Chimeric resistance genes for wheat resistance against pathogens

Abstract

The present invention relates to a chimeric sensor NLR-ID protein obtained by replacing the integrated domain(s) of a native wheat sensor NLR-ID protein by at least one X domain, wherein said X domain binds to (i) a pathogen effector different from those recognized by the replaced integrated domain(s) of said native wheat sensor NLR-ID protein, (ii) an epitope different from the pathogen epitope recognized by the replaced integrated domain(s) of said native wheat sensor NLR-ID protein or (iii) a pathogen-induced plant component different from those recognized by the replaced integrated domain(s) of said native wheat sensor NLR-ID protein. The present invention also relates to the use said chimeric sensor NLR-ID protein for preventing a disease caused by a pathogen in a plant. The present invention also relates to nucleic acids and vectors suitable for obtaining a plant expressing said chimeric sensor NLR-ID protein, thereby conferring to the plant a resistance to a pathogen.

Description

[0001] CHIMERIC RESISTANCE GENES FOR WHEAT RESISTANCE AGAINST PATHOGENS

[0002] Field of the invention

[0003] The present invention relates to methods for obtaining wheat plants resistant to pathogens.

[0004] Background of the invention

[0005] Plant immunity is often triggered by the specific recognition of pathogen effectors by intracellular nucleotide-binding, leucine-rich repeat receptors (NLR). Plant NLRs contain an N-terminal signaling domain that is mostly represented by either a T ol l-i nterleuki n 1 receptor (TIR) domain or a coiled coil (CC) domain.

[0006] Recent studies demonstrated that NLRs with non-canonical domain architectures also play an important role in plant immunity. These natural chimeric immune receptors arise from fusions between NLRs and additional integrated domains (ID) to form NLR-IDs.

[0007] Given that many integrated domains show homology to molecules required for immune responses, integrated domains are generally thought to have derived from plant proteins targeted by pathogen-derived effector proteins. The integrated domains then act as baits for effector recognition within NLRs. In the literature, integrated domains are also described as “integrated decoys” or “integrated sensors”.

[0008] In many cases, single NLR proteins are sufficient for both effector recognition and signaling activation. However, many paired NLRs have now been identified where both proteins are required to confer resistance against pathogens. In pair, the first NLR functionally specializes in detecting the presence of the pathogen (sensor NLR-ID) while the second executes the response (helper or executor NLR). Many sensor NLR-IDs act as baits and, upon effector binding, signal through paired NLRs to trigger defense signaling.

[0009] Liu et al. (2021 , PNAS, vol118, 1-9) inserted mutations in the integrated domain Heavy-metal-associated domain (HMA) of the rice RGA5 NLR-ID protein and obtained a mutated version of the RGA5 NLR-ID protein. Despite the structural similarity between the wild-type and the mutated HMA domains, the modifications led to an enhanced binding affinity to AvrPib, a non-corresponding effector, and blocked the interaction with AVR-Pia, the cognate effector.

[0010] Cesari et al. (2022, Nature communications, 13:1524) engineered the rice RGA5-HMA protein by introducing AVR-PikD binding residues of rice Pikp-1-HMA NLR protein to recognize AVR-PikD, while still recognizing native effectors AVR-Pia and AVR1-CO39. However, according to the authors, said modifications of RG5-HMA failed to confer extended disease resistance specificity against M. oryzae in the obtained transgenic rice plants.

[0011] Marchal et al. (2022, Essay in Biochemistry 66, 527) report several examples of engineering of rice and Arabidopsis NLR-ID proteins. However, the focus of the article is about domain improvement either via structure-guided mutagenesis or via domain shuffling. The authors of Bialas et al. (2021 eLife 10, e66961), in turn, focus on introducing specific amino acid substitutions to the HMA domain, to expand effector recognition.

[0012] Maidment et al. (2023, eLife 2023;12:e81123), focus on exchanging the HMA domain of the rice NLR receptor Pik-1 , that binds the Magnaporthe oryzae effector AVR-Pik, with a non-integrated cytoplasmic rice OsHIPP19 HMA domain, thus expanding effector recognition within the HMA domain family. The authors further introduce amino acid substitutions to the new HMA domain to avoid the autoactivation of the NLR receptor.

[0013] Wheat plants can be affected by many pathogens, in particular viruses, bacteria and fungi, often resulting in significant economic losses. Mainstream approaches to tackle these pathogenic infections in fields are often based on synthetic pesticides that are detrimental to the environment.

[0014] Alternative solutions to these phytosanitary treatments are needed for both economic and environmental considerations.

[0015] Description of the invention

[0016] The inventors created a system composed of a pair of NLR proteins consisting of (i) a helper (or ‘executor’) NLR and (ii) a chimeric sensor NLR-ID comprising a NLR backbone and a variable integrated domain (also referred to as “X domain”), for the development of plant-induced immunity against a wide range of new pathogens.

[0017] The chimeric sensor NLR-ID serves as generic sensor after fusion with a new integrated domain, which determines the specificity of ligand recognition. Advantageously, this pair of NLRs and the variability of the integrated domain can potentially confer resistance to any disease in a plant, for example in wheat.

[0018] By producing plants resistant to pathogens, the present invention offers an attractive and practical alternative to conventional treatments.

[0019] The new integrated domain may be selected from a wide range of possible interactors, for example interactors of pathogen effectors or interactors of pathogen-induced plant components, that can be identified via computational modelling and structural prediction programmes or from interactome studies using biological systems. Non-limitative examples of experimental approaches that can be used to discover new integrated domains are yeast- two-hybrid interaction assays, co-immunoprecipitation, isothermal titration calorimetry, surface plasmon resonance, microscale thermophoresis, fluorescence anisotropy, split enzyme-based systems or resonance energy transfer assays.

[0020] Non-limitative examples of computational modelling and structural prediction programmes that can be used to discover new integrated domains are in silico screening of predicted protein complexes (Evans et al., 2021 bioRxiv March 10, 2022), search for homologies via protein structural alignments (van Kempen et al., 2023 Nature Biotechnology 42; 243) and de novo design of protein structures (Watson et al., 2023 Nature 620, 089).

[0021] In principle, there are no limitations as to the variability of the new integrated domain and therefore, the method disclosed in this application can be extended to a variety of pathosystems.

[0022] Among plant genomes, NLR-IDs occur at low frequency across the NLR phylogeny (established from the conservation of the NB-ARC domain), but a small subset of clades, called Major Integration Clades (MICs), display a much higher proportion of NLR-IDs.

[0023] For example, the inventors found a high diversity of integrated domains in the MIC1 clade located at the Sm1 locus of the wheat chromosome 2B, which indicates repeated integrations of different domains in this clade of NLRs which has gained the capacity to “integrate” new domains. Therefore, the inventors focused their attention to the MIC1- related NLR homeologs in the Sm1 region of the chromosome 2B in their search for sensor NLR-ID candidates.

[0024] In the wheat cultivar Renan genome (Aury et al., 2021, bioRxiv 2021.08.24.457458, release 2. 1 available from December 2021 from the Genoscope repository), the Sm1 locus (see PCT application WO2018 / 069343, particularly figure 2) is located between the positions 2B: 18,874,016 and 2B:19,292,368.

[0025] Thus, document WO2018 / 069343 describes genetic markers delimiting the region containing the Sm1 locus conferring resistance to Orange wheat blossom midge (OWBM) in wheat and reports that this locus contains a NLR (here named TaSmla) and a NLR-ID (here named TaSml b, encoding a protein corresponding to SEQ ID NO: 26 (prior art designation RGA2)). However, this document does not report that these two genes function in pair and are both necessary to confer OWBM resistance.

[0026] It is worth noting that some wheat varieties harbor a single NLR pair (sensor NLR-ID and helper NLR as defined below) in chromosome 2B at said Sm1 locus (for example the varieties Norin 61 , Jagger, ArinaLrForand Mace) whereas other wheat varieties harbor two NLR-ID pairs (among others, Chinese Spring, CDC Stanley, CDC Landmark, and Renan).

[0027] The inventors found a high conservation of the NB-ARC domain across the NLR phylogeny (at least 80% amino acid sequence identity). Based on this observation, the inventors defined three homology clusters of NLR-ID homeologs (named after a representative wheat variety) at the Sm1 locus: the ‘Mace’ cluster, the ‘Renan’ cluster and the ‘ArinaLrFor’ cluster.

[0028] Within the NB-ARC domain, the conserved structural motif 1 subdomain comprises a functional P-loop domain that interacts with ATP and confer to said NB-ARC domain - and ultimately to the NLR-ID protein as a whole - its activity. The inventors grouped all NLR-IDs present at said Sm1 locus within a single cluster of NLR-IDs comprising a motif 1 subdomain of a sequence at least 80% identical to the amino acid sequence SEQ ID NO: 38. Examples of motif 1 subdomain sequences and corresponding consensus sequences drawn from these phylogeny analyses are shown in Table 1 below. Consensus sequence 1 of SEQ ID NO: 43 shows the conserved amino acids in motif 1 subdomain whereas consensus sequence 2 shows the amino acid variations within said motif 1 subdomain in three NLR- IDs representative of the three clusters mentioned above, TaMace2Bb, TaSml b and TaArinaLrFor2Bb, respectively.

[0029] Table 1

[0030] Further molecular, genetic and sequence analyses showed that the genes TaMace2Ba and TaMace2Bb present at the Sm1 locus of chromosome 2B of the wheat variety Mace are genetically linked NLRs in head-to-head orientation. Likewise, the Inventors highlighted that in the wheat variety Renan the genes TaSmla and TaSmlb present at said locus are also genetically linked NLRs in head-to-head orientation.

[0031] In Andersen et al. (2021 Front in Genetics 11 , 898) and Sarris et al. (2016 BMC Biology 14, 8), the authors explore the diversity of NLR-ID in Triticum aestivum cv. Chinese Spring or in plants in general, respectively. However, the authors do not identify the TaMace2Bb NLR-ID gene and do not report any HMA integrated domain as found in the native TaMace2Bb NLR-ID.

[0032] In Bailey et al. (2018 Genome Biology 19, 23), the authors particularly explore NLR- ID genetically linked to paired NLR in grasses. The authors identify 17 tandem NLR / NLR- ID pairs in Triticum aestivum cultivar Chinese spring. However, the authors do not identify the 2 pairs of tandem NLR / NLR-ID in the chromosome 2B at the Sm1 locus. The authors also do not report any HMA integrated domain as found in the native TaMace2Bb NLR-ID. The TaMace2Bb NLR-ID is therefore not described in this article.

[0033] Without being bound by example, the Inventors engineered the native wheat sensor NLR-ID protein TaMace2Bb of the wheat variety Mace to replace its native integrated domain Heavy-Metal-Associated (TaHMA) with an integrated domain isolated from a sensor NLR-ID protein from rice, namely the OsHMA domain, thus swapping effector recognition and promoting the development of new types of immunity in wheat plants.

[0034] This OsHMA domain is known to recognize AVR-Pia, an effector secreted by Magnaporthe oryzae, a fungus responsible for the blast disease in rice.

[0035] The Inventors also engineered said native wheat sensor NLR-ID protein TaMace2Bb of the wheat variety Mace to replace its native integrated domain Heavy-Metal Associated (TaHMA) with an integrated domain isolated from a sensor NLR-ID protein from wheat, namely the LSD-1 -Like zinc-finger protein (TaLOL2), thus swapping effector recognition and promoting the development of new types of immunity in wheat plants.

[0036] The TaLOL2 protein domain (containing zinc-finger domains) is known to recognize PstGSREI , an effector secreted by Puccinia striiformis f. sp. tritici (Pst), a fungus responsible for the stripe rust disease in wheat.

[0037] Transient assays were performed in Nicotiana benthamiana leaves and wheat protoplasts to confirm that these novel chimeric sensor NLR-ID constructs are functional in that they are capable of triggering an effector-dependent cell death (hypersensitive response or HR) leading to plant immunity in the presence of TaMace2Ba, the corresponding paired-NLR helper protein.

[0038] The present invention thus relates to a chimeric sensor NLR-ID protein comprising, from N-ter to C-ter, one coil-coiled (CC) domain, one nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB-ARC domain), one leucine-rich repeat (LRR) domain, one peptide linker and at least one X domain, wherein said CC domain, said NB-ARC domain, and said LRR domain originate from a native wheat sensor NLR-ID protein, wherein said X domain binds to (i) a pathogen effector different from those recognized by the integrated domain or at least one of the integrated domains of said native wheat sensor NLR-ID protein, (ii) an epitope different from the pathogen epitope recognized by the integrated domain or at least one of the integrated domains of said native wheat sensor NLR-ID protein or (iii) a pathogen-induced plant component different from those recognized by the integrated domain or at least one of the integrated domains of said native wheat sensor NLR-ID protein.

[0039] In several embodiments, the peptide linker preferably does not originate from said native wheat sensor NLR-ID protein.

[0040] The present invention particularly relates to a chimeric sensor NLR-ID protein, wherein said chimeric sensor NLR-ID protein is obtained by replacing at least one integrated domain of a native sensor wheat NLR-ID protein by at least one X domain and, optionally, by replacing the peptide linker of said native sensor wheat NLR-ID protein by another peptide linker, wherein said X domain binds to (i) a pathogen effector different from those recognized by said replaced integrated domain(s) of said native wheat sensor NLR-ID protein, (ii) an epitope different from the pathogen epitope recognized by said replaced integrated domain(s) of said native wheat sensor NLR-ID protein or (iii) a pathogen-induced plant component different from those recognized by the replaced integrated domain(s) of said native wheat sensor NLR-ID protein.

[0041] The native wheat sensor NLR-ID protein as defined above is for example protein TaMace2Bb of SEQ ID NO: 2. The native wheat sensor NLR-ID protein as defined above may for example also be protein TaSmlb of SEQ ID NO: 26 or TaArinaLrFor2Bb of SEQ ID NO: 23.

[0042] The NB-ARC domain of the chimeric sensor NLR-ID protein as defined above may comprise a motif 1 subdomain of a sequence at least 80% identical to the amino acid sequence of the motif 1 subdomain domain of TaMace2Bb disclosed as sequence SEQ ID NO: 38.

[0043] The NB-ARC domain of the chimeric sensor NLR-ID protein as defined above preferably comprises a motif 1 subdomain consisting of the consensus sequence SEQ ID NO: 43, more preferably of sequence SEQ ID NO: 44.

[0044] In one embodiment, the sequence of the NB-ARC domain of the chimeric sensor NLR- ID protein as defined above may have at least 80% identity with the amino acid sequence SEQ ID NO: 35. This sequence corresponds to the NB-ARC domain of TaMace2Bb (also referred to as ‘Mace’ cluster).

[0045] In another embodiment, the sequence of the NB-ARC domain of the chimeric sensor NLR-ID protein as defined above may have at least 80% identity with the amino acid sequence SEQ ID NO: 36. This sequence corresponds to the NB-ARC domain of TaSml b (also referred to as ‘Renan’ cluster).

[0046] In yet another embodiment, the sequence of the NB-ARC domain of the chimeric sensor NLR-ID protein as defined above may have at least 80% identity with the amino acid sequence SEQ ID NO: 37. This sequence corresponds to the NB-ARC domain of TaArinaLrFor2Bb (also referred to as ArinaLrForcluster).

[0047] The X domain as defined above may for example be rice OsHMA domain, wheat TaLOL2 domain, wheat TaSnn3 domain, Arabidopsis NAC domain or maize MAP3K domain.

[0048] The peptide linker in the chimeric sensor NLR-ID protein may for example be the wheat peptide linker TaL, the barley peptide linker HvL, the rice peptide linker OsL, the maize peptide linker ZmL or the sorghum peptide linker SbL.

[0049] The present invention also relates to a NLR protein pair comprising: the chimeric sensor NLR-ID protein as defined above, and a helper NLR protein comprising, from N-ter to C-ter, one CC domain, one NB- ARC domain and one LRR domain, wherein said chimeric sensor NLR-ID protein binds to a pathogen effector or a pathogen-induced plant component through its X domain.

[0050] In the NLR protein pair as defined above, the chimeric sensor NLR-ID protein may be obtained by replacing the integrated domain of the native sensor NLR-ID protein TaMace2Bb by at least one X domain and the helper protein may be protein TaMace2Ba.

[0051] In the NLR protein pair as defined above, the chimeric sensor NLR-ID protein may be obtained by replacing at least one integrated domain of the native sensor NLR-ID protein TaSml b by at least one X domain and the helper protein may be protein TaSmla.

[0052] Finally, in the NLR protein pair as defined above, the chimeric sensor NLR-ID protein may be obtained by replacing at least one integrated domain of the native sensor NLR-ID protein TaArinaLrFor2Bb by at least one X domain and the helper protein may be protein TaArinaLrFor2Ba.

[0053] The present invention also relates to a nucleic acid encoding (i) (a) at least one chimeric sensor NLR-ID protein as defined above, (b) at least one X domain of the chimeric sensor NLR-ID protein as defined above or (c) the peptide linker and at least one X domain of the chimeric sensor NLR-ID protein as defined above and (ii), optionally, at least one helper NLR protein of the NLR protein pair as defined above.

[0054] The nucleic acid as defined above may comprise a first nucleic sequence encoding the chimeric sensor NLR-ID protein as defined above and a second nucleic sequence encoding the helper NLR protein as defined above, wherein said first and second sequences are preferably in head-to-head orientation. The present invention also relates to a vector comprising at least one nucleic acid as defined above.

[0055] Another object of the invention is a plant cell, plant or seed, wherein said plant cell, plant or seed comprises at least one chimeric sensor NLR-ID protein as defined above or at least one NLR protein pair as defined above.

[0056] Another object of the invention is a method for obtaining a cell as defined above, wherein said method comprises transforming a cell with at least one nucleic acid as defined above or at least one vector as defined above.

[0057] Another object of the invention is a method for obtaining a plant resistant to a pathogen, wherein said method comprises: transforming a plant cell or plant tissue with at least one nucleic acid as defined above or at least one vector as defined above, to obtain a transformed cell or a transformed tissue, and regenerating a plant from the transformed cell or transformed tissue.

[0058] The present invention also relates to the use of the chimeric sensor NLR-ID protein as defined above or of the NLR protein pair as defined above for preventing a disease caused by a pathogen in a plant.

[0059] Effector, NLR proteins, native sensor NLR-ID protein

[0060] By “effector”, it is herein meant either a pathogen effector or a pathogen-induced plant component.

[0061] By “pathogen effector”, it is herein meant a protein, oligopeptide or peptide produced by a pathogen that alter the structure and function of the plant host cells once in the cytoplasm of said cells, thereby promoting the colonization of the plant tissues by the pathogen. Without being bound by theory, pathogen effectors may be either directly injected into the host by the pathogen secretion systems (as in the case of pathogenic bacteria) or internalized from the extracellular environment by plant cell-dependent endocytosis (as in the case of fungi and oomycetes).

[0062] By “pathogen-induced plant component”, it is herein meant a plant component resulting from pathogen effector-induced modifications in a plant cell or tissue.

[0063] Non limitative examples of pathogen-induced plant components include components produced during the disruption of the cellular processes by a pathogen effector, for example components derived from the alteration of tissue structure, membrane or cell wall integrity, the degradation of membrane receptors or transcription factors, or the disruption of vesicular trafficking. By “NLR protein” it is herein meant Nucleotide-binding domain Leucine-rich Repeat protein, preferably comprising from N-terminal to C-terminal: one coil-coiled (CC) domain, one nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB-ARC domain) and one leucine-rich repeat (LRR) domain. A number of NLR proteins functions as intracellular receptors in a variant of plant immunity known as ‘Effector-Triggered Immunity’ or ETI. A single NLR or, as in the present invention, a couple of diversified NLR proteins, detect pathogen effector proteins either directly by binding the effector, or indirectly by recognizing effector-induced modifications to other plant components. Upon recognition of the pathogen effector, NLR activation and signaling triggers a series of defense responses leading to immunity, closely associated with the Hypersensitive Response (HR), a mechanism used by plants to stop or prevent the spread of infectious pathogens (Balint-Kurti et al, (2019, Molecular Plant Pathology, (20)8 1163-1178).

[0064] By “native sensor NLR-ID protein”, it is herein meant a native NLR protein that detects or recognizes and further signals pathogen effectors, either on its own or as part of a NLR protein pair in plant cells. Recent studies show that NLRs with non-canonical domain architectures play an important role in plant immunity in the presence of helper NLR via their native integrated domain (ID). These natural immune receptors arise from fusions between a NLR protein and one or more additional integrated domains to form NLR-IDs.

[0065] By “NLR protein pair”, it is meant a paired NLRs where both proteins are required to recognize and signal the presence of a pathogen effector, thus conferring resistance against the corresponding pathogen. In a pair, the first NLR is functionally specialized in the detection of the pathogen (“sensor NLR-ID”) while the second executes the response (“helper or executor NLR”).

[0066] By “native protein”, it is herein meant a protein, which is found in nature and has not been genetically modified by genetic manipulations.

[0067] A wheat native sensor NLR-ID protein is thus a sensor NLR-ID protein, which is naturally found in wheat.

[0068] Said wheat native sensor NLR-ID protein preferably comprises, from N-terminal to C- terminal: (i) one coil-coiled (CC) domain, (ii) one nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB-ARC domain), (iii) one leucine-rich repeat (LRR) domain, (iv) one peptide linker, and (v) at least one integrated domain (ID). Wheat sensor NLR-ID proteins generally comprise one or two integrated domains, depending on the variety. In most wheat native sensor NLR-ID proteins comprising two integrated domains, the two integrated domain are in the C-terminal end of the amino acid sequence, linked sequentially to the LRR domain (via a peptide linker). However, some wheat native sensor NLR-ID proteins, such as those found in the wheat varieties ‘Chinese Spring’ or ‘Stanley’, have one integrated domain at the N-terminal end, linked to the CC domain, and one integrated domain at the C-terminal end of the amino acid sequence, linked to the LRR domain (via a peptide linker).

[0069] The coil-coiled (CC) domain, the nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB-ARC domain) and the leucine-rich repeat (LRR) domain of the wheat sensor NLR-ID protein are also herein referred to as the “NLR backbone”.

[0070] Said wheat native sensor NLR-ID protein thus preferably comprise a NLR backbone, a peptide linker and at least one integrated domain (ID).

[0071] By “C-ter”, it is herein meant the C-terminus end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH) (also known as the carboxylterminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus).

[0072] By “N-ter”, it is herein meant the N-terminus start of an amino acid chain (protein or polypeptide), referring to the free amine group (-NH2) located at the end of a polypeptide (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide.

[0073] By “coil-coiled domain” or “CC domain”, it is herein meant the structural motif at the N-terminal end of the NLR protein in which 2-7 alpha-helices are coiled together, in particular like the strands of a rope.

[0074] By “nucleotide-binding and APAF-1 , R protein, CED-4 domain” or “NB-ARC domain”, it is herein meant the highly conserved structural motif of a NLR protein that contains a functional ATPase domain. Its nucleotide-binding state is proposed to regulate activity of the NLR protein as a molecular switch, cycling between ADP (repressed) and ATP (active) bound forms.

[0075] By “motif 1 subdomain” it is herein meant the highly conserved structural subdomain of a NB-ARC domain that contains a P-loop motif and that is 21 amino acid long. The motif 1 subdomain is for example described in Jupe et al., 2012 (BMC Genomics 13, 75). The P- loop is an important domain for the binding of the nucleotide triphosphate as it interacts directly with the phosphate of the bound nucleotide triphosphate, thus conferring to the NB- ARC domain, and ultimately to the NLR itself, its activity (Saraste et al., 1990, Trends in Biochemical Sciences, 15:11 , 430-434,). Motif 1 subdomain is a well-known subdomain of the NB-ARC domain as it is searched by NLR prediction software such as NLR-Parser (Steuernagel et al., 2015, Bioinformatics. 31 (10): 1665-7), NLR-Annotator (Steuernagel et al., 2020, Plant Physiology, 183:2, 468-482) and NLRtracker (Kourelis et al., 2021 , PLoS Biol. 19(10):e3001124).

[0076] Examples of motif 1 subdomains sequences comprised in the NB-ARC domain of sensor NLR-ID TaMace2Bb, sensor NLR-ID TaSml b and sensor NLR-ID TaArinaLrFor2Bb, as well as consensus sequences based on the phylogeny of the domain, are shown in Table 1 above.

[0077] By “leucine-rich repeat domain” or “LRR domain”, it is herein meant the structural motif of the NLR protein composed of repeating 20-30 amino acid stretches rich in the hydrophobic amino acid leucine and that forms an a / p horseshoe fold. In a sensor NLR-ID protein, the LRR domain comprises a common integration domain (CID) at its C-terminal end.

[0078] By “CID domain” or “common integration domain”, it is herein meant a conserved subdomain found in NLR proteins that possess an integrated domain, and that is located in the C-terminus of the NLR backbone, i.e., in the C-terminal of the LRR domain, before the peptide linker.

[0079] A native sensor NLR-ID protein may comprise one or two CID domain(s) in the C- terminus of the LRR domain.

[0080] The CID motif was identified as a conserved motif in an alignment of 55 protein sequences corresponding to NLRs from the MIC1 clade as described in additional file 7 of Bailey et al. (2018, Genome Biol 19, 23). The alignment then was used to train Hidden Markov Models and generate a hmm profile, as described in additional file 8 of Bailey et al. (2018 Genome Biol 19, 23). This hmm profile can be used to detect the presence of the CID motif in any protein sequence.

[0081] The CID domain of TaMace2Bb protein for example consists of sequence SEQ ID NO: 15. Interestingly, TaSml b comprises two CID domains set sequentially in tandem that consists of sequences SEQ ID NO: 41 and SEQ ID NO: 42. TaArinaLrFor2Bb comprises one CID domain, that may consist of SEQ ID NO: 53.

[0082] By “peptide linker”, it is herein meant an amino acid sequence located between the Common Integration Domain (CID) and the integrated domain (ID) of a sensor NLR-ID protein. When a native sensor NLR-ID protein comprises two or more IDs, said protein generally comprises a peptide linker in N-ter of each ID.

[0083] By “integrated domain” or “ID”, it is herein meant a domain of a sensor NLR-ID protein that recognizes directly or indirectly a pathogen effector. The integrated domain may indeed recognize a pathogen effector (i.e., direct recognition) or a pathogen-induced plant component (i.e., indirect recognition). The integrated domain is a non-canonical NLR domain.

[0084] By “native integrated domain” or “native ID”, it is meant an integrated domain of a native sensor NLR-ID protein, which recognizes, directly or indirectly, a pathogen effector. The native integrated domain thus confers effector- recog nition capacity to said native sensor NLR-ID protein. By “non-canonical", it is herein meant a domain of a sensor NLR-ID protein, which is not the CC domain, the NB-ARC domain, nor the LRR domain.

[0085] By the expression “recognize a pathogen effector”, it is herein meant that the integrated domain binds to the pathogen effector or binds to a pathogen-induced plant component.

[0086] The skilled person is able to determine the location of each of these different domains and of the peptide linker in a NLR protein, in particular in a sensor NLR-ID protein, from its amino acid sequence, via bioinformatic tools, for example by looking at the CC, NB-ARC, ID NLR-Tracker RefPlantNLR described in Kourelis et al. (2021 , PLoS Biol 19(10): e3001124) or the LRR predictor tool described in Martin et al. (2020, Genes 2020, 11 , 286).

[0087] For example, in the native sensor NLR-ID protein TaMace2Bb of sequence SEQ ID NO: 2, amino acids 63 to 79 correspond to the coil-coiled (CC) domain, amino acids 115 to 465 correspond to the nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB-ARC domain), wherein amino acids 152 to 172 correspond to the motif 1 subdomain of the NB-ARC domain, amino acids 476 to 799 correspond to the leucine-rich repeat (LRR) domain, wherein amino acids 766 to 799 correspond to the CID, amino acids 800 to 869 correspond to the peptide linker, and amino acids 870 to 943 correspond to the integrated domain.

[0088] For example, in the native sensor NLR-ID protein TaSmlb of sequence SEQ ID NO: 26, amino acids 36 to 107 correspond to the coil-coiled (CC) domain,

[0089] - amino acids 141 to 492 correspond to the nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB-ARC domain), wherein amino acids 176 to 196 correspond to the motif 1 subdomain of the NB-ARC domain, amino acids 503 to 933 correspond to the leucine-rich repeat (LRR) domain, wherein amino acids 805 to 841 correspond to a first CID and amino acids 905 to 933 correspond to a second CID in tandem, amino acids 934 to 1045 correspond to the first peptide linker, amino acids 1046 to 1324 correspond to the first integrated domain, amino acids 1325 to 1356 correspond to the second peptide linker, and amino acids 1357 to 1447 correspond to the second integrated domain.

[0090] For example, in the native sensor NLR-ID protein TaArinaLrFor2Bb of sequence SEQ ID NO: 23, - amino acids 137 to 342 correspond to the nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB-ARC domain), wherein amino acids 155 to 175 correspond to the motif 1 subdomain of the NB-ARC domain,

[0091] - amino acids 526 to 853 correspond to the leucine-rich repeat (LRR) domain, wherein amino acids 821 to 853 correspond to the CID,

[0092] - amino acids 854 to 920 correspond to the peptide linker, and

[0093] - amino acids 921 to 1003 correspond to the integrated domain.

[0094] Said wheat native sensor NLR-ID protein may be selected from the NLR proteins belonging to a Major Integration Clade (MIC) belonging to the same plant or different plant species (intra- or interspecific). Among plant genomes, sensor NLR-ID proteins occur at low frequency across the NLR phylogeny (established from the conservation of the NB-ARC domain), but a small subset of clades, called Major Integration Clades (MICs), display a much higher proportion of NLR-IDs.

[0095] The high diversity of IDs in these MIC clades indicates repeated integrations of different domains in this clade of NLRs which has gained the capacity to “integrate” new domains. Within these MIC clades, there are several examples of closely related NLRs that integrated variable IDs after a common integration sequence, named CID domain, which is found at the C-terminal end of the LRR domain. This conservation in architecture indicates a common ancestry for the NLR backbone and a selection process to integrate variable domains at the CID. The data on the Major Integration Clades are an important source of new integrated domains.

[0096] Non-limitative examples of wheat native sensor NLR-ID proteins include protein TaMace2Bb, protein TaSml b or protein TaArinaLrFor2Bb.

[0097] Protein TaMace2Bb for example consists of sequence SEQ ID NO: 2.

[0098] The integrated domain of TaMace2Bb is the TaHMA domain, for example of sequence SEQ ID NO: 22.

[0099] The protein TaMace2Bb (for example of sequence SEQ ID NO: 2) and its helper NLR protein TaMace2Ba (for example of sequence SEQ ID NO: 1) form a functional NLR pair. As mentioned above, our research disclosed that in the wheat variety Mace the genes TaMace2Bb and TaMace2Ba are located in a head-to-head orientation in the Sm1 locus of chromosome 2B.

[0100] Protein TaSml b for example consists of sequence SEQ ID NO: 26. The protein TaSmlb comprises two integrated domains: 1) a kinase domain (for example of sequence SEQ ID NO: 27) and 2) a Major Sperm Protein (MSP) (for example of sequence of SEQ ID NO: 28), separated by a peptide linker.

[0101] The sensor NLR-ID protein TaSmlb (for example of sequence SEQ ID NO: 26) and its helper NLR protein TaSmla (for example of sequence SEQ ID NO: 25) form a functional NLR pair. As mentioned above, our research also disclosed that in the wheat variety Renan the genes TaSmlb and TaSmla are located in a head-to-head orientation in the Sm1 locus of chromosome 2B.

[0102] Protein TaArinaLrFor2Bb for example consists of sequence SEQ ID NO: 23.

[0103] The protein T aArinaLrFor2Bb (for example of sequence SEQ I D NO: 23) and its helper NLR protein TaArinaLrFor2Ba (for example of sequence SEQ ID NO: 51) form a functional NLR pair. As for the two precedent pairs, our research disclosed that, in the wheat variety ArinaLrFor, the genes TaArinaLrFor2Bb and TaArinaLrFor2Ba are located in a head-to- head orientation in the Sm1 locus of chromosome 2B.

[0104] Limagrain’s patent applications derived from WQ2018069343 do mention the Sm1 locus in chromosome 2 of wheat in relation with resistance to orange wheat blossom midge (OWBM). The authors of Walkoniak et al, 2020 provide a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm1 (TaSmla), a gene associated with insect resistance.

[0105] However, none of these documents describes nor suggests the possibility of a swap of integrated domain(s) in TaMace2Bb, nor in TaSmlb nor in TaArinaLrFor2Bb to extend pathogen recognition to other pathogens or other pathosystems.

[0106] Chimeric sensor NLR-ID protein

[0107] The present invention thus relates to a sensor NLR-ID protein, which is a chimeric sensor NLR-ID protein.

[0108] By “chimeric sensor NLR-ID protein”, it is herein meant an engineered and thus non- naturally occurring sensor NLR-ID protein.

[0109] The chimeric sensor NLR-ID protein of the invention is part of a NLR protein pair as defined above. The other member of said NLR protein pair is a helper NLR protein as defined below.

[0110] The chimeric sensor NLR-ID protein as defined above is preferably obtained from a wheat native sensor NLR-ID protein, by replacing at least one integrated domain from said wheat native sensor NLR-ID protein by at least one X domain and, optionally, by replacing the peptide linker from said native sensor NLR-ID protein by another peptide linker, for example as defined below. If the native wheat sensor NLR-ID protein comprises only one integrated domain, the chimeric sensor NLR-ID protein is obtained by replacing the integrated domain from said wheat native sensor NLR-ID protein by at least one X domain, preferably by one X domain.

[0111] If the native wheat sensor NLR-ID protein comprises at least two integrated domains, the chimeric sensor NLR-ID protein is obtained by replacing at least one integrated domain from said wheat native sensor NLR-ID protein by at least one X domain. For example, one or at least two integrated domains of the wheat native sensor NLR-ID protein may be replaced by one X domain or at least two X domains. For example, each replaced integrated domain is replaced by one X domain. If the wheat native sensor NLR-ID protein comprises two integrated domains and only one integrated domain is replaced, the replaced integrated domain is preferably the integrated domain present in N-ter of the native wheat NLR-ID protein. In a preferred embodiment, the chimeric sensor NLR-ID protein is obtained by replacing all the integrated domains from said wheat native sensor NLR-ID protein by one X domain.

[0112] When the chimeric sensor NLR-ID protein comprises at least two X domains, said X domains may be identical or different.

[0113] When the chimeric sensor NLR-ID protein comprises at least two X domains, the chimeric sensor NLR-ID protein may comprise a peptide linker between two X domains; alternatively, there may be no peptide linker between two X domains.

[0114] The Inventors have surprisingly shown that native wheat sensor NLR-ID proteins from different wheat varieties, which are encoded by at least a gene located at the Sm1 locus of chromosome 2, share at least 80% amino acid sequence identity over the length of their respective motif 1 subdomains (see table 1).

[0115] The NB-ARC domain of the chimeric sensor NLR-ID protein as defined above may comprise a motif 1 subdomain consisting of the consensus sequence SEQ I D NO: 43, more preferably of sequence SEQ ID NO: 44.

[0116] The NB-ARC domain of the chimeric sensor NLR-ID protein as defined above may comprise a motif 1 subdomain at least 80% identical to sequence SEQ ID NO: 38, more preferably at least 85% identity, at least 90% identity, at least 95% identity with sequence SEQ ID NO: 38.

[0117] In one embodiment, the NB-ARC domain of the chimeric sensor NLR-ID protein as defined above may comprise a motif 1 subdomain of sequence SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40. The Inventors have also shown that native wheat sensor NLR-ID proteins from different wheat varieties, which are encoded by at least a gene located at the Sm1 locus of chromosome 2, share at least 80% amino acid sequence identity over the length of their NB-ARC domain, thus defining the three different NLR-ID gene clusters disclosed above.

[0118] The sequence of the NB-ARC domain of the chimeric sensor NLR-ID protein as defined above may thus have at least 80% identity with sequence SEQ ID NO: 35, more preferably at least 85% identity, at least 90% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity with sequence SEQ ID NO: 35. This sequence corresponds to the NB-ARC domain of TaMace2Bb (also referred to as ‘Mace’ cluster).

[0119] The sequence of the NB-ARC domain of the chimeric sensor NLR-ID protein as defined above may for example have at least 80% identity with sequence SEQ ID NO: 36, more preferably at least 85% identity, at least 90% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity with sequence SEQ ID NO: 36. This sequence corresponds to the NB-ARC domain of TaSml b (also referred to as ‘Renan’ cluster).

[0120] The sequence of the NB-ARC domain of the chimeric sensor NLR-ID protein as defined above may for example have at least 80% identity with sequence SEQ ID NO: 37, more preferably at least 85% identity, at least 90% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity with sequence SEQ ID NO: 37. This sequence corresponds to the NB-ARC domain of TaArinaLrFor2Bb (also referred to as ArinaLrFor cluster).

[0121] In one embodiment, the sequence of the NB-ARC domain of the chimeric sensor NLR- ID protein may comprise sequence SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37.

[0122] The chimeric sensor NLR-ID protein as defined above thus for example comprises a NLR backbone as defined above (in particular including a NB-ARC domain as defined above), a peptide linker and at least one X domain as defined below.

[0123] Thus, the chimeric sensor NLR-ID protein as defined above preferably comprises, in a N-ter to C-ter orientation: (i) a coil-coiled (CC) domain, a nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB-ARC domain), a leucine-rich repeat (LRR) domain (comprising a CID domain), which form the NLR backbone, and (ii) a peptide linker and (iii) at least one X domain.

[0124] By "X domain", it is herein meant a protein domain that binds to an epitope from an effector as defined above, i.e. , binds to an epitope of a pathogen effector as defined above or of a pathogen-induced plant component as defined above. The X domain present in a chimeric sensor NLR-ID protein is thus different from the integrated domain or at least one of the integrated domains found in the corresponding native sensor NLR-ID protein sharing the same NLR backbone.

[0125] The X domain as defined above preferably binds to (i) a pathogen effector different from those recognized by the integrated domain or at least one of the integrated domains of the corresponding native wheat sensor NLR-ID protein sharing the same NLR backbone (in particular a pathogen effector different from those recognized by the replaced integrated domain(s)), (ii) an epitope different from the pathogen effector epitope recognized by the integrated domain or at least one of the integrated domains of said corresponding native wheat sensor NLR-ID protein sharing the same NLR backbone (in particular an epitope different from the pathogen effector epitope recognized by the replaced integrated domain(s)) or (iii) a pathogen-induced plant component different from those recognized by the integrated domain or by at least one of the integrated domains of said corresponding native wheat sensor NLR-ID protein sharing the same NLR backbone (in particular a pathogen-induced plant component different from those recognized by the replaced integrated domain(s)).

[0126] When the native wheat sensor NLR-ID protein comprises one and only one integrated domain, the X domain as defined above preferably binds to (i) a pathogen effector different from those recognized by the integrated domain of the corresponding native wheat sensor NLR-ID protein sharing the same NLR backbone, (ii) an epitope different from the pathogen effector epitope recognized by the integrated domain of said corresponding native wheat sensor NLR-ID protein sharing the same NLR backbone or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of said corresponding native wheat sensor NLR-ID protein sharing the same NLR backbone.

[0127] When the native wheat sensor NLR-ID protein comprises at least two integrated domains, the X domain as defined above preferably binds to (i) a pathogen effector different from those recognized by at least one of the integrated domains of the corresponding native wheat sensor NLR-ID protein sharing the same NLR backbone (in particular a pathogen effector different from those recognized by the replaced integrated domain(s)), (ii) an epitope different from the pathogen effector epitope recognized by at least one of the integrated domains of said corresponding native wheat sensor NLR-ID protein sharing the same NLR backbone (in particular an epitope different from the pathogen effector epitope recognized by the replaced integrated domain(s)) or (iii) a pathogen-induced plant component different from those recognized by at least one of the integrated domains of said corresponding native wheat sensor NLR-ID protein sharing the same NLR backbone (in particular a pathogen-induced plant component different from those recognized by the replaced integrated domain(s)).

[0128] The present invention particularly relates to a chimeric sensor NLR-ID protein as defined above preferably comprising, from N-ter to C-ter, one coil-coiled (CC) domain, one nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB-ARC domain), one leucine- rich repeat (LRR) domain, one peptide linker and at least one X domain, wherein said CC domain, said NB-ARC domain, and said LRR domain originate from a native wheat sensor NLR-ID protein, wherein said X domain binds to (i) a pathogen effector different from those recognized by the integrated domain or at least one of the integrated domains of said native wheat sensor NLR-ID protein, (ii) an epitope different from the pathogen effector epitope recognized by the integrated domain or at least one of the integrated domains of said native wheat sensor NLR-ID protein or (iii) a pathogen-induced plant component different from those recognized by the integrated domain or at least one of the integrated domains of said native wheat sensor NLR-ID protein.

[0129] By the expression “said CC domain, said NB-ARC domain, and said LRR domain originate from a native wheat sensor NLR-ID protein", it is herein meant that said domains originate from the same native wheat sensor NLR-ID protein. Said domains may be identical to those of said native wheat sensor NLR-ID protein or differ by amino acid modification(s), such as deletion(s), addition(s) or substitution(s), for example by at least at least 2, at least 4, at least 6, at least 8 or at least 10 modifications. Said domains preferably differ by less than 20 amino acid modification(s), preferably less than 15 amino acid modifications, more preferably less than 10 amino acid modifications.

[0130] A domain which originates from a native wheat sensor NLR-ID protein may for example consists of an amino acid sequence at least 80% identical to those of the corresponding domain of the native wheat sensor NLR-ID protein, preferably at least 85% or at least 90% identical, more preferably at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to those of the corresponding domain of the native wheat sensor NLR-ID protein.

[0131] The X domain as defined above may thus be a pathogen epitope binding domain or a pathogen-induced plant component binding domain.

[0132] By “pathogen epitope”, it is herein meant the part of a pathogen that interacts with the integrated domain of a sensor NLR-ID protein or with the X domain of a chimeric sensor NLR-ID protein. By “pathogen epitope binding domain”, it is herein meant a domain, which specifically binds to a pathogen epitope.

[0133] By “pathogen-induced plant component binding domain”, it is herein meant a domain, which specifically binds to a pathogen-induced plant component as defined above.

[0134] The binding of the X domain of the chimeric sensor NLR-ID protein to its cognate effector allows triggering an immune response, in particular in the form of an hypersensitive response (HR; Balint-Kurti et al, (2019, Molecular Plant Pathology, (20)8 1163-1178).

[0135] The X domain preferably comprises: at least 7 amino acids, preferably at least 15 amino acids, more preferably at least 25 amino acids, and / or at most 600 amino acids, preferably at most 300 amino acids, more preferably at most 150 amino acids.

[0136] The X domain may be a native X domain, a non-native X domain, or a derivative thereof.

[0137] By “native X domain”, it is herein meant that the X domain originates from a native sensor NLR-ID from the same plant or a native sensor NLR-ID found in a different plant. The native X domain may for example be an integrated domain (ID) found in a wheat native sensor NLR-ID protein or an integrated domain (ID) found in a native sensor NLR-ID protein from any other plant, such as for example sorghum, maize, rice, barley, wheat relatives (such as Emmer wheat, Einkorn wheat, Durum wheat), Triticale, oat or rye.

[0138] Non-limitative examples of X domains are ensuing.

[0139] A native X domain as defined above may for example comprise or consist of wheat TaHMA domain, for example of SEQ ID NO: 22, rice OsHMA domain, for example of SEQ ID NO: 12, wheat kinase domain, for example of sequence SEQ ID NO: 27, wheat Major Sperm Protein (MSP) domain, for example of SEQ ID NO: 28, wheat Zinc-finger TaLOL2 domain, for example of sequence SEQ ID NO: 18, wheat kinase and MSP tandem domain (present in TaSnn3-D1 protein), for example of SEQ ID NO: 29, Arabidopsis NAC domain, for example of SEQ ID NO: 30 or maize MAP3K domain, for example of SEQ ID NO: 31.

[0140] OsHMA is the ID of rice OsPia-2 sensor NLR-ID protein. OsHMA recognizes AVR- Pia (for example of sequence SEQ ID NO: 8), an effector secreted by Magnaporthe oryzae, the fungus responsible for the rice blast disease. The wheat TaLO2 protein (containing zinc-finger domains) interacts with the Glycine-Serine-Rich Effector 1 (PstGSREI) effector (for example of sequence SEQ ID NO: 19), an effector secreted by Puccinia striiformis f. sp. tritici (Pst), the fungus responsible for wheat stripe rust disease.

[0141] The wheat TaSnn3-D1 protein (containing a kinase domain and a MSP domain) is involved in the resistance of wheat plants to Stagonospora nodorum, the causing agent of Septoria leaf spot.

[0142] The Arabidopsis AtOREI protein (containing a NAC domain) is a senescence- associated NAC transcription factor known to be targeted by the PeVD1 effector from the soil-borne vascular fungus Verticillium dahliae (Zhang et al., 2021 , Molecular plant, 14:11 , P1901-1917).

[0143] The maize ZmPia-2 protein (containing a MAPK3 domain) is involved in the resistance of maize plants against Colletotrichum graminicola, the causing agent of anthracnose in maize.

[0144] The X domain as defined above may alternatively be a non-native X domain.

[0145] By “non-native X domain”, it is herein meant a protein domain that is not found in native sensor NLR-ID proteins in plants.

[0146] Non-limitative examples of non-native X domain are: an X domain that is not naturally integrated into a NLR protein but is known to interact with a protein domain from a pathogen, for example as disclosed in plant-pathogen or effector interactome studies as described in Tamborski and Krasileva (2000, Annual Review of Plant Biology, Vol. 71 :355- 378) or any other of the 12614 pfam domains described in scientific pfam databases, for example in the INTERPRO database.

[0147] The X domain may also be a derivative of a native or a non-native X domain.

[0148] By “derivative of a native or non-native X domain”, it is herein meant a native or non- native X domain, which has been modified, for example which comprises amino acid modification(s). Such modification(s) can be performed, for example, to modify the three- dimensional structure of the X domain, improve the recognition or binding efficiency of the X domain to its cognate effector, or the overall efficiency of the NLR system.

[0149] A derivative of an X domain (either native or non-native) may for example comprise at least one modification (in particular at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 modifications), for example selected from the group consisting of an amino acid deletion, an amino acid addition and an amino acid substitution, by comparison to said native or non-native domain, while being still able to bind to the effector, at least as efficiently as said native or non-native domain. These modifications may for example allow improving the efficiency of a NLR protein pair, extending the recognition to other classes of effectors or simply reducing the risk of autoactivation of the protein pair, in particular resulting from the domain swap.

[0150] The skilled person can easily determine if a given domain is an X domain able to bind to a determined effector, in particular by assessing the interaction between said given domain and said determined effector, such as in a yeast two-hybrid assay or in a coimmunoprecipitation assay after transient expression in a heterologous system.

[0151] The skilled person can easily determine if a given X domain is an X domain able to trigger an immune response via any of the methods described in the examples below, for example by transient expression of constructs in N. benthamiana leaves or expression in wheat protoplasts.

[0152] The chimeric sensor NLR-ID protein as defined above may be obtained by any method well-known by the skilled person, such as genetic engineering. For example, the chimeric sensor NLR-ID protein may be expressed by a nucleic acid encoding said chimeric protein.

[0153] As defined above, a peptide linker is the amino acid sequence located between the at least one Common Integration Domain (CID) and the integrated domain (ID) of a sensor NLR-ID protein or between two integrated domains of a sensor NLR-ID.

[0154] The peptide linker of the chimeric sensor NLR-ID protein as defined above may be: a native peptide linker, an heterologous peptide linker, or an artificial peptide linker.

[0155] A native peptide linker may be the peptide linker corresponding to the native sensor chimeric NLR-ID protein or the peptide linker corresponding to the X domain, as disclosed below.

[0156] As the CC domain, NB-ARC domain and LRR domain, the peptide linker may be the peptide linker of the native sensor NLR-ID protein, i.e., the peptide linker corresponding to the native sensor NLR-ID protein. For example, if the chimeric sensor NLR-ID protein comprises the NLR backbone of TaMace2Bb protein, the peptide linker may be the peptide linker of the TaMace2Bb protein. As a further example, if the chimeric sensor NLR-ID protein comprises the NLR backbone of TaSmlb protein, the peptide linker may be the peptide linker of the TaSml b protein.

[0157] When the chimeric sensor NLR-ID protein comprises the NLR backbone of a native sensor NLR-ID protein A and a X domain which is the integrated domain of a native sensor NLR-ID protein B, the peptide linker of said chimeric sensor NLR-ID protein may be the peptide linker of said native sensor NLR-ID protein B, i.e., the peptide linker corresponding to the X domain. For example, if the chimeric sensor NLR-ID protein comprises the NLR backbone of TaMace2Bb protein and that the X domain is the wheat TaLOL2 domain of the TaLOL2 protein, the native peptide linker may be the peptide linker of TaLOL2 protein.

[0158] Alternatively, when the chimeric sensor NLR-ID protein comprises the NLR backbone of a native sensor NLR-ID protein A and a X domain which is the integrated domain of a native sensor NLR-ID protein B, the peptide linker of the chimeric sensor NLR- ID protein may be an heterologous peptide linker, i.e. a peptide linker of a sensor NLR-ID protein other than those of said protein A or said protein B or of a sensor NLR-ID protein from a different plant.

[0159] Alternatively, the peptide linker comprised in the chimeric sensor NLR-ID protein is an artificial peptide linker, such as a random peptide linker.

[0160] The peptide linker as defined above may be of any type or any length.

[0161] The peptide linker as defined above preferably comprises: at least 20 amino acids, preferably at least 30 amino acids, more preferably at least 50 amino acids, and / or at most 400 amino acids, preferably at most 300 amino acids, more preferably at most 250 amino acids.

[0162] Non-limitative examples of peptide linkers are the wheat peptide linker TaL of the sensor NLR ID protein TaMace2B, for example of sequence SEQ ID NO: 34, the maize peptide linker ZmL of the sensor NLR ID protein ZmPia-2, for example of sequence SEQ ID NO: 32, the rice peptide linker OsL of the OsPia-2 protein, for example of sequence SEQ ID NO: 14, the barley linker HvL, for example of sequence SEQ ID NO: 21 , or the sorghum peptide linker SbL, for example of sequence SEQ ID NO: 33.

[0163] Thus, in one non-limitative embodiment of the present invention, the chimeric sensor NLR-ID protein comprises a wheat NLR backbone (for example of sequence SEQ ID NO: 3, 24 or 52), a rice peptide linker OsL (for example of sequence SEQ ID NO: 14), and an X domain OsHMA from rice (for example of sequence SEQ ID NO: 12). In another embodiment of the present invention, the chimeric sensor NLR-ID protein comprises a wheat NLR backbone (for example of sequence SEQ ID NO: 3, 24 or 52), a barley peptide linker HvL (for example of sequence SEQ ID NO: 21), and an X domain TaLOL2 from wheat (for example of sequence SEQ ID NO: 18).

[0164] In yet another embodiment of the present invention, the chimeric sensor NLR-ID protein comprises a wheat NLR backbone (for example of sequence SEQ ID NO: 3, 24 or 52), a sorghum peptide linker SbL (for example of sequence SEQ ID NO: 33), and an X domain NAC from Arabidopsis (for example of sequence SEQ ID NO: 30).

[0165] In yet another possible embodiment of the present invention, the chimeric sensor NLR-ID protein comprises a wheat NLR backbone (for example of sequence SEQ ID NO: 3, 24 or 52), a maize peptide linker ZmL (for example of sequence SEQ ID NO: 32), and an X domain ZmMAPK3 from maize (for example of sequence SEQ ID NO: 31).

[0166] In another group of embodiments, the chimeric sensor NLR-ID protein as defined above may comprise, from N-ter to C-ter, a NLR backbone of sequence SEQ ID NO: 3, 24 or 52, a peptide linker as defined above and at least one X domain as defined above.

[0167] Thus, the chimeric sensor NLR-ID protein as defined above may be obtained from a wheat native sensor NLR-ID protein, by replacing both the peptide linker and at least one integrated domain from said wheat native sensor NLR-ID protein by another peptide linker as defined above and by at least one X domain as defined above.

[0168] Thus, in several of the embodiments described above, the chimeric sensor NLR-ID protein as defined above comprises, from N-ter to C-ter, a NLR backbone of sequence SEQ ID NO: 3 (i.e. TaMace2Bb backbone), a peptide linker as defined above and at least one X domain as defined above.

[0169] In one specific embodiment, the chimeric sensor NLR-ID protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 4 (i.e. TaMace2Bb-deltaL / ID-OsL / OsHMA).

[0170] In another specific embodiment, the chimeric sensor NLR-ID protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 17 (i.e. TaMace2Bb-deltaL / ID-HvL / TaLOL2).

[0171] Helper NLR protein

[0172] The present invention also relates to a helper NLR protein. By “helper NLR protein” or “executor NLR protein”, it is herein meant a NLR protein able to be activated by a sensor NLR-ID protein having detected a pathogen effector, thereby triggering a signaling pathway leading to pathogen resistance.

[0173] The helper NLR protein thus functions as central node in immunity as part of the NLR pair described above, by signaling the presence of a pathogen effector, once said effector has been detected by the sensor NLR-ID protein.

[0174] The helper NLR protein as defined above is preferably a wheat helper NLR protein.

[0175] The helper NLR protein as defined above preferably comprises, from N-ter to C-ter, one CC domain, one NB-ARC domain and one LRR domain.

[0176] The CC domain, NB-ARC domain and LRR domain are particularly as defined above in the section “chimeric sensor NLR-ID protein”.

[0177] Contrary to a sensor NLR-ID protein, a helper NLR protein does not comprise an integrated domain as defined above, nor a peptide linker as defined above, nor a common integration domain (CID) as defined above.

[0178] The helper NLR protein as defined above is preferably a native wheat helper NLR protein or a derivative of a native wheat helper protein.

[0179] A wheat native helper NLR protein is thus a helper NLR protein, which is naturally found in wheat.

[0180] A derivative of a helper NLR protein may comprise at least one modification (in particular at least 2, at least 4, at least 6 or at least 10 modifications), for example selected from the group consisting of an amino acid deletion, an amino acid addition or an amino acid substitution, by comparison to said helper NLR protein, while being still able to be activated by a sensor NLR-ID protein (in particular native or chimeric), thereby triggering a signaling pathway leading to pathogen resistance.

[0181] Non-limitative examples of wheat helper NLR protein include protein TaMace2Ba, for example of SEQ ID NO: 1 , protein TaSmla, for example of sequence SEQ ID NO: 25 or protein TaArinaLrFor2Ba, for example of SEQ ID NO: 51.

[0182] The Inventors have also shown that native helper NLR proteins from different wheat varieties, for example those encoded by a TaSmla gene, a TaMace2Ba gene or TaArinaLrFor2Ba gene located at the Sm1 locus on chromosome 2B also share at least 80% identity over the length of the amino acid sequences of their NB-ARC domains.

[0183] NLR protein pair

[0184] The present invention also relates to a NLR protein pair comprising: a chimeric sensor NLR-ID protein as defined above and a helper NLR protein as defined above.

[0185] In the NLR protein pair as defined above, the sensor NLR-ID protein thus recognizes a pathogen effector through its X domain and, upon recognition of said pathogen effector, activates the helper NLR protein, thereby triggering a signaling pathway leading to pathogen resistance.

[0186] The division of recognition and signaling functions in two distinct NLR proteins allows to diversify and to increase the specificity of pathogen effector recognition without compromising the strength of the immune response. Paired NLR proteins may function via negative regulation, whereby one NLR represses the activity of the second and detection of pathogen effectors relieves this repression to initiate immunity. Other paired NLR proteins may also function via receptor cooperation, whereby both receptors are required for a proper effector-triggered activation. The activation of the NLR pair may take place via a protein complex called ‘resistosome’ (Alexander Fdrderer et al, 2022, Current Opinion in Plant Biology). Said resistosome model has been developed to explain why numerous NLRs require oligomerization to function, either in the form of hetero-multimeric complexes or homo-multimeric complexes bound through their N-terminal CO domains.

[0187] A preferred NLR protein pair comprises: a chimeric sensor NLR-ID protein obtained by replacing the integrated domain of the native sensor NLR-ID protein TaMace2Bb (for example TaMace2Bb of sequence SEQ ID NO: 2), by at least one X domain as defined above, wherein said X domain recognizes (i) a pathogen effector different from the effector recognized by the integrated domain of the native sensor NLR-ID protein TaMace2Bb, (ii) an epitope different from those recognized by the integrated domain of the native sensor NLR-ID protein TaMace2Bb, or (iii) a pathogen- induced plant component different from those recognized by the integrated domain of the native sensor NLR-ID protein TaMace2Bb, and protein TaMace2Ba as a helper NLR protein, for example protein TaMace2Ba of sequence SEQ I D NO: 1.

[0188] Another preferred NLR protein pair comprises: a chimeric sensor NLR-ID protein obtained by replacing both the peptide linker and the integrated domain of the native sensor NLR-ID protein TaMace2Bb (for example TaMace2Bb of sequence SEQ ID NO: 2), by another peptide linker as defined above and at least one X domain as defined above, wherein said X domain recognizes (i) a pathogen effector different from the effector recognized by the integrated domain of the native sensor NLR-ID protein TaMace2Bb, (ii) an epitope different from those recognized by the integrated domain of protein TaMace2Bb, or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of the native sensor NLR-ID protein TaMace2Bb, and protein TaMace2Ba as a helper NLR protein, for example protein TaMace2Ba of sequence SEQ I D NO: 1.

[0189] Yet another preferred NLR protein pair comprises: a chimeric sensor NLR-ID protein obtained by replacing at least one integrated domain of the native sensor NLR-ID protein TaSml b (for example TaSmlb of sequence SEQ ID NO: 26), by at least one X domain as defined above, wherein said X domain recognizes (i) a pathogen effector different from the effector recognized by the replaced integrated domain(s) of the native sensor NLR-ID protein TaSml b, (ii) an epitope different from those recognized by the replaced integrated domain(s) of the native sensor NLR-ID protein TaSmlb, or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of the native sensor NLR-ID protein TaSml b, and protein TaSmla as a helper NLR protein, for example protein TaSmla of sequence SEQ ID NO: 25.

[0190] Yet another preferred NLR protein pair comprises: a chimeric sensor NLR-ID protein obtained by replacing both at least one peptide linker and at least one integrated domain of the native sensor NLR-ID protein TaSml b (for example TaSml b of sequence SEQ ID NO: 26), by another peptide linker as defined above and at least one X domain as defined above, wherein said X domain recognizes (i) a pathogen effector different from the effector recognized by the replaced integrated domain(s) of the native sensor NLR-ID protein TaSml b, (ii) an epitope different from those recognized by the replaced integrated domain(s) of the native sensor NLR-ID protein TaSmlb, or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of the native sensor NLR-ID protein TaSmlb, and protein TaSmla as a helper NLR protein, for example protein TaSmla of sequence SEQ ID NO: 25.

[0191] Yet another preferred NLR protein pair comprises: a chimeric sensor NLR-ID protein obtained by replacing the integrated domain of the native sensor NLR-ID protein TaArinaLrFor2Bb (for example TaArinaLrFor2Bb of sequence SEQ ID NO: 23), by at least one X domain as defined above, wherein said X domain recognizes (i) a pathogen effector different from the effector recognized by the replaced integrated domain(s) of the native sensor NLR-ID protein TaArinaLrFor2Bb, (ii) an epitope different from those recognized by the replaced integrated domain(s) of the native sensor NLR-ID protein TaArinaLrFor2Bb, or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of the native sensor NLR-ID protein TaArinaLrFor2Bb, and protein TaArinaLrFor2Ba as a helper NLR protein, for example protein TaArinaLrFor2Ba of sequence SEQ ID NO: 51.

[0192] Yet another preferred NLR protein pair comprises: a chimeric sensor NLR-ID protein obtained by replacing both the peptide linker and the integrated domain of the native sensor NLR-ID protein TaArinaLrFor2Bb (for example TaArinaLrFor2Bb of sequence SEQ ID NO: 23), by another peptide linker as defined above and at least one X domain as defined above, wherein said X domain recognizes (i) a pathogen effector different from the effector recognized by the replaced integrated domain(s) of the native sensor NLR-ID protein TaArinaLrFor2Bb, (ii) an epitope different from those recognized by the replaced integrated domain(s) of the native sensor NLR-ID protein TaArinaLrFor2Bb, or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of the native sensor NLR-ID protein TaArinaLrFor2Bb, and protein TaArinaLrFor2Ba as a helper NLR protein, for example protein TaArinaLrFor2Ba of sequence SEQ ID NO: 51.

[0193] Nucleic acid encoding the chimeric sensor NLR-ID protein and / or the helper NLR protein and vector comprising said nucleic acid.

[0194] The present invention also relates to a nucleic acid encoding (i) (a) at least one chimeric sensor NLR-ID protein as defined above, (b) at least one X domain of a chimeric sensor NLR-ID protein as defined above, or (c) the peptide linker and at least one X domain of a chimeric sensor NLR-ID protein as defined and (ii), optionally, at least one helper NLR protein of the NLR protein pair as defined above.

[0195] The present invention particularly relates to a nucleic acid as defined above, wherein said nucleic acid comprises a first nucleic sequence encoding the chimeric sensor NLR-ID protein of a NLR pair as defined above and a second nucleic sequence encoding the helper NLR protein of said NLR pair.

[0196] The first nucleic acid sequence and the second nucleic acid sequence are each under the control of a promoter and a terminator.

[0197] Any promoter and terminator suitable for expression in plants, for example in wheat, well known by the skilled person may be used.

[0198] Non-limitative examples of the promoter as defined above may be promoter Zmllbi (for example of SEQ ID NO: 5), promoter mas (for example of sequence SEQ ID NO: 9) or promoter 2x35S (for example of sequence SEQ ID NO: 11).

[0199] Non-limitative examples of the terminator as defined above may be terminator SbHSP (for example of sequence SEQ ID NO: 6), terminator AtNos (for example of sequence SEQ ID NO: 16), or terminator CaMV35S for example of sequence SEQ ID NO: 10).

[0200] The present invention also relates to a nucleic acid encoding at least one X domain of a chimeric sensor NLR-ID protein as defined above, said nucleic acid being in particular suitable for replacing the native ID domain or at least one native ID domain of the native sensor NLR-ID protein by said at least one X domain, in particular in a plant cell. Said nucleic acid particularly allows replacing at least one native ID domain of the native sensor NLR-ID protein by said at least one X domain by homologous recombination, for example through CRISPR-Cas technology.

[0201] The present invention also relates to a nucleic acid encoding the peptide linker and at least one_X domain of a chimeric sensor NLR-ID protein as defined above, said nucleic acid being in particular suitable for replacing the native peptide linker and the at least one native ID domain of the native sensor NLR-ID protein by said peptide linker and said at least one X domain, in particular in a plant cell. Said nucleic acid particularly allows replacing the native peptide linker and at least one native ID domain of the native sensor NLR-ID protein by said peptide linker and said at least one X domain by homologous recombination, for example through CRISPR-Cas technology.

[0202] The present invention also relates to a vector comprising at least one nucleic acid as defined above, encoding (i) (a) at least one chimeric sensor NLR-ID protein as defined above, (b) at least one X domain of a chimeric sensor NLR-ID protein as defined above, or (c) the peptide linker and at least X domain of a chimeric sensor NLR-ID protein as defined and (ii), optionally, at least one helper NLR protein of the NLR protein pair as defined above. In the nucleic acids as defined herein, when the chimeric sensor NLR-ID protein comprises one X domain, “a nucleic acid encoding at least one X domain” thus encodes the X domain of the chimeric sensor NLR-ID protein.

[0203] In the above nucleic acids as defined herein, when the chimeric sensor NLR-ID protein comprises at least two X domains, “a nucleic acid encoding at least one X domain” preferably encodes the X domains of the chimeric sensor NLR-ID protein.

[0204] The present invention particularly relates to a vector comprising at least one nucleic acid as defined above, encoding the chimeric sensor NLR-ID protein as defined above and / or the helper NLR protein as defined above.

[0205] The vector as defined above may comprise a nucleic acid as defined above, wherein said nucleic acid comprises a first nucleic sequence encoding the chimeric sensor NLR-ID protein of a NLR pair as defined above and, optionally, a second nucleic sequence encoding the helper NLR protein of said NLR pair.

[0206] The vector as defined above is particularly suitable for expression in a plant cell, for example in wheat.

[0207] The present invention also relates to a vector as defined above comprising a nucleic acid encoding at least one X domain of a chimeric sensor NLR-ID protein as defined above, said vector being particularly suitable for replacing at least one native ID domain of the native sensor NLR-ID protein by said at least one X domain, in particular in a plant cell, in particular by homologous recombination, for example through CRISPR-Cas technology.

[0208] The present invention also relates to a vector as defined above comprising a nucleic acid encoding the peptide linker and at least one X domain of a chimeric sensor NLR-ID protein as defined above, said vector being particularly suitable for replacing the native peptide linker and at least one native ID domain of the native sensor NLR-ID protein by said peptide linker and said at least one X domain, in particular in a plant cell, in particular by homologous recombination, for example through CRISPR-Cas technology.

[0209] The vector as defined above is particularly suitable for expression in wheat.

[0210] The vector as defined above is preferably suitable for integration of said nucleic acid in the genome of a plant, preferably a wheat plant, more preferably at a targeted location in the genome, in particular by homologous recombination.

[0211] As used herein, the term "nucleic acid" can refer to an isolated nucleic acid that is found artificially out of its native environment and is therefore not a product of nature. An isolated nucleic acid or polypeptide may be found in a purified form and / or in a non-native environment such as, for example, in a transgenic cell or bacterium.

[0212] As used herein, ‘genetic engineering’ or ‘recombinant DNA methods’ refer to a process of modifying a target nucleic acid, for example through the integration of exogenous nucleic acid into native nucleic acid, for example by transformation, or through gene editing, in particular CRISPR-Cas technology, TALEN- or ZFN- or meganuclease-mediated editing. DNA recombinant methods thus include the cleavage of a target nucleic acid, and the integration of an exogenous or native sequence via non-homologous end joining (NHEJ) or homologous recombination (HR).

[0213] Non-homologous end joining, and homologous recombination can insert an exogenous nucleic acid or donor polynucleotide into the target nucleic acid cleavage site. A donor polynucleotide can be a sequence that naturally occurs in the genome at a different site than the target nucleic acid cleavage site. A donor polynucleotide can be a sequence that does not naturally occur at the target nucleic acid cleavage site and that is integrated into the genome via NHEJ or HR. A vector can comprise the polynucleotides of the invention.

[0214] The modifications produced to the target DNA due to NHEJ and / or HR can lead to mutation(s) such as deletion(s), insertion(s), substitution(s), for example resulting in gene replacement, transgene insertion, nucleotide deletion, gene disruption, and / or gene mutation. The process of integrating non-native nucleic acid into genomic DNA can be referred to as genome engineering.

[0215] In one preferred embodiment, recombinant DNA methods are used on wheat plant cells deprived of the Sm1 locus of chromosome 2B to introduce both the helper NLR protein TaMace2Ba (for example of sequence SEQ ID NO: 1) and the chimeric sensor NLR-ID protein comprising the TaMace2Bb backbone (for example of sequence SEQ ID NO: 3), a peptide linker as defined above and at least one X domain as defined above.

[0216] In another preferred embodiment, recombinant DNA methods are used on wheat plant cells deprived of the Sm1 locus of chromosome 2B to introduce both the helper NLR protein TaSmla (for example of sequence SEQ ID NO: 25) and the chimeric sensor NLR-ID protein comprising the TaSml b backbone (for example of sequence SEQ ID NO: 24), a peptide linker as defined above and at least one X domain as defined above.

[0217] In another preferred embodiment, recombinant DNA methods are used on wheat plant cells deprived of the Sm1 locus of chromosome 2B to introduce both the helper NLR protein TaArinaLrFor2Ba (for example of sequence SEQ ID NO: 51) and the chimeric sensor NLR- ID protein comprising the TaArinaLrFor2Bb backbone (for example of sequence SEQ ID NO: 52), a peptide linker as defined above and at least one X domain as defined above.

[0218] In yet another preferred embodiment, recombinant DNA methods are used on plant cells from different plant species to introduce the helper NLR protein TaMace2Ba (for example of sequence SEQ ID NO: 1) and the chimeric sensor NLR-ID protein comprising the TaMace2Bb backbone (for example of sequence SEQ ID NO: 3), a peptide linker as defined above and at least one X domain as defined above.

[0219] In another preferred embodiment, recombinant DNA methods are used on plant cells from different plant species to introduce the helper NLR protein TaSmla (for example of sequence SEQ ID NO: 25) and the chimeric sensor NLR-ID protein comprising the TaSml b backbone (for example of sequence SEQ ID NO: 24), a peptide linker as defined above and at least one X domain as defined above.

[0220] In another preferred embodiment, recombinant DNA methods are used on plant cells from different plant species to introduce the helper NLR protein TaArinaLrFor2Ba (for example of sequence SEQ ID NO: 51) and the chimeric sensor NLR-ID protein comprising the TaArinaLrFor2Bb backbone (for example of sequence SEQ ID NO: 52), a peptide linker as defined above and at least one X domain as defined above.

[0221] In yet another preferred embodiment, recombinant DNA methods are used on plant varieties already containing a native sensor NLR-ID gene within the Sm1 locus to swap the native integrated domain or at least one native integrated domain of the native sensor NLR- ID protein (for example TaMace2Bb, in particular of sequence SEQ ID NO: 2, TaSml b, in particular of sequence SEQ ID NO: 26 or TaArinaLrFor2Bb, in particular of sequence SEQ ID NO: 23) by at least one X domain as defined above and, optionally, to swap the native peptide linker by another peptide linker, for example as defined above.

[0222] In some embodiments, the plant cell as defined above comprises a native sensor NLR-ID protein whose motif 1 subdomain has a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical or at least 95% identical to SEQ ID NO: 38.

[0223] In some embodiments, the plant cell as defined above comprises a native sensor NLR-ID protein whose motif 1 subdomain consists of consensus sequence SEQ ID NO: 43, preferably of consensus sequence SEQ ID NO: 44.

[0224] In some embodiments, the plant cell as defined above comprises a native sensor NLR-ID protein whose NB-ARC domain has a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical or at least 95% identical to SEQ ID NO: 35.

[0225] In some embodiments, the plant cell as defined above comprises a native sensor NLR-ID protein whose NB-ARC domain has a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical or at least 95% identical to SEQ ID NO: 36.

[0226] In some embodiments, the plant cell as defined above comprises a native sensor NLR-ID protein whose NB-ARC domain has a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical, or at least 95% identical to SEQ ID NO: 37.

[0227] In some specific embodiments, the plant cell as defined above comprises the native TaMace2Bb sensor NLR-ID protein (for example of sequence SEQ ID NO: 2) and / or the native TaSmlb sensor NLR-ID protein (for example of sequence SEQ ID NO: 26) and / or the native TaArinaLrFor2Bb sensor NLR-ID protein (for example of sequence SEQ ID NO: 23).

[0228] Cell, plant, seed and pathogen

[0229] As used herein, the term "plant" includes reference to whole plants.

[0230] As used herein, the term “plant part” includes plant organs (e.g., leaves, stems, roots... etc.), plant cells or seeds.

[0231] "Plant cell", as used herein may be isolated from or be found in suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, or microspores.

[0232] The class of plants which can be used includes both monocotyledonous and dicotyledonous plants.

[0233] ’Plant’ as used herein also encompasses crop plants and crop plants that are grown and harvested for food or profit: food crops, feed crops, fiber crops, oil crops, ornamental crops, and industrial crops. The term ‘plant’ as used herein also comprises wild relatives of said crop plants, for example wild relatives of wheat plants.

[0234] Said plant may also be any plant, such as wheat, sorghum, maize, rice or barley, as well as wheat relatives (for example Emmer wheat, Einkorn wheat or Durum wheat), triticale, oat or rye.

[0235] The present invention also relates to a plant cell, plant or seed, wherein said plant cell, plant or seed comprises at least one chimeric sensor NLR-ID protein as defined above or at least one NLR protein pair as defined above and wherein said plant is not obtained by means of an essentially biological process.

[0236] The present invention also relates to the progeny of a plant, wherein said progeny comprises at least one chimeric sensor NLR-ID protein as defined above or at least one NLR protein pair as defined above and wherein said plant is not obtained by means of an essentially biological process.

[0237] Said plant or seed is advantageously resistant to a pathogen, for example to rice blast caused by the pathogen Magnaporthe oryzae or wheat stripe rut caused by the pathogen Puccinia striiformis f. sp. tritici (Pst), wherein said pathogen expresses a pathogen effector recognized by the or at least one X domain of the chimeric sensor NLR-ID protein.

[0238] The cells of the plant or seed as defined above preferably comprise at least one nucleic acid as defined above encoding (i) (a) at least one chimeric sensor NLR-ID protein as defined above, (b) at least one X domain of a chimeric sensor NLR-ID protein as defined above, or (c) the peptide linker and at least one X domain of a chimeric sensor NLR-ID protein as defined and (ii), optionally, at least one helper NLR protein of the NLR protein pair as defined above.

[0239] Said nucleic acid is preferably inserted in the genome of the cells, in particular of said plant or seed. Alternatively, said nucleic acids are transiently expressed in plant cells or plant tissues, for example Nicotiana benthamiana cells or wheat protoplasts.

[0240] The plant or seed as defined above is preferably an agronomic plant (crop) or seed.

[0241] By the expression “agronomic plant or seed”, it is herein meant a plant or seed suitable for production on a large scale, in particular for human and animal food or for industrial purposes.

[0242] The cell, plant or seed may be a genetically modified or genetically edited wheat cell, plant or seed.

[0243] By “genetically modified” or “GM”, it is herein particularly meant that the integration of the modification is done at any location in the genome.

[0244] By “genetically edited” or “GE”, it is herein particularly meant that the modification is done at the locus, for example by generating a chimeric sensor NLR-ID from DNA fragments already present in the genome. In a GE cell, plant or seed, the wild type gene, if present in a single copy, for example the wild type gene encoding TaMace2Bb of SEQ ID NO: 2, is no more present. Alternatively, said GE modifications can be done on an artificial locus, wherein said locus comprises the genes of interest as genomic DNA.

[0245] By “plant disease”, it is herein meant the continuous disturbance of plants by causal pathogenic agents (pathogens) that results in an abnormal physiological process that disrupts the plant’s normal structure, growth, function, or other activities. It can be said that the interference of plants with a pathogen causes the physiological or biochemical imbalances that lead to disease (presence and / or spreading of symptoms) or disease resistance.

[0246] By “pathogen”, it is meant any organism or agent, such as fungi, bacteria, protists, nematodes, insects or viruses that cause plant disease. In the present invention, one scope of the inventors is to swap resistance to pathogens, in particular a resistance to pathogens causing serious damage in those plants because of a lack of innate immunity to said pathogens. External symptoms, particularly on leaves, can indicate the nature of the pathogen responsible for the disease and can be used as proxy for the activation (or not) of an immune response by the plant, in particular in the form of hypersensitive response (Balint-Kurti et al, 2019, Molecular Plant Pathology, (20)8 1163-1178). The present invention can also be used for improving resistance of a plant to a pathogen, by allowing the plant to recognize different epitopes of a given pathogen.

[0247] There are several types of effectors in plant-pathogen interactions, based on the mechanism triggered by their presence in the plant cell cytoplasm (for a review see Selin et al. 2016, Front. Microbiol. 7:600). Effectors that elicit an effector-triggered immunity (ETI) response are recognized by plant resistance proteins (R proteins), which are often the intracellular nucleotide-binding leucine rich repeat (NLR) proteins described above. Activation of ETI results in disease resistance and is usually associated with the programmed cell death of cells or tissues known as the hypersensitive response (HR).

[0248] By “hypersensitive response”, or HR, it is meant the localized presence of dead cells or tissue at the infection site after a successful activation of ETI-dependent immunity in plants. The HR response is a proxy for the resistance of said plant against the pathogen that produces the effector protein. HR is part of a host specific gene-for-gene interactions, where an effector, coined Avr (avirulence), is recognized by the cognate R-protein produced by the host plant.

[0249] In the claimed invention, disease resistance may be for example brought about by the presence of (i) the pair of NLR proteins TaMace2Ba (for example of sequence SEQ ID NO: 1) and the chimeric sensor NLR-ID protein comprising the backbone of native sensor NLR- ID protein TaMace2Bb (for example of sequence SEQ ID NO: 3), a peptide linker as defined above and at least one X domain as defined above or (ii) the pair of NLR proteins TaSmla (for example of sequence SEQ ID NO: 25) and the chimeric sensor NLR-ID protein comprising the backbone of native sensor NLR-ID protein TaSml b (for example of sequence SEQ ID NO: 24), a peptide linker as defined above and at least one X domain as defined above, or (iii) the pair of NLR proteins TaArinaLrFor2Ba (for example of sequence SEQ ID NO: 51) and the chimeric sensor NLR-ID protein comprising the backbone of native sensor NLR-ID protein TaArinaLrFor2Bb (for example of sequence SEQ ID NO: 52), a peptide linker as defined above and at least one X domain as defined above.

[0250] By ‘plant disease resistance”, it is meant the physical, biochemical and genetic components that protect plants from specific pathogens, in particular after the infection- induced response of the immune system, as in the case of the invention described herein. Said resistance may be visible in the form of a limited area of cell death on leaves, as part of the HR, or the reduction of pathogen growth on or in the plant (and hence a reduction of disease).

[0251] By “disease tolerance” it is meant plants that exhibit little disease damage despite substantial pathogen levels after infection at similar inoculum levels and in similar environments, when compared with other plants strains or genotypes.

[0252] By “plant disease susceptibility” it is meant plants that exhibit severe disease damage after infection by a pathogen strain at similar inoculum levels and in similar environments, when compared with other plant strains or genotypes.

[0253] Method for obtaining a cell or a plant resistant to a pathogen.

[0254] The present invention also relates to a method for obtaining a plant cell as defined above, wherein said method comprises transforming a plant cell with at least one nucleic acid as defined above or at least one vector as defined above.

[0255] The present invention also relates to a method for obtaining a plant resistant to a pathogen, wherein said method comprises:

[0256] - transforming a plant cell or plant tissue with at least one nucleic acid as defined above and at least one vector as defined above, to obtain a transformed plant cell or a transformed plant tissue,

[0257] - regenerating a plant from the transformed cell or transformed tissue, and

[0258] - optionally, assessing the expression of the chimeric sensor NLR-ID protein by the regenerated wheat plant.

[0259] The plant cell to be transformed may be a protoplast.

[0260] The plant tissue to be transformed may be an apical meristem, cotyledon, embryo, pollen and / or microspores.

[0261] The plant may be a wheat plant, but also, as defined above, any other plant, such as sorghum, maize, rice, barley, wheat relatives (for example Emmer wheat, Einkorn wheat or Durum wheat), triticale, oat or rye. Any technique suitable for plant cell or plant tissue transformation may be used, such as biolistic particle delivery, PEG transformation, electroporation or agrobacterium transgene delivery.

[0262] For agrobacterium transgene delivery, the vector is first transferred into Agrobacterium, to obtain a transformed Agrobacterium and the cell or tissue is then transformed with said transformed Agrobacterium. The Agrobacterium is preferably Agrobacterium tumefaciens.

[0263] When the plant cell or plant tissue to be transformed does not comprise (i.e. does not express) a native sensor NLR-ID protein, nor its paired helper NLR protein, the vector may comprise a nucleic acid encoding the chimeric sensor NLR-ID protein as defined above and the helper NLR protein as defined above. Alternatively, the nucleic acids encoding the chimeric sensor NLR-ID protein as defined above and its paired helper NLR protein as defined above may be provided in two different vectors.

[0264] When the plant cell or plant tissue to be transformed does not comprise (i.e. does not express) a native sensor TaMace2Bb protein, nor the helper TaMace2Ba protein, the vector may comprise (i) optionally, a nucleic acid that encodes the helper NLR protein TaMace2Ba (for example of sequence SEQ ID NO: 1) and (ii) a nucleic acid that encodes the chimeric sensor NLR-ID protein comprising the TaMace2Bb backbone (for example of sequence SEQ ID NO: 3), a peptide linker as defined above and at least one X domain as defined above. Said plant cell or plant tissue may be a wheat plant or wheat cell. Said vector may be suitable for target or non-targeted integration in the genome of the cell.

[0265] When the plant cell or plant tissue to be transformed does not comprise (i.e. does not express) a native sensor TaSmlb protein, nor the helper TaSmla protein, the vector may comprise (i) optionally, a nucleic acid that encodes the helper NLR protein TaSmla (for example of sequence SEQ ID NO: 25) and (ii) a nucleic acid that encodes the chimeric sensor NLR-ID protein comprising the TaSml b backbone (for example of sequence SEQ ID NO: 24), a peptide linker as defined above and at least one X domain as defined above. Said plant cell or plant tissue may be a wheat plant or wheat cell. Said vector may be suitable for target or non-targeted integration in the genome of the cell.

[0266] When the plant cell or plant tissue to be transformed does not comprise (i.e. does not express) a native sensor TaArinaLrFor2Bb protein, nor the helper TaArinaLrFor2Ba protein, the vector may comprise (i) optionally, a nucleic acid that encodes the helper NLR protein TaArinaLrFor2Ba (for example of sequence SEQ ID NO: 51) and (ii) a nucleic acid that encodes the chimeric sensor NLR-ID protein comprising the TaArinaLrFor2Bb backbone (for example of sequence SEQ ID NO: 52), a peptide linker as defined above and at least one X domain as defined above. Said plant cell or plant tissue may be a wheat plant or wheat cell. Said vector may be suitable for target or non-targeted integration in the genome of the cell.

[0267] The nucleic acids encoding the chimeric sensor NLR-ID protein as defined above and the helper NLR protein as defined above may be provided in the same vector or in two different vectors. When at least two vectors are used, transformation into the plant cell or tissue, for example into the cell or tissue, may be done concomitantly or sequentially.

[0268] When the plant cell or plant tissue to be transformed comprises (i.e. expresses) the native sensor NLR-ID protein and its paired helper NLR protein, the nucleic acid encodes (i) the X domain(s) of the chimeric sensor NLR-ID protein as defined above or (ii) the peptide linker and the X domain(s) of the chimeric sensor NLR-ID protein as defined above or the vector comprises (i) a nucleic acid encoding the X domain(s) of the chimeric sensor NLR- ID protein as defined above or (ii) a nucleic acid encoding the peptide linker and the X domain(s) of the chimeric sensor NLR-ID protein as defined above. Said nucleic acid or vector may be suitable for targeted integration at the location of the gene encoding the corresponding native sensor NLR-ID protein, for example via homologous recombination.

[0269] When the nucleic acid encodes the X domain(s) of the chimeric sensor NLR-ID protein as defined above, it preferably replaces the part of the endogenous gene encoding the integrated domain(s) of the native sensor NLR-ID protein, for example by homologous recombination or gene editing. There is therefore only a swap between the native integrated domain(s) of the sensor NLR-ID protein and the X domain(s) in the genome of the cell.

[0270] When the nucleic acid encodes the peptide linker and the X domain(s) of the chimeric sensor NLR-ID protein as defined above, it preferably replaces the part of the endogenous gene encoding the native peptide linker and the integrated domain(s) of the native sensor NLR-ID protein, for example by homologous recombination or gene editing. There is therefore a swap between the native integrated domain(s) and its cognate peptide linker with both the X domain(s) and the peptide linker of the chimeric sensor NLR-ID protein in the genome of the cell.

[0271] The method as defined above may also comprise transforming a plant cell or a plant tissue comprising the native sensor NLR-ID protein and its paired helper NLR protein with a nucleic acid or vector as defined above to express a chimeric sensor NLR-ID protein as defined above. A non-limitative example of such an embodiment may be the transformation of a plant cell or a plant tissue comprising the NLR sensor-1 D, for example TaMace2Bb, TaSml b or TaArinaLrFor2Bb, and the paired NLR helper NLR, for example TaMace2Ba, TaSmla or TaArinaLrFor2Ba, respectively, so that to express a chimeric sensor NLR-ID comprising at least one X domain as defined above, conferring an additional pathogen resistance to the same pathogen or a different pathogen and thus improving pathogen resistance in said plant cell or plant tissue.

[0272] Moreover, when the plant cell or plant tissue to be transformed comprises (i.e. expresses) the paired helper NLR protein, but not the paired native sensor NLR-ID protein, the vector may comprise a nucleic acid encoding the chimeric sensor NLR-ID protein as defined above. In a preferred embodiment, the vector may comprise a nucleic acid encoding a chimeric sensor NLR-ID protein comprising the NLR backbone (for example of sequence SEQ ID NO: 3, SEQ ID NO: 24 or SEQ ID NO: 52), a peptide linker as defined above and at least one X domain as defined above.

[0273] When the vector comprises a nucleic acid encoding at least one nucleic acid encoding (i) (a) the chimeric sensor NLR-ID protein as defined above, (b) at least one X domain of a chimeric sensor NLR-ID protein as defined above, or (c) the peptide linker and at least one X domain of a chimeric sensor NLR-ID protein as defined above and (ii), optionally, the helper NLR protein as defined above, the obtained transformed plant cell or obtained transformed plant tissue thus expresses said chimeric sensor NLR-ID protein and, optionally, said helper NLR protein.

[0274] Regeneration of a plant from a plant cell or plant tissue is well known by the skilled person.

[0275] In particular, the transformed plant cell or plant tissue may be placed in a culture medium suitable for growth.

[0276] The regeneration of a plant from the transformed plant cell or the transformed plant tissue may comprise:

[0277] - growing said transformed plant cell or transformed plant tissue to obtain a callus, and

[0278] - regeneration of shoots from the callus, in particular by somatic embryogenesis.

[0279] The growth of the transformed plant cell into a callus and the regeneration of shoots, in particular by somatic embryogenesis, are carried out in any suitable culture medium comprising plant growth regulators. Induction of somatic embryogenesis can be activated by genes encoding transcription factors, such as BABY BOOM, as described in Horstman et al. (2017, Plant Physiology, Vol. 175, pp. 848-857).

[0280] The regeneration of a plant from the transformed tissue may comprise regeneration of shoots. The regeneration of shoots from the transformed tissue may be carried out in any suitable culture medium comprising plant growth regulators.

[0281] The method may further comprise assessing the expression of said chimeric sensor NLR-ID protein and, optionally, of the helper NLR protein by the regenerated plant.

[0282] Assessing the expression of said chimeric sensor NLR-ID protein and, optionally, of said helper NLR protein by the regenerated plant may be performed by:

[0283] - assessing the resistance of the regenerated plant to a pathogen, wherein said pathogen expresses an effector pathogen recognized by at least one X domain of the chimeric sensor NLR-ID protein,

[0284] - detecting the expression of the chimeric sensor NLR-ID protein by the cells of the regenerated plant, in particular according to any method well-known by the skilled person, such as western-blot or an immunoassay, and / or

[0285] - assessing if the cells of the regenerated plant comprise a nucleic acid encoding said chimeric sensor NLR-ID protein, in particular according to any method well-known by the skilled person, such as sequencing or PCR.

[0286] The regenerated plant expresses said chimeric sensor NLR-ID protein and, optionally, said helper NLR protein if:

[0287] - the regenerated plant is resistant to said pathogen,

[0288] - the cells of the regenerated plant express said chimeric sensor NLR-ID protein, and / or

[0289] - the cells of the regenerated plant comprise a nucleic acid encoding said chimeric sensor NLR-ID protein.

[0290] Use of a chimeric sensor NLR-ID protein or a NLR protein pair

[0291] The present invention also relates to the use of a chimeric sensor NLR-ID protein as defined above or of a NLR protein pair as defined above for preventing a disease caused by a pathogen in a plant.

[0292] Said plant may be a wheat plant, but also, as defined above, any other plant, such as sorghum, maize, rice, barley, wheat relatives (for example Emmer wheat, Einkorn wheat or Durum wheat), triticale, oat or rye.

[0293] The present invention also relates to the use of a chimeric sensor NLR-ID protein as defined above or of a NLR protein pair as defined above for obtaining a plant or a plant seed resistant to a pathogen, in particular a wheat plant or wheat seed. Said plant may be wheat but also any other plant, such as sorghum, maize, rice, barley, wheat relatives (for example Emmer wheat, Einkorn wheat or Durum wheat), triticale, oat or rye.

[0294] Said chimeric sensor NLR-ID protein or NLR protein pair expressed by said plant preferably confers to said plant, in particular a wheat plant, a resistance to a pathogen or increases the tolerance of said plant, in particular a wheat plant, to a pathogen, in comparison with a plant not expressing said chimeric sensor NLR-ID protein or NLR protein pair. Said plant may be wheat but also any other plant, such as sorghum, maize, rice, barley, as well as wheat relatives (for example Emmer wheat, Einkorn wheat or Durum wheat), triticale, oat or rye.

[0295] In the use as defined above, said pathogen expresses a pathogen effector or results in the presence of a pathogen-induced plant component in the plant, wherein said pathogen effector or said pathogen-induced plant component is recognized by the or at least one X domain of the chimeric sensor NLR-ID protein.

[0296] The present invention particularly relates to the use as defined above, wherein said chimeric sensor NLR-ID protein or said NLR protein pair is expressed in a plant, in particular in wheat, in particular in the wheat plant or wheat seed, in other words in the cells of said plant or seed.

[0297] The invention will be further illustrated in the following figures and examples.

[0298] Brief description of the sequences

[0299] SEQ ID NO: 1 corresponds to the amino acid sequence of TaMace2Ba (a wheat helper NLR protein).

[0300] SEQ ID NO: 2 corresponds to the amino acid sequence of TaMace2Bb (a wheat sensor NLR-ID protein).

[0301] SEQ ID NO: 3 corresponds to the amino acid sequence of TaMAce2Bb-deltaL / ID (the NLR backbone of TaMace2Bb).

[0302] SEQ ID NO: 4 corresponds to the amino acid sequence of TaMace2Bb-deltaL / ID- OsL / OsHMA (also tagged ‘TaMace2Bb-deltaL / ID-OsL / HMA’ in text and figures).

[0303] SEQ ID NO: 5 corresponds to the nucleic acid sequence of ZmUbi promoter.

[0304] SEQ ID NO: 6 corresponds to the nucleic acid sequence of SbHSP terminator.

[0305] SEQ ID NO: 7 corresponds to the nucleic acid sequence of mCherry.

[0306] SEQ ID NO: 8 corresponds to the amino acid sequence of AVR-Pia (an effector of rice pathogen) with no signal peptide.

[0307] SEQ ID NO: 9 corresponds to the nucleic acid sequence of mas promoter. SEQ ID NO: 10 corresponds to the nucleic acid sequence of CaMV35S terminator.

[0308] SEQ ID NO: 11 corresponds to the nucleic acid sequence of 2x35S promoter.

[0309] SEQ ID NO: 12 corresponds to the amino acid sequence of the OsHMA domain (ID of rice OsPia-2)

[0310] SEQ ID NO: 13 corresponds to the amino acid sequence of OsPia-2 (a rice sensor NLR-ID protein).

[0311] SEQ ID NO: 14 corresponds to the amino acid sequence of rice peptide linker OsL.

[0312] SEQ ID NO: 15 corresponds to the amino acid sequence of the common integration domain (CID) of TaMace2Bb.

[0313] SEQ ID NO: 16 corresponds to the nucleic acid sequence of AtNos terminator.

[0314] SEQ ID NO: 17 corresponds to the amino acid sequence of TaMace2Bb-deltaL / ID- HvL-TaLOL2.

[0315] SEQ ID NO: 18 corresponds to the amino acid sequence of TaLOL2 domain full length sequence of wheat TraesCS5B02G054000.1).

[0316] SEQ ID NO: 19 corresponds to the amino acid sequence of stripe rust effector PstGSREI (derived from wheat pathogen Puccinia striiformis f. sp. tritici).

[0317] SEQ ID NO: 20 corresponds to the amino acid sequence of barley HORVU5Hr1G001060.1 NLR-ID protein.

[0318] SEQ ID NO: 21 corresponds to the amino acid sequence of the linker HvL (peptide linker of barley HORVU5Hr1G001060.1 NLR-ID protein).

[0319] SEQ ID NO: 22 corresponds to the amino acid sequence of the TaHMA domain (ID of TaMace2Bb).

[0320] SEQ ID NO: 23 corresponds to the amino acid sequence of TaArinaLrFor2Bb (a wheat sensor NLR-ID protein).

[0321] SEQ ID NO: 24 corresponds to the amino acid sequence of NLR backbone of TaSmlb.

[0322] SEQ ID NO: 25 corresponds to the amino acid sequence of TaSmla (a wheat helper NLR protein).

[0323] SEQ ID NO: 26 corresponds to the amino acid sequence of TaSmlb (a wheat sensor NLR-ID protein).

[0324] SEQ ID NO: 27 corresponds to the amino acid sequence of the kinase domain (one of the IDs of TaSmlb).

[0325] SEQ ID NO: 28 corresponds to the amino acid sequence of Major Sperm Protein (MSP) domain (one of the IDs of TaSmlb).

[0326] SEQ ID NO: 29 corresponds to the amino acid sequence of the kinase and Major Sperm Protein tandem domains present in TaSnn3-D1 protein. SEQ ID NO: 30 corresponds to the amino acid sequence of the NAC domain the Arabidopsis AtOREI protein (AT5G39610).

[0327] SEQ ID NO: 31 corresponds to the amino acid sequence of maize MAP3K (ID of maize ZmPia-2).

[0328] SEQ ID NO: 32 corresponds to the amino acid sequence of the maize peptide linker ZmL of ZmPia-2.

[0329] SEQ ID NO: 33 corresponds to the amino acid sequence of peptide linker SbL of the Sorghum bicolor SB02G021226.1 protein (GeneBank XP_002462195.1).

[0330] SEQ ID NO: 34 corresponds to the amino acid sequence of wheat peptide linker TaL of TaMace2Bb.

[0331] SEQ ID NO: 35 corresponds to the amino acid sequence of the NB-ARC domain of wheat TaMace2Bb.

[0332] SEQ ID NO: 36 corresponds to the amino acid sequence of the NB-ARC domain of TaSmlb.

[0333] SEQ ID NO: 37 corresponds to the amino acid sequence of the NB-ARC domain of TaArinaLrFor2Bb.

[0334] SEQ ID NO: 38 corresponds to the amino acid sequence of the motif 1 subdomain of TaMace2Bb.

[0335] SEQ ID NO: 39 corresponds to the amino acid sequence of the motif 1 subdomain of TaSmlb.

[0336] SEQ ID NO: 40 corresponds to the amino acid sequence of the motif 1 subdomain of TaArinaLrFor2Bb.

[0337] SEQ ID NO: 41 corresponds to the amino acid sequence of the first common integration domain (CID) present in TaSmlb.

[0338] SEQ ID NO: 42 corresponds to the amino acid sequence of the second common integration domain (CID) present in TaSmlb.

[0339] SEQ ID NO: 43 corresponds to a consensus sequence of the motif 1 subdomain (wherein each variable amino acid may be any amino acid).

[0340] SEQ ID NO: 44 corresponds to a consensus sequence of the motif 1 subdomain (wherein the alternative amino acids are indicated)

[0341] SEQ ID NO: 45 corresponds to the genomic sequence of TaArinaLrFor2Bb (a wheat sensor NLR-ID protein).

[0342] SEQ ID NO: 46 corresponds to the cDNA sequence of TaArinaLrFor2Bb (a wheat sensor NLR-ID protein).

[0343] SEQ ID NO: 47 corresponds to the genomic sequence of TaMace2Bb (encoding a wheat sensor NLR-ID protein). SEQ ID NO: 48 corresponds to the cDNA sequence of TaMace2Bb (encoding a wheat sensor NLR-ID protein).

[0344] SEQ ID NO: 49 corresponds to the genomic DNA sequence of TaSml b (encoding a wheat sensor NLR-ID protein).

[0345] SEQ ID NO: 50 corresponds to the cDNA sequence of TaSmlb (encoding a wheat sensor NLR-ID protein).

[0346] SEQ ID NO: 51 corresponds to the amino acid sequence of T aArinaLrFor2Ba (a wheat helper NLR protein).

[0347] SEQ ID NO: 52 corresponds to the amino acid sequence of the NLR backbone of TaArinaLrFor2Bb.

[0348] SEQ ID NO: 53 corresponds to the amino acid sequence of the common integration domain (CID) present in TaArinaLrFor2Bb.

[0349] SEQ ID NO: 54 corresponds to the amino acid sequence of Luciferase.

[0350] SEQ ID NO: 55 corresponds to the nucleotide sequence of the promoter 2x35S+TMV.

[0351] SEQ ID NO: 56 corresponds to the nucleotide sequence of the CaMV35S polyA terminator.

[0352] Description of the Figures

[0353] Figure 1 : Structure of the NLR constructs used in Example 1 : native helper NLR TaMace2Ba, designated as “TaMace2Ba” (SEQ ID NO: 1); native sensor NLR-ID TaMace2Bb, designated as “TaMace2Bb” (SEQ ID NO: 2); and chimeric sensor NLR-ID TaMace2Bb without its native linker and without its native ID but with the linker and ID from the rice sensor NLR-ID OsPia-2, designated as “TaMace2Bb-deltaL / ID-OsL / HMA” (SEQ ID NO: 4). The structure of the native rice sensor NLR-ID OsPia-2 (SEQ ID NO: 13) is also presented.

[0354] Figure 2: Effect of combinations of constructs on the immune response in wheat mesophyll leaf protoplasts. Luciferase assay was realized using isolated protoplasts cotransfected with the luciferase reporter construct and the six different combinations of constructs indicated. AVR-Pia, lacking its signal peptide, was used as candidate effector to trigger an immune response. mCherry (SEQ ID NO: 7) was used as a negative control for immune response, as we do not expect any of these combinations of constructs to respond to mCherry. Luciferase activity was determined 16 h post transfection as proxy for cell death.

[0355] For each sample, all of the data points are represented as dots plotted around the luciferase signal for visualization purposes. Each column represents a combination of constructs (labelled on the bottom). Statistical analysis of luciferase signals was performed using a one-way analysis of variance (ANOVA) followed by Tukey multiple comparisons. Adjusted p-value (P) of the test: *** P < 0.001 , ** P < 0.01 , * P < 0.05, n.s. non-significant.

[0356] Figure 3: Swapping the HMA integrated domain of TaMace2Bb with an HMA domain from rice redirects the immune response toward a rice blast effector in Nicotiana benthamiana leaves.

[0357] A. Structure of the NLR constructs used in Example 2: native helper NLR TaMace2Ba, designated as “TaMace2Ba” (SEQ ID NO: 1); native sensor NLR-ID TaMace2Bb, designated as “TaMace2Bb” (SEQ ID NO: 2); chimeric sensor NLR-ID TaMace2Bb without its native peptide linker and without its native ID, designated as “TaMace2Bb-deltaL / ID (SEQ ID NO: 3); and chimeric sensor NLR-ID TaMace2Bb without its native peptide linker and without its native ID but with the peptide linker and ID from the rice sensor NLR-ID OsPia-2, designated as “TaMace2Bb-deltaL / ID-OsL / HMA” (SEQ ID NO: 4). The structure of the native rice sensor NLR-ID OsPia-2 (SEQ ID NO: 13) is also presented.

[0358] B. Cell-death assay scoring represented as raw data points and violin plots for estimated densities. N. benthamiana leaves co- infiltrated with constructs from Figure 3A, and either Luciferase (SEQ ID NO: 54) or AVR-Pia (SEQ ID NO: 8). Cell-death was scored 5 days after infiltration. For each sample, all the data points are represented as dots plotted around the cell death score for visualization purposes. Each column represents a combination of constructs (labelled on the bottom and the right side).

[0359] Figure 1 - 2 legend: NB-ARC = domain present in Nucleotide Binding, APAF-1 , R proteins, and CED-4; CID = Common Integration domain; CC = Coil-coiled domain; HMA = Heavy Metal Associated domain; LRR = Leucine-rich repeat domain; NB = Nucleotide- binding domain; OsL = Linker rice; TaL = Linker wheat. TaLOL2 = wheat LSD-1-Like zinc- finger protein (TaLOL2); In figure 1 : HMA (in white) = wheat Heavy Metal Associated domain; HMA (in grey) = rice Heavy Metal Associated domain.

[0360] Figure 3 legend: NB-ARC = domain present in Nucleotide-Binding, APAF-1 , R proteins, and CED-4; CC = Coil-coiled domain; CID = Common Integration domain; HMA = Heavy Metal Associated domain; LRR = Leucine-rich repeat domain; NB = Nucleotide- binding domain; OsL = Linker rice; TaL = Linker wheat. In figure 3A: HMA (in white) = wheat Heavy Metal Associated domain; HMA (in grey) = rice Heavy Metal Associated domain. Example 1 : Swapping the HMA integrated domain of TaMace2Bb with an HMA domain from rice redirects the immune response toward a rice blast effector in wheat protoplasts

[0361] The following example is offered to illustrate, but not to limit, the claimed subject matter. It is understood that the example and embodiments described herein are for illustrative purposes only, and persons skilled in the art will recognize various reagents or parameters that can be altered without departing from the spirit of the disclosure or the scope of the appended claims.

[0362] Material and Methods

[0363] The experiments below were performed in wheat protoplasts. To determine whether we could prompt AVR-Pia recognition and signalling in wheat protoplasts using an engineered version of TaMace2Bb, we constructed the chimeric TaMace2Bb receptor TaMace2Bb-deltaL / ID-OsL / HMA (SEQ ID NO: 4) where the OsPia-2 HMA domain (SEQ ID NO: 12) along with its own linker (OsL, SEQ ID NO: 14) is integrated in the TaMace2Bb (SEQ ID NO: 3) backbone, after the common integration domain (CID, SEQ ID NO: 15) .

[0364] It was also cloned TaMace2Ba (SEQ ID NO: 1), mCherry (SEQ ID NO: 7) and AVR- Pia with no signal peptide (SEQ ID NO: 8). All these constructs were cloned in a pUC19- derived vector. All the constructs are under the control of an operably linked Zmllbi promoter construct (SEQ ID NO: 5) and an operably linked AtNos terminator construct (SEQ ID NO: 16).

[0365] To examine the function of the engineered TaMace2Bb constructs in wheat, wheat protoplasts were isolated from leaves of wheat seedlings that had been kept in the dark at 25°C and that measure between 9 cm and 15 cm from base to tip, essentially as described in Saur et al. (2019, Plant Methods 15:118). 10.5 x 10A4 wheat (Triticum aestivum cultivar Fielder) protoplasts were co-transfected with 40 ug DNA containing:

[0366] 10 g of luciferase reporter construct (pllbi::PpLUC),

[0367] 10 pg of each NLR construct (and mCherry construct as DNA filler up to 20 pg), and

[0368] 10 pg of either AVR-Pia construct or mCherry construct, by PEG-mediated transformation using the method of Saur et al. (2019, Plant Methods 15:118). 16 h post transfection, luciferase activity was determined using the method of Saur et al. (2019, Plant Methods 15:118).

[0369] Results

[0370] The individual expression of TaMace2Ba (SEQ ID NO: 1) or TaMace2Bb-deltaL / ID-

[0371] OsL / HMA (SEQ ID NO: 4) does not result in a death of wheat protoplasts when expressed in presence of mCherry (SEQ ID NO: 7). Individually, TaMace2Ba is thus not auto-active and the OsL / HMA integration in TaMace2Bb-deltaL / ID-OsL / HMA does not trigger autoactivity.

[0372] However, in wheat protoplasts, the co-expression of both TaMace2Ba (SEQ ID NO: 1) and TaMace2Bb-deltaL / ID-OsL / HMA (SEQ ID NO: 4) is sufficient to induce a cell death in presence of the rice blast effector AVR-Pia (SEQ ID NO: 8), with a difference that is very highly statistically significant from the response observed in presence of mCherry (SEQ ID NO: 7) (see figure 2).

[0373] These observations demonstrate that the chimeric TaMace2Bb-deltaL / ID-OsL / HMA construct encoding the chimeric sensor NLR-ID of SEQ ID NO: 4 can trigger an AVR-Pia- dependent immune response.

[0374] In summary, these results demonstrate that the wheat native sensor NLR-ID TaMace2Bb can be engineered to trigger an immune response after recognition of a cytoplasmic effector deriving from a phytopathogen for which today’s wheat varieties are not known to respond.

[0375] Example 2: Swapping the HMA integrated domain of TaMace2Bb with an HMA domain from rice redirects the immune response toward a rice blast effector in Nicotiana benthamiana leaves (Figure 3A-B) Material and Methods

[0376] To determine whether we could prompt AVR-Pia recognition and signalling in Nicotiana benthamiana leaves using an engineered version of the wheat TaMace2Bb, we constructed the chimeric TaMace2Bb receptor TaMace2Bb-deltaL / ID-OsL / HMA (SEQ ID NO: 4) where the rice OsPia-2 HMA domain (SEQ ID NO: 12) along with its own linker (OsL, SEQ ID NO: 14) is integrated into the TaMace2Bb (SEQ ID NO: 3) backbone, after the common integration domain (CID, SEQ ID NO: 15). We also constructed the chimeric TaMace2Bb receptor TaMace2Bb-deltaL / ID (SEQ ID NO: 3) where no linker and no integrated domain are present after the common integration domain (CID, SEQ ID NO: 15).

[0377] It was also cloned TaMace2Ba (SEQ ID NO: 1), TaMace2Bb (SEQ ID NO: 2), Luciferase (SEQ ID NO: 54) and AVR-Pia with no signal peptide (SEQ ID NO: 8). All these constructs were cloned in a binary vector. All the constructs are under the control of an operably linked CaMV double 35S promoter construct linked to an omega leader upstream the TMVgpl gene of the Tobacco Mosaic Virus (SEQ ID NO: 55) and an operably linked CaMV35S polyA terminator construct (SEQ ID NO: 56).

[0378] Transient gene expression in Nicotiana benthamiana leaves were performed by agroinfiltration according to methods described by van der Hoorn et al. (2000, doi: 10.1094 / MPMI.2000.13.4.439). Briefly, A. tumefaciens strain GV3101 pMP90 carrying binary vectors were inoculated from glycerol stock in LB supplemented with appropriate antibiotics and grown overnight at 28°C until saturation. Cells were harvested by centrifugation at 2000 x g, room temperature for 10 min. Cells were washed once and resuspended in infiltration buffer (10 mM MgCh, 10 mM MES-KOH pH 5.6, 200 pM acetosyringone) to the OD 600 nm = 0.25 in the stated combinations and left to incubate in the dark for 30 minutes at room temperature prior to infiltration into four to five-week-old N. benthamiana leaves. Four to thirteen were inoculated per experiment. Hypersensitive cell death phenotypes were scored 5 days post-infiltration in a range from 0 (no visible necrosis) to 7 (fully confluent necrosis) according to Maqbool et al. (2015, eLife 2015;4:e08709).

[0379] Results

[0380] The co-expression of both TaMace2Ba (SEQ ID NO: 1) and TaMace2Bb (SEQ ID NO: 2) results in a moderate background hypersensitive response when expressed in presence of Luciferase (SEQ ID NO: 54) in N. benthamiana leaves. Luciferase was used here as a negative control for immune response, as we did not expect any of these constructs to respond to Luciferase.

[0381] AVR-Pia is an effector secreted by Magnaporthe oryzae, a fungus responsible for the rice blast disease. In rice, AVR-Pia is perceived by the HMA domain integrated in the NLR- ID OsPia-2 (SEQ ID NO: 13). A moderate background hypersensitive response was also observed when co-expressing both TaMace2Ba (SEQ ID NO: 1) and TaMace2Bb (SEQ ID NO: 2) in presence of AVR-Pia (SEQ ID NO: 8), demonstrating that the TaMace2Ba and TaMace2Bb pair is not sufficient to trigger an immune response in presence of AVR-Pia.

[0382] The co-expression of both TaMace2Ba (SEQ ID NO: 1) and TaMace2Bb-deltaL / ID (SEQ ID NO: 3) does not result in a hypersensitive response when expressed in presence of Luciferase (SEQ ID NO: 54) or AVR-Pia (SEQ ID NO: 8) in N. benthamiana leaves. Hence, removing the integrated domain of TaMace2Bb doesn’t trigger an autoimmune response when the constructs are transiently expressed in this heterologous N. benthamiana system.

[0383] As expected, the co-expression of both TaMace2Ba (SEQ ID NO: 1) and TaMace2Bb- deltaL / ID-OsL / HMA (SEQ ID NO: 4) results only in a weak background hypersensitive response when expressed in presence of Luciferase (SEQ ID NO: 54) in N. benthamiana leaves. Surprisingly, the co-expression of both TaMace2Ba (SEQ ID NO: 1) and TaMace2Bb-deltaL / ID-OsL / HMA (SEQ ID NO: 4) in presence of AVR-Pia (SEQ ID NO: 8) in N. benthamiana leaves is sufficient to trigger a strong immune response. These observations demonstrate that, in presence of TaMace2Ba, the chimeric TaMace2Bb-deltaL / ID-OsL / HMA construct encoding the chimeric sensor NLR-ID of SEQ ID NO: 4 can trigger an AVR-Pia-dependent immune response. On the contrary, in nature the perception of AVR-Pia and the triggering of an AVR-Pia-dependent immune response is only observed in rice plants.

[0384] Overall, these results demonstrate that the wheat native sensor NLR-ID TaMace2Bb does not trigger an immune response to a given cytoplasmic effector (AVR-Pia). On the contrary, a protein chimera comprising a TaMace2Bb backbone and a heterologous ID domain from another plant does perceive this given effector (AVR-Pia). Thus, by swapping the integrated domain of a sensor NLR-ID protein, the inventors succeeded in triggering an immune response to a cytoplasmic effector for which today’s wheat varieties are not known to respond.

Claims

CLAIMS1. A chimeric sensor NLR-ID protein comprising, from N-ter to C-ter, one coil-coiled (CC) domain, one nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB- ARC domain), one leucine-rich repeat (LRR) domain, one peptide linker and at least one X domain, wherein said CC domain, said NB-ARC domain, and said LRR domain originate from a native wheat sensor NLR-ID protein, wherein said X domain binds to (i) a pathogen effector different from those recognized by the integrated domain or by at least one of the integrated domains of said native wheat sensor NLR-ID protein, (ii) an epitope different from the pathogen epitope recognized by the integrated domain or by at least one of the integrated domains of said native wheat sensor NLR-ID protein or (iii) a pathogen-induced plant component different from those recognized by the integrated domain or by at least one of the integrated domains of said native wheat sensor NLR-ID protein.

2. A chimeric sensor NLR-ID protein, wherein said chimeric sensor NLR-ID protein is obtained by replacing at least one integrated domain of a native wheat NLR-ID protein by at least one X domain and, optionally, by replacing the peptide linker of said native sensor wheat NLR-ID protein by another peptide linker, wherein said X domain binds to (i) a pathogen effector different from those recognized by the replaced integrated domain(s) of said native wheat sensor NLR-ID protein, (ii) an epitope different from the pathogen epitope recognized by the replaced integrated domain(s) of said native wheat sensor NLR-ID protein or (iii) a pathogen-induced plant component different from those recognized by the replaced integrated domain(s) of said native wheat sensor NLR-ID protein.

3. The chimeric sensor NLR-ID protein according to claim 1 , wherein the peptide linker does not originate from said native wheat sensor NLR-ID protein.

4. The chimeric sensor NLR-ID protein according to anyone of claims 1 to 3, wherein the NB-ARC domain of said chimeric sensor NLR-ID comprises a motif 1 subdomain of a sequence at least 80% identical to the amino acid sequence SEQ ID NO: 38 and / or comprises a motif 1 subdomain consisting of sequence SEQ ID NO: 43.

5. The chimeric sensor NLR-ID protein according to any one of claims 1 to 4, wherein (i) the X domain is rice OsHMA domain, wheat TaLOL2 domain, wheat TaSnn3 domain, Arabidopsis NAC domain or maize MAP3K domain and / or (ii) the peptide linker is barley peptide linker HvL, rice peptide linker OsL, maize peptide linker ZmL or sorghum peptide linker SbL.

6. The chimeric sensor NLR-ID protein according to any one of claims 1 to 5, wherein the native wheat NLR-ID protein is protein TaMace2Bb of sequence SEQ ID NO: 2, protein TaSmlb of sequence SEQ ID NO: 26 or protein TaArinaLrFor2Bb of sequence SEQ ID NO: 23.

7. A NLR protein pair comprising: the chimeric sensor NLR-ID protein according to any one of claims 1 to 6, and a helper NLR protein comprising, from N-ter to C-ter, one CO domain, one NB- ARC domain and one LRR domain, wherein said chimeric sensor NLR-ID protein binds to a pathogen effector or a pathogen-induced plant component through its X domain.

8. The NLR protein pair according to claim 7, wherein (i) said chimeric sensor NLR- ID protein is obtained by replacing the integrated domain of the native sensor NLR- ID protein TaMace2Bb by at least one X domain and wherein said helper protein is protein TaMace2Ba, (ii) said chimeric sensor NLR-ID protein is obtained by replacing at least one integrated domain of the native sensor NLR-ID protein TaSmlb by at least one X domain and wherein said helper protein is protein TaSmla or (iii) said chimeric sensor NLR-ID protein is obtained by replacing the integrated domain of the native sensor NLR-ID protein TaArinaLrFor2Bb by at least one X domain and wherein the helper protein is protein TaArinaLrFor2Ba.

9. A nucleic acid encoding (i) (a) at least one chimeric sensor NLR-ID protein according to any one of claims 1 to 6, (b) at least one X domain of the chimeric sensor NLR-ID protein according to any one of claims 1 to 6 or (c) the peptide linker and at least one X domain of the chimeric sensor NLR-ID protein as defined in any one of claims 1 to 6 and (ii), optionally, at least one helper NLR protein of the NLR protein pair according to claim 7.

10. The nucleic acid according to claim 9, wherein said nucleic acid comprises a first nucleic sequence encoding the chimeric sensor NLR-ID protein defined in claim 7 or 8 and a second nucleic sequence encoding the helper NLR protein defined in claim 7 or 8, wherein said first and second sequences are in head-to-head orientation.

11. A vector comprising at least one nucleic acid according to claim 9 or 10.

12. A plant cell, plant or seed wherein said plant cell, plant or seed comprises at least one chimeric sensor NLR-ID protein according to any one of claims 1 to 6 or at least one NLR protein pair according to claim 7 or 8.

13. A method for obtaining a plant cell according to claim 12, wherein said method comprises transforming a cell with at least one nucleic acid according to claim 9 or 10 or with at least one vector according to claim 11.

14. A method for obtaining a plant resistant to a pathogen, wherein said method comprises: transforming a plant cell or plant tissue with at least one nucleic acid according to claim 9 or 10 or at least one vector according to claim 11 , to obtain a transformed cell or a transformed tissue, and regenerating a plant from the transformed cell or transformed tissue.

15. Use of the chimeric sensor NLR-ID protein according to any one of claims 1 to 6 or of the NLR protein pair according to claim 7 or 8 for preventing a disease caused by a pathogen in a plant.

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