Chimeric resistance genes for maize resistance against pathogens

Abstract

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Description

[0001] CHIMERIC RESISTANCE GENES FOR MAIZE RESISTANCE AGAINST PATHOGENS

[0002] Field of the invention

[0003] The present invention relates to methods for obtaining maize 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 (HMA) of the rice RGA5 NLR-ID protein and obtained a mutated version of 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-cognate effector, and blocked the interaction with AVR-Pia, the RGA5 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, said modifications of RG5-HMA failed to confer extended disease resistance specificity against M. oryzae in the obtained transgenic rice plants. 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.

[0011] Maize 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.

[0012] Alternative solutions to these phytosanitary treatments are needed out of economic and environmental considerations.

[0013] Description of the invention

[0014] 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.

[0015] The chimeric sensor NLR-ID works as a 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 maize.

[0016] By producing maize plants resistant to pathogens, the present invention offers an attractive and practical alternative to conventional approaches. Besides, the chimeric sensor NLR-ID can also advantageously be used to confer resistant to pathogens in plants other than maize.

[0017] 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.

[0018] 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).

[0019] In principle, there are no limitations as to the variability of the new integrated domain and therefore, the method disclosed in this application could be extended to a variety of pathosystems.

[0020] As way of example, in the series of experiments leading to this invention, the inventors unexpectedly highlighted that the genes ZmPia-1 and ZmPia-2 present in the Rcg1 locus in some maize varieties are genetically linked NLRs in head-to-head orientation. Toman and White (1993, Phytopathology 83:981-986) disclosed two closely linked genes for anthracnose resistance in the maize anthracnose resistant line MP305 but failed to characterize these genes, their exact position or orientation in the Rcg1 locus.

[0021] In another set of experiments, the inventors engineered the native maize sensor NLR- ID protein ZmPia-2 to replace its native ID (a Mitogen- Activated Protein (MAP) Kinase Kinase Kinase (MAP3K)) with a new ID, namely a Heavy-Metal Associated domain (HMA) isolated from a sensor NLR-ID protein from rice. This new domain is known to recognize Avr-Pia, an effector secreted by Magnaporthe oryzae, a fungus responsible for the rice blast disease. Transient assays performed in Nicotiana benthamiana leaves confirm that this novel chimeric sensor NLR-ID construct is functional in that it is capable of triggering an effector-dependent cell death (hypersensitive response or HR) leading to plant immunity in the presence of ZmPia-1 , the corresponding paired-NLR.

[0022] 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 maize sensor NLR-ID protein, wherein said X domain binds to (i) a pathogen effector different from those recognized by the integrated domain of said native maize sensor NLR-ID protein, (ii) an epitope different from the pathogen epitope recognized by the integrated domain of said native maize sensor NLR-ID protein or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of said native maize sensor NLR-ID protein.

[0023] The peptide linker preferably does not originate from said native maize sensor NLR- ID protein.

[0024] The present invention particularly relates to a chimeric sensor NLR-ID protein, wherein said chimeric sensor NLR-ID protein is obtained by replacing the integrated domain of a native sensor maize NLR-ID protein by at least one X domain and, optionally, by replacing the peptide linker of said native sensor maize NLR-ID protein by another peptide linker, wherein said X domain binds to (i) a pathogen effector different from those recognized by the integrated domain of said native maize sensor NLR-ID protein, (ii) an epitope different from the pathogen epitope recognized by the integrated domain of said native maize sensor NLR-ID protein or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of said native maize sensor NLR-ID protein.

[0025] The native maize sensor NLR-ID protein as defined above is for example protein ZmPia-2.

[0026] The X domain as defined above may for example be the rice HMA domain, for example of SEQ ID NO: 12 or the wheat TaLOL2 domain of sequence SEQ ID NO: 20.

[0027] The peptide linker as defined above may for example be the rice peptide linker OsL, for example of SEQ ID NO: 14 or the barley linker HvL of sequence SEQ ID NO: 21.

[0028] 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 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.

[0029] 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 ZmPia-2 by at least one X domain and the helper protein may be protein ZmPia-1.

[0030] The present invention also relates to a nucleic acid encoding (i) (a) at least one chimeric sensor NLR-ID protein as defined above, (b) the X domain(s) of the chimeric sensor NLR-ID protein as defined above or (c) the peptide linker and the X domain(s) 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.

[0031] 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.

[0032] The present invention also relates to a vector comprising at least one nucleic acid as defined above. 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.

[0033] Another object of the invention is a method for obtaining a maize cell as defined above, wherein said method comprises transforming said cell with at least one nucleic acid as defined above or at least one vector as defined above.

[0034] 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.

[0035] 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.

[0036] Effector, NLR proteins, native sensor NLR-ID protein

[0037] By “effector”, it is herein meant either a pathogen effector or a pathogen-induced plant component.

[0038] 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 system (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).

[0039] By “pathogen-induced plant component”, it is herein meant a plant component resulting from pathogen effector-induced modifications in a plant cell or tissue.

[0040] 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.

[0041] 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, detects pathogen effector proteins either directly by binding said pathogen 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).

[0042] 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.

[0043] By “NLR protein pair”, it is meant 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”).

[0044] By “native protein”, it is herein meant a protein, which is found in nature and has not been genetically modified by genetic manipulations.

[0045] A maize native sensor NLR-ID protein is thus a sensor NLR-ID protein, which is naturally found in maize.

[0046] Said maize 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) one integrated domain (ID).

[0047] 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 maize sensor NLR-ID protein are also herein referred to as the “NLR backbone”.

[0048] Said maize native sensor NLR-ID protein thus preferably comprise a NLR backbone, a peptide linker and an integration domain (ID). 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).

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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 / horseshoe fold. In the maize sensor NLR-ID protein, the LRR domain comprises a common integration domain (CID) at its C- terminal end.

[0053] 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.

[0054] 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.

[0055] The CID domain of ZmPia-2 protein for example consists of sequence SEQ ID NO: 15.

[0056] By “peptide linker”, it is herein meant an amino acid sequence located between the common integration domain (CID) and the integrated domain (ID) of the sensor NLR-ID protein. By “integrated domain” or “ID”, it is herein meant the domain of the 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 NLD domain.

[0057] By “native integrated domain” or “native ID”, it is meant the integrated domain of the 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.

[0058] By “non-canonical", it is herein meant a domain of the native sensor NLR-ID protein that is not the CC domain, the NB-ARC domain, nor the LRR domain.

[0059] 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.

[0060] 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 identifying the CC, NB-ARC, LRR and ID domains using the 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).

[0061] For example, in the native sensor NLR-ID protein ZmPia-2 of sequence SEQ ID NO: 2, amino acids 14 to 49 correspond to the coil-coiled (CC) domain, amino acids 160 to 488 correspond to the nucleotide-binding and APAF-1 , R protein, CED-4 domain (NB-ARC domain), amino acids 530 to 929 correspond to the leucine-rich repeat (LRR) domain (wherein amino acids 897 to 929 corresponding to the CID), amino acids 930 to 1147 correspond to the peptide linker, and amino acids 1148 to 1397 correspond to the integrated domain.

[0062] Said maize 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.

[0063] 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.

[0064] Non-limitative examples of maize native sensor NLR-ID protein include protein ZmPia- 2.

[0065] Protein ZmPia-2 for example consists of sequence SEQ ID NO: 2.

[0066] Protein ZmPia-2 allows the recognition of an effector of Colletotrichum graminicola.

[0067] Protein ZmPia-2 and its helper NLR protein ZmPia-1 (for example of sequence SEQ ID NO: 1) form an NLR pair, which allows conferring to maize a resistance to anthracnose caused by the fungus Colletotrichum graminicola.

[0068] The integrated domain of native protein ZmPia-2 is a Mitogen Activated Protein (MAP) kinase kinase kinase (MAP3K) domain, for example of sequence SEQ ID NO: 17.

[0069] ZmPia-1 and ZmPia-2 genes are located in the Rcg1 locus found in some varieties of maize plants, such as MP305 disclosed in Toman and White (1993, Phytopathology 83:981- 986), or the tropical line CML277 disclosed in Hufford et al. (2021 , Science 373 (6555), 655- 662). However, ZmPia-1 and ZmPia-2 are not present in the most frequently used maize variety B73 and were never reported in the literature. Patent EP1874935 B2 by Pioneer identifies the locus Rcg1 in maize variety MP305 in relation with the resistance of maize plants to Colletotrichum graminicola, the fungal pathogen responsible for anthracnose in maize. Toman and White (1993, Phytopathology 83:981-986) disclose that the maize anthracnose resistant line MP305 carries two closely linked genes for anthracnose resistance in the locus Rcg1 but fail to characterize the genes further.

[0070] Patent US10731225 B2 by Pioneer discloses a modified plant with enhanced resistance to Colletotrichum comprising a heterologous polynucleotide encoding a Rcg1 polypeptide (corresponding to ZmPia-1) and a second heterologous polynucleotide encoding a Rcglb polypeptide (corresponding to ZmPia-2). Patent application US20230151382 A1 , also by Pioneer, discloses the integrated domain of the Rcglb protein as a Mitogen-Activated Protein (MAP) kinase kinase kinase (MAP3K) and identified the cognate avr gene product (ANTROT 70 or AvrRcglb) as the effector of Collet otrichum graminicola perceived by the MAP3K domain of Rcgl b via yeast two-hybrid and coimmunoprecipitation. None of these documents suggests that the proteins encoded by the genes ZmPia-1 and ZmPia-2 are forming a functional pair of NLRs. Moreover, they fail to disclose the head-to-head orientation of these genes in the Rcg1 locus, or the possibility to swap the integrated domain of ZmPia-2 to extend pathogen recognition to other pathogens or other pathosystems.

[0071] Chimeric sensor NLR-ID protein

[0072] The present invention thus relates to a sensor NLR-ID protein, which is a chimeric sensor NLR-ID protein.

[0073] By “chimeric sensor NLR-ID protein”, it is herein meant an engineered and thus non- naturally occurring sensor NLR-ID protein.

[0074] 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.

[0075] The chimeric sensor NLR-ID protein as defined above is preferably obtained from a maize native sensor NLR-ID protein, by replacing the integrated domain from said maize 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.

[0076] The chimeric sensor NLR-ID protein as defined above thus for example comprises a NLR backbone as defined above, a peptide linker and at least one X domain as defined below.

[0077] 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, (ii) a peptide linker and (iii) at least one X domain.

[0078] 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 found in the corresponding native sensor NLR-ID protein sharing the same NLR backbone.

[0079] 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 maize 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 maize protein sharing the same NLR backbone or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of the corresponding native maize sensor NLR-ID protein sharing the same NLR backbone.

[0080] In some embodiments, the chimeric sensor NLR-ID protein comprises a single X domain. In such case, the integrated domain from the maize native sensor NLR-ID protein may be replaced by said X domain.

[0081] In some embodiments, the chimeric sensor NLR-ID protein comprises at least two X domains. In such case, the integrated domain from the maize native sensor NLR-ID protein may be replaced by said at least two X domains.

[0082] When the chimeric sensor NLR-ID protein comprises at least two X domains, said X domains may be identical or different. For example, each X domain may recognize a different pathogen effector. Alternatively, each X domain may recognize a different epitope of the same pathogen effector. Alternatively, one X domain may recognize a pathogen effector and the other X domain may recognize a pathogen-induced plant component.

[0083] When the chimeric sensor NLR-ID protein comprises at least two X domains, the chimeric sensor NLR-ID protein may comprise a linker between the X domains; alternatively, there may be no linker between the X domains.

[0084] 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 (for example only one X domain or at least two X domains as defined above), wherein said CC domain, said NB-ARC domain, and said LRR domain originate from a native maize sensor NLR-ID protein, wherein said X domain binds to (i) a pathogen effector different from those recognized by the integrated domain of said native maize sensor NLR-ID protein, (ii) an epitope different from the pathogen effector epitope recognized by the integrated domain of said native maize sensor NLR-ID protein or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of said native maize sensor NLR- ID protein.

[0085] By the expression “said CC domain, said NB-ARC domain, and said LRR domain originate from a native maize sensor NLR-ID protein", it is herein meant that said domains originate from the same native maize sensor NLR-ID protein. Said domains may be identical to those of said native maize 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.

[0086] A domain which originates from a native maize sensor NLR-ID protein may for example consist of an amino acid sequence at least 80% identical to those of the corresponding domain of the native maize 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 maize sensor NLR-ID protein.

[0087] The X domain as defined above may thus be a pathogen epitope binding domain or a pathogen-induced plant component binding domain.

[0088] 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.

[0089] By “pathogen epitope binding domain”, it is herein meant a domain, which specifically binds to a pathogen epitope.

[0090] 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.

[0091] 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).

[0092] 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.

[0093] The X domain may be a native X domain, a non-native X domain, or a derivative thereof.

[0094] 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 a maize integrated domain (ID) found in a maize 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, wheat, rice or barley.

[0095] Non-limitative examples of X domains are ensuing.

[0096] A native X domain may for example comprise or consist of the MAP3K domain of sequence SEQ ID NO: 17, the Heavy Metal-Associated (HMA) domain of SEQ ID NO: 12, or the wheat TaLOL2 domain, for example of sequence SEQ ID NO: 20.

[0097] HMA domain is the ID of rice OsPia-2 sensor NLR-ID protein. HMA domain 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.

[0098] The wheat TaLOL2 (for example of SEQ ID NO: 20) interacts with the Glycine- Serine-Rich Effector 1 (PstGSREI) effector (for example of SEQ ID NO: 22), an effector secreted by Puccinia striiformis, the fungus responsible for wheat stripe rust disease.

[0099] The X domain as defined above may alternatively be a non-native X domain.

[0100] By “non-native X domain”, it is herein meant a protein domain that is not found in native sensor NLR-ID proteins in plants.

[0101] 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.

[0102] The X domain may also be a derivative of a native or a non-native X domain.

[0103] 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.

[0104] 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.

[0105] 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 an heterologous system.

[0106] 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 maize protoplasts.

[0107] 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.

[0108] As defined above, a peptide linker is the amino acid sequence located between the Common Integration Domain (CID) and the integrated domain (ID) of a sensor NLR-ID protein.

[0109] 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.

[0110] 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.

[0111] As the CC domain, NB-ARC domain and LRR domain, the peptide linker may be the peptide linker of the native sensor chimeric NLR-ID protein, i.e., the peptide linker corresponding to the native sensor chimeric NLR-ID protein. For example, if the chimeric sensor NLR-ID protein comprises the NLR backbone of ZmPia-2 protein, the peptide linker may be the peptide linker of the ZmPia-2 protein.

[0112] 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 ZmPia-2 protein and that the X domain is the rice HMA domain of OsPia-2 protein, the native peptide linker may be the peptide linker of OsPia-2 protein.

[0113] 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 maize 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.

[0114] Alternatively, the peptide linker comprised in the chimeric sensor NLR-ID protein is an artificial peptide linker, such as a random peptide linker.

[0115] The peptide linker as defined above may be of any type or any length.

[0116] 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.

[0117] Non-limitative examples of peptide linkers are the maize peptide linker ZmL of the sensor NLR-ID protein ZmPia-2, for example of sequence SEQ ID NO: 18, the rice peptide linker OsL of the OsPia-2 protein, for example of sequence SEQ ID NO: 14, or the barley linker HvL (for example of the HQRVU5Hr1G001060 protein), for example of sequence SEQ ID NO: 21.

[0118] Thus, in one embodiment of the present invention, the chimeric sensor NLR-ID protein comprises a maize NLR backbone (for example of sequence SEQ ID NO: 3), a maize peptide linker ZmL (for example of sequence SEQ ID NO: 18), and an X domain HMA 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 maize NLR backbone (for example of sequence SEQ ID NO: 3), a rice peptide linker OsL (for example of sequence SEQ ID NO: 14), and an X domain HMA from rice (for example of sequence SEQ ID NO: 12).

[0119] In yet another embodiment of the present invention, the chimeric sensor NLR-ID protein comprises a maize NLR backbone (for example of sequence SEQ ID NO: 3), 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: 20).

[0120] The chimeric sensor NLR-ID protein as defined above may thus be obtained from a maize native sensor NLR-ID protein, by replacing both the peptide linker and the integrated domain from said maize native sensor NLR-ID protein by another peptide linker as defined above and by one or at least one X domain as defined above.

[0121] 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., ZmPia-2-deltaL / ID), a peptide linker as defined above and one or at least one X domain as defined above.

[0122] In one embodiment, the chimeric sensor NLR-ID protein comprises or consists of amino acid sequence SEQ ID NO: 4 (i.e., ZmPia-2-deltaL / ID-OsL / HMA) or SEQ ID NO: 19 (i.e., ZmPia-2-deltaL / ID-HvL-TaLOL2).

[0123] Helper NLR protein

[0124] The present invention also relates to a helper NLR protein.

[0125] 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.

[0126] 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.

[0127] The helper NLR protein as defined above is preferably a maize helper NLR protein.

[0128] 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.

[0129] The CC domain, NB-ARC domain and LRR domain are particularly as defined above in the section “chimeric sensor NLR-ID protein”.

[0130] Contrary to a sensor NLR-ID protein, a helper NLR protein preferably 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.

[0131] The helper NLR protein as defined above is preferably a native maize helper NLR protein or a derivative of a native maize helper protein. A maize native helper NLR protein is thus a helper NLR protein, which is naturally found in maize.

[0132] 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.

[0133] Non-limitative examples of maize helper NLR protein include protein ZmPia-1.

[0134] Protein ZmPia-1 for example consists of sequence SEQ ID NO: 1.

[0135] NLR protein pair

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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 ZmPia-2, for example of sequence SEQ ID NO: 2, by an X domain as defined above, wherein said X domain recognizes (i) a pathogen effector different from the Colletotrichum graminicola effector recognized by the integrated domain of the native sensor NLR-ID protein ZmPia-2 ( / .e. MAP3K), (ii) an epitope of Colletotrichum graminicola different from those recognized by the integrated domain of the native sensor NLR-ID protein ZmPia-2 ( / .e. MAP3K), or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of the native sensor NLR-ID protein ZmPia-2 ( / .e. MAP3K), and protein ZmPia-1 as a helper NLR protein, for example protein ZmPia-1 of sequence SEQ ID NO: 1.

[0140] 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 ZmPia-2, for example of sequence SEQ ID NO: 2, by another peptide linker as defined above and an X domain as defined above, wherein said X domain specifically recognizes (i) a pathogen effector different from the Colletotrichum graminicola effector recognized by the integrated domain of the native sensor NLR-ID protein ZmPia-2 ( / .e. MAP3K), (ii) an epitope of Colletotrichum graminicola different from those recognized by the integrated domain of protein ZmPia-2 ( / .e. MAP3K), or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of the native sensor NLR-ID protein ZmPia-2 ( / .e. MAP3K), and protein ZmPia-1 as a helper NLR protein, for example protein ZmPia-1 of sequence SEQ ID NO: 1.

[0141] Nucleic acid encoding the chimeric sensor NLR-ID protein and / or the helper NLR protein and vector comprising said nucleic acid.

[0142] The present invention also relates to a nucleic acid encoding (i) (a) at least one chimeric sensor NLR-ID protein as defined above, (b) the X domain(s) of a chimeric sensor NLR-ID protein as defined above, or (c) the peptide linker and the X domain(s) 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.

[0143] 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. The first nucleic acid sequence and the second nucleic acid sequence are each under the control of a promoter and a terminator.

[0144] Any promoter and terminator suitable for expression in plants, for example in maize, well known by the skilled person may be used.

[0145] 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).

[0146] 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).

[0147] The present invention also relates to a nucleic acid encoding the X domain(s) of a chimeric sensor NLR-ID protein as defined above, said nucleic acid being in particular suitable for replacing the native ID domain of the native sensor NLR-ID protein by said X domain(s), in particular in a plant cell. Said nucleic acid particularly allows replacing the native ID domain of the native sensor NLR-ID protein by said X domain(s) by homologous recombination, for example through CRISPR-Cas technology.

[0148] The present invention also relates to a nucleic acid encoding the peptide linker and the X domain(s) 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 native ID domain of the native sensor NLR-ID protein by said peptide linker and said X domain(s), in particular in a plant cell. Said nucleic acid particularly allows replacing the native peptide linker and the native ID domain of the native sensor NLR-ID protein by said peptide linker and said X domain(s) by homologous recombination, for example through CRISPR-Cas technology.

[0149] 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) the X domain(s) of a chimeric sensor NLR-ID protein as defined above, or (c) the peptide linker and the X domain(s) 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.

[0150] 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.

[0151] 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.

[0152] The vector as defined above is particularly suitable for expression in a plant cell, for example in a maize cell.

[0153] In the nucleic acids as defined herein, when the chimeric sensor NLR-ID protein comprises only one X domain, “a nucleic acid encoding the X domain(s)” thus encodes the X domain of the chimeric sensor NLR-ID protein.

[0154] 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 the X domain(s)” encodes at least one of the X domains of the chimeric sensor NLR-ID protein and, preferably, encodes the X domains of the chimeric sensor NLR-ID protein.

[0155] The present invention also relates to a vector as defined above comprising a nucleic acid encoding the X domain(s) of a chimeric sensor NLR-ID protein as defined above, said vector being particularly suitable for replacing the native ID domain of the native sensor NLR-ID protein by said X domain(s), in particular in a plant cell, in particular by homologous recombination, for example through CRISPR-Cas technology.

[0156] The present invention also relates to a vector as defined above comprising a nucleic acid encoding the peptide linker and the X domain(s) of a chimeric sensor NLR-ID protein as defined above, said vector being particularly suitable for replacing the native peptide linker and the native ID domain of the native sensor NLR-ID protein by said peptide linker and said X domain(s), in particular in a plant cell, in particular by homologous recombination, for example through CRISPR-Cas technology.

[0157] The vector as defined above is particularly suitable for expression in maize.

[0158] The vector as defined above is preferably suitable for integration of said nucleic acid in the genome of a plant, preferably a maize plant, more preferably at a targeted location in the genome, in particular by homologous recombination.

[0159] 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.

[0160] 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).

[0161] Through non-homologous end joining, and homologous recombination an exogenous nucleic acid or donor polynucleotide can be inserted 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.

[0162] 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.

[0163] In one preferred embodiment, recombinant DNA methods are used on maize plant cells deprived of the Rcg1 locus to introduce both the helper NLR protein ZmPia-1 (for example of sequence SEQ ID NO: 1) and the chimeric sensor NLR-ID protein comprising the ZmPia-2 backbone (for example of sequence SEQ ID NO: 3), a peptide linker as defined above and at least one X domain as defined above.

[0164] In another preferred embodiment, recombinant DNA methods are used on plant cells from different species to introduce the helper NLR protein ZmPia-1 (for example of sequence SEQ ID NO: 1) and the chimeric sensor NLR-ID protein comprising the ZmPia-2 backbone (for example of sequence SEQ ID NO: 3), a peptide linker as defined above and at least one X domain as defined above.

[0165] In yet another preferred embodiment, recombinant DNA methods are used on plant varieties already containing the Rcg1 locus to swap the native MAP3K integrated domain of the native sensor NLR-ID protein ZmPia-2 (for example of sequence SEQ ID NO: 2) 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.

[0166] In some embodiments, the plant cells contain the native ZmPia-2 sensor NLR-ID protein as disclosed in SEQ ID NO: 2.

[0167] Cell, plant, seed and pathogen

[0168] As used herein, the term “plant” includes reference to whole plants. As used herein, the term “plant part” includes plant organs (e.g., leaves, stems, roots... etc.), plant cells or seeds.

[0169] “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.

[0170] The class of plants which can be used includes both monocotyledonous and dicotyledonous plants.

[0171] “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 varieties of said crop plants, such as for example sweetcorn in the case of maize plants, and wild relatives of said crop plants, for example wild relatives of maize plants.

[0172] Said plant may be maize but also any other plant, such as sorghum, wheat, rice, barley, triticale, oat or rye.

[0173] 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.

[0174] 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.

[0175] Said maize plant or seed is advantageously resistant to a pathogen, for example to Colletotrichum graminicola or to a pathogen different from Colletotrichum graminicola, wherein said pathogen expresses a pathogen effector recognized by the X domain of the chimeric sensor NLR-ID protein.

[0176] 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) the X domain(s) of a chimeric sensor NLR-ID protein as defined above, or (c) the peptide linker and the X domain(s) 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.

[0177] 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 maize protoplasts.

[0178] The plant or seed as defined above is preferably an agronomic plant (crop) or seed. 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.

[0179] The cell, plant or seed may be a genetically modified or genetically edited cell, plant or seed.

[0180] By “genetically modified” or “GM”, it is herein particularly meant that the integration of the modification is done at any location in the genome.

[0181] 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 ZmPia-2 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.

[0182] 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.

[0183] 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 from a resistance to Colletotrichum graminicola, the causative agent of anthracnose in maize, to a resistance to any other pathogen, as defined above. 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.

[0184] 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).

[0185] 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. In the claimed invention, disease resistance is for example brought about by the presence of the pair of NLR proteins ZmPia-1 (of sequence SEQ ID NO: 1) and the chimeric sensor NLR-ID protein comprising the backbone of native sensor NLR-ID protein ZmPia-2 (of sequence SEQ ID NO: 3), a peptide linker as defined above and at least one X domain as defined above.

[0186] 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).

[0187] 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.

[0188] 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.

[0189] Method for obtaining a plant cell or a plant resistant to a pathogen.

[0190] 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.

[0191] The present invention also relates to a method for obtaining a plant resistant to a pathogen, wherein said method comprises:

[0192] - 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,

[0193] - regenerating a plant from the transformed cell or transformed tissue, and - optionally, assessing the expression of the chimeric sensor NLR-ID protein by the regenerated plant.

[0194] The plant cell to be transformed may be a protoplast.

[0195] The plant tissue to be transformed may be an apical meristem, cotyledon, embryo, pollen and / or microspores.

[0196] The plant may be maize, but also, as defined above, any other plant, such as sorghum, wheat, rice, barley, triticale, oat or rye.

[0197] 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.

[0198] For agrobacterium transgene delivery, the vector is first transferred into Agrobacterium, to obtain a transformed Agrobacterium and the plant cell or plant tissue is then transformed with said transformed Agrobacterium. The Agrobacterium is preferably Agrobacterium tumefaciens.

[0199] When the plant cell or plant tissue to be transformed does not comprise ( / .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.

[0200] When the plant cell or plant tissue to be transformed does not comprise ( / .e. does not express) a native ZmPia-2 protein, nor the helper ZmPia-1 protein, the vector may comprise (i) optionally, a nucleic acid that encodes the helper NLR protein ZmPia-1 (for example of sequence SEQ ID NO: 1) and (ii) a nucleic acid that encodes the chimeric ZmPia-2 protein comprising the ZmPia-2 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 cell or tissue may be of any plant as defined above. Said plant cell or plant tissue may be a maize cell or maize tissue. Said vector may be suitable for target or non-targeted integration in the genome of the cell.

[0201] The nucleic acids encoding the chimeric ZmPia-2 protein as defined above and the ZmPia-1 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 maize cell or tissue, may be done concomitantly or sequentially.

[0202] When the plant cell or plant tissue to be transformed comprises ( / .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.

[0203] 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 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 of the sensor NLR-ID protein and the X domain(s) in the genome of the cell.

[0204] 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 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 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.

[0205] 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-ID ZmPia-2 and the paired NLR helper NLR ZmPia-1 with a chimeric sensor NLR-ID comprising at least one X domain as defined above, conferring an additional pathogen resistance beyond that of Colletotrichum graminicola or improving pathogen resistance to Colletotrichum graminicola in said plant cell or plant tissue.

[0206] 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 ZmPia-2 backbone (for example of sequence SEQ ID NO: 3), a peptide linker as defined above and at least one X domain as defined above.

[0207] 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) the X domain(s) of a chimeric sensor NLR-ID protein as defined above, or (c) the peptide linker and the X domain(s) of a chimeric sensor NLR-ID protein as defined 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.

[0208] Regeneration of a plant from a plant cell or plant tissue is well known by the skilled person.

[0209] In particular, the transformed plant cell or plant tissue may be placed in a culture medium suitable for plant growth.

[0210] The regeneration of a plant from the transformed plant cell or the transformed plant tissue may comprise:

[0211] - growing said transformed plant cell or transformed plant tissue to obtain a callus, and

[0212] - regeneration of shoots from the callus, in particular by somatic embryogenesis.

[0213] 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).

[0214] The regeneration of a plant from the transformed tissue may comprise regeneration of shoots.

[0215] The regeneration of shoots from the transformed tissue may be carried out in any suitable culture medium comprising plant growth regulators.

[0216] The method may further comprise assessing the production of said chimeric sensor NLR-ID protein and, optionally, of the helper NLR protein by the regenerated plant.

[0217] Assessing the production of said chimeric sensor NLR-ID protein and, optionally, of said helper NLR protein by the regenerated plant may be performed by: - assessing the resistance of the regenerated plant to a pathogen, wherein said pathogen expresses an effector pathogen recognized by the or at least one X domain of the chimeric sensor NLR-ID protein,

[0218] - detecting the production 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

[0219] - 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.

[0220] The regenerated plant produces said chimeric sensor NLR-ID protein and, optionally, said helper NLR protein, if:

[0221] - the regenerated plant is resistant to said pathogen,

[0222] - the cells of the regenerated plant express said chimeric sensor NLR-ID protein, and / or

[0223] - the cells of the regenerated plant comprise a nucleic acid encoding said chimeric sensor NLR-ID protein.

[0224] Said plant may be maize but also any other plant, such as sorghum, wheat, rice, or barley, triticale, oat or rye.

[0225] Use of a chimeric sensor NLR-ID protein or a NLR protein pair

[0226] 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.

[0227] Said plant may be maize, but also any other plant, such as sorghum, wheat, rice, barley, triticale, oat or rye.

[0228] The maize plant may be any variety of maize, such as sweetcorn,

[0229] 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 maize plant or maize seed. Said plant may be maize, but also any other plant, such as sorghum, wheat, rice, barley, triticale, oat or rye. The maize plant may be any variety of maize, such as sweetcorn,

[0230] Said chimeric sensor NLR-ID protein or NLR protein pair expressed by said plant preferably confers to said plant, in particular a maize plant, a resistance to a pathogen or increases the tolerance of said plant, in particular a maize 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 maize (including varieties thereof such as sweetcorn), but also any other plant, such as sorghum, wheat, rice, barley, triticale, oat or rye.

[0231] 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.

[0232] 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 maize, in particular in the maize plant or maize seed, in other words in the cells of said plant or seed.

[0233] The invention will be further illustrated in the following figures and examples.

[0234] Brief description of the sequences

[0235] SEQ ID NO: 1 corresponds to the amino acid sequence of ZmPia-1 (a maize helper NLR protein).

[0236] SEQ ID NO: 2 corresponds to the amino acid sequence of ZmPia-2 (a maize sensor NLR-ID protein).

[0237] SEQ ID NO: 3 corresponds to the amino acid sequence of ZmPia-2-deltaL / ID (the NLR backbone of ZmPia-2 as defined above).

[0238] SEQ ID NO: 4 corresponds to the amino acid sequence of ZmPia-2-deltaL / ID- OsL / HMA.

[0239] SEQ ID NO: 5 corresponds to the nucleic acid sequence of ZmUbi promoter.

[0240] SEQ ID NO: 6 corresponds to the nucleic acid sequence of SbHSP terminator.

[0241] SEQ ID NO: 7 corresponds to the amino acid sequence of mCherry.

[0242] SEQ ID NO: 8 corresponds to the amino acid sequence of AVR-Pia (an effector of rice pathogen Magnaporthe oryzae) with no signal peptide and an additional N-terminal methionine.

[0243] SEQ ID NO: 9 corresponds to the nucleic acid sequence of mas promoter.

[0244] SEQ ID NO: 10 corresponds to the nucleic acid sequence of CaMV35S terminator.

[0245] SEQ ID NO: 11 corresponds to the nucleic acid sequence of 2x35S promoter.

[0246] SEQ ID NO: 12 corresponds to the amino acid sequence of the OsHMA domain (ID derived from rice OsPia-2)

[0247] SEQ ID NO: 13 corresponds to the amino acid sequence of OsPia-2 (a rice sensor NLR-ID protein). SEQ ID NO: 14 corresponds to the amino acid sequence of rice peptide linker OsL.

[0248] SEQ ID NO: 15 corresponds to the amino acid sequence of common integration domain (CID) present in ZmPia-2.

[0249] SEQ ID NO: 16 corresponds to the nucleic acid sequence of AtNos terminator.

[0250] SEQ ID NO: 17 corresponds to the amino acid sequence of the maize MAP3K (ID of maize ZmPia-2).

[0251] SEQ ID NO: 18 corresponds to the amino acid sequence of the maize peptide linker ZmL of ZmPia-2.

[0252] SEQ ID NO: 19 corresponds to the amino acid sequence of ZmPia-2-deltaL / ID-HvL- TaLOL2.

[0253] SEQ ID NO: 20 corresponds to the amino acid sequence of TaLOL2 domain (ID derived from wheat LOL2 protein).

[0254] SEQ ID NO: 21 corresponds to the amino acid sequence of the linker HvL (peptide linker of barley NLR-ID protein).

[0255] SEQ ID NO: 22 corresponds to the amino acid sequence of the PstGSREI effector.

[0256] SEQ ID NO: 23 corresponds to the genomic sequence of ZmPia-1 (encoding a maize helper NLR protein) from maize line CML277.

[0257] SEQ ID NO: 24 corresponds to the cDNA sequence of ZmPia-1 (encoding a maize helper NLR protein).

[0258] SEQ ID NO: 25 corresponds to the genomic sequence of ZmPia-2 (encoding a maize helper NLR protein).

[0259] SEQ ID NO: 26 corresponds to the cDNA sequence of ZmPia-2 (encoding a maize helper NLR protein).

[0260] Description of the Figures

[0261] Figure 1 : Design of the constructs used to assess anthracnose resistance in transgenic maize plants.

[0262] A. Structure of the proteins assessed in Example 1 : native helper NLR ZmPia-1 , designated as “ZmPia-1” (SEQ ID NO: 1); native sensor NLR-ID ZmPia-2, designated as “ZmPia-2” (SEQ ID NO: 2); engineered sensor NLR-ID ZmPia-2 without its native linker and its native ID, designated as “ZmPia-2 backbone ZmPia-2-deltaL / ID” (SEQ ID NO: 3); and chimeric sensor NLR-ID ZmPia-2 without its native linker and its native ID but with a rice linker and a rice HMA ID, designated as “ZmPia-2-deltaL / ID-OsL / HMA” (SEQ ID NO: 4).

[0263] B. Combinations of proteins assessed in Example 1 : native helper NLR ZmPia-1 coexpressed with sensor NLR-ID ZmPia-2 either in a native version (combination #1), in an engineered version (combination #2) or in a chimeric version (combination #3) as described above.

[0264] Figure 2: Swapping the MAP3K integrated domain of ZmPia-2 with an HMA domain from rice redirects the immune response toward the rice blast effector AVR-Pia in Nicotiana benthamiana leaves.

[0265] A. Structure of the NLR proteins assessed in Example 2: native helper NLR ZmPia-1 , designated as “ZmPia-1” (SEQ ID NO: 1); native sensor NLR-ID ZmPia-2 containing its native ZmL linker and native MAP3K domain, designated as “ZmPia-2” (SEQ ID NO: 2); chimeric sensor NLR-ID ZmPia-2 without its native linker and its native ID but with a linker and a ID from the rice sensor NLR-ID OsPia-2, designated as “ZmPia-2-deltaL / ID- OsL / HMA” (SEQ ID NO: 4); and native sensor NLR-ID OsPia-2 (SEQ ID NO: 13).

[0266] B. Effect of the different combinations of proteins on the immune response characterized by a hypersensitive cell death response (HR) at the infiltration site on Nicotiana benthamiana leaves. Representative N. benthamiana leaves co-infiltrated with constructs from Figure 2A, and either AVR-Pia or mCherry, photographed 5 days after infiltration. The infiltration site for each construct is labelled on the picture and circled with dashed lines. The fluorescent protein mCherry was used as a negative control for immune response, as we did not expect any of these proteins to respond to mCherry,

[0267] C. Cell-death assay scoring represented as raw data points and violin plots for estimated densities. For each sample, all of 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 sides).

[0268] D. Statistical analysis of HR scores using an estimation graphic displaying bootstrap estimations of HR scores distributions. Qualitative cell-death scoring from autofluorescence (presented in panel C) was analyzed using the estimation methods described in Ho et al. (2019, Nat Methods 16, 565-566) and visualized with estimation graphics using the besthr R library vO.3.2 described in MacLean D. (2019, Besthr Zenodo).

[0269] Briefly, in this process all autofluorescence (cell-death) scores in samples under comparison were ranked, irrespective of sample. The mean ranks of the control (ZmPia-1 + mCherry) and test samples were taken and a bootstrap process was begun on ranked test data, in which samples of equal size to the experiment were replaced and the mean rank calculated. After 100 bootstrap samples, rank means were calculated, a distribution of the mean ranks was drawn and its 2.5 and 97.5 quantiles calculated. 95% confidence intervals of the test means are considered to be different when they do not overlap. Figure 1 - 2 legend: ARC = domain present in 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; MAP3K = MAP kinase kinase kinase domain; NB = Nucleotide-binding domain; OsL = Linker Rice; ZmL = Linker maize.

[0270] Figure 3: A. Design of the constructs; B. Results. Legend: ARC = domain present in APAF-1 , R proteins, and CED-4 domain; CID = Common Integration domain; CC = Coil- coiled domain; ID = zf-LSD1 domain found in barley HCRVU5Hr1G001060.1 (Bailey et al. 2018, Genome Biol 19, 23), LRR = Leucine-rich repeat domain; MAP3K = MAP kinase kinase kinase domain; NB = Nucleotide-binding domain; HvL = barley linker found in HGRVU5Hr1G001060.1 (Bailey et al. 2018, Genome Biol 19, 23); ZmL = Linker maize; TaLOL.= wheat Lesion Simulating Disease (LSD) One-Like 2 (TaLOL2) domain.

[0271] EXAMPLES

[0272] Example 1 : The integrated domain (ID) of ZmPia-2 is necessary to confer anthracnose resistance (Figure 1)

[0273] Material and Methods

[0274] Four constructs were tested in three combinations (see Figure 1). ZmPia-1 (SEQ ID NO: 1) and ZmPia-2 (SEQ ID NO: 2) are both necessary to confer maize resistance against anthracnose caused by the fungus Colletotrichum graminicola (see for example patent EP1874935 B1). To determine if the integrated domain (ID) MAPK3 found in ZmPia- 2 is a necessary component to confer resistance to anthracnose, the function of ZmPia-2- deltaL / ID (SEQ ID NO: 3) and ZmPia-2-deltaL / ID-OsL / HMA (SEQ ID NO: 4) were assessed. ZmPia-2-deltaL / ID is a truncated version of ZmPia-2, where both the integrated domain and its linker have been removed. ZmPia-2-deltaL / ID-OsL / HMA (SEQ ID NO: 4) is an engineered version of ZmPia-2 where the native linker and the native integrated domain were swapped with an unrelated linker and domain from rice (object of the invention).

[0275] These four constructs were cloned in a binary vector under the control of an operably linked ZmUbi promoter (SEQ ID NO: 5) and an operably linked SbHSP terminator (SEQ ID NO: 6). Using Agrobacterium-mediated transformation of immature embryos (Ishida et al, 2007 Nature Protocols), the maize variety A188 was stably transformed with constructs encoding either ZmPia-1 (SEQ ID NO: 1), ZmPia-2 (SEQ ID NO: 2), ZmPia-2- deltaL / ID (SEQ ID NO: 3) or ZmPia-2-deltaL / ID-OsL / HMA (SEQ ID NO: 4). Immature embryos were also co-transformed, with two constructs encoding either ZmPia-1 (SEQ ID NO: 1) and ZmPia-2 (SEQ ID NO: 2), ZmPia-1 (SEQ ID NO: 1) and ZmPia-2- deltaL / ID (SEQ ID NO: 3) or ZmPia-1 (SEQ ID NO: 1) and ZmPia-2-deltaL / ID-OsL / HMA (SEQ ID NO: 4).

[0276] Plants were regenerated from calli and T 1 plant from independent transformation events carrying a unique T-DNA for either construct were selected. T1 plants were subjected to Colletotrichum graminicola infection and disease was scored using the method described in Romanek et al., 2017 (Crop Breeding and Applied Biotechnology 17: 390-398, 2017 Brazilian Society of Plant Breeding. http: / / dx.doi.org / 10.1590 / 1984- 70332017v17n4a58).

[0277] Results

[0278] The non-transformed maize variety A188 is susceptible to anthracnose caused by the fungus Colletotrichum graminicola. Transformants expressing either ZmPia-1 (SEQ ID NO: 1), ZmPia-2 (SEQ ID NO: 2), ZmPia-2-deltaL / ID (SEQ ID NO: 3) or ZmPia-2- deltaL / ID-OsL / HMA (SEQ ID NO: 4) individually also remain susceptible to anthracnose.

[0279] On the contrary, stable co-expression of ZmPia-1 and ZmPia-2 in transgenic A188 maize is sufficient to confer resistance against maize anthracnose.

[0280] Crucially, the carboxy-terminal MAP3K domain integrated in the ZmPia-2 protein is necessary to confer resistance against maize anthracnose as the stable co-expression of either ZmPia-1 and ZmPia-2-deltaL / ID, or ZmPia-1 and ZmPia-2-deltaL / ID-OsL / HMA- genes in transgenic A188 maize does not confer resistance against maize anthracnose. Swapping the MAP3K integrated domain of ZmPia-2 with an HMA domain from rice redirects the immune response toward a rice blast effector in Nicotiana benthamiana (Figure 2A-D)

[0281] Material and methods

[0282] Fused to canonical NLR domains, integrated domains (IDs) 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 consequence, an ID would determine the specificity of the detected ligand, and swapping ID could redirect the recognition against a different pathogen.

[0283] In the maize variety CML277, the ZmPia-2 gene encodes a resistance gene consisting of a NLR backbone (composed of a CC domain, a NB-ARC domain, a LRR domain, and a common integration domain (CID)) followed, in carboxy-terminal position, by a linker region and an integrated domain (ID) consisting of a Mitogen Activated Protein (MAP) kinase kinase kinase (MAPKKK or MAP3K). To determine whether a chimeric ZmPia-2 could trigger a ZmPia-1 -dependent immune response after recognition of an avirulent effector not originating from Colletotrichum graminicola, the function of a chimeric receptor containing the NLR backbone of ZmPia-2 and both the linker (OsL, SEQ ID NO: 14) and the Heavy Metal- Associated integrated domain (HMA, SEQ ID NO: 12) derived from the Oryza sativa OsPia- 2 receptor was evaluated. In rice, the sensor NLR-ID OsPia-2 perceives AVR-Pia, an effector secreted by Magnaporthe oryzae. The full sequence of the OsPia-2 receptor is shown in SEQ ID NO: 13.

[0284] To determine whether we could prompt AVR-Pia recognition using an engineered version of ZmPia-2, it was constructed one chimeric ZmPia-2 receptor (ZmPia-2-deltaL / ID- OsL / HMA (SEQ ID NO: 4) where the OsPia-2 linker and HMA domain are integrated after the CID of the ZmPia-2 backbone (see Figure 2A for construct design). It was also cloned sequences encoding ZmPia-1 (SEQ ID NO: 1), ZmPia-2 (SEQ ID NO: 2), mCherry (SEQ ID NO: 7) and AVR-Pia with no signal peptide (SEQ ID NO: 8) (see Figure 2 A). All these constructs were cloned in a binary vector. The ZmPia-1 construct has an operably linked mas promoter (SEQ ID NO: 9) and an operably linked CaMV35S terminator (SEQ ID NO: 10). All the other constructs have an operably linked 2x35S promoter (SEQ ID NO: 11) and an operably linked CaMV35S terminator (SEQ ID NO: 10).

[0285] 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. Two leaves from three plants were inoculated per experiment (n = 6). 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). Chlorophyll fluorescence is used as a proxy for the intensity of the HR response in those cells or tissues.

[0286] Results

[0287] As shown in Figure 2B, Figure 2C and Figure 2D, the individual expression of ZmPia- 1 (SEQ ID NO: 1), ZmPia-2 (SEQ ID NO: 2) or ZmPia-2-deltaL / ID-OsL / HMA (SEQ ID NO: 4) does not generally result in a hypersensitive response when expressed in presence of mCherry (SEQ ID NO: 7) in N. benthamiana leaves. The fluorescent mCherry protein was used here as a negative control for immune response, as we do not expect any of these constructs to respond to mCherry. Individually, the ZmPia pair members are thus not autoactive and the OsHMA integration in ZmPia-2-deltaL / ID-OsL / HMA (SEQ ID NO: 4) does not trigger auto-activity. As expected, no response was observed when ZmPia-1 , ZmPia-2 or ZmPia-2-deltaL / ID-OsL / HMA were co-expressed with AVR-Pia, as we predict both helper NLR (ZmPia-1 , SEQ ID NO: 1) and sensor NLR-ID (ZmPia-2, SEQ ID NO: 2) are required to signal the presence of the effector.

[0288] However, co-expressing both ZmPia-1 (SEQ ID NO: 1) and ZmPia-2 (SEQ ID NO: 2) in presence of mCherry (SEQ ID NO: 7) resulted in a hypersensitive response. The ZmPia pair is thus activated in the absence of AVR-Pia in N. benthamiana. A quantitative assessment of the assay results is shown in Figure 2C and Figure 2D. HR scoring presented as dot plots and violin plots for AVR-Pia or mCherry mean HR scoring measured in N. benthamiana leaves 5 days post- infiltration.

[0289] In ZmPia-2-deltaL / ID-OsL / HMA (SEQ ID NO: 4), the OsHMA domain (SEQ ID NO: 12) is integrated with its own linker OsL (SEQ ID NO: 14), identified in OsPia-2 (SEQ ID NO: 13), between the common integration domain (CID) of the original ZmPia-2 protein (SEQ ID NO: 15) and the integrated OsHMA domain (SEQ ID NO: 12).

[0290] Co-expressing ZmPia-1 (SEQ ID NO: 1) and ZmPia-2-deltaL / ID-OsL / HMA (SEQ ID NO: 4) in presence of mCherry (SEQ ID NO: 7) reduced the auto-activity of the engineered ZmPia pair. Crucially, the co-expression of ZmPia-1 (SEQ ID NO: 1) and ZmPia-2-deltaL / ID- OsL / HMA (SEQ ID NO: 4) in presence of AVR-Pia (SEQ ID NO: 8) allowed to observe an increased level of immune response specific to the presence of AVR-Pia (see Figure 2B and Figure 2C). The statistical analysis (Figure 2D) indicates that these two responses are statistically different as the 95% confidence intervals of both resampled distributions are not overlapping. Swapping the MAP3K integrated domain of ZmPia-2 with a wheat protein interacting with the fungal effector PstGSREI redirects the immune response towards the effector PstGSREI in maize mesophyll leaf protoplasts (Figure 3)

[0291] Material and Methods

[0292] To determine whether a chimeric ZmPia-2 could trigger a ZmPia-1-dependent immune response after recognition of a candidate effector not originating from Colletotrichum graminicola, the functions of a chimeric receptor containing the NLR backbone of ZmPia-2 fused to TaLOL2, a wheat protein interacting with the Puccinia striiformis effector PstGSREI , was evaluated.

[0293] The wheat LESION SIMULATING DISEASE (LSD) One-Like 2 (TaLOL2) protein contains three zinc-finger domains. TaLOL2 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 (Qi et al., 2019 Molecular Plant 12, 1624).

[0294] To determine whether we could prompt PstGSREI recognition using an engineered version of ZmPia-2, it was constructed the chimeric ZmPia-2 receptor ZmPia-2-deltaL / ID- HvL / TaLOL2 (SEQ ID NO: 19) where the HvL linker (SEQ ID NO: 21) and TaLOL2 domain (SEQ ID NO: 20) are integrated after the CID of the ZmPia-2 backbone (see Figure 3A for construct design).

[0295] It was also cloned sequences encoding ZmPia-1 (SEQ ID NO: 1), ZmPia-2 (SEQ ID NO: 2), mCherry (SEQ ID NO: 7) and PstGSREI with no signal peptide (SEQ ID NO: 22). All these constructs were cloned in a pUC19-derived vector. All the constructs are under the control of an operably linked ZmUbi promoter construct (SEQ ID NO: 5) and an operably linked AtNos terminator construct (SEQ ID NO: 16).

[0296] To examine the function of the engineered ZmPia-2 constructs in maize, maize protoplasts were isolated from leaves of maize 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 maize (Zea mays cultivar A188) protoplasts were co-transfected with 40 ug DNA containing:

[0297] 10 pg of luciferase reporter construct (pUbi::PpLUC),

[0298] 10 pg of each NLR construct (and mCherry construct as DNA filler up to 20 pg), and

[0299] 10 pg of either PstGSREI 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).

[0300] Results

[0301] As shown in Figure 3B (first and second columns), the individual expression of ZmPia- 1 (SEQ ID NO: 1) or ZmPia-2-deltaL / ID-HvL / TaLOL2 (SEQ ID NO: 19) does not result in a death of maize protoplasts when expressed in presence of mCherry (SEQ ID NO: 7). Thus, taken individually, ZmPia-1 is not auto-active and the integration of the HvL / TaLOL2 domain into ZmPia-2-deltaL / ID-HvL / TaLOL2 does not trigger auto activity.

[0302] The co-expression of both ZmPia-1 (SEQ ID NO: 1) and ZmPia-2-deltaL / ID- HvL / TaLOL2 (SEQ ID NO: 19) leads to a partial auto-activity in presence of mCherry (third column). However, an increased level of immune response is observed specifically in presence of PstGSREI (fourth column).

[0303] These observations demonstrate that the pair ZmPia-1 and chimeric ZmPia-2-deltaL / ID- HvL / TaLOL2 can trigger a PstGSREI dependent immune response. In summary, these results demonstrate that the maize native sensor NLR-ID ZmPia-2 can be engineered with effector interacting domains to trigger an immune response after recognition of a cytoplasmic effector not originating from Colletotrichum graminicola.

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 maize sensor NLR-ID protein, wherein said X domain binds to (i) a pathogen effector different from those recognized by the integrated domain of said native maize sensor NLR-ID protein, (ii) an epitope different from the pathogen epitope recognized by the integrated domain of said native maize sensor NLR-ID protein or (iii) a pathogen-induced plant component different from those recognized by the integrated domain of said native maize sensor NLR-ID protein.

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

4. The chimeric sensor NLR-ID protein according to anyone of claims 1 to 3, wherein the native maize sensor NLR-ID protein is protein ZmPia-2 of sequence SEQ ID NO: 2.

5. The chimeric sensor NLR-ID protein according to any one of claims 1 to 4, wherein (i) the X domain is the rice HMA domain of sequence SEQ ID NO: 12 or the wheat TaLOL2 domain of sequence SEQ ID NO: 20 and / or (ii) the peptide linker is therice peptide linker OsL of SEQ ID NO: 14 or the barley linker HvL of sequence SEQ ID NO: 21.

6. A NLR protein pair comprising: the chimeric sensor NLR-ID protein according to any one of claims 1 to 5, 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.

7. The NLR protein pair according to claim 6, wherein said chimeric sensor NLR-ID protein is obtained by replacing the integrated domain of the native sensor NLR- ID protein ZmPia-2 by at least one X domain and wherein said helper protein is protein ZmPia-1.

8. A nucleic acid encoding (i) (a) at least one chimeric sensor NLR-ID protein according to any one of claims 1 to 5, (b) the X domain(s) of the chimeric sensor NLR-ID protein according to any one of claims 1 to 5 or (c) the peptide linker and the X domain(s) of the chimeric sensor NLR-ID protein as defined in any one of claims 1 to 5 and (ii), optionally, at least one helper NLR protein of the NLR protein pair according to claim 6 or 7.

9. The nucleic acid according to claim 8, wherein said nucleic acid comprises a first nucleic sequence encoding the chimeric sensor NLR-ID protein defined in claim 6 or 7 and a second nucleic sequence encoding the helper NLR protein defined in claim 6 or 7, wherein said first and second sequences are in head-to-head orientation.

10. A vector comprising at least one nucleic acid according to claim 8 or 9.

11. 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 5 or at least one NLR protein pair according to claim 6 or 7.

12. A method for obtaining a cell according to claim 11 , wherein said method comprises transforming a cell with at least one nucleic acid according to claim 8 or 9 or at least one vector according to claim 10.

13. 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 8 or 9 or at least one vector according to claim 10, to obtain a transformed cell or a transformed tissue, and - regenerating a plant from the transformed cell or transformed tissue.

14. Use of the chimeric sensor NLR-ID protein according to any one of claims 1 to 5 or of the NLR protein pair according to claim 6 or 7 for preventing a disease caused by a pathogen in a plant.