (Kim et al., 2017; Gaudelli et al. 2017). Alternatively, in this method, sgRNAs can be used in conjunction with a template or donor DNA construct to introduce a targeted SNP or mutation (particularly one of the substitutions described herein) into the NRC gene. In this embodiment, the introduction of a template DNA strand (followed by cleavage by the sgRNA in double-stranded DNA) can be used to generate specific targeted mutations (i.e., SNPs) in genes using homologous recombination repair. As a further alternative, prime editing can be used to introduce specific mutations (Anzalone et al., 2019). In this case, a catalytically inactive Cas9 endonuclease is fused to an engineered reverse transcriptase programmed with a prime editing guide RNA (pegRNA) specific to the target site and encoding the desired edit. Using this method, nucleotides encoding the conserved HD1-1 domain can be mutated, thereby generating one of the substitutions described above in the amino acid sequence.

[0115] After targeted genome editing, the amplification products can be analyzed for the presence of mutations in NLR genes, particularly in the HD1-1 domain, using rapid, high-throughput screening methods. Mutations can be identified and screened for improved immunity to pathogen effectors, resulting in stable, non-segregating plant lines. In this way, mutants can be identified that have one or more mutations in NLR genes, particularly in the HD1-1 region, and therefore have improved immunity to a given pathogen compared to control plants.

[0116] In one embodiment, after targeted genome editing, mutant NLR proteins are transiently expressed in N. benthamiana to confirm that the mutants are insensitive to suppression (i.e., revert). This can be determined using a programmed cell death assay that activates by self-activating mutations or by sensor + non-pathogenic effector proteins. In a further embodiment, proof-of-concept experiments are carried out in crop plants, such as soybeans.

[0117] Also within the scope of the present invention are plants obtained or obtainable by the above-mentioned methods, or seeds or other reproductive organs obtained or obtainable from the above-mentioned plants having a functional mutation in at least one endogenous NLR gene (preferably NRC2 and / or NRC3).

[0118] In one embodiment, the progeny plants are stably transformed with the CRISPR construct and contain the exogenous polynucleotide genetically maintained in the plant cells. The method may include a step to confirm stable integration of the construct. The method may also include the additional step of recovering seeds or other reproductive organs from the selected progeny plants.

[0119] In one example, the plant may not have mutations in any other genes, i.e., it may only have mutations in one or more of the NLR proteins described above.

[0120] In another aspect of the present invention, there is provided a method for conferring or improving pathogen resistance in a plant, the method comprising introducing at least one of the above-described mutations into at least one nucleic acid sequence encoding an NLR (nucleotide-binding domain and leucine-rich repeat-containing) protein; the NLR protein comprises an HD1 domain; and the at least one mutation is a mutation of one or more nucleotides at the N-terminal portion of the HD1 domain, as described above.

[0121] In another aspect of the present invention, there is provided a method for conferring or improving pathogen resistance in a plant, the method comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein; the NLR protein comprises an HD1 domain; and the NLR protein comprises one or more amino acid mutations at the N-terminal portion of the HD1 domain.

[0122] In another aspect of the present invention, there is also provided a method for producing a plant with improved pathogen resistance, the method comprising introducing at least one of the above-described mutations into at least one nucleic acid sequence encoding an NLR (nucleotide-binding domain and leucine-rich repeat-containing) protein; the NLR protein comprises an HD1 domain; and the at least one mutation is a mutation of one or more nucleotides at the N-terminal portion of the HD1 domain, as described above.

[0123] In another aspect of the present invention, there is also provided a method for producing a plant with improved pathogen resistance, the method comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein; the NLR protein comprises an HD1 domain; and the NLR protein comprises one or more amino acid mutations at the N-terminal portion of the HD1 domain.

[0124] "Conferring or improving pathogen resistance" means that at least one of pathogen / pest growth or fitness is reduced or fecundity is improved in a plant carrying the mutant NLR described above compared to a wild-type or control plant. A reduction or improvement, as used herein, can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the level of pathogen / pest growth or fitness in a control or wild-type plant, or compared to the fecundity of a control or wild-type plant.

[0125] A "pathogen" may include any disease-causing agent, particularly a disease-causing organism such as a bacterium, a virus, a nematode, or a fungus. A pathogen infects a plant and causes disease, and may reduce yield or otherwise damage the plant, for example, by the action of a toxin. A "pest" may include an organism such as an animal, insect, or nematode. A "pest" may infect a plant with a pathogen or damage the plant directly (e.g., by sucking sap, boring holes in stems or fruit, and / or cutting roots, stems, and leaves).

[0126] Alternatively, the conferring or improving of pathogen resistance may result in an increase in yield or seed yield, and as such, the conferring or improving of pathogen resistance can be measured by measuring an increase in yield or seed yield.

[0127] The term "yield" generally refers to a measurable production of economic value, typically related to a specified crop, area, and time period. Individual plant parts directly contribute to yield based on their number, size, and / or weight. Actual yield is calculated based on the area (m²) for a given crop in a given year. 2 ), which is the total production (including both harvested and estimated production) divided by the area of ​​land (m 2 ) is calculated by dividing by

[0128] Preferably, the increased yield includes at least one of an increased number and / or weight of seeds, an increased number of pods per plant (if the plant contains pods), an increased thousand kernel weight (TKW), an increased biomass, an increased fresh weight, and an increased growth, preferably an increased root growth. The increased yield is an increase compared to a control or wild-type plant. For example, the yield may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to a wild-type or control plant.

[0129] In another aspect of the present invention, there is provided a method for improving plant immunity, comprising introducing at least one of the above-described mutations into at least one nucleic acid sequence encoding an NLR (nucleotide-binding domain and leucine-rich repeat-containing) protein; the NLR protein comprises an HD1 domain; and the at least one mutation is a mutation of one or more nucleotides at the N-terminal portion of the HD1 domain, as described above.

[0130] In another aspect of the present invention, there is provided a method for improving plant immunity, comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, as described herein; the NLR protein comprising an HD1 domain; and the NLR protein comprising one or more amino acid mutations at the N-terminal portion of the HD1 domain.

[0131] In one embodiment, the nucleic acid construct expresses a nucleic acid sequence encoding an NLR protein, wherein the NLR protein is selected from SEQ ID NO: 37, 38, 39, 44, 45, 52, 53, 54, 55, 56, 57, 58, or 59, or a functional variant or homolog thereof, and wherein preferably the regulatory sequence is operably linked to a regulatory sequence.

[0132] In another aspect of the present invention, there is also provided a method for producing a plant with improved immunity, comprising introducing at least one of the mutations described above into at least one nucleic acid sequence encoding an NLR (nucleotide-binding domain and leucine-rich repeat-containing) protein; the NLR protein comprising an HD1 domain; and the at least one mutation being a mutation of one or more nucleotides at the N-terminal portion of the HD1 domain, as described above.

[0133] In another aspect of the present invention, there is also provided a method for producing a plant with improved immunity, comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein; said NLR protein comprising an HD1 domain; and said NLR protein comprising one or more amino acid mutations at the N-terminal portion of said HD1 domain, as described herein.

[0134] In one embodiment, the nucleic acid construct expresses a nucleic acid sequence encoding an NLR protein, wherein the NLR protein is selected from SEQ ID NO: 37, 38, 39, 44, 45, 52, 53, 54, 55, 56, 57, 58, or 59, or a functional variant or homolog thereof, and wherein preferably the regulatory sequence is operably linked to a regulatory sequence.

[0135] The improvement in plant immunity can be measured by any method known in the art. In one embodiment, the improvement in immunity can be measured by measuring cell death in the presence of a pathogen or pathogen effector compared to cell death in a wild-type plant or a control plant. Examples of cell death assays that can be performed are described in the Examples.

[0136] By "improved" is meant at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 95% improvement in immunity compared to a wild-type or control plant.

[0137] In another aspect of the present invention, there is provided a method for generating a modified NLR protein, wherein a pathogen effector is unable to bind to said NLR protein or the pathogen effector has a reduced ability to bind to said NLR protein. "Binding" is defined above. Preferably, the method comprises introducing at least one of the above-described mutations into the N-terminal portion of an NLR protein, as described above.

[0138] The method comprises: a. Selecting plant parts; b. transforming at least one cell of the plant part of step (a) with at least one CRISPR construct or sgRNA molecule, wherein said CRISPR construct or sgRNA molecule targets the NRC gene and introduces at least one mutation in the HD1-1 region, as described above; c. regenerating at least one plant from the transfected cell(s); d. Selecting one or more plants obtained by step (c) that exhibit at least one mutation in the HD1-1 region. may also include:

[0139] In one embodiment, the method may include obtaining a DNA sample from the transformed plant and performing DNA amplification to detect at least one mutation in the HD1-1 region. In further embodiments of any of the methods described herein, the method may further include at least one or more steps of evaluating the phenotype of the genetically modified plant and measuring at least one of immunity to a given pathogen. In other words, the method may include screening the plant for a desired phenotype.

[0140] Alternatively, more general mutagenesis methods can be used to introduce at least one mutation into the HD1-1 region of the NLR gene. These methods include both physical mutagenesis and chemical mutagenesis. Those skilled in the art will understand that other means can be used to generate such mutants, and methods for mutagenesis and polynucleotide modification are well known in the art. For example, see Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; US Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and the references cited herein.

[0141] In another embodiment, the mutagenesis is physical mutagenesis, such as the application of ultraviolet radiation, X-rays, gamma rays, fast or thermal neutrons, or fast or thermal protons. The targeted population can then be screened to identify substitution mutations in the HD1-1 region of the NLR gene.

[0142] In another embodiment of the various aspects of the invention, the method includes inducing mutations in the plant population with a mutagen. The mutagen may be fast neutron irradiation or a chemical mutagen, such as the following non-limiting list: ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), triethylmelamine (1'EM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N'-nitro-nitrosoguanidine (MNNG). The mutagen may be selected from the group consisting of nitrosoguanidine, 2-aminopurine, 7,12-dimethylbenz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (such as diepoxyoctane (DEO) and diepoxybutane (BEB)), 2-methoxy-6-chloro-9[3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170), and formaldehyde. Again, the targeted population can then be screened to identify one of the above mutations in the HD1-1 region of the NLR gene.

[0143] In another embodiment, the method used to generate and analyze mutations is TILLING (targeting induced local lesions in genomes). In an alternative embodiment, the method used to generate and analyze mutations is EcoTILLING. EcoTILLING is a branching technique similar to TILLING, except that its purpose is to reveal natural mutations in a given population, rather than induced mutations. The EcoTILLING method was first described in Comai et al. 2004.

[0144] In another embodiment, the method utilizes oligonucleotide directed mutagenesis (ODM).

[0145] Genetically modified plants of the present invention can also be obtained by introducing any of the sequences of the present invention by crossbreeding, for example, by pollinating a wild-type or control plant with pollen from a genetically modified plant described herein, or by pollinating the pistil of a plant described herein with other pollen that has not been transformed or genetically modified as described in the present invention.

[0146] In a further aspect of the present invention, there is provided a plant obtained or obtainable by the above-described method. In a further aspect, there is provided a seed or other reproductive organ obtained or obtainable from said plant. Progeny plants obtained from said seeds or other reproductive organs, as well as seeds or other reproductive organs obtained from said progeny plants, are also within the scope of the present invention.

[0147] In another aspect of the present invention, there is provided a method for screening a population of plants to identify and / or select plants that exhibit pathogen resistance or that will exhibit improved pathogen resistance, the method comprising detecting at least one polymorphism (or a mutation as described above) in the NRC2 and / or NRC3 gene in the plant or plant reproductive cells (wherein preferably the polymorphism is a polymorphism in the N-terminal portion of the HD1 domain of the NRC2 and / or NRC3 gene); and selecting the plant.

[0148] Suitable tests for assessing the presence of polymorphisms will be well known to those skilled in the art, including, but not limited to, DNA sequencing (e.g., amplicon sequencing, resistance gene enrichment sequencing (RenSeq), or primer-based Sanger sequencing), isozyme electrophoresis, RFLP (Restriction Fragment Length Polymorphism), RAPD (Randomly Amplified Polymorphic DNA), AP-PCR (Arbitrarily Primed Polymerase Chain Reaction), DAF (DNA Amplification Fingerprinting), SCAR (Sequence Characterized Amplified Region), AFLP (Amplified Fragment Length Polymorphism), SSR (Simple Sequence Repeat, also known as microsatellite), and SNPs (Single Nucleotide Polymorphisms). In one embodiment, Kompetitive Allele Specific PCR (KASP) genotyping is used.

[0149] In one embodiment, the method comprises: a) obtaining a nucleic acid sample from the plant; and b) nucleic acid amplification of NRC2 and / or NRC3 alleles using one or more primer pairs; Includes.

[0150] In a further embodiment, the method may further comprise introgressing or crossing a chromosomal region comprising at least one of the above-described NRC polymorphisms into a second plant or plant germ cell to produce an introgressed plant or plant germ cell.

[0151] As used herein, a "pathogen effector" may refer to any protein of pathogen origin that functions in a host (plant) for the benefit of the pathogen. In particular, a "pathogen effector" includes any pathogen effector capable of binding to an NLR protein, e.g., the HD1-1 region of NRC2 and / or NRC3. Examples of such pathogen effectors include SPRYSEC10 (SS10), SPRYSEC34 (SS34), and SPRYSEC15 (SS15) from Globodera rostochiensis, a pathogen of the potato cyst nematode. Other examples include AVRcap1b and PITG-15278, which are RXLR-WY / LWY domain containing effectors from Phytophthora infestans. Preferably, the pathogen effector is SS15. Preferably, the pathogen is a potato cyst nematode.

[0152] In one embodiment, the plant is a crop plant. Crop plant refers to any plant grown on a commercial scale for human or animal consumption or use. In another embodiment, the plant is Arabidopsis, Nicotiana benthamiana, Nicotiana tabacum, Medicago truncatula, or other suitable model organisms used in plant research.

[0153] The plant may be a dicotyledonous or monocotyledonous plant.

[0154] The dicotyledonous plant may be selected from a family including, but not limited to, Asteraceae, Brassicaceae (e.g., Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae, or Fabaceae), Malvaceae, Rosaceae, or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, watermelon, pumpkin, cabbage, tomato, potato, yam, pepper, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, fava bean, pea, lentil, peanut, chickpea, apricot, pear, peach, grape, or citrus species. In one embodiment, the plant is oilseed rape.

[0155] Plants also include biofuel and bioenergy crops such as rapeseed / canola, sugarcane, sweet sorghum, Panicum virgatum (switchgrass), flax, lupine and willow, poplar, poplar hybrids, Miscanthus, or gymnosperms (e.g., loblolly pine). Plants also include silage crops (corn), pasture or fodder (grasses, clover, sanfoin, alfalfa), fiber (e.g., cotton, flax), building materials (e.g., pine, oak), pulpwood (e.g., poplar), feeder stock for the chemical industry (e.g., high erucic acid rape, linseed) and recreational feeder stock (e.g., turf grass for golf courses), ornamental plants for public and private gardens (e.g., snapdragons, petunias, roses, geraniums, Nicotiana sp.), and household plants and cut flowers (African violet, begonia, chrysanthemum, geranium, coleus spider plant, dracaena, rubber tree).

[0156] The monocotyledonous plant may be selected from the families Arecaceae, Amaryllidaceae, or Poaceae, for example. For example, the plant may be a cereal such as wheat, rice, barley, maize, oat, sorghum, rye, millet, buckwheat, turfgrass, rye, sugarcane, or Festuca species, or a crop such as onion, leek, yam, or banana.

[0157] Preferably, the plant is a crop plant. Crop plant refers to any plant that is cultivated on a commercial scale for human or animal consumption or use. Preferred plants are corn, wheat, rice, rapeseed, sorghum, soybean, potato, tomato, grape, barley, pea, bean, broad bean, lettuce, cotton, sugarcane, sugar beet, broccoli or other Brassica vegetables, or poplar.

[0158] In a preferred embodiment, the plant is selected from the Solanaceae family. In one example, the plant is potato (Solanum tuberosum), eggplant (Solanum melongena), petunia (Petunia spp., e.g., Petunia x hybrida or Petunia hybrida), grape physalis (Physalis philadelphica), grape physalis (Physalis peruviana), Physalis sp., woody nightshade (Solanum dulcamara), garden huckleberry (Solanum scabrum), gboma eggplant (Solanum macrocarpon), pepper (Capsicum spp.; e.g., Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), tomato (Solanum lycopersicum or Lycopersicon esculentum), tobacco (Nicotiana spp., e.g., N. tabacum, N. benthamiana), Solanum americanum, Solanum demissum, Solanum stoloniferum, Solanum papita, Solanum bulbocastanum, Solanum edinense, Solanum schenckii, Solanum hjertingii, Solanum venturi, Solanum mochiquense, Solanum chacoense, and Solanum pimpinellifolium (Solanum pimpinelli folium). Preferred Solanaceae plants are agriculturally cultivated Solanaceae plants, including, but not limited to, potato, tomato, physalis grandiflora, eggplant, pepper, tobacco, physalis grandiflora, and petunia.

[0159] The term "plant" as used herein encompasses whole plants and progeny of said plants, as well as plant parts (including seeds, fruits, young leaves, stems, leaves, roots (including tubers), flowers, tissues, and organs), including each of the aforementioned inventive variations. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores. The present invention also extends to harvestable parts of the inventive plants described herein, including but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers, and bulbs.

[0160] The term "reproductive organs" includes seeds and other reproductive organs of plants, such as tubers.

[0161] In the most preferred embodiment, the plant part or harvestable product is a seed or grain.Therefore, in a further aspect of the present invention, the seed or grain that grows on the genetically modified plant described herein is provided.Therefore, in one aspect of the present invention, a seed is provided, wherein the seed comprises at least one of the above-mentioned mutations in the NLR gene.Also provided are progeny plants obtained from the seed, and seeds obtained from the progeny plants.

[0162] In all aspects of the present invention, the control plant used herein is a plant that has not been modified by the method of the present invention. Thus, in one embodiment, the control plant does not have one or more mutations in the NLR gene described herein. In one embodiment, the control plant is a wild-type plant. The control plant is typically of the same plant species and preferably has the same genetic background as the modified plant. The control plant may also be a wild-type plant that has the same mutations as the present invention (except for the specific mutation that is believed to result in the phenotype of the present invention). For example, the control plant contains another copy of the wild-type gene instead of the edited gene of the present invention.

[0163] While the above disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including how to make and use the invention and the best mode thereof, the following examples are provided to further enable those skilled in the art to practice the invention and to provide a complete description thereof. However, those skilled in the art will understand that the specific aspects of these examples should not be construed as limiting the invention, and that the scope of the invention should be grasped from the claims appended to this disclosure and their equivalents. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in light of the present disclosure.

[0164] Unless the context requires otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0165] The invention will now be illustrated in the following non-limiting examples. [Example]

[0166] Pathogens suppress NLRs to counteract immunity. Here, we show that cyst nematode effectors inhibit resistosome formation of the helper NLR immunity protein NRC2 through direct binding, thereby physically blocking the molecular rearrangements required for activation. This results in suppression of immune signaling and disease resistance. By combining structural approaches and leveraging NLR diversity, we engineered single-amino acid point mutants of the helper NLR that evade parasite suppression. This engineered helper NLR restored the activity of the cyst nematode disease resistance protein. This provides a novel strategy for editing immune receptor genes to restore hidden / suppressed disease resistance in crop genomes.

[0167] This study provides an approach to rationally design NLRs that circumvent pathogen suppression. By relieving pathogen suppression of helper NLRs such as NRC2, it is possible to "revive" hidden upstream sensors that are normally disabled by interference with downstream signaling partners.

[0168] [SS15 directly inhibits NRC2 resistosome formation] NRC2 or NRC4 was transiently expressed in leaves of nrc2 / 3 / 4 CRISPR KO Nicotiana benthamiana plants, along with its upstream sensor Rx and effector SS15. A BN-PAGE-based readout of NRC resistosome formation was utilized. Biochemical analysis employed mutants of NRC2 and NRC4 (NRC2 and NRC4, respectively) with mutations in the N-terminal MADA motif that abolish cell death induction without compromising receptor activation, oligomerization, or localization. EEE and NRC4 AAANext, we activated the Rx-NRC system by coexpressing PVX CP (potato virus X coat protein)-GFP or free GFP as an inactive control. In the absence of SS15, both NRC2 and NRC4 oligomerize upon effector-induced activation mediated by their upstream sensors. However, in the presence of SS15, Rx / CP-activated NRC2 fails to oligomerize and appears as a ∼240 kDa band co-migrating with SS15. Inactive NRC2 coexpressed with SS15 also migrates as a ∼240 kDa band, which migrates slower than inactive NRC2 in the absence of SS15, suggesting the formation of an NRC-SS15 complex in vivo (Figure 1B). We also observed that coexpression of SS15 not only blocked NRC2 oligomerization but also prevented the previously reported shift of activated NRC2 from the cytoplasm to the plasma membrane (PM) and the formation of NRC2 PM puncta upon Rx / CP activation. We conclude that SS15 acts as a direct protein-mediated inhibitor of NRC2 by directly binding to the NB-ARC domain of NRC2 and preventing the formation of signal-competent oligomeric resistosomes, thereby suppressing immune signaling.

[0169] The HD1-1 region of the NB-ARC domain determines SS15 binding and inhibition of NRC. We generated a series of NRC2-NRC4 chimeric proteins in the SS15-binding NB-ARC domain (Figures 2A-2B) and subsequently analyzed them for gain or loss of SS15 binding via in planta co-immunoprecipitation. NRC4, a chimeric mutant of NRC4 harboring the N-terminal portion of the HD1 region of NRC2 (herein referred to as the "HD1-1" region), was also identified. 2HD1-1 We identified NRC4, which acquires binding to SS15 (Fig. 2C). 2HD1-1is susceptible to inhibition by SS15 and fails to oligomerize and induce cell death in the presence of SS15 (Fig. 2D, dashed black circle; Fig. 2E). We conclude that SS15 binds to the HD1-1 region and that binding to HD1-1 is sufficient for this effector to act as a direct inhibitor of NRC resistosome formation and programmed cell death.

[0170] [Identification of the SS15-NRC binding interface enables manipulation of NRC2 for pathogen suppression evasion] We hypothesized that identifying key residues mediating the interaction with the HD1-1 region could enable NRC2 to evade SS15-mediated immunosuppression. To this end, we generated crystals of SS15 in complex with the NB-ARC domain of NRC1, an NRC2 homolog that is also suppressed by SS15, and obtained X-ray diffraction data at 4 Å resolution (Figure 3A and Figure 4). We demonstrated that SS15 binds to a loop in HD1-1 that connects the NB domain to the HD1 and WHD domains, providing independent evidence that the SS15-NRC interaction is mediated by this region. This loop has previously been shown to act as a "hinge" that allows the NB domain to move relative to the HD1 and WHD domains (Figure 4). By binding to and immobilizing this hinge, SS15 likely eliminates the conformational change critical for NLR activation.

[0171] Furthermore, because SS15 can inhibit NRC1, NRC2, and NRC3, we leveraged the high degree of conservation characteristic of plant NB-ARC domains to narrow down the list of residues within the binding interface that support this interaction. We obtained a shortlist of residues within the HD1-1 region that are similar in NRC1, NRC2, and NRC3 (Figure 3B). Combining information from the cocrystal structure and alignment allowed us to select 13 candidate residues in NRC2 for testing by mutagenesis (Figure 3B). Each of these residues was mutated to the corresponding amino acid found in NRC4, and these NRC2 mutants were screened for sensitivity to SS15 inhibition in cell death assays. We identified at least two mutants, NRC2, that were activated by Rx / CP and induced cell death. E316P and NRC2 D317K We found that NRC2 was no longer inhibited by SS15 (Fig. 3C). Furthermore, we tested all 13 single amino acid mutants for binding to SS15 by in planta co-immunoprecipitation. D317K and NRC2 E316P We found that Rx / CP-activated NRC2 exhibited reduced binding to SS15 compared to NRC2 (Figure 3D), consistent with the observation that SS15 was unable to inhibit this mutant (Figure 3C). We concluded that residues E316 and D317 are important for SS15-mediated inhibition of NRC2, and that mutating residue D317 in particular allows Rx / CP-activated NRC2 to avoid binding and inhibition by SS15.

[0172] We next tested whether these two SS15 escape mutants maintained correct functionality along with other previously examined NRC2-dependent sensor NLRs. E316P and NRC2 D317KWe performed complementation assays in nrc2 / 3 / 4 CRISPR KO N. benthamiana plants using SS15 to test these helpers by activating a panel of agronomically important sensor NLRs that mediate resistance to diverse pathogens. This panel included the potato cyst nematode R genes and the Rx paralog Gpa2, as well as various oomycete and bacterial resistance proteins. In the absence of SS15, both NRC2 mutants mediate cell death when activated by all NRC2-dependent sensors tested (Figure 5A). In the presence of SS15, NRC2 E316P The mutant was able to escape SS15 repression when activated by Rx but not by all other sensors tested. D317K The mutant was able to escape SS15 repression regardless of the sensor NLR used to activate it. D317K were selected for follow-up biochemical experiments using the previously described BN-PAGE-based resistosome formation assay. Unlike Rx / CP-activated NRC2, Rx / CP-activated NRC2 D317K NRC2 oligomerized in the presence of SS15 and showed no in vivo complex formation with the inhibitor (Fig. 5B). D317K It is concluded that upon activation by multiple agriculturally important sensor NLRs, SS15 can completely evade immunosuppression while retaining the ability to oligomerize and mediate cell death.

[0173] [Consideration] In this study, we revealed that parasite effectors have evolved as inhibitors of helper NLRs by directly binding to the HD1 region of the NB-ARC domain and preventing resistosome formation. SS15 binds to and immobilizes a critical hinge loop in the NB-ARC domain of these NLRs, restricting the movement of the NB domain relative to the HD1 and WHD domains and preventing immune receptor activation. Notably, while SS15 can bind to and inhibit NRC2, we generated a chimeric NRC2-NRC4 mutant, which, together with structural information, helped identify the binding interface. Mutation experiments at this interface revealed a single amino acid NRC2 (NRC2) that evades SS15 inhibition without compromising receptor signaling ability. D317K ) mutant was successfully created. D317K The mutant was able to support signaling by the NRC2-dependent sensor even in the presence of SS15.

[0174] The approach described here can restore hidden or suppressed resistance proteins and enhance disease resistance. Furthermore, single-amino acid NRC2 mutants can be generated in-locus using gene editing techniques in agriculturally important crop species, enabling the adoption of this technology in countries where transgenic approaches are not feasible. In this study, we developed a novel approach to achieve robust immunity by engineering NLRs that evade parasite suppression. This approach can also be applied to other plant NLR immune receptors that are directly targeted by parasite effectors. This technology has the potential to open a new era in disease resistance breeding.

[0175] [Sequence List] SEQ ID NO: 1; Nicotiana benthamiana NRC2a HD1 domain (NbNRC2a-HD1), amino acid sequence; GenBank accession number: ALQ52761. PHDLKFLTEDESWILLEKKVFHKDKCPPELELSGKSIAKKCNGLPLAIVVIAGALIGKGKTSREWKQVDESVGEHLINK

[0176] SEQ ID NO: 2; Nicotiana benthamiana NRC2a HD1-1 domain (NbNRC2a-HD1-1), amino acid sequence; GenBank accession number: ALQ52761. PHDLKFLTEDESWILLEKKVFHKDKCPPELELSGKSIAK

[0177] SEQ ID NO: 3; Nicotiana benthamiana NRC2a RNBS-C motif, amino acid sequence; Genbank accession number: ALQ52761. HDLKFLTEDESWILLEKKVF

[0178] SEQ ID NO: 4; Nicotiana benthamiana NRC2b HD1 domain (NbNRC2b-HD1), amino acid sequence; GenBank accession number: ALQ52762. PHDLKFLTEDESWILLEKKVFHKDKCPPELELSGKSIAKKCNGLPLAIVVIAGALIGKGKTSREWKQVDESVGEHLINK

[0179] SEQ ID NO: 5; Nicotiana benthamiana NRC2b HD1-1 domain (NbNRC2b-HD1-1), amino acid sequence; GenBank accession number: ALQ52762. PHDLKFLTEDESWILLEKKVFHKDKCPPELELSGKSIAK

[0180] SEQ ID NO: 6; Nicotiana benthamiana NRC2b RNBS-C motif, amino acid sequence; GenBank accession number: ALQ52762. HDLKFLTEDESWILLEKKVF

[0181] SEQ ID NO: 7; Nicotiana benthamiana NRC3 HD1 domain (NbNRC3-HD1), amino acid sequence; GenBank accession number: QER78240. PHDLKFLTENESWELLEKRVFHKEKCPFELELPGKSIAKKCRGLPLAIVVIAGALIGKGKTTREWELVADSVGEHLINR

[0182] SEQ ID NO: 8; Nicotiana benthamiana NRC3 HD1-1 domain (NbNRC3-HD1-1), amino acid sequence; GenBank accession number: QER78240. PHDLKFLTENESWELLEKRVFHKEKCPFELELPGKSIAK

[0183] SEQ ID NO: 9; Nicotiana benthamiana NRC3 RNBS-C motif, amino acid sequence; Genbank accession number: QER78240. HDLKFLTENESWELLEKRVF

[0184] SEQ ID NO: 10; Nicotiana benthamiana, NRC4 HD1 domain (NbNRC4-HD1), amino acid sequence; GenBank accession number: QER78241. PHDLKFLTPKESFELLVKRVFGKKPCPKDLVGHGESIAGKCGGVPLAVVVIAGALRGRPNTSDWIRVERNVVQHLYTNS

[0185] SEQ ID NO: 11; Nicotiana benthamiana, NRC4 HD1-1 domain (NbNRC4-HD1), amino acid sequence; GenBank accession number: QER78241. PHDLKFLTPKESFELLVKRVFGKKPCPKDLVGHGESIAG

[0186] SEQ ID NO: 12; Nicotiana benthamiana, NRC4 RNBS-C motif, amino acid sequence; GenBank accession number: QER78241. HDLKFLTPKESFELLVKRVF

[0187] SEQ ID NO: 13: Solanum tuberosum NRC2 HD1 domain (StNRC2-HD1), amino acids; NCBI Reference Sequence: XP_006359790.1. PHDLKFLSEDESWILLEKKVFHKDKCPPELVVPSGKSIAKKCKGLPLAIVVIAGALIGKGKTPREWKQVDDSVSEHLINR

[0188] SEQ ID NO: 14; Solanum tuberosum NRC2 HD1-1 domain (StNRC2-HD1-1), amino acids; NCBI reference sequence: XP_006359790.1. PHDLKFLSEDESWILLEKKVFHKDKCPPELVVPSGKSIAK

[0189] SEQ ID NO: 15; Solanum tuberosum NRC2 RNBS-C motif, amino acids; NCBI reference sequence: XP_006359790. HDLKFLSEDESWILLEKKVF

[0190] SEQ ID NO:16; Solanum tuberosum NRC3 HD1 domain (StNRC3-HD1), amino acids; NCBI reference sequence: XM_006362512.2 PHDLKFLTEDESWELLEKKVFHKEKCPPELELPGKSIAEKCMGLPLAIVVIAGALIGKGKTTREWELVAASVREHLINR

[0191] SEQ ID NO: 17; Solanum tuberosum NRC3 HD1-1 domain (StNRC3-HD1-1), amino acids; NCBI Reference Sequence: XM_006362512.2 PHDLKFLTEDESWELLEKKVFHKEKCPPELELPGKSIAE

[0192] SEQ ID NO: 18; Solanum tuberosum NRC3 RNBS-C motif, amino acids; NCBI reference sequence: XM_006362512.2 DLKFLTEDESWELLEKKVF

[0193] SEQ ID NO: 19; Solanum tuberosum NRC4 HD1 domain (StNRC4-HD1), amino acids; NCBI Reference Sequence: XP_006367034.1 PHVLKFLKTEESFELLVMRVFGKGSCPNELVVTGKKIAEKCGGVPLVVVVIAGALRGRSDKKDWERVDKNVVQHFGEHT

[0194] SEQ ID NO: 20; Solanum tuberosum NRC4 HD1-1 domain (StNRC4-HD1-1), amino acids; NCBI Reference Sequence: XP_006367034.1 PHVLKFLKTEESFELLVMRVFGKGSCPNELVVTGKKIAE

[0195] SEQ ID NO: 21; Solanum tuberosum NRC4 RNBS-C motif, amino acids; NCBI Reference Sequence: XP_006367034.1 HELKFLEKEESFELLVMRVF

[0196] SEQ ID NO: 22; Solanum lycopersicum, NRC1 HD1 domain (SlNRC1-HD1), amino acid sequence; NCBI reference sequence: NP_001234202. PHDLKFLTDEESWILLEKRAFHKAKCLPELETNGKSIARKCKGLPLAIVVIAGALIGKSKTIKEWEQVDQSVGEHFINR

[0197] SEQ ID NO: 23; Solanum lycopersicum, NRC1 HD1-1 domain (SlNRC1-HD1-1), amino acid sequence; NCBI reference sequence: NP_001234202. PHDLKFLTDEESWILLEKRAFHKAKCLPELETNGKSIAR

[0198] SEQ ID NO: 24; Solanum lycopersicum, NRC1 RNBS-C motif, amino acid sequence; NCBI reference sequence: NP_001234202. HDLKFLTDEESWILLEKRA

[0199] SEQ ID NO: 25; Solanum lycopersicum, NRC2 HD1 domain (SlNRC2-HD1), amino acid sequence; NCBI reference sequence: XP_004248798. PHDLKFLTEDESWILLEKKVFHKDKCPPELVLSGKSIAKKCKGLPLAIVVIAGALIGKGKTPREWKQVDDSVSEHLINR

[0200] SEQ ID NO: 26; Solanum lycopersicum, NRC2 HD1-1 domain (SlNRC2-HD1-1), amino acid sequence; NCBI reference sequence: XP_004248798. PHDLKFLTEDESWILLEKKVFHKDKCPPELVLSGKSIAK

[0201] SEQ ID NO: 27; Solanum lycopersicum, NRC2 RNBS-C motif, amino acid sequence; NCBI reference sequence: XP_004248798. HDLKFLTEDESWILLEKKVF

[0202] SEQ ID NO: 28; Solanum lycopersicum, NRC3 HD1 domain (SlNRC3-HD1), amino acid sequence; NCBI reference sequence: XP_004238948. PHDLKFLTKDESWELLEKKVFHKEKCPPELELPGISIAEKCMGLPLAIVVIAGALIGKGKTTREWELVAASVGEHLINR

[0203] SEQ ID NO: 29; Solanum lycopersicum, NRC3 HD1-1 domain (SlNRC3-HD1-1), amino acid sequence; NCBI Reference Sequence: XP_004238948. PHDLKFLTKDESWELLEKKVFHKEKCPPELELPGISIAE

[0204] SEQ ID NO: 30; Solanum lycopersicum, NRC3 RNBS-C motif, amino acid sequence; NCBI Reference Sequence: XP_004238948. HDLKFLTKDESWELLEKKVF

[0205] In SEQ ID NOS: 32, 34, 36, 41, 43, 47, 49, and 51, bolded bases represent the NB-ARC (HD1) domain, underlined bases represent the NB-ARC1 (HD1-1) subdomain, and italics represent the RNBS-C motif. In SEQ ID NOS: 31, 33, 35, 40, 42, 46, 48, and 50, underlined bases represent the NB-ARC1 (HD1-1) subdomain. In SEQ ID NOS: 37, 38, 39, 44, 45, and 52-59, the mutated residues are underlined and highlighted in the individual sequences.

[0206] SEQ ID NO: 31; Nicotiana benthamiana, NbNRC2a cDNA; GenBank accession number: KT936525. ATGGCGAACGTTGCGGTGGAGTTTCTGGTGCAGAACTTGATGCAGCTGCTAAGAGACAATGCGGAGCTGATTGTTGGAGTTAAGGATTCGGCTGAGAGTCTGCTTCAAGATCTCAATGATTTCAACGCTTTTCTCAAGCAAACTGCTAAGTCCCGCACTGAGAACGATGTCCACAAAGAATTGGTGAAGAAAATTAAGACTGTGGTCAACTCTGCTGAAGATGCCATTGATAAGTTTGTGATTGAGGCTAAGCTTCACAAGGACAAAGGGGTTGGCAGATTTGTGGATGTTAAGCATTATAAAAGAGTGTATGATGTAGCAGGGGAGATTAAAACTATTAGAGACAAAGTCAAAGAAATTCGTCTCAATAATGCACTTGACCTTCAGGCCCTTCAAGATGAAGATCAATCTGCCAAAGGTGTCCAAGAAAGAAAGCCTCCGGTGGTAGAGGAAGATGATGTGGTTGGATTTGAAGAGGAAGCAGATAAAGTAATCAACCGTCTTCTGGGAGGATCGAGTGGACTAGAAGTTGTTCCAGTTGTTGGAATGCCTGGTCTCGGCAAAACGACACTGGCAAATAAGATTTACAAGCATCCTGACATCGGGTACCAGTTTTTTACTCGCATTTGGGTTTATGTTTCTCAATCATACAGGAGAAGAGAATTATTTCTCAACATCATCAGCAAATTCACTCGCAATACCAAACAATACCACGATATGTGTGAGGAGGATTTAGCTGATGAAATAGAAGATTTTTTGGGCAAGGGAGGGAAATACTTGATTGTCTTGGATGATGTATGGTCTCCTGACGCTTGGGAACGTATCAGAATAGCTTTCCCAAACAACAACAAATCCAATAGAATATTATTGACCACCCGAGATAGCAAAGTTGCTAAGCAATGCAAGCAGTGCATTGGTATA CCTCATGATTTAAAATTTCTGACTGAAGATGAAAGTTGGATTTTACTGGAGAAGAAAGTTTTCCATAAAGATAAATGTCCTCCTGAATTGGAACTATCTGGAAAGAGCATAGCCAAA

[0207] SEQ ID NO: 32; Nicotiana benthamiana, NbNRC2a amino acid sequence; GenBank accession number: ALQ52761.1. JPEG2025539497000002.jpg110166

[0208] SEQ ID NO: 33; Nicotiana benthamiana, NbNRC2b cDNA; GenBank accession number: KT936526. ATGGCGAACGTTGCGGTGGAATTTTTGGTGCAGAACTTGATGCAGCTGCTGAGGGACAATGCGGAGCTGATTATTGGGGTTAAGGATTCGGCTGAGAGCCTGCTTCAAGATCTCAATGATTTCAACGCTTTTCTCAAGCAAGCTGCCAAGTCCCGCATTGAGAACGATGTCCACAAAGAATTGGTGAAGAAAATTAGACAATGGTCAACTCTGCTGAAGATGCCATTGATAAGTTTGTAATTGAGGCTAAGCTTCACAAGGACAAAGGGGTTGGCAGATTTGTAGATGTTAAGCATTATAAAAGAGTGTATGATGTAGCAGCCGAGATCAAAGGTATCAGAGAAAAGTGAAAGAAATCCGTCAGAATAATGCCCTTGACCTTCAAGCCCTTCAAGATGAAGATCAATCTGCCAAAGGTGTCGAAGAAAGAAAGCCTCCAGTGGTAGAGGAAGATGATG TGGTTGGATTTGAAGAGGAAGCAGATAAAGTAATCAACCGTCTTCTGGGAGGATTGAGTGGACTAGAAGTTGTTCCAGTTGTTGGAATGCCTGGTCTCGGCAAAACGACGCTAGCAAATAAGATTTACAAGCATCCTGACATCGGGTACCAGTTTTTTACTCGCATTTGGGTTTATGTTTCTCAATCATACAGGAGAAGAGAATTATTTCTCAACATCATCAGCAAATTC ACTCGAAATACCAAACAATACCATGATATGTGTGAGGAGGATTTAGCTGATGAAATACAAGATTTTTTGGGCAAAGGAGGAAAATACTTGATTGTCTTGGATGATGTATGGTCTCCTGACGCTTGGGAACACATCAAAATAGCTTTCCCGAACAACAACAAATCCAATAGAATATTATTGACCACTCGAGATAGCAAAGTTGCTAAGCAATGCAAGCAGTGCATTGGTATA CCTCATGATTTAAAATTTCTGACTGAAGATGAAAGTTGGATTTTACTGGAGAAGAAAGTTTTCCATAAAGATAAATGTCCTCCTGAATTGGAACTATCTGGAAAGAGCATAGCCAAA

[0209] SEQ ID NO: 34; Nicotiana benthamiana, NbNRC2b cDNA; GenBank accession number: ALQ52762. JPEG2025539497000003.jpg108166

[0210] SEQ ID NO: 35; Nicotiana benthamiana, NbNRC3 cDNA; GenBank accession number: MK692736. ATGGCAGATGTAGCAGCAGATGTAGCAGTAAAATTCCTAGTAGAAAACTTGATGCAATTATTAATCGACAACGCTGACTTGATTATTGGTATAAAGGGTGAAGTTGAAAATTTACTACAAGATCTGAATGATTTCAATGCTTTTCTCAAACAAGCAGCTAAATCAAGGAGAGACAATGAAGTCTTGAAATCACTAGTAAAGAAGATAAGGAAAGTGGTAAATGATGCTGAAGATTCAATTGATAAGTTTGTGATTGAAGCTAAAAGACATGACGATAAAAATAAATTTGCTCAGTGGTTTCATCTTACTCATGTTGCTAGAGCAAAAGGGGTTGCTGATGAGATTAAAACTATAAGGGAAAGGGTGAAGGAAATTCGCCAAAATGATGCTTATGGACTTCAAGCTATAACTTCTTATGATAATTTCAACCAAGGTGCTCAGGAGAGGAAGGTCCCTGTAG TTGAGGAAGACGACGTGGTAGGCTTTGATGACGAAGCGAAAACTGTAATTGATCGCCTCATTGGAGGATCAGATTATGTTGTGCCGGTCGTTGGTATGCCTGGTCTTGGAAAAACAACTTTGGCATATAAGATTTTCAAGGATTCCACAGTTGAGTATGAGTTTTTCAACCGCATATGGGTATATGTCTCTCAATCATTCAACAGAAGGGAAATATTTCTCAACATCATCAGCAAATTCACTCGAAACACCAAACAATACCATGATACACCAGAGGAGGAATTAGCAAATGAAATAAAGGAGCTGCTTGGGAAGGGTGGGAAATATCTTGTTGTTTTAGATGATGTGTGGACAAGAGAAGCTTGGGATCGCATTAAAATTGCTTTCCCCAATAATAATAAACGGAATAGAGTCTTGATGACTACTAGACAAAACAATGTGGCTAAGTCTTGCAACGATAAA CCTCATGATCTAAAGTTTTTGACTGAAAATGAAAGTTGGGAGCTACTTGAGAAGAGAGTTTTTCACAAGGAAAAATGTCCATTTGAGTTAGAATTACCTGGAAAAAGTATAGCCAAA

[0211] SEQ ID NO: 36; Nicotiana benthamiana, NbNRC3 amino acid sequence; GenBank accession number: QER78240. JPEG2025539497000004.jpg110164

[0212] SEQ ID NO: 37; Nicotiana benthamiana, NRC2a D317K amino acid. MANVAVEFLVQNLMQLLRDNAELIVGVKDSAESLLQDLNDFNAFLKQTAKSRTENDVHKELVKKIKTVVNSAEDAIDKFVIEAKLHKDKGVGRFVDVKHYKRVYDVAGEIKTIRDKVKEIRLNNALDLQALQDEDQSAKGVQERKPPVVEEDDVVGFE EEADKVINRLLGGSSGLEVVPVVGMPGLGKTTLANKIYKHPDIGYQFFTRIWVYVSQSYRRRELFLNIISKFTRNTKQYHDMCEEDLADEIEDFLGKGGKYLIVLDDVWSPDAWERIRIAFPNNNKSNRILLTTRDSKVAKQCKQCIGIPHDLKFLTE KESWILLEKKVFHKDKCPPELELSGKSIAKKCNGLPLAIVVIAGALIGKGKTSREWKQVDESVGEHLINKDQPENCNKLVQLSYDRLSYDLKACFLYCGAFPGGFEIPAWKLIRLWIAEGFIQYKGHLSLECKAEDNLNDLINRNLMVMQRTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGAEQYFPGKRELATYRRLCIHSSVLEFISTKPSGEHVRSFLSFSLKKIEMPSVDIPTIPKGFPLLRVFDVESINFSRFSKEFFQLYHLRYIAFSSDTI KIIPKHIGELWNIQTLIINTQQRSLDIQANIWNMERLRHLHTNSSAKLPVPVTPRSSKPLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKIAVLLEPNKSLLNKNVKKLESLENLKLINDSSQTGKGLRLPPYIFP TKLRKLSLVDTWLEWNDMSILGQMEHLEVLKLKENGFMGECWESVGGFCSLLVLWIERTDLVSWKASADHFPRLKHLVLICCDKLKEIPIGLADIRSFQVMELQNSTKTAAISARGIRDKKDKQTQEGTNNNGFKLSIFPPDL

[0213] sequence number 38; Nicotiana benthamiana, NRC2b D317K amino acid. MANVAVEFLVQNLMQLLRDNAELIIGVKDSAESLLQDLNDFNAFLKQAAKSRIENDVHKELVKKIKTMVNSAEDAIDKFVIEAKLHKDKGVGRFVDVKHYKRVYDVAAEIKGIREKVKEIRQNNALDLQALQDEDQSAKGVEERKPPVVEEDDVVGFEEEADKVINRLLGGLSGLEVPVVVGMPGLGKTTLANKIYKHPDIGYQFFTRIWVYVSQSYRRRELFLNIISKFTRNTKQYHDMCEEDLADEIQDFLGKGGKYLIVLDDVWSPDAWEHIKIAFPNNNKSNRILLTTRDSKVAKQCKQCIGIPHDLKFLTEK ESWILLEKKVFHKDKCPPELELSGKSIAKKCNGLPLAIVVIAGALIGKGKTSREWKQVDESVGEHLINKDQPENCNKLVQLSYDRLSYDLKACFLYCGAFPGGFEIPAWKLIRLWIAEGFIQYKGHLSLECKAEDNLNDLINRNLMVMQRTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGAEQYFPGKRELATYRRLCIHSSVLEFISTKPSGEHVRSFLSFSLKKVEMPSVDIPTIPKGFPLLRVFDVESINFSRFSKEFFQLYHLRYIAFSSDTI KIIPKHIGELWNIQTLIINTQQHSLDIQANIWNMARLRHLHTNSSAKLPVPVTPRSSKPLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKVAVLLEPNKSLLNKNVKKLESLENLKLINDSSQTGKGLRLPPYIFP TKLRKLSLVDTWLEWNDMSILGQMEHLEVLKLKENGFMGECWESVGGFCSLLVLWIERTDLVSWKASADHFPRLKHLVLICCDKIKEIPIGLADIHSFQVMELQNSTKTAAISAREIRDKKDKQTQEGTNNNGFKLSIFPPDL

[0214] sequence number 39; Nicotiana benthamiana, NRC3 N317K amino acid. MADVAADVAVKFLVENLMQLLIDNADLIIGIKGEVENLLQDLNDFNAFLKQAAKSRRDNEVLKSLVKKIRKVVNDAEDSIDKFVIEAKRHDDKNKFAQWFHLTHVARAKGVADEIKTIRERVKEIRQNDAYGLQAITSYDNFNQGAQERKVPVVEEDDVVGFDDEAKTVIDRLIGGSDYVVPVVGMPGLGKTTLAYKIFKDSTVEYEFFNRIWVYVSQSFNRREIFLNIISKFTRNTKQYHDTPEEELANEIKELLGKGGKYLVVLDDVWTREAWDRIKIAFNNNKRNRVLMTTRQNNVAKSCNDKPHDLKFLTEK ESWELLEKRVFHKEKCPFELELPGKSIAKKCRGLPLAIVVIAGALIGKGKTTREWELVADSVGEHLINRDPENCKKLVQMSYDRLPYDLKACFLYCGAFPGGSEISAQKLICLWIAEGFIQYQGPLTLEDIAEDHLNDLVNRNLVMVMKRSSSGQIKTCRVHDMLHEFCRHEAMMEENLFQEIKRGQEHSFPEKQELASYRRLCIHSSVSEFLSTKPFAEHVRSFLCFASKKFEMPLGEIPAIPRAFPLLRVLDAESIKSIFSRFSREFFKLFHLRYIAFSTDSIMT IPTNIGNLWNVQTLIIETQQGTLDIKADIWNMTRLRHVCINASATLPSPKRPKSSKDNLVNRCLQTLSTIAPECCTAEVFTRTPNLKLGVRGKIDALLETSKDGSSSGLFSNIGKLDCLEKLKLVNDTRQSRKQLHLPPAYIFPQKLKKLTLIDTWFEWKDMSILGLLEYLEVLKLKKENAFRGQSWEPEDSGFPRLQVLWIERTDLSSWKASSGNFPRLKCLVLIACDNLKELPAELADVENLQLMELQSTSVSAAKSARAILKKKQQKVGSGFKLSVFPPDLGL

[0215] sequence number 40; Solanum tuberosum StNRC2, cDNA. NCBI reference sequence: XM_006359728.2 ATGGCGAACGTAGCAGTAGAATTTCTCGTTGAGAACTTGATGCAGCTGCTGCGGGACAACGCAGAGCTAATTAGTGGAGTTAAAGAGGCTGCTGAGAGTCTACTTCAAGATCTAAATGATTTCAACGCCTTTCTTAAGCAAGCTGCCAAGTGCCACATCAACGAGAACGAAGTTCTGAGAGAACTCGTCAAGAAAATCAGAACAGTGGTTAACTCTGCTGAAGATGCT ATTGATAAATTTGTGATTGAAGCTAAGCTACACAAGGATAAGGGTATGACCAGAGTATTAGACCTTCCCCATTATAATAAGGTCAGGGAGGTAGCTGGTGAGATCAAAGCTATACGAAACAAAGTCAAAGAGATCAGACAGAATGATGCCATTGGACTTCAGGCCCTTCAAGATGATGATTCATCTGCCAGAGGTTTCGAAGAAAGAAAGCCTCCAGTGGTAGAGGAAG ATGATGTGGTTGGATTTGACGAAGAAGCAGATATTGTAATCAAACGCCTTCTTGGAGAATCAAATCGTCTAGAAGTTGTTCCAGTTGTTGGTATGCCTGGTCTCGGCAAAACGACCCTAGCAAATAAAATATACAAGCATCCTAAAATCGGGTATGAATTTTTTACTCGTATTTGGGTTTATGTATCTCAATCATACAGGAGAAGAGAATTATTTCTCAACATCATCAGCAAATTCACTCGAAATACGAAACAATATCATGGGATGTGTGAGGAGGATTTGGCTGATGAAATACAAGAATTCTTGGGAAAGGGAGGAAAATACTTGGTTGTCTTGGATGATGTATGGTCGGATGAAGCTTGGGAACGTATCAAGATAGCTTTCCCAAATAACAACAAACCGAATCGAGTATTGTTGACCACTCGAGATTCCAAAGTGGCTAAACAATGCACTCCCATA CCTCATGATTTAAAATTTCTGAGTGAAGATGAAAGTTGGATATTACTGGAGAAGAAGGTTTTTCACAAGGATAAATGTCCTCCTGAATTGGTGGTACCATCTGGGAAGAGCATAGCAAAA

[0216] SEQ ID NO: 41; Solanum tuberosum StNRC2, amino acid sequence; NCBI Reference Sequence: XP_006359790.1 JPEG2025539497000005.jpg111164

[0217] SEQ ID NO: 42; Solanum tuberosum StNRC3, cDNA; NCBI Reference Sequence: XM_006362512.2 ATGGCGGATGTAGCAGTAAAGTTTTTAGTAGAAAATTTGATGCAATTACTAATCGACAACGCCGATTTGATTCTCGGAATCAAAGGCGAAGTTGAAAATCTACTCCGAGATCTCAATGACTTCAATGCTTTCCTCAAACAAGCTGCTAAATCCCGCAGGGAAAATGAGGTTTTGAAAGAAATGGTGAAGAAAATCAGAAAAGTGGTGAATGATGCTGAGGATTCAATTGATAAATTTGTGATTGAAGCTAAGAGACATGATGATAAAAACAAATTTGCTCAATGGTTTCATATTACTCATGTGGCTAGAGCAAAAGGGGTAGCAGATGAGATCAAAAGTATAAAGGAAAGAGTGAAGGAAATTAGAGAAAATGACGCTTATGGCCTTCAAGCGATAACTTTAGATGATAATTTCAATAGAGGTGATGAAGAGAGGAAGGCCCCTGTAGTTGAGGAAG ATGATGTGGTTGGTTTTGATGATGAAGCAAAAATTGTAATTGATCGTCTCATTGGAGGATCAGATTATGTTGAGGTTGTGCCAGTTGTTGGTATGCCTGGTCTTGGTAAAACAACTTTGGCATATAAGATTTACAAGGATCCGAAGGTTGAGTATGAGTTCTTCACCCGCGTTTGGGTATATGTCTCTCAAACGTTCAAGAGAAGGGAAATATTTCTCAACATTATCAGCAAGTTCACTCGAAACACCAAACAATATCATGATACACCAGAGGATGACTTAGCAAATGAAGTGAAGGAGCTTCTTGGAAAAGGTGGAAAATATCTTATTGTTTTGGATGATGTGTGGACGATGGAAGCTTGGGATCGTATCAAAATTGCTTTCCCTAATAATGGTAAACGGAATAGAGTGTTGATGACCACGAGAGAATCAAATGTGGCTAAGTGTTGTAATGATAAA CCTCATGATCTTAAGTTTTTAACTGAAGATGAAAGTTGGGAGCTACTTGAGAAGAAGGTTTTTCACAAGGAAAAGTGTCCACCTGAGTTAGAATTGCCCGGGAAAAGTATAGCTGAA

[0218] SEQ ID NO: 43; Solanum tuberosum StNRC3, amino acids; NCBI Reference Sequence: XP_006362574.1 JPEG2025539497000006.jpg111162

[0219] SEQ ID NO: 44; Solanum tuberosum NRC2 D315K , amino acids. MANVAVEFLVENLMQLLRDNAELISGVKEAAESLLQDLNDFNAFLKQAAKCHINENEVLRELVKKIRTVVNSAEDAIDKFVIEAKLHKDKGMTRVLDLPHYKRVREVAGEIKAIRNKVKEIRQNDAIGLQALQDDDSSARGFEERKPPVVEEDDVVG FDEEADIVIKRLLGESNRLEVVPVVGMPGLGKTTLANKIYKHPKIGYEFFTRIWVYVSQSYRRREFLNIISKFTRNTKQYHGMCEEDLADEIQEFLGKGGKYLVVLDDVWSDEAWERIKIAFPNNNKPNRVLLTTRDSKVAKQCTPIPHDLKFLSE KESWILLEKKVFHKDKCPPELVVPSGKSIAKKCKGLPLAIVVIAGALIGKGKTPREWKQVDDSVSEHLINRDHPENCNKLVQMSYDRLPYDLKACFLYCSAFPGGFQIPAWKLIRLWIAEGFIQYKGHLSLECKGEDNLNDLI NRNLVMVMERTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGSEYYFPGKRELSTYRRLCIHSSVLDFISTKPSAEHVRSFLSSKKIEMPSADIPTIPKGFPLLRVLDVESINFSRFSKEFYQLYHLRYVAFSSDSI KILPKLMGELWNIQTIIINTQQRTLDIQANIWNMERLRHLHTNSSAKLPVPAPKNSKVTLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKIAVLLDNKSAVSLKNVKRLEYLENLKLINDSSIQTGKLRLPAYIFP TKLRKLTLLDTWLEWKDMSILGQLEHLEVLKMKENGFTGESWESTGGFCSLLVLWIERTNLVTWKASADDFPRLKHLVLICCDYLKEVPIALADIRSFQVMMLQNSTKTAAISRQIQAKKDNQTQQGTKNIAFKLSIFPPDL

[0220] sequence number 45; Solanum tuberosum NRC3 D315K , amino acid. MADVAVKFLVENLMQLLIDNADLILGIKGEVENLLRDLNDFNAFLKQAAKSRRENEVLKEMVKKIRKVVNDAEDSIDKFVIEAKRHDDKNKFAQWFHITHVARAKGVADEIKSIKERVKEIRENDAYGLQAITLDDNFNRGDEERKAPVVEEDDVVG FDDEAKIVIDRLIGGSDYVEVVPVGMPGLGKTTLAYKIYKDPKVEYEFFTRVWVYVSQTFKREIFLNIISKFTRNTKQYHDTPEDDLANEVKELLGKGGKYLIVLDDVWTMEAWDRIKIAFPNNGKRNRVLMTTRESNVAKCCNDKPHDLKFLTE KESWELLEKKVFHKEKCPPLELELPGKSIAEKCMGLPLAIVVIAGALIGKGKTTREWELVAASVREHLINRDPENCKKLVQMSYDRLPYDLKACFLYCGAFPGGSQIPAKKLIRLWIAEGFIQYQGPLALEDVAEDHLNDLVNRNLVMVMQRSCSGQIKICRVHDMLHEFCRHEAMTEEDLFQEIKQGQERSFPGKQELATYRRLCIHSGVPEFLSTKPSGEHVRSFLCVGSKKIDMPPNEIPSIPKAFPLLRVLDAESIKFNRFSREFFKLFHLRYIALSTDKIKTIPV DFGNLWNVQTLIVETQEATLDIKADIWNMTRLRHVCTNASATLPSTKRPKSSKGNLVNRCLQTLSTIAPECCTAEVFTRTPNLKLGVRGKIDALLETSKDGSGSVLFSNIGKLACLEYLKLVNDTRISSKPLHLPPAYIFPQKLKKLSLVDTWFEWKDMSILGLLPDLEVLKLKENAFKGQSWEQEDGGFPRLQVLWIERTDLTSWKASSGNFPRLKHLALISCDKLEELPAELADVKNLQLIELQSSSESAARSARAILKRNQEKEQEGDKGTGFKLSIFPHDLGL

[0221] sequence number 46; Solanum lycopersicum, SlNRC1 cDNA; NCBI reference sequence: NM_001247273. ATGGTTGATGTAGGGGTTGAATTTCTGTTAGAGAACTTGAAGCAATTGGTACTGGACAATGTGGAGTTAATCGGAGGAGCTAAAGATGAAATCGAGAATCTGCGTGATGATTTGAGTGAATTCAATGCCTTTCTCAAGCAAGCTGCAATGGTCCGCAGCGAAAACCCAGTTCTCAAAGAACTAGTGAGGAGTATCAGAAAAGTGGTGAATCGTGCTGAAGATGCTGTTGATAAATTTGTAATTGAAGCTAAAGTTCATAAAGACAAAGGGTTTAAAGGGGTTTTCGATAAACCTGGACATTATAGAAGAGTGAGGGATGCAGCTGTGGAGATTAAAGGTATCAGAGATAAAATGAGAGAAATTCGGCAAAATAAGGCACATGGCCTTCAGGCTCTACTTCAAGATCATGATGATTCAATCAGCAGAGGTGGAGAAGAGAGACAGCCTCCTGTGGTTGAGG AAGATGATGTGGTGGGCTTTGACGATGAGGCGCAGACGGTAATCGACCGTCTTCTTGAAGGATCAGGTGATTTAGAGGTTATTCCAGTAGTTGGAATGCCTGGTCTTGGCAAAACTACACTAGCCACTAAGATCTTCAAGCATCCGAAGATTGAGTACGAGTTCTTTACTAGACTTTGGCTTTACGTTTCCCAATCATACAAGACAAGAGAATTATATCTTAACATCATCAGTAAATTCACCGGAAACACCAAACATTGCCGTGATATGTCTGAAAAGGATTTAGCTCTTAAGGTACAAGAGATTTTGGAAGAAGGAGGAAAATACTTGATTGTCTTGGATGATGTCTGGTCGACAGATGCTTGGGATCGTATCAAGATTGCTTTCCCGAAAAATGACAAGGGCAATAGAGTATTGTTGACTACTCGAGACCACCGTGTTGCAAGATATTGCAATAGGAGT CCACATGATTTAAAATTTCTGACTGATGAAGAGAGTTGGATTTTACTGGAGAAAAGAGCTTTTCACAAAGCTAAATGTCTCCCCGAATTGGAAACAAACGGAAAAAGCATAGCCAGG

[0222] SEQ ID NO: 47; Solanum lycopersicum, SlNRC1 amino acid sequence; NCBI reference sequence: NP_001234202. JPEG2025539497000007.jpg111162

[0223] SEQ ID NO: 48; Solanum lycopersicum, SlNRC2 cDNA; NCBI Reference Sequence: XM_004248750. ATGGCGAACGTAGCAGTGGAATTTCTGGTTGAGAACTTGATGCAGTTGCTGCGGGACAACGTAGAGCTAATTAGTGGAGTTAAAGAGGCTGCTGAGAGTCTACTTCAAGATCTAAATGATTTCAATGCTTTTCTTAAGCAAGCTGCCAAGTGTCACATCAACGAGAACGAAGTCCTCAGAGAACTTGTTAAGAAAATAAGAACAGTCGTTAACTCTGCTGAAGATGCT ATTGATAAATTTGTTATTGAAGCTAAGCTACACAAGGATAAGGGTGTCACCAGAGTATTAGATCTTCCCCATTATAAAAGAGTCAAAGAGGTAGCTGGTGAGATCAAAGCTATACGAAACAAAGTCAGAGAGATCCGACAGACTGATGCCATTGGACTTCAGGCCCTTCAAGATGATGATTTATCTGCTAGAGGTTCCGAAGAAAGAAAGCCTCCAGTGGTAGAGGAAG ATGACGTGGGTTGGATTTGACGAAGAAGCAGATATTGTAATCAACCGCCTTCTTGGAGAATCGAATCATCTAGAAGTTGTTCCAGTTGTTGGTATGCCTGGTCTCGGCAAAACGACCCTAGCAAATAAGATTTACAAGCATCCTAAAATCGGGTATGAGTTTTTTACTCGTATTTGGGTTTATGTGTCTCAATCATACAGGAGAAGAGAATTATTTCTCAACATCATCAGCAAATTCACTCGAAATACAAAACAATATCATGGGATGTGTGAGGAGGATTTGGCTGATGAAATACAAGAATTCTTGGGAAAGGGAGGAAAATACTTGGTTGTGTTGGATGACGTATGGTCGGATGAAGCTTGGGAACGTATCAAGATAGCATTCCCAAATAACAACAAACCGAATCGAGTATTGTTGACCACTCGAGATTCCAAAGTGGCTAAACAATGCAATCCCATA CCTCATGATCTAAAATTTCTGACTGAAGATGAAAGTTGGATATTACTGGAGAAGAAGGTTTTTCACAAGGACAAATGTCCTCCTGAATTGGTACTATCTGGAAAGAGCATAGCAAAA

[0224] SEQ ID NO: 49; Solanum lycopersicum, SlNRC2 amino acid sequence; NCBI Reference Sequence: XP_004248798. JPEG2025539497000008.jpg110162

[0225] SEQ ID NO: 50; Solanum lycopersicum, SlNRC3, cDNA; NCBI Reference Sequence: XM_004238900.4. ATGGCGGATGTAGCAGTAAAGTTCTTATTAGAAAATTTGACGCAGTTACTAATCGACAACGCCGATTTGATTCTCGGAATCCAAGGCGAAGTTGAAAATCTACTCACAGATCTCAATTACTTTAATGCTTTCCTCAAAGAAGCTGCTAAATCCCGCAGGGAAAATGAGGTTTTGAAAGAATTGGTGAAGAAAATCAGAAAAGTTGTGAACGATGCTGAAGATTCGATTGATAAATTTGTGGTTGAAGCTAAGAGACATGATGATAAAAACAAATTTGCTCAATGGTTTCATATTACTCATGTGGCTAGAGCCAAAGGGGTAGCGGATGAGATCAAAAGTATAAGGGAAAGAGTGAAGGAAATTAGAGATAATGACGCTTATGGCCTTCAAGCTATAACTTTGGATGATAATTTCAACAGAGGTGACGAAGAGAGGAAGGCCCCTGTAGTTGAGGAAG ATGATGTGGTTGGTTTTGATGATGAAGCAAAAACTGTAATTGATCGTCTCATCGGAGGATCAGACTATGTTGAGGTTGTGCCAGTTGTTGGTATGCCTGGTCTTGGTAAAACAACTTTGGCATATAAGATTTACAAGGATCCAAAGGTTGAGTATGAGTTCTTCACCCGCGTTTGGGTATATGTCTCTCAAACATTCAAGAGAAGGGAAATATTTCTCAACATTATCAGCAAGTTTACTCGAAACACCAAACAATATGATGATACACCAGAGGATGACTTAGCAAATGAAGTGAAGGAGCTTCTTGGAAAAGGTGGAAAATATCTTATTGTTTTGGATGATGTGTGGACGATGGAAGCTTGGGATCGTATCAAAATTGCTTTCCCTAATAATGGTAAACGAAATAGAGTGTTGATGACCACGAGACAATCAAATGTGGCGAAGCGTTGTAATGATAAA CCTCATGATCTTAAGTTTTTAACAAAAGATGAAAGTTGGGAGCTACTTGAGAAGAAGGTTTTTCACAAGGAAAAGTGTCCACCTGAGTTAGAATTACCCGGGATAAGTATAGCCGAA

[0226] SEQ ID NO: 51; Solanum lycopersicum, SlNRC3, amino acid sequence; NCBI reference sequence: XP_004238948. JPEG2025539497000009.jpg111162

[0227] SEQ ID NO: 52; Solanum lycopersicum NRC1 D316K , amino acids. MVDVGVEFLLENLKQLVLDNVELIGGAKDEIENLRDDLSEFNAFLKQAAMVRSENPVLKELVRSIRKVVNRAEDAVDKFVIEAKVHKDKGFKGVFDKPGHYRRVRDAAVEIKGIRDKMREIRQNKAHGLQALLQDHDDSISRGGEERQPPVVEEDDV VGFDDEAQTVIDRLLEGSGDLEVIPVVGMPGLGKTTLATKIFKHPKIEYEFFTRLWLYVSQSYKTRELYLNIISKFTGNTKHCRDMSEKDLALKVQEILEEGGKYLIVLDDVWSTDAWDRIKIAFPKNDKGNRVLLTTRDHRVARYCNRSPHDLKFLT KEESWILLEKRAFHKAKCLPELETNGKSIARKCKGLPLAIVVIAGALIGKSKTIKEWEQVDQSVGEHFINRDQPNSCDKLVRMSYDVLPYDWKACFLYFGTFPRGYLIPARKLIRLWIAEGFIQYRGDLSPECKAEEYLNELVN RNLVMVMQRTVDGQIKTCRVHDMLYEFCWQEATTEENLFHEVKFGGEQSVREVSTHRRLCIHSSVVEFISKPSGEHVRSFLCFSPEKIDTPPTVSANISKAFPLLRVFDTESIKINRFCKEFFQLYHLRYIAFSFDSIKVIP KHVGELWNVQTLIVNTQQINLDIQADILNMPRRLRHLLTNTSAKLPALANPKTSKTTLVNQSLQTLSTIAPESCTEYVLSRAPNLKKLGIRGKIAKLMEPSQSVLLNNVKRLQFLENLKLINVGQIDQTQLRLPPASIFPTKLR KLTLLDTWLEWDDMSVLKQLENLQVLKLKDNAFKGENWELNDGGFPFLQVLCIERANLVSWNASGDHFPRLKHLHISCDKLEKIPIGLADICSLQVMDLRNSTKSAAKSAREIQAKKKNKLQPAKSQKFELSVFPPDSDVQTAS

[0228] sequence number 53; Solanum lycopersicum NRC2 D315K , amino acid MANVAVEFLVENLMQLLRDNVELISGVKEAAESLLQDLNDFNAFLKQAAKCHINEVEVLRELVKKIRTVVNSAEDAIDKFVIEAKLHKDKGVTRVLDLPHYKRVKEVAGEIKAIRNKVREIRQTDAIGLQALQDDDLSARGSEERKPPVVEEDDVVGFDEAADIVINRLGESNHLEVVPVVGMPGLGKTTLANKIYKHPKIGYEFFTRIWVYVSQSYRRRELFLNIISKFTRNTKQYHGMCEEDLADEIQEFLGKGGKYLVVLDDVWSDEAWERIKIAFPNNNKPNRVLLTTRDSKVAKQCNPIPHDLKFLTE KESWILLEKKVFHKDKCPPELVLSGKSIAKKCKGLPLAIVVIAGALIGKGKTPREWKQVDDSVSEHLINRDHPENCNKLVQMSYDRLPYDLKACFLYCSAFPGGFQIPAWKLIRLWIAEGFIQYKGHLSLECKGEDNLNDLINRNLVMVMERTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGSEQYFPGKRELSTYRRLCIHSSVLDFFSTKPSAEHVRSFLSFSSSKKIEMPSADIPTIPKGFPLLRVLDVESINFSRFSREFYQLYHLRYVAFSSDSIK ILPKLMGELWNIQTIIINTQQRTLDIQANIWNMERLRHLHTNSSAKLPVPAPKNSKVTLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKISVLLDNKSAASLKNVKRLEYLENLKLINDSSIQTSKLRLPPAYIFP TKLRKLTLLDTWLEWKDMSILGQLEHLEVLKMKENGFSGESWESTGGFCSLLVLWIERTNLVSWKASADDFPRLKHLVLICCDNLKEVPIALADIRSFQVMMLQNSTKTAAISRQIQAKKDNQTQQGTKNIAFKLSIFPPDL

[0229] sequence number 54; Solanum lycopersicum NRC3 D315K , amino acid MADVAVKFLLENLTQLLIDNADLILGIQGEVENLLTDLNYFNAFLKEAAKSRRENEVLKELVKKIRKVVNDAEDSIDKFVVEAKRHDDKNKFAQWFHITHVARAKGVADEIKSIRERVKEIRDNDAYGLQAITLDDFNRGDEERKAPVVEEDDVVGFDDEAKTVIDRLIGGSDYVEVVPVVGMPGLGKTTLAYKIYKDPKVEYEFTRVWVYVSQTFKRREIFLNIISKFTRNTKQYDDTPEDDLANEVKELLGKGGKYLIVLDDVWTMEAWDRIKIAFPNNGKRNRVLMTTRQSNVAKRCNDKPHDLKFLTK DESWELLEKKVFHKEKCPPELELPGISIAEKCMGLPLAIVVIAGALIGKGKTTREWELVAASVGEHLINRDPENCKKLVQMSYDRLPYDLKACFLYCGAFPGGSQIPAKKLIRLWIAEGFIQYQGPLALEDVAEDHLNDLVNRNL VMVTQRSCSGQIKTCRVHDMLHEFCRHEAMMEENLFQEIKQGQERSFPGKQELATYRRLCIQSLIPEFLSMKPSGEHVRSFLCVGSKKIDMPPNEIPSIPKAFPLLRVLDAESIKFSRFSREFFKLFHLRYIALSTDKIKTIPA DFGNLWNIQTLIVETQQATLDIKADIWNMTRLRHVCTNASATLPSTKRPKSSKDNLVNRCLQTLSTIAPECCTAEVFTRTPNLKKLGVRGKIDALLESSKDGSGSGLFSNIGKLGCLEYLKLVNDTRLSSKPLHLPPAYIFPQK LKKLSLVDTWFEWKDMSILGLLPELEVLKLKENAFKGQSWEQEDGGFPRLQVLWIERTDLTSWKASSGNFPRLKHLALISCDKLEELPAELADVKNLQLIELQSSSESAARSARAILKRNQEKEQDGDKGTGFKLSIFPHDLGL

[0230] SEQ ID NO: 55; Solanum tuberosum NRC2 S317K , amino acids. MANVAVEFLVENLMQLLRDNAELISGVKEAAESLLQDLNDFNAFLKQAAKCHINENEVLRELVKKIRTVVNSAEDAIDKFVIEAKLHKDKGMTRVLDLPHYKRVREVAGEIKAIRNKVKEIRQNDAIGLQALQDDDSSARGFEERKPPVVEEDDVVGF DEEADIVIKRLLGESNRLEVVPVVGMPGLGKTTLANKIYKHPKIGYEFFTRIWVYVSQSYRRREFLNIISKFTRNTKQYHGMCEEDLADEIQEFLGKGGKYLVVLDDVWSDEAWERIKIAFPNNNKPNRVLLTTRDSKVAKQCTPIPHDLKFLSEDEK WILLEKKVFHKDKCPPELVVPSGKSIAKKCKGLPLAIVVIAGALIGKGKTPREWKQVDDSVSEHLINRDHPENCNKLVQMSYDRLPYDLKACFLYCSAFPGGFQIPAWKLIRLWIAEGFIQYKGHLSLECKGEDNLNDLINR NLVMVMERTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGSEQYFPGKRELSTYRRLCIHSSVLDFISTKPSAEHVRSFLSFSSKIEMPSADIPTIPKGFPLLRVLDVESINFSRFSKEFYQLYHLRYVAFSSDSIK ILPKLMGELWNIQTIIINTQQRTLDIQANIWNMERLRHLHTNSSAKLPVPVAKNSKVTLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKIAVLLDNKSAVSLNKNVKRLEYLENLKLINDSSIQTGKLRLPAYIFP TKLRKLTLLDTWLEWKDMSILGQLEHLEVLKMKENGFTGESWESTGGFCSLLVLWIERTNLVTWKASADDFPRLKHLVLICCDYLKEVPIALADIRSFQVMMLQNSTKTAAISRQIQAKKDNQTQQGTKNIAFKLSIFPPDL

[0231] sequence number 56; Solanum tuberosum NRC3 S317K , amino acid. MADVAVKFLVENLMQLLIDNADLILGIKGEVENLLRDLNDFNAFLKQAAKSRRENEVLKEMVKKIRKVVNDAEDSIDKFVIEAKRHDDKNKFAQWFHITHVARAKGVADEIKSIKERVKEIRENDAYGLQAITLDDNFNRGDEERKAPVVEEDDVVGF DDEAKIVIDRLIGGSDYVEVVPVGMPGLGKTTLAYKIYKDPKVEYEFFTRVWVYVSQTFKREIFLNIISKFTRNTKQYHDTPEDDLANEVKELLGKGKGYLIVLDVWTMEAWDRIKIAFPNNGKRNRVLMTTRESNVAKCCNDKPHDLKFLTEDEK WELLEKKVFHKEKCPPELELPGKSIAEKCMGLPLAIVVIAGALIGKGKTTREWELVAASVREHLINRDPENCKKLVQMSYDRLPYDLKACFLYCGAFPGGSQIPAKKLIRLWIAEGFIQYQGPLALEDVAEDHLNDLVNRNLV MVMQRSCSGQIKICRVHDMLHEFCRHEAMTEEDLFQEIKQGQERSFPGKQELATYRRLCIHSGVPEFLSTKPGSGEHVRSFLCVGSKKIDMPPNEIPSIPKAFPLLRVLDAESIKFNRFSREFFKLFHLRYIALSTDKIKTIPVD FGNLWNVQTLIVETQEATLDIKADIWNMTRLRHVCTNASATLPSTKRPKSSKGNLVNRCLQTLSTIAPECCTAEVFTRTPNLKLGVRGKIDALLETSKDGSGSVLFSNIGKLACLEYLKLVNDTRISSKPLHLPPAYIFPQKLKKLSLVDTWFEWKDMSILGLLPDLEVLKLKENAFKGQSWEQEDGGFPRLQVLWIERTDLTSWKASSGNFPRLKHLALISCDKLEELPAELADVKNLQLIELQSSSESAARSARAILKRNQEKEQEGDKGTGFKLSIFPHDLGL

[0232] sequence number 57; Solanum lycopersicum NRC1 E317K , amino acid. MVDVGVEFLLENLKQLVLDNVELIGGAKDEIENLRDDLSEFNAFLKQAAMVRSENPVLKELVRSIRKVVNRAEDAVDKFVIEAKVHKDKGFKGVFDKPGHYRVRDAAVEIKGIRDKMREIRQNKAHGLQALLQDHDDSISRGGEERQPPVVEEDDVV GFDDEAQTVIDRLLEGSGDLEVIPVVGMPGLGKTTLATKIFKHPKIEYEFFTRLWLYVSQSYKTRELYLNIISKFTGNTKHCRDMSEKDLALKVQEILEEGKGYLIVLDDVWSTDAWDRIKIAFPKNDKGNRVLLTTRDHRVARYCNRSPHDLKFLTD K ESWILLEKRAFHKAKCLPELETNGKSIARKCKGLPLAIVVIAGALIGKSKTIKEWEQVDQSVGEHFINRDQPNSCDKLVRMSYDVLPYDWKACFLYFGTFPRGYLIPARKLIRLWIAEGFIQYRGDLSPECKAEEYLNELVNRNLVMVMQRTVDGQIKTCRVHDMLYEFCWQEATTEENLFHEVKFGGEQSVREVSTHRRLCIHSSVVEFISKKPSGEHVRSFLCFSPEKIDTPPTTVSANISKAFPLLRVFDTESIKINRFCKEFFQLYHLRYIAFSFDSIKVIP KHVGELWNVQTLIVNTQQINLDIQADILNMPRRLRHLLTNTSAKLPALANPKTSKTTLVNQSLQTLSTIAPESCTEYVLSRAPNLKKLGIRGKIAKLMEPSQSVLLNNVKRLQFLENLKLINVGQIDQTQLRLPPASIFPTKLR KLTLLDTWLEWDDMSVLKQLENLQVLKLKDNAFKGENWELNDGGFPFLQVLCIERANLVSWNASGDHFPRLKHLHISCDKLEKIPIGLADICSLQVMDLRNSTKSAAKSAREIQAKKKNKLQPAKSQKFELSVFPPDSDVQTAS

[0233] sequence number 58; Solanum lycopersicum NRC2 S317K , amino acid MANVAVEFLVENLMQLLRDNVELISGVKEAAESLLQDLNDFNAFLKQAAKCHINEVEVLRELVKKIRTVVNSAEDAIDKFVIEAKLHKDKGVTRVLDLPHYKRVKEVAGEIKAIRNKVREIRQTDAIGLQALQDDDLSARGSEERKPPVVEEDDVVGFDEADIVINRLGESNHLEVVPVVGMPGLGKTTLANKIYKHPKIGYEFFTRIWVYVSQSYRRRELFLNIISKFTRNTKQYHGMCEEDLADEIQEFLGKGGKYLVVLDDVWSDEAWERIKIAFPNNNKPNRVLLTTRDSKVAKQCNPIPHDLKFLTEDE K WILLEKKVFHKDKCPPELVLSGKSIAKKCKGLPLAIVVIAGALIGKGKTPREWKQVDDSVSEHLINRDHPENCNKLVQMSYDRLPYDLKACFLYCSAFPGGFQIPAWKLIRLWIAEGFIQYKGHLSLECKGEDNLNDLINRN LVMVMERTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGSSEQYFPGKRELSTYRRLCIHSSVLDFFSTKPSAEHVRSFLSFSSKIEMPSADIPTIPKGFPLLRVLDVESINFSRFSREFYQLYHLRYVAFSSDSIKI LPKLMGELWNIQTIIINTQQRTLDIQANIWNMERLRHLHTNSSAKLPVPAPKNSKVTLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKISVLLDNKSAASLKNVKRLEYLENLKLINDSSIQTSKLRLPPAYIFPT KLRKLTLLDTWLEWKDMSILGQLEHLEVLKMKENGFSGESWESTGGFCSLLVLWIERTNLVSWKASADDFPRLKHLVLICCDNLKEVPIALADIRSFQVMMLQNSTKTAAISRQIQAKKDNQTQQGTKNIAFKLSIFPPDL

[0234] sequence number 59; Solanum lycopersicum NRC3 S317K , amino acid MADVAVKFLLENLTQLLIDNADLILGIQGEVENLLTDLNYFNAFLKEAAKSRRENEVLKELVKKIRKVVNDAEDSIDKFVVEAKRHDDKNKFAQWFHITHVARAKGVADEIKSIRERVKEIRDNDAYGLQAITLDDNFNRGDEERKAPVVEEDDVVGF DDEAKTVIDRLIGGSDYVEVVPVVGMPGLGKTTLAYKIYKDPKVEYEFFTRVWVYVSQTFKRREIFLNIISKFTRNTKQYDDTPEDDLANEVKELLGKGGKYLIVLDDVWTMEAWDRIKIAFPNNGKRNRVLMTTRQSNVAKRCNDKPHDLKFLTKDE K WELLEKKVFHKEKCPPELELPGISIAEKCMGLPLAIVVIAGALIGKGKTTREWELVAASVGEHLINRDPENCKKLVQMSYDRLPYDLKACFLYCGAFPGGSQIPAKKLIRLWIAEGFIQYQGPLALEDVAEDHLNDLVNRNLVMVTQRSCSGQIKTCRVHDMLHEFCRHEAMMEENLFQEIKQGQERSFPGKQELATYRRLCIQSLIPEFLSMKPSGEHVRSFLCVGSKKIDMPPNEIPSIPKAFPLLRVLDAESIKFSRFSREFFKLFHLRYIALSTDKIKTIPAD FGNLWNIQTLIVETQQATLDIKADIWNMTRLRHVCTNASATLPSTKRPKSSKDNLVNRCLQTLSTIAPECCTAEVFTRTPNLKKLGVRGKIDALLESKDGSGSGLFSNIGKLGCLEYLKLVNDTRLSSKPLHLPPAYIFPQK LKKLSLVDTWFEWKDMSILGLLPELEVLKLKENAFKGQSWEQEDGGFPRLQVLWIERTDLTSWKASSGNFPRLKHLALISCDKLEELPAELADVKNLQLIELQSSSESAARSARAILKRNQEKEQDGDKGTGFKLSIFPHDLGL

Claims

1. A genetically modified plant, plant part thereof, or plant cell, wherein the plant, plant part, or plant cell comprises at least one mutation in at least one nucleic acid sequence encoding a nucleotide-binding domain and a leucine-rich repeat-containing (NLR) protein, the NLR protein comprising an HD1 domain, and the at least one mutation is a mutation of one or more amino acids in the N-terminal portion of the HD1 domain.

2. The plant, plant part thereof, or plant cell of claim 1, wherein the at least one mutation reduces or prevents inhibition by pathogen effectors of the initiation of an immune response by an NLR protein.

3. 1. A genetically modified plant, plant part thereof, or plant cell, wherein the plant, plant part, or plant cell comprises at least one mutation in at least one nucleic acid sequence encoding a nucleotide-binding domain and a leucine-rich repeat-containing (NLR) protein, wherein the NLR protein initiates an immune response pathway, and wherein the at least one mutation reduces or prevents inhibition of the initiation of the immune response pathway by the NLR protein by pathogen effectors.

4. 4. The plant, plant part thereof, or plant cell of claim 3, wherein the NLR protein initiates an immune response by interacting with one or more downstream signaling partners, and the at least one mutation preserves the interaction.

5. A genetically modified plant, plant part thereof, or plant cell, which expresses a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, said NLR protein comprising an HD1 domain, and said NLR protein comprising one or more amino acid mutations in the N-terminal portion of said HD1 domain.

6. 10. A genetically modified plant, plant part thereof, or plant cell according to any one of the preceding claims, wherein the NLR protein is a nucleotide-binding domain and leucine-rich repeat-containing required for cell-death (NRC) protein.

7. 7. The genetically modified plant, plant part thereof, or plant cell of claim 6, wherein the at least one mutation reduces or prevents inhibition by pathogen effectors of oligomerization of the NRC protein into complexes to initiate an immune response.

8. 10. A genetically modified plant, plant part thereof, or plant cell according to any one of the preceding claims, wherein the at least one mutation reduces or prevents binding between the NLR protein and the pathogen effector.

9. A genetically modified plant, plant part thereof, or plant cell described in any one of the preceding claims, wherein the N-terminal portion of the HD1 domain comprises not more than the N-terminal 42 amino acids of the HD1 domain, more preferably not more than the N-terminal 38 amino acids of the HD1 domain.

10. A genetically modified plant, plant part thereof, or plant cell described in any one of the preceding claims, wherein the N-terminal portion of the HD1 domain comprises the sequence defined in SEQ ID NO: 2, 5, 8, 14, 17, 23, 26 or 29, or a variant or fragment thereof.

11. 11. The genetically modified plant, plant part thereof, or plant cell of claim 10, wherein the variant has at least 60% overall sequence identity to SEQ ID NO: 2, 5, 8, 14, 17, 23, 26, or 29.

12. 10. A genetically modified plant, plant part thereof, or plant cell according to any one of the preceding claims, wherein said at least one mutation is in the RNBS-C motif of said HD1 domain.

13. 13. The genetically modified plant, plant part thereof, or plant cell of claim 12, wherein the RNBS-C motif comprises the sequence defined in SEQ ID NO: 3, 6, 9, 15, 18, 24, 27, or 30, or a variant or fragment thereof.

14. 14. The genetically modified plant, plant part thereof, or plant cell of claim 13, wherein the mutant has at least 60% overall sequence identity to the sequence defined in SEQ ID NO: 3, 6, 9, 15, 18, 24, 27, or 30.

15. 10. A genetically modified plant, plant part thereof, or plant cell according to any one of the preceding claims, wherein the mutation is a substitution.

16. 16. The genetically modified plant, plant part thereof, or plant cell of claim 15, wherein the substitution is at one or more positions in the NLR amino acid sequence selected from 315, 316, or 317 (or homologous positions in homologous sequences) of any of SEQ ID NOs: 32, 34, 36, 41, 43, 47, 49, or 51, or a homolog or functional variant thereof.

17. 17. The genetically modified plant, plant part thereof, or plant cell of claim 16, wherein the substitution is a substitution with a hydrophilic amino acid and / or a positively charged amino acid.

18. 18. A genetically modified plant, plant part thereof or plant cell according to claim 16 or 17, wherein the substitution is at position D317 or at a homologous position in a homologous sequence, preferably the substitution is D317K.

19. 16. A genetically modified plant, plant part thereof, or plant cell according to any one of claims 1 to 15, wherein the mutation is a substitution that replaces all or part of the N-terminal portion of the HD1 domain with a corresponding portion in a second NLR protein, and the second NLR protein is not inhibited by the pathogen effector.

20. 20. The genetically modified plant, plant part thereof, or plant cell of claim 19, wherein the mutation is a substitution that replaces all or most of the RNBS-C motif with a corresponding RNBS-C motif in a second NLR protein, and the second NLR protein is not inhibited by the pathogen effector.

21. 6. The genetically modified plant, plant part thereof, or plant cell of claim 5, wherein the nucleic acid construct expresses a nucleic acid sequence encoding an NLR protein, the NLR protein being selected from SEQ ID NOs: 37, 38, 39, 44, 45, 52, 53, 54, 55, 56, 57, 58, or 59, and preferably the regulatory sequence is operably linked to a regulatory sequence.

22. 10. A genetically modified plant, plant part thereof or plant cell according to any one of the preceding claims, wherein the pathogen is potato cyst nematode.

23. 10. A genetically modified plant, plant part thereof, or plant cell according to any one of the preceding claims, wherein the pathogen effector is SPRYSEC15.

24. 10. A genetically modified plant, plant part thereof, or plant cell according to any one of the preceding claims, wherein the plant is a monocotyledonous or dicotyledonous plant.

25. 10. A genetically modified plant, plant part thereof or plant cell according to any one of the preceding claims, wherein the plant is a crop plant, preferably a Solanaceae plant.

26. 10. A genetically modified plant, plant part thereof, or plant cell according to any one of the preceding claims, wherein the NLR protein is NRC1 and / or NRC2 and / or NRC3.

27. 10. A genetically modified plant part according to any one of the preceding claims, wherein the plant part is a seed or a grain.

28. A method for conferring or improving pathogen resistance in a plant, the method comprising introducing at least one mutation into at least one nucleic acid sequence encoding a nucleotide-binding domain and leucine-rich repeat-containing (NLR) protein, the NLR protein comprising an HD1 domain, and the at least one mutation being a mutation of one or more amino acids in the N-terminal portion of the HD1 domain.

29. 1. A method for conferring or improving pathogen resistance in a plant, the method comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, the NLR protein comprising an HD1 domain, and the NLR protein comprising one or more amino acid mutations in the N-terminal portion of the HD1 domain.

30. A method for producing a plant with improved pathogen resistance, the method comprising introducing at least one mutation into at least one nucleic acid sequence encoding a nucleotide-binding domain and a leucine-rich repeat-containing (NLR) protein, the NLR protein comprising an HD1 domain, and the at least one mutation being a mutation of one or more amino acids in the N-terminal portion of the HD1 domain.

31. A method for producing a plant with improved pathogen resistance, the method comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, the NLR protein comprising an HD1 domain, and the NLR protein comprising one or more amino acid mutations in the N-terminal portion of the HD1 domain.

32. A method for producing a modified NLR protein in which a pathogen effector is unable to bind to the modified NLR protein or has a reduced ability to bind to the modified NLR protein, the method comprising introducing at least one mutation into at least one nucleic acid sequence encoding an NLR protein, the NLR protein comprising an HD1 domain, and the at least one mutation being a mutation of one or more amino acids in the N-terminal portion of the HD1 domain.

33. The method of any of claims 28 to 32, wherein the at least one mutation reduces or prevents inhibition by pathogen effectors of oligomerization of the NLR protein into complexes to initiate an immune response.

34. The method of any of claims 28 to 33, wherein the at least one mutation reduces or prevents binding between the NLR protein and the pathogen effector.

35. The method of any one of claims 28 to 34, wherein the N-terminal portion of the HD1 domain comprises the N-terminal 42 amino acids or less of the HD1 domain, more preferably the N-terminal 38 amino acids or less of the HD1 domain.

36. 36. The method of any of claims 28 to 35, wherein the N-terminal portion of the HD1 domain comprises the sequence defined in SEQ ID NO: 2, 5, 8, 14, 17, 23, 26 or 29 or a variant or fragment thereof.

37. 37. The method of claim 36, wherein the variant has at least 60% overall sequence identity to SEQ ID NO: 2, 5, 8, 14, 17, 23, 26 or 29.

38. 38. The method of any one of claims 28 to 37, wherein the at least one mutation is in the RNBS-C motif of the HD1 domain.

39. 39. The method of claim 38, wherein the RNBS-C motif comprises the sequence defined in SEQ ID NO: 3, 6, 9, 15, 18, 24, 27 or 30, or a variant or fragment thereof.

40. 40. The method of claim 39, wherein the variant has at least 60% overall sequence identity to the sequence defined in SEQ ID NO: 3, 6, 9, 15, 18, 24, 27 or 30.

41. The method of any one of claims 28 to 40, wherein the mutation is a substitution.

42. The method of any one of claims 28 to 41, wherein the substitution is a substitution with a hydrophilic amino acid and / or a positively charged amino acid.

43. The method of any of claims 28 to 42, wherein the substitution is at position D317 or at a homologous position in a homologous sequence, preferably the substitution is D317K.

44. The method of any of claims 28 to 40, wherein the mutation is a substitution that replaces all or most of the N-terminal portion of the HD1 domain with a corresponding portion of the HD1 domain in a second NLR protein, wherein the second NLR protein is not inhibited by the pathogen effector.

45. 45. The method of claim 44, wherein the mutation is a substitution that replaces all or most of the RNBS-C motif with a corresponding RNBS-C motif in a second NLR protein, and the second NLR protein is not inhibited by the pathogen effector.

46. 32. The method of claim 29 or 31, wherein the nucleic acid construct expresses a nucleic acid sequence encoding an NLR protein, wherein the NLR protein is selected from SEQ ID NO: 37, 38, 39, 44, 45, 52, 53, 54, 55, 56, 57, 58 or 59 or a functional variant or homologue thereof, and preferably the regulatory sequence is operably linked to a regulatory sequence.

47. The method of any one of claims 28 to 46, wherein the pathogen is a potato cyst nematode.

48. The method of any one of claims 28 to 47, wherein the pathogen effector is SPRYSEC15.

49. The method of any one of claims 28 to 48, wherein the plant is a monocotyledonous or dicotyledonous plant.

50. The method according to any one of claims 28 to 49, wherein the plant is a crop plant, preferably a Solanaceae plant.

51. The method of any of claims 28 to 50, wherein the NLR protein is NRC1 and / or NRC2 and / or NRC3.

52. A plant obtained or obtainable by the method according to any one of claims 28 to 51.

53. A method for screening a population of plants and identifying and / or selecting plants that exhibit pathogen resistance or improved pathogen resistance, the method comprising detecting at least one polymorphism in the NRC2 and / or NRC3 gene in the plant or plant germplasm, preferably wherein the polymorphism is in the N-terminal portion of the HD1 domain of the NRC2 and / or NRC3 gene.

54. 54. The method of claim 53, wherein the N-terminal portion of the HD1 domain comprises the N-terminal 42 amino acids or less of the HD1 domain, more preferably the N-terminal 38 amino acids or less of the HD1 domain.

55. An isolated nucleotide-binding domain and leucine-rich repeat-containing (NLR) protein, wherein the NLR protein comprises a sequence selected from SEQ ID NOs: 37, 38, 39, 44, 45, 52, 53, 54, 55, 56, 57, 58, and 59, or a functional variant or homolog thereof.

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