Method for regulating immune function of plants

Abstract

The present invention relates to genetically altered plants, parts thereof, and plant cells comprising one or more mutations in one or more NLR proteins, such as helper NLR proteins NRC2 and / or NRC3, and methods of providing or improving immunity of a plant to a pathogen or pest by introducing one or more mutations into one or more NLR genes.

Description

Technical Field

[0001] The present invention relates to genetically altered plants, parts thereof and plant cells comprising one or more mutations in one or more NLR proteins (e.g., the accessory NLR proteins NRC2 and / or NRC3), and methods for providing or improving plant immunity to pathogens or pests by introducing one or more mutations into one or more NLR genes. Background Art

[0002] Plant pests and diseases threaten agricultural productivity and food quality, posing a clear and imminent danger to our food system. Global crop yields can be lost up to 30% due to pathogens and pests, costing hundreds of billions of dollars in lost food production. This loss is enough to feed billions of people. By 2050, it is estimated that global food production will need to increase by approximately 70% to feed the projected population.

[0003] Outbreaks caused by plant diseases are increasing in frequency due to global trade, climate change, and the tendency of plant pathogens to overwhelm the disease resistance painstakingly bred into crop plants. Current crop disease management strategies rely on chemical control, but many commonly used fungicides / pesticides face increasing regulation due to environmental and health concerns. The most sustainable strategy for managing plant pathogens and pests is to breed crops with broad-spectrum disease resistance. However, current approaches to breeding for disease resistance can be slow and inefficient and do not fully leverage our fundamental understanding of plant immunity.

[0004] One sustainable approach to combating pathogens is through genetic improvement of crops. Although plants possess a genetic toolkit for combating disease, the ability of pathogens to adapt to and evade the plant immune system limits resistance breeding. Plants defend themselves against parasites through specialized disease-resistance proteins encoded as immune sensors that activate plant immunity upon sensing pathogens. However, some pathogens evade or suppress plant disease resistance, limiting the effectiveness of these immune sensors in agriculture. Engineering disease resistance is an alternative strategy but has historically been limited by our superficial understanding of the underlying mechanisms.

[0005] Therefore, there is a need to develop new genetic mechanisms to provide disease resistance in plants, especially in commercially important crops.

[0006] Nucleotide-binding and leucine-rich repeat (NLR)-type intracellular immune receptors are essential components of innate immunity in plants and animals. They mediate the intracellular recognition of pathogens and subsequently initiate a series of immune responses to combat infection. NLRs can be activated by virulence proteins (termed effectors) secreted by pathogens and delivered into host cells to modulate host physiology. A hallmark of NLR activation in plants and animals is their oligomerization into higher-order immune complexes, termed resistosomes or inflammasomes, respectively. These complexes initiate immune signaling through a variety of mechanisms, often leading to a form of programmed cell death, termed the hypersensitive response (HR) in plants or 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 found 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 inhibitors of NLR-mediated immunity. In some cases, adapted pathogens deploy effectors that directly or indirectly interfere with NLR signaling to suppress immune activation through a variety of strategies. The precise mechanisms by which pathogen effectors can impair NLR-mediated immunity to promote disease remain largely unknown. Furthermore, although several strategies for engineering novel effector recognition specificities in NLRs have been proposed in recent years, methods to mitigate the effects of effector-mediated NLR immunosuppression remain lacking. Summary of the Invention

[0007] "The technology described herein makes it possible to engineer disease-resistant crops with minimal genomic changes, thereby increasing food production while minimizing the use of chemicals. Until now, identifying methods to improve disease resistance using modern breeding techniques has been challenging, so the invention presented herein represents a beneficial tool to support farmers in their fight against plant diseases and aid their efforts to feed the world's population."

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

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

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

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

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

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

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

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

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

[0017] In one embodiment, the NLR protein initiates the immune response by interacting with one or more downstream signaling partners, and wherein the at least one mutation preserves the interaction.

[0018] In another aspect of the present invention, a genetically altered plant, plant part thereof, or plant cell is provided, wherein the plant, part thereof, or plant cell expresses a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, wherein the NLR protein comprises an HD1 domain, and wherein the NLR protein comprises a mutation in one or more amino acids in the N-terminal portion of the HD1 domain.

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

[0020] In one embodiment, the at least one mutation reduces or prevents inhibition of the oligomerization of the NRC protein into a complex that initiates an immune response by a pathogen effector.

[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 no more than the N-terminal 42 amino acids of the HD1 domain, more preferably no more than the N-terminal 38 amino acids of the HD1 domain.

[0023] In one embodiment, the N-terminal portion of the HD1 domain comprises a sequence as defined by 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 located in the RNBS-C motif of the HD1 domain. Preferably, the RNBS-C motif comprises a sequence as defined by 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 defined by SEQ ID NO: 3, 6, 9, 15, 18, 24, 27 or 30.

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

[0026] Preferably, the mutation is a substitution at one or more positions in the amino acid sequence, wherein the position is selected from position 315, 316 or 317 of any one of SEQ ID NO: 32, 34, 36, 41, 43, 47, 49 or 51 or a homologue or functional variant thereof. Alternatively, the substitution is a homologous position in a homologous sequence.

[0027] In one embodiment, the substitution is to 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 or a homologous position in a homologous sequence of SEQ ID NO: 32 or 34 or a homolog or functional variant thereof.

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

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

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

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

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

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

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

[0037] In one embodiment, the mutation replaces 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 wherein preferably the regulatory sequence is operably linked to the 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 monocot or a dicot. 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 a 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, a method for screening a plant population and identifying and / or selecting plants that exhibit pathogen resistance or improved pathogen resistance is provided, the method comprising detecting at least one polymorphism in the NRC2 and / or NRC3 gene in the plant or plant germplasm, wherein preferably the polymorphism is located in the N-terminal part of the HD1 domain of the NRC2 and / or NRC3 gene.

[0045] In one embodiment, the N-terminal portion of the HD1 domain comprises no more than the N-terminal 42 amino acids of the HD1 domain, more preferably no more than the N-terminal 38 amino acids of the HD1 domain.

[0046] In another aspect of the present invention, an isolated protein containing a nucleotide binding domain and a leucine-rich repeat sequence (NLR) is provided, wherein the NLR protein comprises a sequence selected from SEQ ID NO: 37, 38, 39, 44, 45, 52, 53, 54, 55, 56, 57, 58, 59 or a functional variant or homolog thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The invention is further described in the following non-limiting drawings:

[0049] Figure 1 :SS15 directly inhibits NRC2 formation.

[0050] (A) Schematic diagram of the NRC immune receptor network, comprising multiple sensor NLRs (Prf, Gpa2, Rx, R1, and Rpi-blb2) and their downstream auxiliary NLRs, NRC2 and NRC4. Effector-triggered activation of one or more of these sensors leads to oligomerization of downstream auxiliary proteins and resistance body formation. The potato nematode (Globodera rostochiensis) effector SS15 can directly inhibit NRC2 by directly binding to the NB-ARC domain. (B) BN-PAGE assay using inactive and activated Rx together with NRC2 or NRC4 in the absence or presence of SS15. C-terminally V5-tagged Rx and C-terminally 4xMyc-tagged NRC2 EEE or NRC4 AAA Coexpressed with free GFP or C-terminally GFP-tagged PVX CP (potato virus X coat protein). These effector-sensor-accessory combinations were co-infiltrated with either a 6xHA-mCherry fusion protein or an N-terminally 6xHA-tagged SS15. Total protein extracts were analyzed in parallel by native and denaturing PAGE and immunoblotted with the corresponding antisera indicated below. Approximate molecular weights (kDa) of the proteins are indicated on the left. Experiments were repeated three times with similar results.

[0051] Figure 2 : The HD1-1 region of the NB-ARC domain determines the association and inhibition of SS15 on NRC.

[0052] (A) Schematic diagram of the NRC domain structure, highlighting the internalization of the NB-ARC domain in the NRC2-NRC4 chimeric protein (i.e., NRC42HD1-1 and NRC4 2HD1-2 ). The association (+) or non-association (-) between these NLR immune receptors and SS15, as determined by in planta co-immunoprecipitation, is detailed on the right. (B) A zoomed-in view of the amino acid sequence alignment between AtZAR1, NRC2, and NRC4 (N. benthamiana) focused on the HD1 region of the NB-ARC domain. The predicted secondary structure is shown above the alignment. Well-characterized motifs in this region, such as RNBS-C and GLPL, are underlined below the alignment. (C) Co-immunoprecipitation (Co-IP) assay between SS15 and chimeric NRC2-NRC4 variants. C-terminally 4xMyc-tagged NRC proteins were transiently co-expressed with N-terminally 4xHA-tagged SS15. IP (MycIP) was performed using agarose beads conjugated to a Myc antibody. Total protein extracts were immunoblotted with the corresponding antisera indicated on the left. The approximate molecular weight (kDa) of the proteins is shown on the right. Ponceau red dye (PS) was used as a Rubisco loading control. The experiment was repeated three times with similar results. (D) Photographs of representative leaves of N. benthamiana nrc2 / 3 / 4 knockout (KO) plants showing the interaction of Rx and PVX CP with NRC2, NRC4, and two NRC2-NRC4 chimeras (NRC4 2HD1-1 and NRC4 2HD1-2 ) HR after co-expression. These effector-sensor-accessory protein combinations were co-expressed with free mCherry-6xHA fusion protein (EV) or with N-terminally 4xHA-tagged SS15. (E) BN-PAGE assay using inactive and activated Rx together with NRC4 or NRC2-NRC4 chimeric proteins in the absence or presence of SS15. C-terminally V5-tagged Rx and C-terminally 4xMyc-tagged NRC4 AAA or NRC4 AAA-2HD1-1 Coexpressed with free GFP or C-terminally GFP-tagged PVX CP. These effector-sensor-accessory combinations were co-infiltrated with mCherry-6xHA fusion protein or N-terminally 4xHA-tagged SS15. Total protein extracts were run in parallel on native and denaturing PAGE and immunoblotted with the corresponding antisera indicated on the left. Approximate molecular weights (kDa) of the proteins are indicated on the left. Experiments were repeated three times with similar results.

[0053] Figure 3 : Identification of the SS15-NRC binding interface enables engineering of NRC2 to escape pathogen inhibition.

[0054] (A)SS15-NRC1 NB-ARCStructure of the complex. (B) Alignment of the HD1-1 regions of AtZAR1, NRC1 (tomato), NRC2, NRC3, and NRC4 (Nicotiana benthamiana). Candidate residues were screened based on the interface identified in the co-crystal structure of SS15 with the NRC1 NB-ARC domain, as well as features that are conserved in NRC1, NRC2, and NRC3 but not in NRC4 and AtZAR1. Thirteen NRC2 variants were generated by mutating individual candidate positions to the corresponding amino acids in NRC4 (detailed below the alignment). (C) Photographs of representative leaves from N. benthamiana nrc2 / 3 / 4 knockout plants showing HR following co-expression of Rx and PVX CPs with NRC2 or the different NRC2 variants generated. These effector-sensor-accessory protein combinations were co-expressed with free mCherry-6xHA fusion protein (EV) or with N-terminally 4xHA-tagged SS15. (D) Co-immunoprecipitation (Co-IP) assay between SS15 and NRC2 variants. C-terminally 4xMyc-tagged NRC2 variants were transiently co-expressed with N-terminally 4xHA-tagged SS15. IP was performed using agarose beads conjugated to a Myc antibody (Myc IP). Total protein extracts were immunoblotted with the corresponding antisera indicated on the left. The approximate molecular weight (kDa) of the proteins is shown on the right. Ponceau red dye (PS) was used as a Rubisco loading control. The experiment was repeated three times with similar results.

[0055] Figure 4 :SS15 and NRC1 NB-ARC Crystal structure of the complex.

[0056] (A) Electron density map showing SS15 and NRC1 in the asymmetric unit of tomato NB-ARC Relative orientation and arrangement of SS15 and NRC1. 2Fo-Fc map with 1σ contour. (B) SS15 and NRC1 NB-ARC Two possible interfaces between SS15 and full-length NRC1 were revealed by crystal packing. The two interfaces (Interface 1 and Interface 2) are labeled (left). Modeling of the two potential binding interfaces of SS15 in complex with full-length NRC1 shows steric clashes between the CC domain of NRC1 and SS15, making Interface 2 unlikely to be biologically relevant in the full-length context (right). (C) SS15-NRC1 NB-ARC A zoomed-in view of the interaction interface relative to the ATP binding site within the NB-ARC domain of NRC1. The phosphate moiety of ATP is oriented in the opposite direction to the SS15 binding interface (shown as a ball-and-stick model), indicating that SS15 is unlikely to displace bound ATP or prevent ATP hydrolysis. (D) SS15-NRC1 NB - ARCThe structure of AtZAR1 is superimposed on the NB-ARC domain in the inactive, intermediate, and active conformations of AtZAR1. Visualizing these three states reveals the trajectory of the NB domain relative to the HD1 and WHD domains as it transitions from the inactive state to the activated state. Without wishing to be bound by theory, SS15 binding at the critical hinge region between the NB and HD1-WHD domains may immobilize this loop, preventing these critical intramolecular rearrangements and thus NLR activation.

[0057] Figure 5 :Engineering NRC2 D317K Accessory proteins support immune signaling by multiple sensor NLRs in the presence of SS15.

[0058] (A) Photographs of representative leaves from N. benthamiana nrc2 / 3 / 4 knockout plants showing the interaction of different NRC2-dependent sensors NLR with NRC2 and NRC2 in the absence or presence of SS15. E316P or NRC2 D317K HR after co-expression. (B) Inactive and activated Rx were co-expressed with NRC2 or NRC2 in the absence or presence of SS15. D317K BN-PAGE assay was performed together. C-terminal V5-tagged Rx and C-terminal 4xMyc-tagged NRC2 EEE or NRC2 EEE-D317K Coexpressed with free GFP or C-terminally GFP-tagged PVX CP. These effector-sensor-accessory combinations were co-infiltrated with either a 6xHA-mCherry fusion protein or an N-terminally 4xHA-tagged SS15. Total protein extracts were analyzed by native and denaturing PAGE in parallel and immunoblotted with the corresponding antisera indicated below. Approximate molecular weights (kDa) of the proteins are indicated on the left. Experiments were repeated three times with similar results. Detailed Description of the Invention

[0060] The present invention will now be further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Unless otherwise clearly stated to the contrary, each aspect so defined may be combined with any other one or more aspects. In particular, any feature designated as preferred or advantageous may be combined with any other one or more features designated as preferred or advantageous.

[0061] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, which are well known to those skilled in the art. Such techniques are fully explained in the literature.

[0062] As used herein, the words "nucleic acid," "nucleic acid sequence," "nucleotide," "nucleic acid molecule," or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogs of DNA or RNA generated using nucleotide analogs. It can 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 mRNA or protein products. These terms also encompass genes. The terms "gene" or "gene sequence" are broadly used to refer to DNA nucleic acids associated with biological functions. Thus, a gene can include introns and exons in a genomic sequence, or can include only coding sequences as in a cDNA, and / or can include a combination of cDNA and regulatory sequences.

[0063] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length linked together by peptide bonds.

[0064] For the purposes of the present invention, "genetically altered plants" are plants that have been genetically altered compared to naturally occurring wild-type (WT) plants. In one embodiment, genetically altered plants are plants that have been altered using a mutagenesis method (e.g., targeted genome modification or genome editing) compared to naturally occurring wild-type (WT) plants. In one embodiment, the plant genome has been altered using a mutagenesis method compared to the wild type. Such plants have altered phenotypes as described herein, such as enhanced immunity to pathogens. Thus, in this example, these phenotypes are conferred by the presence of an altered plant genome (e.g., a mutation in at least one gene encoding an NLR gene). In particular, aspects of the present invention relate to recombinant DNA technology and exclude embodiments based solely on the production of plants by traditional breeding methods.

[0065] According to all aspects of the present invention, including the methods below and including the plants, methods and uses described below, the term "regulatory sequence" is used interchangeably herein with "promoter", and all terms should be understood in the broad context to refer to regulatory nucleic acid sequences that are capable of affecting the expression of the sequences to which they are linked. The term "regulatory sequence" also encompasses synthetic fusion molecules or derivatives that confer, activate or enhance expression of a nucleic acid molecule in a cell, tissue or organ.

[0066] In one embodiment, the promoter may be a constitutive promoter or a strong promoter. Alternatively, the promoter may be a tissue-specific promoter.

[0067] A "constitutive promoter" refers to a promoter that is transcriptionally active in at least one cell, tissue, or organ at most (but not necessarily all) stages of growth and development and under most environmental conditions. Examples of constitutive promoters include the cauliflower mosaic virus promoter (CaMV35S or 19S), the rice actin promoter, the maize ubiquitin promoter, the rubisco small subunit, the maize or alfalfa H3 histone, OCS, SAD1 or 2, GOS2, or any promoter that provides enhanced expression.

[0068] A "strong promoter" refers to a promoter that results in increased or overexpression of a gene. Examples of strong promoters include, but are not limited to, CaMV-35S, CaMV-35Somega, Arabidopsis thaliana ubiquitin UBQ1, rice ubiquitin, actin, or maize alcohol dehydrogenase 1 promoter (Adh-1).

[0069] As used herein, the term "operably linked" refers to a functional connection between a promoter sequence and a gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.

[0070] In one embodiment, the progeny plant is stably transformed with the nucleic acid construct described herein and comprises an exogenous polynucleotide that is heritably maintained in the plant cell. The method may include the step of verifying that the construct is stably integrated. The method may also include the additional step of collecting seeds from the selected progeny plant.

[0071] In one aspect of the present disclosure, a genetically altered plant, plant part or plant cell thereof is provided, wherein the plant, plant part or plant cell comprises at least one mutation in at least one nucleic acid sequence encoding a protein comprising a nucleotide binding domain and a leucine-rich repeat (NLR).

[0072] In an alternative aspect of the present invention, a genetically altered plant, plant part thereof, or plant cell is provided, wherein the plant, part thereof, or plant cell expresses a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, wherein the NLR protein comprises an HD1 domain, and wherein the NLR protein comprises a mutation in one or more amino acids in the N-terminal portion of the HD1 domain, as described herein.

[0073] 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 the regulatory sequence.

[0074] NLRs belong to the signaling ATPase (STAND) superfamily with multiple domains. They typically exhibit a tripartite domain structure consisting of an N-terminal signaling domain, a central nucleotide binding domain, and a C-terminal superstructure-forming repeat. The central domain, called NB-ARC (a nucleotide-binding adaptor shared by APAF-1, plant R proteins, and CED-4) or NACHT (shared by NAIP2, C2TA, HET-E, and TP1) in plant and animal NLRs, is characteristic of this protein family and plays a key role as a molecular switch, mediating the conformational changes required for activation. It consists of a nucleotide-binding domain (NB) in NB-ARC, a helical domain (HD1), and a winged helix domain (WHD), with the NACHT domain exhibiting a second helical domain (HD2). A variety of NLR activation and signaling strategies have been found in nature. In some cases, a singleton NLR protein can mediate both elicitor perception and subsequent immune signaling (19). However, some NLRs can function as receptor pairs or in higher-order configurations 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 the case with the Solanaceae NRC immune receptor network, which is composed of multiple sensor NLRs that require a series of downstream helper NLRs called NRCs (NLRs required for cell death) to successfully initiate immune signaling. The NRC network can encompass up to half of the NLRome in some Solanaceae species and plays a key role in mediating immunity against a variety of plant pathogens, including oomycetes, bacteria, viruses, nematodes, and insects.

[0075] As a result of a coevolutionary arms race with their hosts, a variety of plant and metazoan pathogens have evolved effectors that can interfere with host NLR signaling to promote disease through different strategies. In some cases, effectors can indirectly inhibit NLR-mediated immunity by interfering with host proteins that regulate NLR signaling or act downstream of NLR signaling (15, 17, 21, 22). In other cases, some effectors have evolved to interact directly with NLRs to inhibit their function (15, 16, 23). One example is the potato cyst nematode effector SS15, which can inhibit signaling mediated by auxiliary NLRs, namely NRC1, NRC2, and NRC3, by directly binding to the central NB-ARC domain of auxiliary NLRs (15)( Figure 1 A).

[0076] Thus, the genetically altered plant, plant part or plant cell thereof may comprise at least one mutation in at least one nucleic acid sequence encoding a sensor NLR, a singleton NLR or an auxiliary NLR, such as an NRC protein (e.g., NRC2 or NRC3). Singleton NLRs and auxiliary NLRs may be collectively referred to as performer NLRs.

[0077] By "at least one mutation in at least one nucleic acid sequence encoding an NLR" is meant that when an NLR gene is present in more than one copy or homologous genes (having the same or slightly different sequences), at least one mutation is present in at least one (endogenous) gene. In one embodiment, all genes are mutated. Additionally or alternatively, by "at least one mutation in at least one nucleic acid sequence encoding an NLR" is meant that the nucleic acid sequence of one or more NLR proteins is mutated. For example, only NRC2 (and at least one or all homologous genes) is mutated. Alternatively, only NRC3 (and at least one or all homologous genes) is mutated. Or both NRC2 and NRC3 are mutated (and at least one or all homologous genes thereof). Preferably, the NLR is not NRC4.

[0078] An "endogenous" nucleic acid or gene can refer to a native or natural sequence in the genome of a plant.

[0079] In one embodiment, the NLR may be at least one of NRCl, NRC2, or NRC3.

[0080] The nucleic acid sequence of NRC1 may encode the NRC1 protein as defined 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.

[0081] The nucleic acid sequence of NRC2 may encode an NRC2 protein as defined in SEQ ID NO: 32, 34, 41 or 49, or a functional variant or homolog 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 homolog thereof.

[0082] The nucleic acid sequence of NRC3 may encode an NRC3 protein as defined by 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.

[0083] 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. Such terms are used interchangeably herein.

[0084] The HD1 domain is a conserved domain that contains one or more conserved motifs. Preferably, the HD1 domain contains an RNBS-C and / or GLPL motif. However, in particular, the HD1 domain can be considered to be a domain having 50 to 100 amino acids, more preferably about 60 to 90 amino acids, and especially about 80 amino acids, and containing an RNBS-C motif.

[0085] The RNBS (resistance nucleotide binding site)-C motif may comprise the following consensus sequence: LxxxExWxLF), wherein the leucines at positions 0 and 8 are highly conserved. In further embodiments, the RNBS-C motif may comprise an amino acid sequence as defined by SEQ ID NO: 3, 6, 9, 15, 18, 24, 27 or 30, or a functional fragment or variant thereof.

[0086] As about Figure 2 As described, we found that only the N-terminal half of the HD1 domain is responsible for binding pathogen effectors and, furthermore, mutations in this region specifically result in NLR proteins that can escape pathogen effector inhibition while retaining full functionality (e.g., can oligomerize and mediate cell death when activated by effector or sensor NLRs).

[0087] Thus, 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 comprising one or more mutations may comprise no more than the N-terminal 42 amino acids of the HD1 domain (e.g., amino acids 1 to 42). Preferably, the N-terminal portion of the HD1 domain may comprise no more than the N-terminal 38 amino acids of the HD1 domain (e.g., amino acids 1 to 38). Preferably, the N-terminal portion of the HD1 domain comprises or consists of an RNBS-C motif as described above. The N-terminal portion of HD1 may also be referred to herein as HD1-1.

[0088] The HD1-1 region may comprise or consist of a nucleic acid sequence encoding an amino acid sequence as defined 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 RNBS-C motif as described above. Preferably, the at least one mutation is located in the RNBS-C motif.

[0089] As used herein in relation to any sequence defined herein, the term "variant" or "functional variant" refers to a variant gene sequence or portion of said gene sequence that retains the biological function of the intact non-variant sequence. Thus, in the context of an NLR protein, a functional variant may be a variant that is capable of mediating an immune response, such as a hypersensitivity reaction (HR). For an NRC protein, a functional variant may be capable of oligomerization to initiate an immune response. In the context of HD1 or HD1-1, a functional variant may be a variant that promotes oligomerization of the NRC protein to initiate an immune response.

[0090] Functional variants also include variants of the gene of interest, which have sequence changes that do not affect function, such as in non-conserved residues. Variants that are substantially identical to the wild-type sequence shown herein are also encompassed, i.e., only some sequence variations (e.g., in non-conserved residues), and have biological activity (e.g., ability to oligomerize and cause cell death). It is well known in the art that changes in the nucleic acid sequence that result in different amino acids being produced at a given site that does not affect the functional properties of the encoded polypeptide. For example, the codon for the amino acid alanine (hydrophobic amino acid) can be replaced by a codon encoding another residue with lower hydrophobicity (e.g., glycine) or a residue with higher hydrophobicity (e.g., valine, leucine, or isoleucine). Similarly, it is also expected that a negatively charged residue will be replaced by another negatively charged residue (e.g., aspartic acid is replaced by glutamic acid), or a positively charged residue will be replaced by another positively charged residue (e.g., lysine is replaced by arginine), resulting in functionally equivalent products. It is expected that changes in the nucleotides that result in changes in the N-terminal and C-terminal portions of the polypeptide molecule will not change the activity of the polypeptide. Each proposed modification is well within the routine skill of the art, as is determining that the biological activity of the encoded product is retained.

[0091] As used in any aspect of the invention described herein, a "variant" or "functional variant" has 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity.

[0092] As used herein, the term "homologue" also refers to NLR gene orthologues from other plant species. Suitable homologues can be identified by sequence comparison and identification of conserved domains as described above. Predictors are available in the art for identifying such sequences. The function of a homologue can be identified as described herein, and those skilled in the art are therefore able to confirm the function, for example, when overexpressed in a plant.

[0093] Homologs may also have (in increasing order of priority) 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 to an amino acid sequence cited herein or a nucleic acid sequence cited herein. Functional variants of the NLR gene homologues as defined above are also within the scope of the present invention.

[0094] If the nucleotides in two nucleic acid sequences or polypeptides or the respective sequences of amino acid residues are identical when the maximum correspondence as described below is compared, then the two nucleic acid sequences or polypeptides are said to be "identical". The percentage of the term "identical" or "identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned to achieve maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual comparison and visual inspection. When the percentage of sequence identity is used to refer to a protein or peptide, it should be appreciated that non-identical residue positions are usually different because of conservative amino acid substitutions, in which amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), and therefore do not change the functional properties of the molecule. When sequences are different in conservative substitutions, the sequence identity percentage can be adjusted upwards to correct the conservative nature of the substitution. The method for carrying out this adjustment is well known to those skilled in the art. For sequence comparison, usually a sequence is used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters. Non-limiting examples of algorithms suitable for determining sequence identity percentages and sequence similarity are BLAST and BLAST 2.0 algorithms. The overall sequence identity of a variant can be determined using any number of sequence alignment programs known in the art. For example, Emboss Stretcher from EMBL-EBI can be used: https: / / www.ebi.ac.uk / Tools / psa / emboss_stretcher / (using default parameters: for proteins, paired output format, matrix = BLOSUM62, open gap = 1, extended gap = 1; for nucleotides, paired output format, matrix = DNAfull, open gap = 16, extended gap = 4).

[0095] It will be appreciated by those skilled in the art that the homologous positions in suitable homologues and these sequences can be identified by sequence comparison (e.g., BLAST, comparison) and identification of conserved domains. Phylogenetic tree analysis using nucleotide or amino acid sequences can be used to establish the orthologues of NLR genes. Predictors for identifying such sequences exist in this area. The functions of homologues can be identified as described herein, and those skilled in the art can therefore confirm the functions, for example, by assaying programmed cell death. Once homologue sequences are identified, homologue positions or, as used herein, "corresponding positions in homologue sequences" can therefore be determined by performing sequence alignments. For example, potato (S. tuberosum) or Nicotiana benthamiana (N. benthamiana) NRC2 or NRC3 sequences can be used as query sequences (i.e., one of the sequences defined by SEQ ID NO:31 to 36 or 40 to 43) to identify homologues in the target plant genome.

[0096] In some embodiments, the present invention and nucleotide sequence as described herein can be used to separate corresponding sequences from other organisms (particularly other plants, for example crop plants). In this way, methods such as PCR, hybridization can be used to identify this type of sequence based on the sequence homology with sequence described herein. When identifying and separating homologues, the topological structure and characteristic domain structure (for example, there is RNBS-C motif) of the sequence can also be considered. Sequence can be separated based on itself and the sequence identity of the whole sequence or its segment. In hybridization techniques, all or part of the known nucleotide sequence is used as a probe, and this probe selectively hybridizes with other corresponding nucleotide sequences existing in the cloned genomic DNA fragment or cDNA fragment (i.e. genome or cDNA library) colony from selected plants. The hybridization probe can be a genomic DNA fragment, cDNA fragment, RNA fragment or other oligonucleotide, and can be used with a detectable group or any other detectable marker mark. Methods for preparing probes for hybridization and constructing cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989), Molecular Cloning: A Library Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0097] Hybridization of such sequences can be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is meant conditions in which the degree to which a probe hybridizes to its target sequence is detectably higher than the degree to which it hybridizes to other sequences (e.g., at least 2 times relative to background). Stringent conditions are sequence-dependent and can vary under different circumstances. By controlling the stringency of hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probe hybridization). Alternatively, stringent conditions can be adjusted to allow for some mismatches in the sequence, thereby detecting a lower degree of similarity (heterologous probe hybridization). Typically, the probe length is less than about 1000 nucleotides, preferably less than 500 nucleotides.

[0098] Typically, stringent conditions are those in which the salt concentration is less than about 1.5 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is typically less than 24 hours, typically about 4 to 12 hours. Stringent conditions can also be achieved by adding destabilizing agents such as formamide.

[0099] In a further embodiment, a variant as used herein may comprise a nucleic acid sequence encoding an NLR polypeptide as defined herein, which is capable of hybridizing to a nucleic acid sequence as defined herein under stringent conditions as defined herein.

[0100] In one embodiment, a genetically altered plant, plant part or plant cell thereof is provided, wherein the plant, plant part or plant cell comprises at least one mutation in at least one nucleic acid sequence encoding at least one NLR protein (preferably NRC1, NRC2 and / or NRC3),

[0101] The NRC1 gene comprises or consists of the following nucleic acid sequence:

[0102] a. a nucleic acid sequence encoding a polypeptide as defined in one of SEQ ID NO: 47; or

[0103] b. a nucleic acid sequence as defined in one of SEQ ID NO: 46; or

[0104] 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 (a) or (b); or

[0105] d. a nucleic acid sequence encoding an NRC2 polypeptide as defined herein, which is capable of hybridizing under stringent conditions as defined herein with the nucleic acid sequence of any one of (a) to (c); and

[0106] The NRC2 gene comprises or consists of the following nucleic acid sequence:

[0107] e. a nucleic acid sequence encoding a polypeptide as defined in one of SEQ ID NO: 32, 34, 41 or 49; or

[0108] f. a nucleic acid sequence as defined in one of SEQ ID NO: 31, 33, 40 or 48; or

[0109] 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 (e) or (f); or

[0110] h. a nucleic acid sequence encoding an NRC2 polypeptide as defined herein, which is capable of hybridizing under stringent conditions as defined herein with the nucleic acid sequence of any one of (e) to (g); and

[0111] The NRC3 gene comprises or consists of the following nucleic acid sequence:

[0112] i. a nucleic acid sequence encoding a polypeptide as defined in one of SEQ ID NO: 36, 43 or 51; or

[0113] j. a nucleic acid sequence as defined in one of SEQ ID NO: 35, 42 or 50; or

[0114] 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 (a) or (b); or

[0115] 1. A nucleic acid sequence encoding an NRC3 polypeptide as defined herein, which is capable of hybridizing under stringent conditions as defined herein to the nucleic acid sequence of any one of (a) to (c).

[0116] Preferably, the at least one mutation introduced into at least one NLR protein and in particular into the HD1-1 region of an NLR protein results in a corresponding amino acid sequence change or mutation of the NLR protein. Figure 5 As shown, when the NLR protein is an NRC protein, the at least one mutation preferably reduces or prevents the inhibition of pathogen effectors on the oligomerization of the NRC protein into a complex for initiating an immune response. In other words, the pathogen effectors are no longer able to inhibit the function (immune activation) of the NRC protein. The oligomerization of the NRC protein can be measured, for example, by BN-PAGE.

[0117] More preferably, the at least one mutation reduces or prevents the binding between the NLR protein and the pathogen effector. The binding between the NLR protein and the pathogen effector can be determined by any conventional method in the art, such as the immunoprecipitation described in the Examples.

[0118] Importantly, the mutation does not affect the function of the NLR protein. In one example, the at least one mutation reduces or prevents the inhibition of the pathogen effector on the NLR protein to initiate the immune response pathway. In the case of the NRC protein, the NRC protein is (still) able to oligomerize and / or mediate an immune response (e.g., a cell death response). In one example, the mutation retains (or does not change) the downstream interactions / signaling pathways of the natural NLR protein. For example, when the natural NLR protein initiates an immune response by one or more interactions with one or more downstream molecules, the mutant NLR protein initiates an immune response by the same interaction.

[0119] like Figure 3 and Figure 5 As shown, we also demonstrated that mutations in particular in the HD1-1 region maintained upstream signaling with the sensor NLR, escaped inhibition by pathogen effectors, and impaired binding of pathogen effectors to NLRs (i.e., reduced or no binding to NLRs).

[0120] The at least one mutation introduced into the at least one nucleic acid sequence encoding at least one NRC protein may be selected from the following types of mutations:

[0121] 1. A "missense mutation," which is a change in a nucleic acid sequence that results in the substitution of one amino acid for another;

[0122] 2. "Nonsense mutation" or "stop codon mutation", which is a change in the nucleic acid sequence that results in the introduction of a premature stop codon and thus terminates translation (producing a truncated protein); in plants, the translation stop codon can be selected from "TGA" (UGA in RNA), "TAA" (UAA in RNA) and "TAG" (UAG in RNA); therefore, any nucleotide substitution, insertion, deletion that results in one of these codons appearing in the mature mRNA being translated (in reading frame) will terminate translation;

[0123] 3. "Insertion mutations" of one or more nucleotides or one or more amino acids, which are caused by the addition of one or more codons in the coding sequence of a nucleic acid;

[0124] 4. "Deletion mutation" of one or more nucleotides or one or more amino acids, which is caused by deleting one or more codons in the coding sequence of the nucleic acid;

[0125] 5. "Frameshift mutations," which result in the nucleic acid sequence being translated in a different reading frame downstream of the mutation. Frameshift mutations can have a variety of causes, such as insertions, deletions, or duplications of one or more nucleotides;

[0126] 6. "Splice site" mutations, which are mutations that result in nucleotide insertions, deletions, or substitutions at splice sites (i.e., splice acceptor or splice donor mutations);

[0127] Wherein preferably, any one or more of the above-mentioned mutations result in a reduction or elimination of the binding of the NLR protein to the pathogen effector. As described above, by "binding" is meant a direct or indirect association with each other. In other words, the NLR and pathogen effector are immunoprecipitated when expressed in plants. Compared with the amount of binding between the wild-type NLR and the pathogen effector (e.g., which can be measured by co-ip), 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%, at least 95%. Alternatively, the interaction between the NLR and the pathogen effector cannot be detected--in other words, binding is eliminated.

[0128] Preferably, the one or more mutations are substitution mutations. For example, the mutations may be any substitution in HD1-1, or more preferably any substitution in the RNBS-C motif of HD1-1.

[0129] like Figure 3 As shown, the substitution is at one or more positions in the amino acid sequence, wherein the position is selected from position 315, 316 or 317 of any one of SEQ ID NO: 32, 34, 36, 41, 47, 49 or 51 or a homologue or functional variant thereof. Alternatively, the substitution is at a homologous position in a homologous sequence.

[0130] Preferably, the substitution is with a hydrophilic and / or positively charged amino acid. In one embodiment, the substitution is with an amino acid having opposite biochemical properties - for example, but not limited to, lysine for aspartic acid (positive charge replaced by negative charge), serine for alanine (polar replaced by non-polar).

[0131] 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.

[0132] In one embodiment, the substitution is a D317K substitution or a homologous position in a homologous sequence of SEQ ID NO: 32 or 34 or a homolog or functional variant thereof.

[0133] In one embodiment, the substitution is an N317K substitution or a homologous position in a homologous sequence of SEQ ID NO: 36 or a homolog or functional variant thereof.

[0134] In one embodiment, the substitution is a D315K substitution or a homologous position in a homologous sequence of SEQ ID NO: 41, 43, 49 or 51, or a homolog or functional variant thereof.

[0135] In one embodiment, the substitution is a D316K substitution or a homologous position in a homologous sequence of SEQ ID NO: 47 or a homolog or functional variant thereof.

[0136] In one embodiment, the substitution is an S317K substitution or a homologous position in a homologous sequence of SEQ ID NO: 41, 43, 49 or 51, or a homolog or functional variant thereof.

[0137] In one embodiment, the substitution is an E317K substitution or a homologous position in a homologous sequence of SEQ ID NO: 47 or a homolog or functional variant thereof.

[0138] Preferably, if Figure 5 As shown, the substitution is preferably at position D317 of SEQ ID NO: 32 or 34 or a homologous position in a homologous sequence, wherein preferably the substitution is D317K.

[0139] Homologous positions, or as used herein, "corresponding positions in homologous sequences," can be determined by performing a sequence alignment after homologous sequences have been identified. For example, a BLAST search can be performed against the genome of the plant of interest using a potato or Nicotiana benthamiana NRC1, NRC2, or NRC3 sequence as a query sequence (i.e., one of the sequences defined by SEQ ID NO: 32, 34, 36, 41, 43, 47, 49, or 51) to identify homologs.

[0140] Alternatively, the mutation replaces all or a substantial portion of the HD1-1 region with a corresponding HD1-1 region in a second NLR protein, wherein the second NLR protein is not inhibited by the pathogen effector.

[0141] The mutation may also replace 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.

[0142] By "uninhibited" is meant that the pathogen effector is unable or insignificantly bound to the second NLR protein, or that the pathogen effector does not inhibit or prevent the second NLR protein from eliciting an immune response (e.g., cell death). In one example, the second NLR protein can be NRC4.

[0143] The nucleic acid sequence of the NRC4 RNBS-C motif may encode the RNBS-C motif as defined in SEQ ID NO: 12 or a homologue or functional variant thereof.

[0144] In one embodiment, the mutation is introduced using targeted genome editing. In other words, in one embodiment, the present invention relates to methods and plants generated by genetic engineering methods as described above, and does not encompass naturally occurring varieties or plants produced by traditional breeding methods.

[0145] Targeted genome modification or targeted genome editing is a genome engineering technology that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events.

[0146] In a preferred embodiment, the genome editing method used according to aspects of the present invention is CRISPR. Alternatively, TALEN can be used for targeted genome editing.

[0147] In a preferred embodiment of any aspect of the invention described herein, the sgRNA can be used with a modified Cas9 protein, such as a nickase Cas9 or nCas9 or a "deactivated" Cas9 (dCas9), fused to a "base editor"—e.g., an enzyme such as a deaminase (e.g., cytidine deaminase), TadA (tRNA adenosine deaminase), or an ADAR or APOBEC. These enzymes are capable of replacing one base with another. The result is that the DNA is not deleted, but a single base substitution is made (Kim et al., 2017; Gaudelli et al., 2017). Alternatively, the method can use sgRNA together 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 template DNA strand is introduced after sgRNA-mediated double-stranded DNA cleavage, which can be used to generate specific targeted mutations (i.e., SNPs) in the gene using homology-directed repair. As a further alternative, lead editing can be used to introduce specific mutations (Anzalone et al., 2019). Here, a catalytically impaired Cas9 endonuclease is fused to an engineered reverse transcriptase that is programmed by a lead editing guide RNA (pegRNA) that is both specific for the target site and encodes the desired edit. Using the above approach, we can mutate the nucleotides encoding the conserved HD1-1 domain, resulting in one of the above-mentioned substitutions in the amino acid sequence.

[0148] Once targeted genome editing has been performed, the amplified products can be analyzed using rapid high-throughput screening procedures to detect the presence of mutations in NLR genes (particularly in the HD1-1 domain). Once mutations are identified and screened for increased immunity to pathogen effectors, stable, non-segregating plant lines can be generated. Thus, mutants can be identified that have one or more mutations in NLR genes (particularly in the HD1-1 region) and therefore have increased immunity to a given pathogen compared to control plants.

[0149] In one embodiment, a targeted genome editing was performed to transiently express a mutant in an NLR protein in Nicotiana benthamiana to confirm that the mutant was insensitive to inhibition (i.e., resurrected). This can be determined using a programmed cell death assay, i.e., activated by its own active mutation or activated by a sensor + avirulent effector protein. In a further embodiment, proof-of-concept experiments were performed in crop plants (e.g., soybeans).

[0150] Plants obtained or obtainable by such methods, as well as seeds or other propagation material obtained or obtainable from such plants, which carry a functional mutation in at least one endogenous NLR gene, preferably NRC2 and / or NRC3, are also within the scope of the present invention.

[0151] In one embodiment, the progeny plant is stably transformed with the CRISPR construct and comprises an exogenous polynucleotide that is heritably maintained in the plant cell. The method may include the step of verifying that the construct is stably integrated. The method may also include the additional step of collecting seeds or other propagation material from the selected progeny plant.

[0152] In one example, the plant may not comprise a mutation introduced into any other gene. In other words, a mutation is only introduced into one or more NLR proteins as described above.

[0153] In another aspect of the present invention, a method for providing or improving pathogen resistance in a plant is provided, the method comprising introducing any one of the above mutations into at least one nucleic acid sequence encoding a protein comprising a nucleotide binding domain and a leucine-rich repeat (NLR), wherein the NLR protein comprises an HD1 domain; and wherein the at least one mutation is a mutation of one or more nucleotides in the N-terminal portion of the HD1 domain, as described above.

[0154] In another aspect of the present invention, a method for providing or improving pathogen resistance in a plant is provided, the method comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, wherein the NLR protein comprises an HD1 domain, and wherein the NLR protein comprises a mutation in one or more amino acids in the N-terminal portion of the HD1 domain.

[0155] In another aspect of the present invention, a method for producing a plant with improved pathogen resistance is also provided, the method comprising introducing at least one of the above-mentioned mutations into at least one nucleic acid sequence encoding a protein containing a nucleotide binding domain and a leucine-rich repeat sequence (NLR), wherein the NLR protein comprises an HD1 domain; and wherein the at least one mutation is a mutation of one or more nucleotides in the N-terminal portion of the HD1 domain, as described above.

[0156] In another aspect of the present invention, a method for producing a plant with improved pathogen resistance is provided, the method comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, wherein the NLR protein comprises an HD1 domain, and wherein the NLR protein comprises a mutation in one or more amino acids in the N-terminal portion of the HD1 domain.

[0157] By "providing or improving pathogen resistance" is meant that at least one of pathogen / pest growth, fitness, or productivity is reduced in a plant carrying a mutant NLR as described above, compared to a wild-type or control plant. As used herein, the reduction or improvement 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% relative to the level of pathogen / pest growth or fitness in a control or wild-type plant, or relative to the productivity of a control or wild-type plant.

[0158] "Pathogens" may include any disease-causing agent, particularly pathogenic organisms (such as bacteria, viruses, nematodes, and fungi). Pathogens infect plants and cause disease, and may, for example, reduce yield or otherwise damage the plant, for example, through the action of toxins. "Pests" may include organisms such as animals, insects, or nematodes, and may infect plants with the pathogen or directly damage the plant (for example, by sucking sap, boring into stems and fruits, and cutting roots, stems, and leaves).

[0159] Alternatively, providing or improving pathogen resistance may result in an increase in yield or seed yield. Thus, providing or improving pathogen resistance may be measured by measuring an increase in yield or seed yield.

[0160] The term "yield" generally refers to a measurable product of economic value, usually associated with a specific crop, area, and time period. Individual plant parts directly influence yield based on their number, size, and / or weight. Actual yield is the annual yield per square meter of a crop and is determined by dividing total yield (including harvested and assessed yield) by the number of square meters planted.

[0161] Preferably, the increase in yield comprises at least one of: an increase in seed number and / or weight, an increase in the number of pods per plant (if the plant contains pods), an increase in 1000-kernel weight (TKW), an increase in biomass, an increase in fresh weight, and an increase in growth (preferably root growth). Yield is increased relative to a control or wild-type plant. For example, 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.

[0162] In another aspect of the present invention, a method for improving plant immunity is provided, the method comprising introducing at least one of the above mutations into at least one nucleic acid sequence encoding a protein containing a nucleotide binding domain and a leucine-rich repeat sequence (NLR), wherein the NLR protein comprises an HD1 domain; and wherein the at least one mutation is a mutation of one or more nucleotides in the N-terminal portion of the HD1 domain, as described above.

[0163] In another aspect of the present invention, a method for improving plant immunity is provided, wherein the method comprises introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, as described herein, wherein the NLR protein comprises an HD1 domain, and wherein the NLR protein comprises a mutation in one or more amino acids in the N-terminal portion of the HD1 domain.

[0164] 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 the regulatory sequence.

[0165] In another aspect of the present invention, a method for producing a plant with improved immunity is also provided, the method comprising introducing at least one of the above-mentioned mutations into at least one nucleic acid sequence encoding a protein containing a nucleotide binding domain and a leucine-rich repeat sequence (NLR), wherein the NLR protein comprises an HD1 domain; and wherein the at least one mutation is a mutation of one or more nucleotides in the N-terminal portion of the HD1 domain, as described above.

[0166] In another aspect of the present invention, a method for producing a plant with improved immunity is also provided, wherein the method comprises introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, wherein the NLR protein comprises an HD1 domain, and wherein the NLR protein comprises a mutation in one or more amino acids in the N-terminal portion of the HD1 domain, as described herein.

[0167] 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 the regulatory sequence.

[0168] Improvement in plant immunity can be measured by any technique in the art. In one embodiment, improvement in immunity can be measured by measuring cell death in the presence of a pathogen or pathogen effector compared to cell death in wild-type or control plants. Examples of cell death assays that can be performed are described in the Examples.

[0169] By "improved" is meant that immunity is improved 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%, at least 95% compared to wild type or control plants.

[0170] In another aspect of the present invention, a method for producing an altered NLR protein is provided, wherein a pathogen effector is unable to bind to the NLR protein or has reduced binding ability to the NLR protein. "Binding" is as defined above. Preferably, the method comprises introducing at least one of the above-mentioned mutations into the N-terminal portion of the NLR protein, as described above.

[0171] The method may include:

[0172] a. Selecting a part of the plant;

[0173] b. transforming at least one cell of a part of the plant of paragraph (a) with at least one CRISPR construct or sgRNA molecule, wherein the CRISPR construct or sgRNA molecule targets the NRC gene and introduces at least one mutation into the HD1-1 region as described above;

[0174] c. regenerating at least one plant derived from the transfected cell or cells;

[0175] d. selecting one or more plants obtained according to paragraph (c) which exhibit at least one mutation in the HD1-1 region.

[0176] 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 of the steps of evaluating the phenotype of the genetically altered plant and measuring at least one immunity to a given pathogen. In other words, the method may include the step of screening for plants exhibiting a desired phenotype.

[0177] Alternatively, more conventional mutagenesis methods can be used to introduce at least one mutation into the HD1-1 region of the NLR gene. These methods include physical and chemical mutagenesis. One skilled in the art will appreciate that other methods can be used to generate such mutants, and methods of mutagenesis and polynucleotide alterations 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; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and references cited therein.

[0178] In another embodiment, the mutagenesis is physical mutagenesis, such as application of ultraviolet radiation, X-rays, gamma rays, fast or thermal neutrons, or protons.The target population can then be screened to identify substitution mutations in the HD1-1 region of the NLR gene.

[0179] In another embodiment of the various aspects of the invention, the method comprises mutagenizing the plant population with a mutagen. The mutagen can be fast neutron irradiation or a chemical mutagen selected from the following non-limiting list: ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), triethylmelamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine , N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12-dimethylbenz[a]anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, busulfan, diepoxyalkylene oxides (diepoxyoctane (DEO), diepoxybutylene (BEB), etc.), 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170), or formaldehyde. Similarly, the target population can then be screened to identify one of the above mutations in the HD1-1 region of the NLR gene.

[0180] In another embodiment, the method for creating and analyzing a sudden change is the local lesion (TILLING) induced in the targeted genome. In an alternative embodiment, the method for creating and analyzing a sudden change is EcoTILLING. EcoTILLING is a molecular technique similar to TILLING, except that its target is to disclose the natural variation in a given colony, rather than inducing a sudden change. The first publication of the EcoTILLING method is recorded in Comai et al. (2004).

[0181] In another embodiment, the method uses oligonucleotide-directed mutagenesis (ODM).

[0182] Genetically modified plants of the present invention can also be obtained by hybrid transfer of any sequence of the present invention, for example, by pollinating wild-type or control plants with pollen from genetically modified plants described herein, or by pollinating pistils of plants described herein with other pollen that has not been transformed or genetically modified as described herein.

[0183] In another aspect of the present invention, a plant obtained or obtainable by the above method is provided. In a further aspect, seeds or other propagation materials obtained or obtainable from said plant are provided. The scope of the present invention also includes offspring plants obtained from said seeds or other propagation materials, as well as seeds or other propagation materials obtained from said offspring plants.

[0184] In another aspect of the present invention, a method for screening a plant population and identifying and / or selecting plants that exhibit pathogen resistance or improved pathogen resistance is provided, the method comprising detecting at least one polymorphism (or mutation as described above) in the NRC2 and / or NRC3 gene in the plant or plant germplasm, wherein preferably the polymorphism is located in the N-terminal part of the HD1 domain of the NRC2 and / or NRC3 gene; and selecting the plant.

[0185] Suitable tests for assessing the presence of polymorphisms are well known to those skilled in the art and include, but are not limited to, DNA sequencing (e.g., amplicon sequencing, RenSeq (resistance gene enrichment sequencing), or Sanger sequencing using primers), isozyme electrophoresis, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), random primer polymerase chain reaction (AP-PCR), DNA amplification fingerprinting (DAF), sequence-characteristic amplified regions (SCARs), amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs, also known as microsatellite markers), and single nucleotide polymorphisms (SNPs). In one embodiment, competitive allele-specific PCR (KASP) genotyping is used.

[0186] In one embodiment, the method comprises:

[0187] a) obtaining a nucleic acid sample from the plant; and

[0188] b) performing nucleic acid amplification of the NRC2 and / or NRC3 alleles using one or more primer pairs.

[0189] In further embodiments, the method may further comprise introgressing or crossing a chromosomal region comprising at least one of the NRC polymorphisms described above into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.

[0190] As used herein, a "pathogen effector" may refer to any protein derived from a pathogen that acts in the host (plant) to benefit the pathogen. In particular, this includes any pathogen effector that is capable of binding to an NLR protein, and for example to the HD1-1 region of NRC2 and / or NRC3. Examples of such pathogen effectors include SPRYSEC10 (SS10), SPRYSEC34 (SS34), SPRYSEC15 (SS15) from the potato cyst nematode pathogen (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 potato cyst nematode.

[0191] In one embodiment, the plant is a crop plant. By crop plant is meant any plant grown on a commercial scale for human or animal consumption or use. In another embodiment, the plant is a suitable model organism used in Arabidopsis, Nicotiana benthamiana, Nicotiana tabacum, Medicago truncatula, or other plant research.

[0192] The plant may be a dicot or a monocot.

[0193] The dicotyledonous plant can 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 can 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, grapevine, or citrus species. In one embodiment, the plant is rapeseed.

[0194] Also included are biofuel and bioenergy crops such as rapeseed / canola, sugarcane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupine and willow, poplar, poplar hybrids, Miscanthus or gymnosperms such as loblolly pine. Also included are crops used for silage (corn), grazing or feed (grass, clover, donkey grate, alfalfa), fiber (such as cotton, flax), building materials (such as pine, oak), pulping (such as poplar), feedstock for the chemical industry (such as high-erucic acid rapeseed, linseed), and feedstock for institutional uses (such as turf grass for golf courses), ornamental plants for public and private gardens (such as snapdragons, petunias, roses, geraniums, Nicotiana species), and household plants and cut flowers (African violets, begonias, chrysanthemums, geraniums, coleus, spider plants, dracaena, rubber plants).

[0195] The monocotyledonous plant may, for example, be selected from the following families: Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop (such as wheat, rice, barley, corn, oats, sorghum, rye, millet, buckwheat), a lawn grass, Italian ryegrass, sugarcane or a Festuca species, or a crop plant (such as onion, leek, yam or banana).

[0196] Preferably, the plant is a crop plant. By crop plant is meant any plant grown 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, kidney bean, broad bean, lettuce, cotton, sugarcane, sugar beet, broccoli or other vegetable Brassica plants, or poplar.

[0197] In a preferred embodiment, the plant is selected from the Solanaceae family. In one example, the plant can be selected from potato (Solanum tuberosum), eggplant (Solanum melongena), petunia (Petunia spp., such as Petunia x hybrida or Petunia hybrida), tomatillo (Physalis philadelphica), capsicum (Physalis peruviana), Physalis sp., woody nightshade (Solanum dulcamara), nightshade (Solanum scabrum), gboma eggplant (Solanum macrocarpon), pepper (Capsicum spp.; such as Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), tomato (Solanum lycopersicum or Lycopersiconesculentum), tobacco (Nicotiana spp., such as N. tabacum, Nicotiana 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. Preferred Solanaceae plants are agriculturally grown Solanaceae plants, including, but not limited to, potato, tomato, tomatillo, eggplant, pepper, tobacco, cape gooseberry, and petunia.

[0198] As used herein, the term "plant" encompasses whole plants and progeny of plants, as well as plant parts (including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs), each of which comprises a mutation as described above. 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 plants of the present invention as described herein, but is not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs.

[0199] The term "propagation material" encompasses seeds and other vegetative propagation material, such as tubers.

[0200] In a most preferred embodiment, the plant part or harvestable product is a seed or grain. Thus, in a further aspect of the present invention, seeds or grain produced by a genetically altered plant as described herein are provided. Accordingly, in one aspect of the present invention, seeds are provided, wherein the seeds comprise at least one of the above-described mutations in an NLR gene. Progeny plants obtained from the seeds and seeds obtained from the progeny are also provided.

[0201] According to all aspects of the present invention, as used herein, a control plant is a plant that has not been modified according to the methods of the present invention. Thus, in one embodiment, the control plant does not have one or more mutations in the NLR gene as described herein. In one embodiment, the control plant is a wild-type plant. The control plant typically belongs to the same plant species as the modified plant, preferably has the same genetic background. The control plant can also be a wild-type plant, which has the same changes as the present invention, but does not contain the specific changes that are believed to provide the phenotype. For example, the control plant contains another copy of the WT gene instead of the edited gene.

[0202] Although the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods of making and using the present invention and the best mode thereof, the following examples are provided to further enable those skilled in the art to practice the present invention and to provide a complete written description thereof. However, it will be understood by those skilled in the art that the specific contents of these examples should not be interpreted as limiting the present invention, and the scope of the present invention should be understood from the claims appended hereto and their equivalents. In view of this disclosure, various other aspects and embodiments of the present invention will be apparent to those skilled in the art.

[0203] Unless the context dictates otherwise, the descriptions and definitions of the features above are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

[0204] The invention will now be described by way of the following non-limiting examples. Example

[0205] Pathogens inhibit NLRs to eliminate immunity. Here, we demonstrate that cyst nematode effectors inhibit resistosome formation of the auxiliary NLR immunity protein NRC2 by directly binding, which in turn physically prevents the intramolecular rearrangements required for activation. This leads to the inhibition of immune signaling and disease resistance. By combining structural approaches and leveraging NLR diversity, we engineered single amino acid point mutants of the auxiliary NLR that escape parasite inhibition. The engineered auxiliary NLR restored the activity of the cyst nematode resistance protein. This represents a new strategy for editing immune receptor genes to restore recessive / ineffective disease resistance in crop genomes.

[0206] We provide a method for the rational design of NLRs that evade pathogen inhibition. Relieving pathogen inhibition of accessory NLRs, such as NRC2, allows for the "restoration" of cryptic upstream sensors that are normally inactivated by interference with their downstream signaling partners.

[0207] SS15 directly inhibits NRC2 resistance body formation

[0208] NRC2 or NRC4, along with their upstream sensor Rx and effector SS15, were transiently expressed in leaves of nrc2 / 3 / 4 CRISPR-knockout Nicotiana benthamiana plants. A BN-PAGE-based readout was used for NRC resistosome formation. For biochemical analysis, we used NRC2 and NRC4 variants with mutations in their N-terminal MADA motifs (NRC2 and NRC4, respectively). EEE and NRC4 AAA ), the mutation abolished cell death induction without affecting receptor activation, oligomerization or localization. We then activated the Rx-NRC system by co-expressing PVX CP (potato virus X coat protein)-GFP or co-expressing free GFP (as an inactive control). In the absence of SS15, both NRC2 and NRC4 oligomerize upon effector-triggered activation mediated by their upstream sensors. However, when SS15 is present, Rx / CP-activated NRC2 fails to oligomerize and appears as a band of approximately 240 kDa, which co-migrates with SS15. Inactive NRC2 co-expressed with SS15 also migrates as a band of approximately 240 kDa, which migrates more slowly than inactive NRC2 in the absence of SS15, indicating the formation of an NRC2-SS15 complex in vivo ( Figure 1B). We also observed that coexpression of SS15 not only blocked NRC2 oligomerization but also prevented the previously reported translocation of activated NRC2 from the cytoplasm to the plasma membrane (PM), as well as the formation of NRC2 PM-associated puncta upon Rx / CP activation. We conclude that SS15 acts as a direct protein inhibitor of NRC2 to inhibit immune signaling by directly binding to the NB-ARC domain of NRC2 to block the formation of signaling-competent oligomeric antipathosomes.

[0209] The HD1-1 region of the NB-ARC domain determines SS15 association and inhibition of NRC

[0210] We generated a series of NRC2-NRC4 chimeric proteins in which SS15 binds to the NB-ARC domain ( Figure 2 A-2B), and we subsequently determined its association with SS15 by in planta coimmunoprecipitation. We identified a chimeric variant of NRC4 2HD1-1 , which carries the N-terminal portion of the HD1 region of NRC2 (referred to herein as the "HD1-1" region), this variant associates with SS15 ( Figure 2 C) NRC4 2HD1-1 Susceptible to SS15 inhibition, unable to oligomerize and trigger cell death in the presence of SS15 ( Figure 2 D, black dashed circle; Figure 2 E) We conclude that SS15 binds to the HD1-1 region and that binding to the HD1-1 region is sufficient for this effector to function as a direct inhibitor of NRC resistosome formation and programmed cell death.

[0211] Identification of the SS15-NRC binding interface enables engineering of NRC2 to evade pathogen inhibition

[0212] We hypothesized that if we identified the key residues that mediate the interaction with the HD1-1 region, we could mutate them to allow NRC2 to escape immunosuppression by SS15. To this end, we prepared crystals of SS15 in complex with the NB-ARC domain of the NRC2 homolog NRC1 (which is also inhibited by SS15) and obtained X-ray diffraction data, which we interpreted to Resolution( Figure 3 A, Figure 4 We determined that SS15 binds to a loop in HD1-1 that connects the NB domain to the HD1 and WHD domains, providing orthogonal evidence that the SS15-NRC interaction is mediated by this region. This loop was previously shown to act as a "hinge," allowing the NB domain to move relative to the HD1 and WHD domains ( Figure 4By binding to and securing this hinge, SS15 may abrogate conformational changes that are crucial for NLR activation.

[0213] Furthermore, since SS15 can inhibit NRC1, NRC2, and NRC3, we took advantage of the high conservation of plant NB-ARC domains to narrow down the list of residues within the binding interface that could support this interaction. We screened the HD1-1 region for residues that are similar in NRC1, NRC2, and NRC3 ( Figure 3 B). Combining the cocrystal structure and alignment information allowed us to select 13 candidate residues to be tested by mutagenesis in NRC2 ( Figure 3 B) We mutated each of these residues to the corresponding amino acids found in NRC4 and screened these NRC2 variants for sensitivity to SS15 inhibition in a cell death assay. This revealed that at least two variants of NRC2 E316P and NRC2 D317K , which triggers cell death when activated by Rx / CP and is no longer inhibited by SS15 ( Figure 3 C) We also tested the association of all 13 single amino acid mutants with SS15 by in planta co-immunoprecipitation and found that NRC2 D317K and NRC2 E316P showed reduced association with SS15 ( Figure 3 D), which is consistent with the observation that SS15 cannot inhibit this variant ( Figure 3 C) Consistent with this finding, we conclude that residues E316 and D317 are critical for SS15-mediated NRC2 inhibition, and in particular, mutating residue D317 allows Rx / CP-activated NRC2 to escape SS15 association and inhibition.

[0214] We next tested whether these two SS15-escaping variants retained normal functionality with other previously characterized NRC2-dependent sensor NLRs. We did this by using NRC2 E316P and NRC2 D317K This was tested in complementation assays in nrc2 / 3 / 4 CRISPR-knockout Nicotiana benthamiana plants, activating these accessory proteins with a panel of agronomically important sensors, NLRs, that mediate resistance to a variety of pathogens. This panel includes the potato cyst nematode R gene and its Rx paralog, Gpa2, as well as various oomycete and bacterial resistance proteins. In the absence of SS15, both NRC2 variants mediated cell death when activated by all tested NRC2-dependent sensors ( Figure 5 A) In the presence of SS15, NRC2 E316P The variant escaped SS15 inhibition when activated by Rx but not by all other tested sensors.D317K The variants were able to escape SS15 inhibition regardless of the sensor NLR activated. Therefore, we selected NRC2 D317K Subsequent biochemical studies were performed using the BN-PAGE-based assay for pathogenic body formation discussed above. D317K It can still oligomerize in the presence of SS15 and does not appear to form an in vivo complex with the inhibitor ( Figure 5 B). We conclude that NRC2 D317K It is able to completely evade SS15-mediated immunosuppression and retains the ability to oligomerize and mediate cell death upon activation by multiple agronomically important NLR sensors.

[0215] discuss

[0216] Our studies reveal how parasite effectors have evolved to act as inhibitors of accessory NLRs by directly binding to the HD1 region of the NB-ARC domain of accessory NLRs to prevent resistance body formation. By binding and immobilizing a critical hinge loop in the NB-ARC domain of these NLRs, SS15 restricts movement of the NB domain relative to the HD1 and WHD domains, thereby preventing immune receptor activation. Remarkably, while SS15 can bind to and inhibit NRC2, we generated chimeric NRC2-NRC4 variants, which, together with structural information, helped us identify the binding interface. Mutational studies of this interface allowed us to generate single-amino acid NRC2 (NRC2 D317K ) variants that escape SS15 inhibition without impairing receptor signaling capacity. D317K The variant now supports signaling of any NRC2-dependent sensor even in the presence of SS15.

[0217] Using the methods described here, recessive or inactivated resistance proteins can be restored to enhance disease resistance. Furthermore, single-amino acid NRC2 variants can be generated in situ using gene editing in agronomically important crop species, making this technology feasible for deployment in countries where transgenic approaches are not feasible. In this work, we developed a novel approach to achieve stable immunity by engineering NLRs that evade parasite inhibition. This approach can be applied to other plant NLR immune receptors that are directly targeted by parasite effectors. This technology holds the promise of ushering in a new era of breeding for disease resistance.

[0218] Sequence Listing

[0219] SEQ ID NO: 1; Nicotiana benthamiana NRC2a HD1 domain (NbNRC2a-HD1), amino acid; GenBank accession number: ALQ52761.

[0220] PHDLKFLTEDESWILLEKKVFHKDKCPPELELSGKSIAKKCNGLPLAIVVIAGALIGKGKTSREWKQVDESVGEHLINK

[0221] SEQ ID NO: 2; Nicotiana benthamiana NRC2a HD1-1 domain (NbNRC2a-HD1-1), amino acid; GenBank accession number: ALQ52761.

[0222] PHDLKFLTEDESWILLEKKVFHKDKCPPELELSGKSIAK

[0223] SEQ ID NO: 3; Nicotiana benthamiana NRC2a RNBS-C motif, amino acid; GenBank accession number: ALQ52761.

[0224] HDLKFLTEDESWILLEKKVF

[0225] SEQ ID NO: 4; Nicotiana benthamiana NRC2b HD1 domain (NbNRC2b-HD1), amino acid; GenBank Accession No.: ALQ52762.

[0226] PHDLKFLTEDESWILLEKKVFHKDKCPPELELSGKSIAKKCNGLPLA IVVIAGALIGKGKTSREWKQVDESVGEHLINK

[0227] SEQ ID NO: 5; Nicotiana benthamiana NRC2b HD1-1 domain (NbNRC2b-HD1-1), amino acid; GenBank Accession No.: ALQ52762.

[0228] PHDLKFLTEDESWILLEKKVFHKDKCPPELELSGKSIAK

[0229] SEQ ID NO: 6; Nicotiana benthamiana NRC2b RNBS-C motif, amino acid; GenBank accession number: ALQ52762.

[0230] HDLKFLTEDESWILLEKKVF

[0231] SEQ ID NO: 7; Nicotiana benthamiana NRC3 HD1 domain (NbNRC3-HD1), amino acid; GenBank accession number: QER78240.

[0232] PHDLKFLTENESWELLEKRVFHKEKCPFELELPGKSIAKKCRGLPLA IVVIAGALIGKGKTTREWELVADSVGEHLINR

[0233] SEQ ID NO: 8; Nicotiana benthamiana NRC3 HD1-1 domain (NbNRC3-HD1-1), amino acid; GenBank accession number: QER78240.

[0234] PHDLKFLTENESWELLEKRVFHKEKCPFELELPGKSIAK

[0235] SEQ ID NO: 9; Nicotiana benthamiana NRC3 RNBS-C motif, amino acid; GenBank accession number: QER78240.

[0236] HDLKFLTENESWELLEKRVF

[0237] SEQ ID NO: 10; Nicotiana benthamiana, NRC4 HD1 domain (NbNRC4-HD1), amino acids; GenBank Accession No.: QER78241.

[0238] PHDLKFLTPKESFELLVKRVFGKKPCPKDLVGHGESIAGKCGGVPL AVVVIAGALRGRPNTSDWIRVERNVVQHLYTNS

[0239] SEQ ID NO: 11; Nicotiana benthamiana, NRC4 HD1-1 domain (NbNRC4-HD1), amino acid; GenBank Accession No.: QER78241.

[0240] PHDLKFLTPKESFELLVKRVFGKKPCPKDLVGHGESIAG

[0241] SEQ ID NO: 12; Nicotiana benthamiana, NRC4 RNBS-C motif, amino acid; GenBank Accession No.: QER78241.

[0242] HDLKFLTPKESFELLVKRVF

[0243] SEQ ID NO: 13; Potato NRC2 HD1 domain (StNRC2-HD1), amino acids; NCBI Reference Sequence: XP_006359790.1.

[0244] PHDLKFLSEDESWILLEKKVFHKDKCPPELVVPSGKSIAKKCKGLPL AIVVIAGALIGKGKTPREWKQVDDSVSEHLINR

[0245] SEQ ID NO: 14; Potato NRC2 HD1-1 domain (StNRC2-HD1-1), amino acids; NCBI Reference Sequence: XP_006359790.1.

[0246] PHDLKFLSEDESWILLEKKVFHKDKCPPELVVPSGKSIAK

[0247] SEQ ID NO: 15; Potato NRC2 RNBS-C motif, amino acids; NCBI Reference Sequence: XP_006359790.

[0248] HDLKFLSEDESWILLEKKVF

[0249] SEQ ID NO: 16; Potato NRC3 HD1 domain (StNRC3-HD1), amino acids; NCBI Reference Sequence: XM_006362512.2

[0250] PHDLKFLTEDESWELLEKKVFHKEKCPPELELPGKSIAEKCMGLPLA IVVIAGALIGKGKTTREWELVAASVREHLINR

[0251] SEQ ID NO: 17; Potato NRC3 HD1-1 domain (StNRC3-HD1-1), amino acids; NCBI Reference Sequence: XM_006362512.2

[0252] PHDLKFLTEDESWELLEKKVFHKEKCPPELELPGKSIAE

[0253] SEQ ID NO: 18; Potato NRC3 RNBS-C motif, amino acids; NCBI Reference Sequence: XM_006362512.2

[0254] DLKFLTEDESWELLEKKVF

[0255] SEQ ID NO: 19; Potato NRC4 HD1 domain (StNRC4-HD1), amino acids; NCBI Reference Sequence: XP_006367034.1

[0256] PHVLKFLKTEESFELLVMRVFGKGSCPNELVVTGKKIAEKCGGVPL VVVVIAGALRGRSDKKDWERVDKNVVQHFGEHT

[0257] SEQ ID NO: 20; Potato NRC4 HD1-1 domain (StNRC4-HD1-1), amino acids; NCBI Reference Sequence: XP_006367034.1

[0258] PHVLKFLKTEESFELLVMRVFGKGSCPNELVVTGKKIAE

[0259] SEQ ID NO: 21; Potato NRC4 RNBS-C motif, amino acids; NCBI Reference Sequence: XP_006367034.1

[0260] HELKFLEKEESFELLVMRVF

[0261] SEQ ID NO: 22; Tomato, NRC1 HD1 domain (S1NRC1-HD1), amino acids; NCBI Reference Sequence: NP_001234202.

[0262] PHDLKFLTDEESWILLEKRAFHKAKCLPELETNGKSIARKCKGLPLA IVVIAGALIGKSKTIKEWEQVDQSVGEHFINR

[0263] SEQ ID NO: 23; Tomato, NRC1 HD1-1 domain (S1NRC1-HD1-1), amino acids; NCBI Reference Sequence: NP_001234202.

[0264] PHDLKFLTDEESWILLEKRAFHKAKCLPELETNGKSIAR

[0265] SEQ ID NO: 24; Lycopersicon esculentum, NRC1 RNBS-C motif, amino acids; NCBI Reference Sequence: NP_001234202.

[0266] HDLKFLTDEESWILLEKRA

[0267] SEQ ID NO: 25; Tomato, NRC2 HD1 domain (S1NRC2-HD1), amino acids; NCBI Reference Sequence: XP_004248798.

[0268] PHDLKFLTEDESWILLEKKVFHKDKCPPELVLSGKSIAKKCKGLPLA IVVIAGALIGKGKTPREWKQVDDSVSEHLINR

[0269] SEQ ID NO: 26; Tomato, NRC2 HD1-1 domain (S1NRC2-HD1-1), amino acids; NCBI Reference Sequence: XP_004248798.

[0270] PHDLKFLTEDESWILLEKKVFHKDKCPPELVLSGKSIAK

[0271] SEQ ID NO: 27; Lycopersicon esculentum, NRC2 RNBS-C motif, amino acids; NCBI Reference Sequence: XP_004248798.

[0272] HDLKFLTEDESWILLEKKVF

[0273] SEQ ID NO: 28; Tomato, NRC3 HD1 domain (S1NRC3-HD1), amino acids; NCBI Reference Sequence: XP_004238948.

[0274] PHDLKFLTKDESWELLEKKVFHKEKCPPELELPGISIAEKCMGLPLA IVVIAGALIGKGKTTREWELVAASVGEHLINR

[0275] SEQ ID NO: 29; Tomato, NRC3 HD1-1 domain (S1NRC3-HD1-1), amino acids; NCBI Reference Sequence: XP_004238948.

[0276] PHDLKFLTKDESWELLEKKVFHKEKCPPELELPGISIAE

[0277] SEQ ID NO: 30; Lycopersicon esculentum, NRC3 RNBS-C motif, amino acids; NCBI Reference Sequence: XP_004238948.

[0278] HDLKFLTKDESWELLEKKVF

[0279] In SEQ ID NOs: 32, 34, 36, 41, 43, 47, 49, and 51: bold bases indicate the NB-ARC (HD1) domain, underlined bases indicate the NB-ARC1 (HD1-1) subdomain, and italics indicate the RNBS-C motif. In SEQ ID NOs: 31, 33, 35, 40, 42, 46, 48, and 50: underlined bases indicate the NB-ARC1 (HD1-1) subdomain. In SEQ ID NOs: 37, 38, 39, 44, 45, and 52-59: mutated residues are underlined and highlighted in the individual sequences.

[0280] SEQ ID NO: 31; Nicotiana benthamiana, NbNRC2a cDNA; GenBank accession number: KT936525.

[0281] ATGGCGAACGTTGCGGTGGAGTTTCTGGTGCAGAACTTGATGCAGCTGCTAAGAGACAATGCGGAGCTGATTGTTGGAGTTAAGGATTCGGCTGAGAGTCTGCTTCAAGATCTCAATGATTTCAACGCTTTTCTCAAGCAAACTGCTAAGTCCCGCACTGAGAACGATGTCCACAAAGAATTGGTGAAGAAAATTAAGACTGTGGTCAACTCTGCTGAAGATGCCATTGATAAGTTTGTGATTGAGGCTAAGCTTCACAAGGACAAAGGGGTTGGCAGATTTGTGGATGTTAAGCATTATAAAAGAGTGTATGATGTAGCAGGGGAGATTAAAACTATTAGAGACAAAGTCAAAGAAATTCGTCTCAATAATGCACTTGACCTTCAGGCCCTTCAAGATGAAGATCAATCTGCCAAAGGTGTCCAAGAAAGAAAGCCTCCGGTGGTAGAGGAAGATGATGTGGTTGGATTTGAAGAGGAAGCAGATAAAGTAATCAACCGTCTTCTGGGAGGATCGAGTGGACTAGAAGTTGTTCCAGTTGTTGGAATGCCTGGTCTCGGCAAAACGACACTGGCAAATAAGATTTACAAGCATCCTGACATCGGGTACCAGTTTTTTACTCGCATTTGGGTTTATGTTTCTCAATCATACAGGAGAAGAGAATTATTTCTCAACATCATCAGCAAATTCACTCGCAATACCAAACAATACCACGATATGTGTGAGGAGGATTTAGCTGATGAAATAGAAGATTTTTTGGGCAAGGGAGGGAAATACTTGATTGTCTTGGATGATGTATGGTCTCCTGACGCTTGGGAACGTATCAGAATAGCTTTCCCAAACAACAACAAATCCAATAGAATATTATTGACCACCCGAGATAGCAAAGTTGCTAAGCAATGCAAGCAGTGCATTGGTATA CCTCATGATTTAAAATTTCTGACTGAAGATGAAAGTTGGATTTTACTGGAGAAGAAAGT TTTCCATAAAGATAAATGTCCTCCTGAATTGGAACTATCTGGAAAGAGCATAGCCAAA

[0282] SEQ ID NO: 32; Nicotiana benthamiana, NbNRC2a amino acid; GenBank accession number: ALQ52761.1.

[0283]

[0284] SEQ ID NO: 33; Nicotiana benthamiana, NbNRC2b cDNA; GenBank accession number: KT936526.

[0285] ATGGCGAACGTTGCGGTGGAATTTTTGGTGCAGAACTTGATGCAGCTGCTGAGGGACAATGCGGAGCTGATTATTGGGGTTAAGGATTCGGCTGAGAGCCTGCTTCAAGATCTCAATGATTTCAACGCTTTTCTCAAGCAAGCTGCCAAGTCCCGCATTGAGAACGATGTC CACAAAGAATTGGTGAAGAAAATTAAGACAATGGTCAACTCTGCTGAAGATGCCATTGATAAGTTTGTAATTGAGGCTAAGCTTCACAAGGACAAAGGGGTTGGCAGATTTGTAGATGTTAAGCATTATAAAAGAGTGTATGATGTAGCAGCCGAGATCAAAGGTATCAGAG AAAAAGTGAAAGAAATCCGTCAGAATAATGCCCTTGACCTTCAAGCCCTTCAAGATGAAGATCAATCTGCCAAAGGTGTCGAAGAAAGAAAGCCTCCAGTGGTAGAGGAAGATGATGTGGTTGGATTTGAAGAGGAAGCAGATAAAGTAATCAACCGTCTTCTGGGAGGATTGAGTGGACTAGAAGTTGTTCCAGTTGTTGGAATGCCTGGTCTCGGCAAAACGACGCTAGCAAATAAGATTTACAAGCATCCTGACATCGGGTACCAGTTTTTTACTCGCATTTGGGT TTTATGTTTCTCAATCATACAGGAGAAGAGAATTATTTCTCAACATCATCAGCAAATTCACTCGAAATACCAAACAATACCATGATATGTGTGAGGAGGATTTAGCTGATGAAATACAAGATTTTTTGGGCAAAGGAGGAAAATA CTTGATTGTCTTGGATGATGTATGGTCTCCTGACGCTTGGGAACACATCAAAATAGCTTTCCCGAACAACAACAAATCCAATAGAATATTATTGACCACTCGAGATAGCAAAGTTGCTAAGCAATGCAAGCAGTGCATTGGTATA CCTCATGATTTAAAATTTCTGACTGAAGATGAAAGTTGGATTTTACTGGAGAAGAAAG TTTTCCATAAAGATAAATGTCCTCCTGAATTGGAACTATCTGGAAAGAGCATAGCCAAAAAATGTAATGGACTACCCCTTGCGATTGTTGTTATTGCAGGAGCACTAATTGGGAAAGGTAAGACATCAAGAGAGTGGAAACAAGTGGATGAGAGTGTGGGTGAACACCTCATAAATAAAGACCAGCCTGAGAATTGTAACAAATTGGTGCAACTGAGTTATGATCGCTTGTCTTATGACTTGAAAGCATGTTTTTTATATTGTGGTGCATTTCCCGGAGGCTTTGAGATCCCTGCTTGGAAGTTAATCCGTTTGTGGATCGCAGAAGGGTTCATACAGTATAAAGGCCACTTATCTCTTGAGTGTAAAGCAGAGGATAACTTGAATGATCTCATCAACAGGAATCTAGTGATGGTAATGCAAAGGACATCTGATGGGCAAATCAAAACATGTCGTCTTCATGACATGTTGCACGAGTTTTGCAG ACAAGAGGCGATGAAGGAAGAAAATCTTTTCCAAGAAATAAAATTAGGTGCTGAGCAATATTTCCCAGGAAAACGGGAACTAGCCACCTACCGTCGCTTATGCATTCATTCCTCAGTTTTGGAATTTATCTCTACAAAGCCCTCAGGTGAACATGTCAGGTCATTCTTATCTTTTTCTTTAAAAAAGGTCGAGATGCCATCTGTCGACATCCC AACCATACCAAAAGGCTTCCCGTTGCTGAGGGTTTTTGATGTCGAGTCCATCAACTTCAGTCGCTTTTCCAAGGAGTTTTTCCAGTTATATCATTTGAGGTATATTGCTTTCTCATCTGACACAATCAAGATCATTCCTAAACACATTGGAGAACTGTGGAACATCCAAACACTCATAATTAACACGCAACAACACTCTCTTGATATCCAAGCAAACATATGGAATATGGCACGACTAAGGCATCTGCACACTAACTCTTCTGCTAAATTGCCTGTTCCTGTGACCCCAAGAAGTAGTAAAGTTCCTTTGGTAAATCAAAGCCTGCAAACTCTCTCCACCATTGCTCCCGAAAGCTGCACAGAAGAAGTGTTTGCAAGGACTCCAAACCTGAAAAAGTTGGGTATTCGTGGGAAAGTTGCTGTGCTTCTTGAACCTAATAAGTCATTGTTAAAAAATGTGAAGAAACTAGAATCCCTTGAAAACTTGAAGCTGATAAATGATAGTAGTCAAACAGGAAAAGGGTTACGCCTTCCACCCTCATATATATTTCCCACGAAGTTGAGGAAGCTATCTTTAGTAGATACCTGGCTGGAGTGGAACGATATGTCTATATTGGGGCAGATGGAACACCTTGAAGTTCTGAAGCTGAAAGAAAATGGGTTTATGGGAGAATGCTGGGAGTCAGTTGGAGGTTTTTGTTCCCTATTGGTGTTGTGGATTGAAAGGACAGACTTAGTTTCTTGGAAAGCATCAGCTGATCACTTTCCAAGACTTAAGCATCTTGTTCTCATCTGCTGCGATAAGATTAAGGAAATCCCCATTGGCCTGGCTGATATACACAGCTTCCAAGTGATGGAGTTGCAAAACTCCACCAAAACAGCAGCAATATCTGCACGGGAAATACGAGACAAAAAAGACAAGCAAACTCAAGAGGGGACTAACAACAATGGGTTCAAGCTCTCTATATTCCCTCCCGATCTCTGA

[0286] SEQ ID NO:34; Nicotiana benthamiana, NbNRC2b cDNA; GenBank accession number: ALQ52762.

[0287]

[0288]

[0289] SEQ ID NO:35; Nicotiana benthamiana, NbNRC3 cDNA; GenBank accession number: MK692736.

[0290] ATGGCAGATGTAGCAGCAGATGTAGCAGTAAAATTCCTAGTAGAAAACTTGATGCAATTATTAATCGACAACGCTGACTTGATTATTGGTATAAAGGGTGAAGTTGAAAATTTACTACAAGATCTGAATGATTTCAATGCTTTTCTCAAACAAGCAGCTAAATCAAGGAGAGACAATGAAGTCTTGAAATCACTAGTAAAGAAGATAAGGAAAGTGGTAAATGATGCTGAAGATTCAATTGATAAGTTTGTGATTGAAGCTAAAAGACATGACGATAAAAATAAATTTGCTCAGTGGTTTCATCTTACTCATGTTGCTAGAGCAAAAGGGGTTGCTGATGAGATTAAAACTATAAGGGAAAGGGTGAAGGAAATTCGCCAAAATGATGCTTATGGACTTCAAGCTATAACTTCTTATGATAATTTCAACCAAGGTGCTCAGGAGAGGAAGGTCCCTGTAGTTGAGGAAGACGACGTGGTAGGCTTTGATGACGAAGCGAAAACTGTAATTGATCGCCTCATTGGAGGATCAGATTATGTTGTGCCGGTCGTTGGTATGCCTGGTCTTGGAAAAACAACTTTGGCATATAAGATTTTCAAGGATTCCACAGTTGAGTATGAGTTTTTCAACCGCATATGGGTATATGTCTCTCAATCATTCAACAGAAGGGAAATATTTCTCAACATCATCAGCAAATTCACTCGAAACACCAAACAATACCATGATACACCAGAGGAGGAATTAGCAAATGAAATAAAGGAGCTGCTTGGGAAGGGTGGGAAATATCTTGTTGTTTTAGATGATGTGTGGACAAGAGAAGCTTGGGATCGCATTAAAATTGCTTTCCCCAATAATAATAAACGGAATAGAGTCTTGATGACTACTAGACAAAACAATGTGGCTAAGTCTTGCAACGATAAA CCTCATGATCTAAAGTTTTTGACTGAAAATGAAAGTTGGGAGCTACTTGAGAAGAGAGT TTTTCACAAGGAAAAATGTCCATTTGAGTTAGAATTACCTGGAAAAAGTATAGCCAAA

[0291] SEQ ID NO: 36; Nicotiana benthamiana, NbNRC3 amino acid; GenBank accession number: QER78240.

[0292]

[0293] SEQ ID NO: 37; Nicotiana benthamiana, NRC2a D317K Amino acids.

[0294] MANVAVEFLVQNLMQLLRDNAELIVGVKDSAESLLQDLNDFNAFLKQTAKSRTENDVHKELVKKIKTVVNSAEDAIDKFVIEAKLHKDKGVGRFVDVKHYKRVYDVAGEIKTIRDKVKEIRLNNALDLQALQDEDQSAKGVQERKPPVVEEDDVVGFE EEADKVINRLLGGSSGLEVVPVVGMPGLGKTTLANKIYKHPDIGYQFFTRIWVYVSQSYRRRELFLNIISKFTRNTKQYHDMCEEDLADEIEDFLGKGGKYLIVLDDVWSPDAWERIRIAFPNNNKSNRILLTTRDSKVAKQCKQCIGIPHDLKFLTE KESWILLEKKVFHKDKCPPELELSGKSIAKKCNGLPLAIVVIAGALIGKGKTSREWKQVDESVGEHLINKDQPENCNKLVQLSYDRLSYDLKACFLYCGAFPGGFEIPAWKLIRLWIAEGFIQYKGHLSLECKAEDNLNDLINRNLVMVMQRTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGAEQYFPGKRELATYRRLCIHSSVLEFISTKPSGEHVRSFLSFSLKKIEMPSVDIPTIPKGFPLLRVFDVESINFSRFSKEFFQLYHLRYIAFSSDTIKIIPKHIGELWNIQ TLIINTQQRSLDIQANIWNMERLRHLHTNSSAKLPVPVTPRSSKVPLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKIAVLLEPNKSLLKNVKKLESLENLKLINDSSQTGKGLRLPPSYIFPTKLRKLSLVDTWLEWNDMSILGQMEHLEVLKLKENGFMGECWESVGGFCSLLVLWIERTDLVSWKASADHFPRLKHLVLICCDKLKEIPIGLADIRSFQVMELQNSTKTAAISARGIRDKKDKQTQEGTNNNGFKLSIFPPDL

[0295] SEQ ID NO:38; Nicotiana benthamiana, NRC2b D317K Amino acids.

[0296] MANVAVEFLVQNLMQLLRDNAELIIGVKDSAESLLQDLNDFNAFLKQAAKSRIENDVHKELVKKIKTMVNSAEDAIDKFVIEAKLHKDKGVGRFVDVKHYKRVYDVAAEIKGIREKVKEIRQNNALDLQALQDEDQSAKGVEERKPPVVEEDDVVGFEEEADKVINRLLGGLSGLEVVPVVGMPGLGKTTLANKIYKHPDIGYQFFTRIWVYVSQSYRRRELFLNIISKFTRNTKQYHDMCEEDLADEIQDFLGKGGKYLIVLDDVWSPDAWEHIKIAFPNNNKSNRILLTTRDSKVAKQCKQCIGIPHDLKFLTE KESWILLEKKVFHKDKCPPELELSGKSIAKKCNGLPLAIVVIAGALIGKGKTSREWKQVDESVGEHLINKDQPENCNKLVQLSYDRLSYDLKACFLYCGAFPGGFEIPAWKLIRLWIAEGFIQYKGHLSLECKAEDNLNDLINRNLVMVMQRTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGAEQYFPGKRELATYRRLCIHSSVLEFISTKPSGEHVRSFLSFSLKKVEMPSVDIPTIPKGFPLLRVFDVESINFSRFSKEFFQLYHLRYIAFSSDTIKIIPKHIGELWNIQTLIINTQQHSLDIQANIWNMARLRHLHTNSSAKLPVPVTPRSSKVPLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKVAVLLEPNKSLLKNVKKLESLENLKLINDSSQTGKGLRLPPSYIFPTKLRKLSLVDTWLEWNDMSILGQMEHLEVLKLKENGFMGECWESVGGFCSLLVLWIERTDLVSWKASADHFPRLKHLVLICCDKIKEIPIGLADIHSFQVMELQNSTKTAAISAREIRDKKDKQTQEGTNNNGFKLSIFPPDL

[0297] SEQ ID NO:39; Nicotiana benthamiana, NRC3 N317K Amino acids.

[0298] MADVAADVAVKFLVENLMQLLIDNADLIIGIKGEVENLLQDLNDFNAFLKQAAKSRRDNEVLKSLVKKIRKVVNDAEDSIDKFVIEAKRHDDKNKFAQWFHLTHVARAKGVADEIKTIRERVKEIRQNDAYGLQAITSYDNFNQGAQERKVPVVEEDDVVGFDDEAKTVIDRLIGGSDYVVPVVGMPGLGKTTLAYKIFKDSTVEYEFFNRIWVYVSQSFNRREIFLNIISKFTRNTKQYHDTPEEELANEI KELLGKGGKYLVVLDDVWTREAWDRIKIAFPNNNKRNRVLMTTRQNNVAKSCNDKPHDLKFLTE KESWELLEKRVFHKEKCPFELELPGKSIAKKCRGLPLAIVVIAGALIGKGKTTREWELVADSVGEHLINRDPENCKKLVQMSYDRLPYDLKACFLYCGAFPGGSEISAQKLICLWIAEGFIQYQGPLTLEDIAEDHLNDLVNRNLVMVMKRSSSGQIKTCRVHDMLHEFCRHEAMMEENLFQEIKRGQEHSFPEKQELASYRRLCIHSSVSEFLSTKPFAEHVRSFLCFASKKFEMPLGEIPAIPRAFPLLRVLDAESIKFSRFSREFFKLFHLRYIAFSTDSIMTIPTNIGNLWNVQTLIIETQQGTLDIKADIWNMTRLRHVCINASATLPSPKRPKSSKDNLVNRCLQTLSTIAPECCTAEVFTRTPNLKKLGVRGKIDALLETSKDGSSSGLFSNIGKLDCLEKLKLVNDTRQSRKQLHLPPAYIFPQKLKKLTLIDTWFEWKDMSILGLLEYLEVLKLKENAFRGQSWEPEDSGFPRLQVLWIERTDLSSWKASSGNFPRLKCLVLIACDNLKELPAELADVENLQLMELQSTSVSAAKSARAILKKKQQKVGSGFKLSVFPPDLGL

[0299] SEQ ID NO:40; Potato StNRC2, cDNA. NCBI Reference Sequence: XM_006359728.2

[0300] ATGGCGAACGTAGCAGTAGAATTTCTCGTTGAGAACTTGATGCAGCTGCTGCGGGACAACGCAGAGCTAATTAGTGGAGTTAAAGAGGCTGCTGAGAGTCTACTTCAAGATCTAAATGATTTCAACGCCTTTCTTAAGCAAGCTGCCAAGTGCCACATCAACGAGAACGAAGTTCTGAGAGAACTCGTCAAGAAAAT CAGAACAGTGGTTAACTCTGCTGAAGATGCTATTGATAAATTTGTGATTGAAGCTAAGCTACACAAGGATAAGGGTATGACCAGAGTATTAGACCTTCCCCATTATAAAGGTCAGGGAGGTAGCTGGTGAGATCAAAGCTATACGAAACAAAGTCAAAGAGATCAGACAGAATGATGCCATTGGACTTCAGGCCCT TCAAGATGATGATTCATCTGCCAGAGGTTTCGAAGAAAGAAAGCCTCCAGTGGTAGAGGAAGATGATGTGGTTGGATTTGACGAAGAAGCAGATATTGTAATCAAACGCCTTCTTGGAGAATCAAATCGTCTAGAAGTTGTTCCAGTTGTTGGTATGCCTGGTCTCGGCAAAACGACCCTAGCAAATAAAATATACAAGCATCCTAAAATCGGGTATGAATTTTTTACTCGTATTTGGGTTTATGTATCTCAATCATACAGGAGAAGAGAATTATTTCTCAACATCATCAGCAAATTCACTCGAAATACGAAACAATATCATGGGATGTGTGAGGAGGATTTGGCTGATGAAATACAAGAATTCTTGGGAAAGGGAGGAAAATACTTGGTTGTCT TGGATGATGTATGGTCGGATGAAGCTTGGGAACGTATCAAGATAGCTTTCCCAAATAACAACAAACCGAATCGAGTATTGTTGACCACTCGAGATTCCAAAGTGGCTAAACAATGCACTCCCATA CCTCATGATTTAAAATTTCTGAGTGAAGATGAAAGTTGGATATTACTGGAGAAGAAGGTTTTTC ACAAGGATAAATGTCCTCCTGAATTGGTGGTACCATCTGGGAAGAGCATAGCAAAA

[0301] SEQ ID NO:41; Potato StNRC2, amino acid; NCBI Reference Sequence: XP_006359790.1

[0302]

[0303] SEQ ID NO: 42; Potato StNRC3, cDNA; NCBI Reference Sequence: XM_006362512.2

[0304] ATGGCGGATGTAGCAGTAAAGTTTTTAGTAGAAAATTTGATGCAATTACTAATCGACAACGCCGATTTGATTCTCGGAATCAAAGGCGAAGTTGAA AATCTACTCCGAGATCTCAATGACTTCAATGCTTTCCTCAAACAAGCTGCTAAATCCCGCAGGGAAAATGAGGTTTTGAAAGAAATGGTGAAGAAAATCAGAAAAGTGGTGAATGATGCTGAGGATTCAATTGATAAATTTGTGATTGAAGCTAAGAGACATGATGATAAAAACAAATTTGCTCAATGGTTTCATATTACTCATGTGGCTAGAGCAAAAGGGGTAGCAGATGAGATCAAAAGTATAAAGGAAAGAGTGAAGGAAATTAGAGAAAATGACGCTTATGGCCTTCAAGCGATAACTTTAGATGATAATTTCAATAGAGGTGATGAAGAGAGGAAGGCCCCTGTAGTTGAGGAAGATGATGTGGTTGGTTTTGATGATGAAGCAAAAATTGTAATTGATCGTC TCATTGGAGGATCAGATTATGTTGAGGTTGTGCCAGTTGTTGGTATGCCTGGTCTTGGTAAAACAACTTTGGCATATAAGATTTACAAGGATCCGAAGGTTGAGTATGAGTTCTTCACCCGCGTTTGGGTATATGTCTCTCAAACGTTCAAGAGAAGGGAAATATTTCTCAACATTATCAGCAAGTTCACTCGAAACACCAAACAATATCATGATACACCAGAGGATGACTTAGCAAATGAAGTGAAGGAGCTTCTTGGAAAAGGTGGAAAATATCTTATTGTTTTGGATGATGTGTGGACGATGGAAGCTTGGGATCGTATCAAAATTGCTTTCCCTAATAATGGTAAACGGAATAGAGTGTTGATGACCACGAGAGAATCAAATGTGGCTAAGTGTTGTAATGATAAA CCTCATGATCTTAAGTTTTTAACTGAAGATGAAAGTTGGGAGCTACTTGAGAAGAAGGTTTTTC ACAAGGAAAAGTGTCCACCTGAGTTAGAATTGCCCGGGAAAAGTATAGCTGAA

[0305] SEQ ID NO: 43; Potato StNRC3, amino acid; NCBI Reference Sequence: XP_006362574.1

[0306]

[0307]

[0308] SEQ ID NO:44; Potato NRC2 D315K , amino acids.

[0309] MANVAVEFLVENLMQLLRDNAELISGVKEAAESLLQDLNDFNAFLKQAAKCHINENEVLRELVKKIRTVVNSAEDAIDKFVIEAKLHKDKGMTRVLDLPHYKRVREVAGEIKAIRNKVKEIRQNDAIGLQALQDDDSSARGFEERKPPVVEEDDVVG FDEEADIVIKRLLGESNRLEVVPVVGMPGLGKTTLANKIYKHPKIGYEFFTRIWVYVSQSYRRRELFLNIISKFTRNTKQYHGMCEEDLADEIQEFLGKGGKYLVVLDDVWSDEAWERIKIAFPNNNKPNRVLLTTRDSKVAKQCTPIPHDLKFLSE KESWILLEKKVFHKDKCPPELVVPSGKSIAKKCKGLPLAIVVIAGALIGKGKTPREWKQVDDSVSEHLINRDHPENCNKLVQMSYDRLPYDLKACFLYCSAFPGGFQIPAWKLIRLWIAEGFIQYKGHLSLECKGEDNLNDLINRNLVMVMERTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGSEQYFPGKRELSTYRRLCIHSSVLDFISTKPSAEHVRSFLSFSSKKIEMPSADIPTIPKGFPLLRVLDVESINFSRFSKEFYQLYHLRYVAFSSDSIKILPKLMGELWNIQTIIINTQQRTLDIQANIWNMERLRHLHTNSSAKLPVPVAPKNSKVTLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKIAVLLDNKSAVSLKNVKRLEYLENLKLINDSSIQTGKLRLPPAYIFPTKLRKLTLLDTWLEWKDMSILGQLEHLEVLKMKENGFTGESWESTGGFCSLLVLWIERTNLVTWKASADDFPRLKHLVLICCDYLKEVPIALADIRSFQVMMLQNSTKTAAISARQIQAKKDNQTQQGTKNIAFKLSIFPPDL

[0310] SEQ ID NO:45; Potato NRC3 D315K , amino acids.

[0311] MADVAVKFLVENLMQLLIDNADLILGIKGEVENLLRDLNDFNAFLKQA AKSRRENEVLKEMVKKIRKVVNDAEDSIDKFVIEAKRHDDKNKFAQWFHITHVARAKGVADEIKSIKERVKEIRENDAYGLQAITLDDNFNRGDEERKAPVVEEDDVVGFDDEAKIVIDRLIGGSDYVEVVPVVGMPGLGKTTLAYKIYKDPKVEYEFFTRVWVYVSQTFKRREIFLNIISKFTRNTKQYHDTPEDDLANEVKELLGKGGKYLIVLDDVWTMEAWDRIKIAFPNNGKRNRVLMTTRESNVAKCCNDKPHDLKFLTE KESWELLEKKVFHKEKCPPELELPGKSIAEKCMGLPLAIVVIAGALIGKGKTTREWELVAASVREHLINRDPENCKKLVQMSYDRLPYDLKACFLYCGAFPGGSQIPAKKLIRLWIAEGFIQYQGPLALEDVAEDHLNDLVNRNLVMVMQRSCSGQIKICRVHDMLHEFCRHEAMTEEDLFQEIKQGQERSFPGKQELATYRRLCIHSGVPEFLSTKPSGEHVRSFLCVGSKKIDMPPNEIPSIPKAFPLLRVLDAESIKFNRFSREFFKLFHLRYIALSTDKIKTIPVDFGNLWNVQTLIVETQEATLDIKADIWNMTRLRHVCTNASATLPSTKRPKSSKGNLVNRCLQTLSTIAPECCTAEVFTRTPNLKKLGVRGKIDALLETSKDGSGSVLFSNIGKLACLEYLKLVNDTRISSKPLHLPPAYIFPQKLKKLSLVDTWFEWKDMSILGLLPDLEVLKLKENAFKGQSWEQEDGGFPRLQVLWIERTDLTSWKASSGNFPRLKHLALISCDKLEELPAELADVKNLQLIELQSSSESAARSARAILKRNQEKEQEGDKGTGFKLSIFPHDLGL

[0312] SEQ ID NO:46; Tomato, SlNRC1 cDNA; NCBI Reference Sequence: NM_001247273.

[0313] ATGGTTGATGTAGGGGTTGAATTTCTGTTAGAGAACTTGAAGCAATTGGTACTGGACAATGTGGAGTTAATCGGAGGAGCTAAAGATGAAATCGAGAATCTGCGTGATGATTTGAGTGAATTCAATGCCTTTCTCAAGCAAGCTGCAATGGTCCGCAGCGAAAACCCAGTTCTCAAAGAACTAGTGAGGAGTATCAGAAAAGTGGTGAATCGTGCTGAAGATGCTGTTGATAAATTTGTAATTGAAGCTAAAGTTCATAAAGACAAAGGGTTTAAAGGGGTTTTCGATAAACCTGGACATTATAGAAGAGTGAGGGATGC AGCTGTGGAGATTAAAGGTATCAGAGATAATAAGAGAGAAATTCGGCAAAATAAGGCACATGGCCTTCAGGCTCTACTTCAAGATCATGATGATTCAATCAGCAGAGGTGGAGAAGAGAGACAGCCTCCTGTGGTTGAGGAAGATGATGTGGTGGGCTTT GACGATGAGGCGCAGACGGTAATCGACCGTCTTCTTGAAGGATCAGGTGATTTAGAGGTTATTCCAGTAGTTGGAATGCCTGGTCTTGGCAAAACTACACTAGCCACTAAGATCTTCAAGCATCCGAAGATTGAGTACGAGTTCTTTACTAGACTTTGGC TTTACGTTTCCCAATCATACAAGACAAGAGAATTATATCTTAACATCATCAGTAAATTCACCGGAAACACCAAACATTGCCGTGATATGTCTGAAAAGGATTTAGCTCTTAAGGTACAAGAGATTTTGGAAGAAGGAGGA AAATACTTGATTGTCTTGGATGATGTCTGGTCGACAGATGCTTGGGATCGTATCAAGATTGCTTTCCCGAAATTGACAAGGGCAATAGAGTATTGTTGACTACTCGAGACCACCGTGTTGCAAGATATTGCAATAGGAGT CCACATGATTTAAAATTTCTGACTGATGAAGAGAGTTGGATTTTACTGGAGAAAAGAG CTTTTCACAAAGCTAAATGTCTCCCCGAATTGGAAACAAACGGAAAAAGCATAGCCAGG

[0314] SEQ ID NO: 47; Tomato, S1NRC1 amino acid; NCBI Reference Sequence: NP_001234202.

[0315]

[0316] SEQ ID NO: 48; Tomato, S1NRC2 cDNA; NCBI Reference Sequence: XM_004248750.

[0317] ATGGCGAACGTAGCAGTGGAATTTCTGGTTGAGAACTTGATGCAGT TGCTGCGGGACAACGTAGAGCTAATTAGTGGAGTTAAAGAGGCTGCTGAGAGTCTACTTCAAGATCTAAATGATTTCAATGCTTTTCTTAAGCAAGCTGCCAAGTGTCACATCAACGAGAACGAAGTCCTCAGAGAACTTGTTAAGAAAATAAGAACAGTCGTTAACTCTGCTGAAGATGCTATTGATAAATTTGTTATTGAAGCTAAGCTACACAA GGATAAGGGTGTCACCAGAGTATTAGATCTTCCCCATTATAAAAGAGTCAAAGAGGTAGCTGGTGAGATCAAAGCTATACGAAACAAAGTCAGAGAGATCCGACAGACTGATGCCATTGGACTTCAGGCCCTTCAAGATGATGATTTATCTGCTAGAGGTTCCGAAGAAAGAAAGCCTCCAGTGGTAGAGGAAGATGACGTGGTTGGATTTGACGAA GAAGCAGATATTGTAATCAACCGCCTTCTTGGAGAATCGAATCATCTAGAAGTTGTTCCAGTTGTTGGTATGCCTGGTCTCGGCAAAACGACCCTAGCAAATAAGATTTACAAGCATCCTAAAATCGGGTATGAGTTTTTTACTCGTATTTGGGTTTATGTGTCTCAATCATACAGGAGAAGAGAATTATTTCTCAACATCATCAGCAAATTCACTCGAAATACAAAACAATATCATGGGATGTGTGAGGAGGATTTGGCTGATGAAATACAAGAATTCTTGGGAAAGGGAGGAAAATACTTGGTTGTGTTGGATGACGTATGGTCGGATGAAGCTTGGGAACGTATCAAGATAGCATTCCCAAATAACAACAAACCGAATCGAGTATTGTTGACCACTCGAGATTCCAAAGTGGCTAAACAATGCAATCCCATA CCTCATGATCTAAAATTTCTGACTGAAGATGAAAGTTGGATATTACTGGAGAAGAAGGTTTTTC ACAAGGACAAATGTCCTCCTGAATTGGTACTATCTGGAAAGAGCATAGCAAAA

[0318] SEQ ID NO: 49; Tomato, S1NRC2 amino acid; NCBI Reference Sequence: XP_004248798.

[0319]

[0320]

[0321] SEQ ID NO: 50; Tomato, S1NRC3, cDNA; NCBI Reference Sequence: XM_004238900.4.

[0322] ATGGCGGATGTAGCAGTAAAGTTCTTATTAGAAAATTTGACGCAGTTACTAATCGACAACGCCGATTTGATTCTCGGAATCCAAGGCGAAGTTGAAAATCTACTCACAGATCTCAATTACTTTAATGCTTTCCTCAAAGAAGCTGCTAAATCCCGCAGGGAAAATGAGGTTTTGAAAGAATTGGTGAAGAAAATCAGAAAAGTTGTGAACGATGCTGAAGATTCGATTGATAAATTTGTGGTTGAAGCTAAGAGACATGATGATAAAAACAAATTTGCTCAATGGTTTCATATTACTCATGTGGCTAGAGCCAAAGGGGTAGCGGATGAGATCAAAAGTATAAGGGAAAGAGTGAAGGAAATTAGAGATAATGACGCTTATGGCCTTCAAGCTATAACTTTGGATGATAATTTCAACAGAGGTGACGAAGAGAGGAAGGCCCCTGTAGTTGAGGAAG ATGATGTGGTTGGTTTTGATGATGAAGCAAAAACTGTAATTGATCGTCTCATCGGAGGATCAGACTATGTTGAGGTTGTGCCAGTTGTTGGTATGCCTGGTCTTGGTAAAACAACTTTGGCATATAAGATTTACAAGGATCCAAAGGTTGAGTATGAGTTCTTCACCCGCGTTTGGGTATATGTCTCTCAAACATTCAAGAGAAGGGAAATATTTCTCAACATTATCAGCAAGTTTACTCGAAACACCAAACAATATGATGATACACCAGAGGATGACTTAGCAAATGAAGTGAAGGAGCTTCTTGGAAAAGGTGGAAAATATCTTATTGTTTTGGATGATGTGTGGACGATGGAAGCTTGGGATCGTATCAAAATTGCTTTCCCTAATAATGGTAAACGAAATAGAGTGTTGATGACCACGAGACAATCAAATGTGGCGAAGCGTTGTAATGATAAA CCTCATGATCTTAAGTTTTTAACAAAAGATGAAAGTTGGGAGCTACTTGAGAAGAAGGTTTTTCA CAAGGAAAAGTGTCCACCTGAGTTAGAATTACCCGGGATAAGTATAGCCGAA

[0323] SEQ ID NO:51; Tomato, SlNRC3, Amino Acid; NCBI Reference Sequence: XP_004238948.

[0324]

[0325] SEQ ID NO:52; Tomato NRC1 D316K , Amino Acid.

[0326] MVDVGVEFLLENLKQLVLDNVELIGGAKDEIENLRDDLSEFNAFLKQAAMVRSENPVLKELVRSIRKVVNRAEDAVDKFVIEAKVHKDKGFKGVFDKPGHYRRVRDAAVEIKGIRDKMREIRQNKAHGLQALLQDHDDSISRGGEERQPPVVEEDDVVGFDDEAQTVIDRLLEGSGDLEVIPVVGMPGLGKTTLATKIFKHPKIEYEFFTRLWLYVSQSYKTRELYLNIISKFTGNTKHCRDMSEKDLALKVQEILEEGGKYLIVLDDVWSTDAWDRIKIAFPKNDKGNRVLLTTRDHRVARYCNRSPHDLKFLT KEESWILLEKRAFHKAKCLPELETNGKSIARKCKGLPLAIVVIAGALIGKSKTIKEWEQVDQSVGEHFINRDQPNSCDKLVRMSYDVLPYD WKACFLYFGTFPRGYLIPARKLIRLWIAEGFIQYRGDLSPECKAEEYLNELVNRNLVMVMQRTVDGQIKTCRVHDMLYEFCWQEATTEENLFHEVKFGGEQSVREVSTHRRLCIHSSVVEFISKKPSGEHVRSFLCFSPEKIDTPPTVSANISKAFPLLRVFDTESIKINRFCKEFFQLYHLRYIAFSFDSIKVIPKHVGELWNVQTLIVNTQQINLDIQADILNMPRLRHLLTNTSAKLPALANPKTSKTTLVNQSLQTLSTIAPESCTEYVLSRAPNLKKLGIRGKIAKLMEPSQSVLLNNVKRLQFLENLKLINVGQIDQTQLRLPPASIFPTKLRKLTLLDTWLEWDDMSVLKQLENLQVLKLKDNAFKGENWELNDGGFPFLQVLCIERANLVSWNASGDHFPRLKHLHISCDKLEKIPIGLADICSLQVMDLRNSTKSAAKSAREIQAKKNKLQPAKSQKFELSVFPPDSDVQTAS

[0327] SEQ ID NO:53; Tomato NRC2 D315K , amino acids

[0328] MANVAVEFLVENLMQLLRDNVELISGVKEAAESLLQDLNDFNAFLKQAAKCHINENEVLRELVKKIRTVVNSAEDAIDKFVIEAKLHKDKGVTRVLDLPHYKRVKEVAGEIKAIRNKVREIRQTDAIGLQALQDDDLSARGSEERKPPVVEEDDVVGFDEEADIVINRLLGESNHLEVVPVVGMPGLGKTTLANKIYKHPKIGYEFFTRIWVYVSQSYRRRELFLNIISKFTRNTKQYHGMCEEDLADEIQEFLGKGGKYLVVLDDVWSDEAWERIKIAFPNNNKPNRVLLTTRDSKVAKQCNPIPHDLKFLTE KESWILLEKKVFHKDKCPPELVLSGKSIAKKCKGLPLAIVVIAGALIGKGKTPREWKQVDDSVSEHLINRDHPENCNKLVQMSYDRLPYDLKACFLYCSAFPGGFQIPAWKLIRLWIAEGFIQYKGHLSLECKGEDNLNDLINRNLVMVMERTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGSEQYFPGKRELSTYRRLCIHSSVLDFFSTKPSAEHVRSFLSFSSKKIEMPSADIPTIPKGFPLLRVLDVESINFSRFSREFYQLYHLRYVAFSSDSIKILPKLMGELWNIQTIIINTQQRTLDIQANIWNMERLRHLHTNSSAKLPVPVAPKNSKVTLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKISVLLDNKSAASLKNVKRLEYLENLKLINDSSIQTSKLRLPPAYIFPTKLRKLTLLDTWLEWKDMSILGQLEHLEVLKMKENGFSGESWESTGGFCSLLVLWIERTNLVSWKASADDFPRLKHLVLICCDNLKEVPIALADIRSFQVMMLQNSTKTAAISARQIQAKKDNQTQQGTKNIAFKLSIFPPDL

[0329] SEQ ID NO:54; Tomato NRC3 D315K , amino acids

[0330] MADVAVKFLLENLTQLLIDNADLILGIQGEVENLLTDLNYFNAFLKEAA KSRRENEVLKELVKKIRKVVNDAEDSIDKFVVEAKRHDDKNKFAQWFHITHVARAKGVADEIKSIRERVKEIRDNDAYGLQAITLDDNFNRGDEERKAPVVEEDDVVGFDDEAKTVIDRLIGGSDYVEVVPVVGMPGLGKTTLAYKIYKDPKVEYEFFTRVWVYVSQTFKRREIFLNIISKFTRNTKQYDDTPEDDLANEVKELLGKGGKYLIVLDDVWTMEAWDRIKIAFPNNGKRNRVLMTTRQSNVAKRCNDKPHDLKFLTK DESWELLEKKVFHKEKCPPELELPGISIAEKCMGLPLAIVVIAGALIGKGKTTREWELVAASVGEHLINRDPENCKKLVQMSYDRLPYDLKACFLYCGAFPGGSQIPAKKLIRLWIAEGFIQYQGPLALEDVAEDHLNDLVNRNLVMVTQRSCSGQIKTCRVHDMLHEFCRHEAMMEENLFQEIKQGQERSFPGKQELATYRRLCIQSLIPEFLSMKPSGEHVRSFLCVGSKKIDMPPNEIPSIPKAFPLLRVLDAESIKFSRFSREFFKLFHLRYIALSTDKIKTIPADFGNLWNIQTLIVETQQATLDIKADIWNMTRLRHVCTNASATLPSTKRPKSSKDNLVNRCLQTLSTIAPECCTAEVFTRTPNLKKLGVRGKIDALLESSKDGSGSGLFSNIGKLGCLEYLKLVNDTRLSSKPLHLPPAYIFPQKLKKLSLVDTWFEWKDMSILGLLPELEVLKLKENAFKGQSWEQEDGGFPRLQVLWIERTDLTSWKASSGNFPRLKHLALISCDKLEELPAELADVKNLQLIELQSSSESAARSARAILKRNQEKEQDGDKGTGFKLSIFPHDLGL

[0331] SEQ ID NO:55; Potato NRC2 S317K , amino acids.

[0332] MANVAVEFLVENLMQLLRDNAELISGVKEAAESLLQDLNDFNAFLKQAAKCHINENEVLRELVKKIRTVVNSAEDAIDKFVIEAKLHKDKGMTRVLDLPHYKRVREVAGEIKAIRNKVKEIRQNDAIGLQALQDDDSSARGFEERKPPVVEEDDVVGFDEEADIVIKRLLGESNRLEVVPVVGMPGLGKTTLANKIYKHPKIGYEFFTRIWVYVSQSYRRRELFLNIISKFTRNTKQYHGMCEEDLADEIQEFLGKGGKYLVVLDDVWSDEAWERIKIAFPNNNKPNRVLLTTRDSKVAKQCTPIPHDLKFLSEDE KWILLEKKVFHKDKCPPELVVPSGKSIAKKCKGLPLAIVVIAGALIGKGKTPREWKQVDDSVSEHLINRDHPENCNKLVQMSYDRLPYDLKACFLYCSAFPGGFQIPAWKLIRLWIAEGFIQYKGHLSLECKGEDNLNDLINRNLVMVMERTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGSEQYFPGKRELSTYRRLCIHSSVLDFISTKPSAEHVRSFLSFSSKKIEMPSADIPTIPKGFPLLRVLDVESINFSRFSKEFYQLYHLRYVAFSSDSIKILPKLMGELWNIQTIIINTQQRTLDIQANIWNMERLRHLHTNSSAKLPVPVAPKNSKVTLVNQSL QTLSTIAPESCTEEVFARTPNLKKLGIRGKIAVLLDNKSAVSLKNVKRLEYLENLKLINDSSIQTGKLRLPPAYIFPTKLRKLTLLDTWLEWKDMSILGQLEHLEVLKMKENGFTGESWESTGGFCSLLVLWIERTNLVTWKASADDFPRLKHLVLICCDYLKEVPIALADIRSFQVMMLQNSTKTAAISARQIQAKKDNQTQQGTKNIAFKLSIFPPDL

[0333] SEQ ID NO:56; Potato NRC3 S317K , amino acids.

[0334] MADVAVKFLVENLMQLLIDNADLILGIKGEVENLLRDLNDFNAFLKQAAKSRRENEVLKEMVKKIRKVVNDAEDSIDKFVIEAKRHDDKNKFAQWFHITHVARAKGVADEIKSIKERVKEIRENDAYGLQAITLDDNFNRGDEERKAPVVEEDDVVGFDDEAKIVIDRLIGGSDYVEVVPVVGMPGLGKTTLAYKIYKDPKVEYEFFTRVWVYVSQTFKRREIFLNIISKFTRNTKQYHDTPEDDLANEVKELLGKGGKYLIVLDDVWTMEAWDRIKIAFPNNGKRNRVLMTTRESNVAKCCNDKPHDLKFLTEDE KWELLEKKVFHKEKCPPELELPGKSIAEKCMGLPLAIVVIAGALIGKGKTTREWELVAASVREHLINRDPENCKKLVQMSYDRLPYDLKACFLYCGAFPGGSQIPAKKLIRLWIAEGFIQYQGPLALEDVAEDHLNDLVNRNLVMVMQRSCSGQIKICRVHDMLHEFCRHEAMTEEDLFQEIKQGQERSFPGKQELATYRRLCIHSGVPEFLSTKPSGEHVRSFLCVGSKKIDMPPNEIPSIPKAFPLLRVLDAESIKFNRFSREFFKLFHLRYIALSTDKIKTIPVDFGNLWNVQTLIVETQEATLDIKADIWNMTRLRHVCTNASATLPSTKRPKSSKGNLVNRCLQTLSTIAPECCTAEVFTRTPNLKKLGVRGKIDALLETSKDGSGSVLFSNIGKLACLEYLKLVNDTRISSKPLHLPPAYIFPQKLKKLSLVDTWFEWKDMSILGLLPDLEVLKLKENAFKGQSWEQEDGGFPRLQVLWIERTDLTSWKASSGNFPRLKHLALISCDKLEELPAELADVKNLQLIELQSSSESAARSARAILKRNQEKEQEGDKGTGFKLSIFPHDLGL

[0335] SEQ ID NO:57; Tomato NRC1 E317K , amino acids.

[0336] MVDVGVEFLLENLKQLVLDNVELIGGAKDEIENLRDDLSEFNAFLKQAAMVRSENPVLKELVRSIRKVVNRAEDAVDKFVIEAKVHKDKGFKGVFDKPGHYRRVRDAAVEIKGIRDKMREIRQNKAHGLQALLQDHDDSISRGGEERQPPVVEEDDVVGFDDEAQTVIDRLLEGSGDLEVIPVVGMPGLGKTTLATKIFKHPKIEYEFFTRLWLYVSQSYKTRELYLNIISKFTGNTKHCRDMSEKDLALKVQEILEEGGKYLIVLDDVWSTDAWDRIKIAFPKNDKGNRVLLTTRDHRVARY CNRSPHDLKFLTD KESWILLEKRAFHKAKCLPELETNGKSIARKCKGLPLAIVVIAGALIGKSKTIKEWEQVDQSVGEHFINRDQPNSCDKLVRMSYDVLPYDWKACFLYFGTFPRGYLIPARKLIRLWIAEGFIQYRGDLSPECKAEEYLNELVNRNLVMVMQRTVDGQIKTCRVHDMLYEFCWQEATTEENLFHEVKFGGEQSVREVSTHRRLCIHSSVVEFISKKPSGEHVRSFLCFSPEKIDTPPTVSANISKAFPLLRVFDTESIKINRFCKEFFQLYHLRYIAFSFDSIKVIPKHVGELWNVQTLIVNTQQINLDIQADILNMPRLRHLLTNTSAKLPALANPKTSKTTLVNQSLQTLSTIAPESCTEYVLSRAPNLKKLGIRGKIAKLMEPSQSVLLNNVKRLQFLENLKLINVGQIDQTQLRLPPASIFPTKLRKLTLLDTWLEWDDMSVLKQLENLQVLKLKDNAFKGENWELNDGGFPFLQVLCIERANLVSWNASGDHFPRLKHLHISCDKLEKIPIGLADICSLQVMDLRNSTKSAAKSAREIQAKKNKLQPAKSQKFELSVFPPDSDVQTAS

[0337] SEQ ID NO:58; Tomato NRC2 S317K , amino acids

[0338] MANVAVEFLVENLMQLLRDNVELISGVKEAAESLLQDLNDFNAFLKQAAKCHINENEVLRELVKKIRTVVNSAEDAIDKFVIEAKLHKDKGVTRVLDLPHYKRVKEVAGEIKAIRNKVREIRQTDAIGLQALQDDDLSARGSEERKPPVVEEDDVVGFDEEADIVINRLLGESNHLEVVPVVGMPGLGKTTLANKIYKHPKIGYEFFTRIWVYVSQSYRRRELFLNIISKFTRNTKQYHGMCEEDLADEIQEFLGKGGKYLVVLDDVWSDEAWERIKIAFPNNNKPNRVLLTTRDSKVAKQCNPIPHDLKFLTEDE KWILLEKKVFHKDKCPPELVLSGKSIAKKCKGLPLAIVVIAGALIGKGKTPREWKQVDDSVSEHLINRDHPENCNKLVQMSYDRLPYDLKACFLYCSAFPGGFQIPAWKLIRLWIAEGFIQYKGHLSLECKGEDNLNDLINRNLVMVMERTSDGQIKTCRLHDMLHEFCRQEAMKEENLFQEIKLGSEQYFPGKRELSTYRRLCIHSSVLDFFSTKPSAEHVRSFLSFSSKKIEMPSADIPTIPKGFPLLRVLDVESINFSRFSREFYQLYHLRYVAFSSDSIKILPKLMGELWNIQTIIINTQQRTLDIQANIWNMERLRHLHTNSSAKLPVPVAPKNSKVTLVNQSLQTLSTIAPESCTEEVFARTPNLKKLGIRGKISVLLDNKSAASLKNVKRLEYLENLKLINDSSIQTSKLRLPPAYIFPTKLRKLTLLDTWLEWKDMSILGQLEHLEVLKMKENGFSGESWESTGGFCSLLVLWIERTNLVSWKASADDFPRLKHLVLICCDNLKEVPIALADIRSFQVMMLQNSTKTAAISARQIQAKKDNQTQQGTKNIAFKLSIFPPDL

[0339] SEQ ID NO:59; Tomato NRC3 S317K , amino acids

[0340] MADVAVKFLLENLTQLLIDNADLILGIQGEVENLLTDLNYFNAFLKEAAKSRRENEVLKELVKKIRKVVNDAEDSIDKFVVEAKRHDDKNKFAQWFHITHVARAKGVADEIKSIRERVKEIRDNDAYGLQAITLDDNFNRGDEERKAPVVEEDDVVGFDDEAKTVIDRLIGGSDYVEVVPVVGMPGLGKTTLAYKIYKDPKVEYEFFTRVWVYVSQTFKRREIFLNIISKFTRNTKQYDDTPEDDLANEVKELLGKGGKYLIVLDDVWTMEAWDRIKIAFPNNGKRNRVLMTTRQSNVAKRCNDKPHDLKFLTKDE KWELLEKKVFHKEKCPPELELPGISIAEKCMGLPLAIVVIAGALIGKGKTTREWELVAASVGEHLINRDPENCKKLVQMSYDRLPYDLKACFLYCGAFPGGSQIPAKKLIRLWIAEGFIQYQGPLALEDVAEDHLNDLVNRNLVMVTQRSCSGQIKTCRVHDMLHEFCRHEAMMEENLFQEIKQGQERSFPGKQELATYRRLCIQSLIPEFLSMKPSGEHVRSFLCVGSKKIDMPPNEIPSIPKAFPLLRVLDAESIKFSRFSREFFKLFHLRYIALSTDKIKTIPAD FGNLWNIQTLIVETQQATLDIKADIWNMTRLRHVCTNASATLPSTKRPKSSKDNLVNRCLQTLSTIAPECCTAEVFTRTPNLKKLGVRGKIDALLESKDGSGSGLFSNIGKLGCLEYLKLVNDTRLSSKPLHLPPAYIFPQK LKKLSLVDTWFEWKDMSILGLLPELEVLKLKENAFKGQSWEQEDGGFPRLQVLWIERTDLTSWKASSGNFPRLKHLALISCDKLEELPAELADVKNLQLIELQSSSESAARSARAILKRNQEKEQDGDKGTGFKLSIFPHDLGL

Claims

1. A genetically altered plant, plant part or plant cell thereof, wherein the plant, plant part or plant cell comprises at least one mutation in at least one nucleic acid sequence encoding a protein comprising a nucleotide binding domain and a leucine-rich repeat (NLR), wherein the NLR protein comprises an HD1 domain, and wherein 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 or plant cell thereof according to claim 1, wherein the at least one mutation reduces or prevents inhibition of an immune response initiated by a pathogen effector against the NLR protein.

3. A genetically altered plant, plant part or plant cell thereof, wherein the plant, plant part or plant cell comprises at least one mutation in at least one nucleic acid sequence encoding a protein comprising a nucleotide binding domain and a leucine-rich repeat (NLR), 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 a pathogen effector by the NLR protein.

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

5. A genetically altered plant, plant part or plant cell thereof, wherein the plant, plant part or plant cell expresses a nucleic acid construct comprising a nucleic acid sequence encoding a mutant NLR protein, wherein the NLR protein comprises an HD1 domain, and wherein the NLR protein comprises a mutation in one or more amino acids in the N-terminal portion of the HD1 domain.

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

7. The genetically altered plant, plant part thereof or plant cell according to claim 6, wherein the at least one mutation reduces or prevents inhibition of the oligomerization of the NRC protein into a complex that initiates an immune response by pathogen effectors.

8. The genetically altered plant, plant part 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. The genetically altered plant, plant part or plant cell thereof according to any one of the preceding claims, wherein the N-terminal part of the HD1 domain comprises no more than the N-terminal 42 amino acids of the HD1 domain, more preferably no more than the N-terminal 38 amino acids of the HD1 domain.

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

11. The genetically altered plant, plant part or plant cell according to 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. The genetically altered plant, plant part or plant cell thereof according to any one of the preceding claims, wherein the at least one mutation is located in the RNBS-C motif of the HD1 domain.

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

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

15. The genetically altered plant, plant part or plant cell according to any one of the preceding claims, wherein the mutation is a substitution.

16. The genetically altered plant, plant part or plant cell according to claim 15, wherein the substitution is at one or more positions in the NLR amino acid sequence, wherein the position is selected from position 315, 316 or 317 (or a homologous position in a homologous sequence) of any one of SEQ ID NO: 32, 34, 36, 41, 43, 47, 49 or 51 or a homolog or functional variant thereof.

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

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

19. The genetically altered plant, plant part or plant cell according to any one of claims 1 to 15, wherein the mutation is a replacement of all or part of the N-terminal portion of the HD1 domain with the corresponding portion of a second NLR protein, wherein the second NLR protein is not inhibited by the pathogen effector.

20. The genetically altered plant, plant part or plant cell according to claim 19, wherein the mutation replaces 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.

21. The genetically altered plant, plant part or plant cell according to claim 5, wherein the nucleic acid construct expresses a nucleic acid sequence encoding an NLR protein, wherein the NLR protein is selected from the group consisting of SEQ ID NO: 37, 38, 39, 44, 45, 52, 53, 54, 55, 56, 57, 58 or 59, and wherein preferably the regulatory sequence is operably linked to a regulatory sequence.

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

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

24. The genetically altered plant, plant part or plant cell thereof according to any one of the preceding claims, wherein the plant is a monocot or a dicot.

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

26. The genetically altered plant, plant part or plant cell thereof according to any one of the preceding claims, wherein the NLR protein is NRCl and / or NRC2 and / or NRC3.

27. The genetically altered plant part of any one of the preceding claims, wherein the plant part is a seed or a grain.

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

29. A method of providing 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, wherein the NLR protein comprises an HD1 domain, and wherein the NLR protein comprises a mutation in one or more amino acids 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 protein comprising a nucleotide binding domain and a leucine-rich repeat (NLR), wherein the NLR protein comprises an HD1 domain; and wherein the at least one mutation is 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, wherein the NLR protein comprises an HD1 domain, and wherein the NLR protein comprises a mutation in one or more amino acids in the N-terminal portion of the HD1 domain.

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

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

34. The method of any one 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 no more than the N-terminal 42 amino acids of the HD1 domain, more preferably no more than the N-terminal 38 amino acids of the HD1 domain.

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

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

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

40. The method of claim 39, wherein the variant has at least 60% overall sequence identity to the sequence defined by 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 to a hydrophilic amino acid and / or a positively charged amino acid.

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

44. The method of any one of claims 28 to 40, wherein the mutation is a replacement of all or a substantial portion 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. The method of any one of claims 44, wherein the mutation replaces 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.

46. ​​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 the group consisting of 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 the regulatory sequence is preferably operably linked to a regulatory sequence.

47. The method of any one of claims 28 to 46, wherein the pathogen is 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 monocot or a dicot.

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

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

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

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

54. The method of claim 53, wherein the N-terminal portion of the HD1 domain comprises no more than the N-terminal 42 amino acids of the HD1 domain, more preferably no more than the N-terminal 38 amino acids of the HD1 domain.

55. An isolated protein containing a nucleotide binding domain and a leucine-rich repeat (NLR), wherein the NLR protein comprises a sequence selected from SEQ ID NO: 37, 38, 39, 44, 45, 52, 53, 54, 55, 56, 57, 58, 59 or a functional variant or homolog thereof.

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