Methods of conferring disease immunity in plants

Abstract

The present invention relates to fusion proteins comprising pathogen-originated protease cleavage site and autoactive NLR protein (or NLR fragment), as well as uses of these fusion proteins to provide or improve plant resistance against pathogens in plants.

Description

[0001] M&C PC933865LU

[0002] 1

[0003] Methods of conferring disease immunity

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to chimeric proteins comprising pathogen-originated protease cleavage sites and an autoactive intracellular nucleotide-binding and leucine-rich repeat immune receptors (NLR) protein (or NLR fragment), as well as uses of these chimeric proteins to provide or improve plant resistance against pathogens in plants.

[0006] BACKGROUND TO THE INVENTION

[0007] The challenge to feed an increasing global population requires sustainable food production under changing climates. Plant pathogens and pests have devastating effects on crop growth and yields. As such, there is a constant need to develop disease resistance crops.

[0008] Plant disease resistance can be broadly classified into (i) complete resistance with visible hypersensitive response (HR) (classic complete resistance) or without visible HR (ER, extreme resistance), and (ii) partial resistance, sometimes showing systemic HR (SHR) with systemic necrosis due to pathogen spread.

[0009] Most resistance genes (R-genes) encode intracellular nucleotide-binding and leucine-rich repeat immune receptors (NLRs) that play a central role in plant immunity. Plant NLRs include Toll-like / interleukin-1 receptor resistance (TIR) domain-containing NLRs (TNLs) and coiled-coil (CC) domain-containing NLRs (CNLs) and RESISTANCE TO POWDERY MILDEW 8-like CC (CCR) domain-containing NLRs (RNLs). Many TNLs and CNLs specifically recognize pathogen- derived effector proteins and then induce multiple defense responses including HR, also known as programmed cell death at pathogen infection sites. RNLs function downstream of TNLs and some CNLs, and are involved in immune signal transduction. Another subclass of NLRs possess unconventional integrated domains such as the heavy metal-associated (HMA) domain which is involved in pathogen effector detection, and often require other conventional NLRs for immune activation after effector detection.

[0010] However, a single NLR detects only a limited number of effectors, thus conferring narrow-spectrum or race-specific resistance to pathogens. Thus, expanding the

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[0013] recognition specificities of NLRs through site-directed mutagenesis and domain shuffling is highly desirable and may lead to the development of new disease resistance. However, current NLR engineering approaches are constrained by rapid evolution of plant pathogens in the field and may lack durability upon deployment.

[0014] In one example of bioengineering plant disease resistance the NLR protein RPS5 is activated for immunity only after a second host protein PBS1, acting as a decoy, is cleaved by pathogen-secreted proteases. PBS1 can be engineered as a new decoy to expand the NLR recognition specificity for disease resistance. However, many plant species lack the known RPS5 or analogous NLR, thus limiting the use of this "decoy engineering" approach.

[0015] There therefore exists a need to improve methods of engineering NLR proteins to obtain broad-spectrum pathogen resistance.

[0016] SUMMARY OF THE INVENTION

[0017] Pathogens pose threats to crop production and global food security. Bioengineering resistance crops remains one of the most daunting challenges in agriculture. We describe an innovative strategy to engineer plant disease resistance by expressing a chimeric protein containing a blocking peptide / protein such as a flexible polypeptide, coupled with at least one pathogen-originated protease cleavage sites (PCS) in-frame fused to the N-terminus of an autoactive NLR immune receptor (Figure 1a). We have shown that upon invasion, pathogen-originated specific proteases cleave the inactive chimeric proteins to form free autoactive NLR proteins that are then able to trigger broadspectrum plant disease resistance. We have demonstrated that such engineered NLR proteins confer plant resistance against multiple pathogens, specifically multiple potyviruses. Given many across-kingdom pathogenic organisms encode proteases, this strategy can be exploited to control viruses, bacteria, oomycetes, fungi, insects and pests in plants.

[0018] Accordingly, in one aspect of the invention there is provided a fusion (or chimeric) protein comprising an immune receptor protein or fragment thereof, and at least one pathogen-originated protease cleavage site (PCS).

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[0021] In one embodiment, the immune receptor protein is a plant receptor protein. By plant immune receptor protein is meant any cell-surface receptor which detects extracellular pathogen-associated or microbe-associated molecular patterns (PAMPs / MAMPs) and damage signals (DAMPs) to trigger a pattern-triggered immunity (PTI), and any intracellular receptor which detects intracellular pathogen effectors to initiate effector-triggered immunity (ETI). Examples of cell-surface plant immune receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs). Examples of intracellular plant immune receptor proteins include nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins.

[0022] More preferably, the immune receptor protein is a nucleotide-binding domain and leucine-rich repeat-containing (NLR) protein or its N-terminal fragment thereof. In one embodiment, the NLR protein or the N-terminal fragment of NLR comprises or consists of a CC domain, CCR domain or a MADA motif. Where the immune receptor is a NLR or N-terminal fragment thereof, the fusion (or chimeric) protein may be referred to herein as a “fusion (or chimeric) NLR” or “fusion (or chimeric) N-terminal fragment of NLR”.

[0023] The chimeric protein of the invention, and preferably the fusion (or chimeric) NLR or fusion (or chimeric) N-terminal fragment of NLR thereof may further comprise at least one blocking peptide, wherein the blocking peptide prevents the free NLR or N-terminal fragment of NLR thereof from eliciting a hypersensitive response (HR).

[0024] In one embodiment, the blocking peptide comprises at least one residue, wherein preferably the blocking peptide is selected from FLAG, GFP, YFP, HA and Myc or a fragment thereof.

[0025] In one embodiment, at least one PCS and optionally at least one blocking peptide are at the N-terminus of the NLR protein or the N-terminal fragment of NLR thereof.

[0026] In one embodiment, the NLR or N-terminal fragment of NLR thereof is an autoactive variant.

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[0029] In one embodiment, the chimeric protein of the invention further comprises at least one pathogen effector and / or at least one protein that confers abiotic stress tolerance or resistance.

[0030] The pathogen of the pathogen-originated protease may be selected from at least one plant pest, such as a virus, bacteria, oomycete, insect or fungus. In one example the virus is selected from the Potyviridae family. In another example the pathogen is selected from one of the pathogens listed in T ables 1 , 2 or 3.

[0031] In another aspect of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding the fusion (or chimeric) protein of the invention.

[0032] In another aspect of the invention there is provided a vector comprising the nucleic acid molecule of the invention operably linked to at least one regulatory sequence.

[0033] In another aspect of the invention there is provided a genetically altered plant, part thereof or plant cell comprising the fusion (or chimeric) protein of the invention, the nucleic acid molecule the invention or the vector of the invention.

[0034] In another aspect of the invention, there is provided a method of providing or improving pathogen resistance in a plant, the method comprising providing a plant with the fusion (or chimeric) protein of the invention, the nucleic acid molecule of the invention or the vector of the invention.

[0035] In another aspect of the invention there is provided a method of producing a plant with improved pathogen resistance, the method comprising introducing and expressing the fusion (or chimeric) protein of the invention, the nucleic acid molecule of the invention or the vector of the invention.

[0036] In another aspect of the invention, there is provided a method of producing a plant with improved pathogen resistance, the method comprising introducing at least one mutation in at least one nucleic acid sequence encoding a nucleotide-binding domain and leucine-rich repeat-containing (NLR) protein, wherein the mutation is the addition of one or more nucleic acid sequences encoding a pathogen-originated protease cleavage site (PCS)

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[0039] and / or a blocking peptide, as described herein. Preferably, the at least one mutation is introduced using gene editing, such as but not limited to, CRISPR.

[0040] In another aspect of the invention, there is provided a plant obtained or obtainable by the method of the invention.

[0041] In a further aspect of the invention, there is provided a plant protection product comprising the fusion (or chimeric) protein of the invention, the nucleic acid molecule of the invention or the vector of the invention.

[0042] DESCRIPTION OF THE FIGURES

[0043] The invention is further described in the following non-limiting figures:

[0044] Figure 1 shows that HA-PCSPVY-aTm-22confers complete resistance against multiple potyviruses. (a) Design to bioengineer plant immunity via pathogen-derived protease activity to activate auto-inhibited NLRs. * represents autoactivation mutation, (b) Structure of the potyviral polyprotein. Arrow indicates the proteolytic cleavage sites of potyviral proteases P1, HC-Pro and Nla / Nla-Pro. Mature viral proteins are indicated, (c) The N-terminal amino acid (aa) sequence of HA-PCSPVY-aTm-22. It comprises a randomly designed flexible blocking peptide containing HA tags (highlighted red) and PVY Nla / Nla-Pro cleavage site (PCSPVY) sequence (underlined). Tm-22amino-acid residues without the first methionine are highlighted purple. Red arrow indicates the cleavage site, (d) Co-expression of HA-PCSPVY-aTm-22and PVY Nla caused cell death. The individual or combined constructs were transiently expressed in N. benthamiana by agroinfiltration. The photograph was taken at 2 days post agroinfiltration. Immunoblotting confirmed successful expression of HA-PCSPVY-aTm-22and NlaPVY-Myc with nonagroinfiltrated leaf tissues as negative control, e, f, HA-PCSPVY-aTm-22(e) and HA-PCSPVY-aTm-22-HA (f) can be cleaved by Myc-Nla-ProPVY. Engineered NLRs were coexpressed with N-terminal Myc tagged wild-type (WT) Nla-ProPVYand its proteaseinactive mutant C151A at the left or right half of same leaves. Leaf tissues without agroinfiltration (NA) and expressing empty vector (EV) were used as negative controls. Total protein was isolated and analyzed. NLR proteins and proteases were detected by anti-HA or anti-Myc antibody, respectively. Black and red arrows indicate positions of the HA-PCSPVY-aTm-22-HA, and its C-terminal cleaved product i.e. aTm-22-HA. Loading of

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[0047] total protein samples for analyses are shown (lower panel). Sizes and positions of protein markers are indicated, (g) PVY-GFP-infected WT and resistant transgenic plants under normal light and long-wavelength ultra-violet (UV) light at 21 days post infection (dpi), (h) No PVY RNA was detected by RT-PCR in systemic leaves of resistant transgenic HA-PCSPVY-aTm-22T1 plants infected with PVY-GFP at 21-dpi. Numbers 1-4 means 4 samples from 4 different transgenic plants, (i), (k), (m), (o), Transgenic HA-PCSPVY-aTm-22T1 plants conferred resistance against TuMV-GFP (i), PPV-GFP (k), PepMoV (m) and ChiVMV (o). Plants were photographed at 21-dpi. (j), (I), (n), (p), No viral RNAs were detected by RT-PCR at 21-dpi in the systemic leaves of resistant transgenic HA-PCSPVY-aTm-22T1 plants inoculated with TuMV-GFP (j), PPV-GFP (I), PepMoV (n) and ChiVMV (p). Numbers 1-3 means 3 samples from 3 different transgenic plants. The bar is 5 cm in figure (g), (i), (k), (m) and (o).

[0048] Figure 2 shows that HA-PCSPVY-aAtNRG1.1 conferred resistance against multiple potyviruses. (a) The N-terminal amino acid (aa) sequences of HA-PCSPVY-aAtNRG1.1 and HA-PCSPVY-aNbNRG1. They comprise blocking peptide containing HA tags (highlighted red) and PCSPVYsequence (underlined) and N-terminal aAtNRGI.1 aminoacid residues (highlighted purple) or N-terminal aNbNRGI amino-acid residues (highlighted blue). Red arrow indicates the cleavage site, (b) HA-PCSPVY-aAtNRG1 induced strong cell death when co-expressed with NlaPVY-Myc. Photographs were taken at 2 days post agroinfiltration. Immunoblotting confirmed expression of HA-PCSPVY-aAtNRG1.1 and HA-PCSPVY-aNbNRG1 proteins, (c), (d), HA-PCSPVY-aAtNRG1.1 (c) and HA-PCSPVY-aAtNRG1.1-HA (d) were cleaved by Myc-Nla-ProPVY. Engineered NLRs were co-expressed with N-terminal Myc tagged wild-type (WT) Nla-ProPVYand its protease-inactive C151A mutant at the left or right half of same leaves. Leaf tissues without agroinfiltration (NA) and expressing empty vector (EV) were used as negative controls. Total protein was isolated and analyzed. NLR proteins and proteases were detected by anti-HA or anti-Myc antibody, respectively. Black or red arrow indicates position of the HA-PCSPVY-aAtNRG1.1-HA, and the C-terminal cleaved product aAtNRGI.1 -HA. e, WT and T1 transgenic plants with extreme resistance (ER) or classic complete resistance (HR) were photographed at 21-dpi by PVY-GFP. The bar is 5 cm. f, No PVY RNA was detected by RT-PCR in systemic leaves of T1 transgenic HA-PCSPVY-aAtNRG1.1 plants infected with PVY-GFP at 23-dpi. g, T1 transgenic plant co-infected with 4 potyviruses TuMV-GFP, PPV-GFP, PepMoV and ChiVMV. Wild-type plants

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[0051] infected with each individual virus was used as controls. Plants were photographed at 21 -dpi. The bar is 5 cm. h, No viral RNA was detected by RT-PCR in systemic leaves of T1 transgenic HA-PCSPVY-aAtNRG1.1 plants co-infected with the 4 potyviruses at 21-dpi.

[0052] Figure 3 shows that HA-PCSTEV-PCSPVY-aAtNRG1.1 confers resistance against PVY and TEV. (a) N-terminal amino acid sequences of HA-PCSTEV-PCSPVY-aAtNRG1.1. It contains a randomly designed flexible peptide along with dual protease cleavage sites to block aNLR activity. Red amino-acid sequences represent HA tag. Both TEV Nla cleavage site (PCSTEVhighlighted grey) and PCSPVY(highlighted blue) are underlined. Purple amino-acid residues are the AtNRG1.1 protein sequence without the first methionine. Arrow indicates the cleavage position, (b) Transient co-expression of HA-PCSTEV-PCSPVY-aAtNRG1.1 and Nla from either TEV or PVY induced cell death. The representative photograph was taken at 2 days post agroinfiltration. Immunoblotting confirmed expression of HA-PCSTEV-PCSPVY-aAtNRG1.1, NlaPVY-Myc and NlaTEV-Myc. c, d, HA-PCSTEV-PCSPVY-aAtNRG1.1 (c) and HA-PCSTEV-PCSPVY-aAtNRG1.1-HA (d) was cleaved by Myc-Nla-ProPVYand Myc-Nla-ProPVY. The engineered aNLR was coexpressed with wild-type Myc-Nla-ProPVY(or Myc-Nla-ProTEV) in one half leaf, and with its C151 A mutant in another half of the same leaf. Total protein was isolated and analyzed. The engineered NLR proteins and proteases were detected by anti-HA and anti-Myc antibody, respectively. Black arrow indicates position of full-length HA-PCSTEV-PCSPVY-aAtNRG1.1, and red one indicates that of C-terminal cleavage product (d). e, WT and transgenic plants infected with PVY-GFP were photographed under normal light and UV light at 21 -dpi . Bar is 5 cm. f, RT-PCR showed that no PVY RNA was detected in the systemic leaves of transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants at 23 dpi. g, Infection of WT and transgenic plants with TEV-GFP. Photographs were taken at 21 -dpi. Bar is 5 cm. h, RT-PCR showed that no TEV RNA was detected in the systemic leaves of resistant HA-PCSTEV-PCSPVY-aAtNRG1.1 plants at 21 dpi.

[0053] Figure 4 shows HA-PCSSMV-aAtNRG1.1 conferred resistance against soybean mosaic virus, a, The N-terminal amino acid (aa) sequence of HA-PCSSMV-aAtNRG1.1. It comprises blocking peptide containing HA tags (highlighted red), PCSSMVsequence (underlined), and N-terminal aAtNRG1.1 amino-acid residues (highlighted purple). Red arrow indicates the cleavage site, b, HA-PCSSMV-aAtNRG1.1 induced strong cell death

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[0056] when co-expressed with NlaSMV-Myc, but not NlaPVY-Myc. Photographs were taken at 2 days post agroinfiltration, c, d, HA-PCSSMV-aAtNRG1.1 (c) and HA-PCSSMV-aAtNRG1.1-HA (d) were cleaved by NlaSMV-Myc. Engineered NLRs were co-expressed with NlaSMV-Myc and NlaPVY-Myc at the left or right half of same N. benthamiana leaves. Leaf tissues without agroinfiltration (NA) and expressing empty vector (EV) were used as negative controls. Total protein was isolated and analyzed. NLR proteins and proteases were detected by anti-HA or anti-Myc antibody, respectively. Black and red arrows indicate positions of the HA-PCSSMV-aAtNRG1.1-HA and its C-terminal cleaved product aAtNRG1.1-HA. (e) the topmost systemic leaves under normal and 440-460 nm light at 25 dpi of SMV-eGFP. Both non-inoculated WT and inoculated transgenic T1 soybean plants have developed 5 trifoliate leaves, and their 5th trifoliate leaves were shown. The infected WT soybean plant only developed 4 trifoliate leaves, and the 4th trifoliate leave was shown, (f) Analysis of HA-PCSSMV-aAtNRG1.1 protein and SMV RNA at 25 days post-inoculation (dpi) in T1 transgenic soybean plants, g, the inoculated leaves of SMV-eGFP were shown. Bar is 2 cm.

[0057] Figure 5 shows expression and traits of transgenic plants of HA-PCSPVY-aTm-22. a, Transient expression of either NlaPVY-Myc or NlaTuMV-Pro-Myc caused cell death in TO transgenic HA-PCSPVY-aTm-22plants (lines #3 and #5), but not in wild-type plants at 2 days post agroinfiltration, b, Successful expression of Myc-tagged proteins was confirmed by immunoblotting. Loading of total protein samples for analyses are shown (lower panel). Sizes and positions of protein markers are indicated, c, T1 transgenic HA-PCSPVY-aTm-22plants of two independent lines showed no developmental defects, d, No significant trait difference between wild-type and transgenic plants. Height and fresh weight were measured when the plants are 3 months old, and seed setting indicated by numbers of seed pods were measured when the plants are 4 months old. ns means no significance as determined by Student's t tests (n=6). Data are represented as mean ± s.d. e, Transgene and protein expression of the HA-PCSPVY-aTm-22chimeric protein were detected by genomic PCR (upper panel) and immunoblotting (middle panel) in three independent individuals of T1 plants from two independent transgenic lines #3 and #5. Loading of total protein samples for analyses are shown (lower panel). Sizes and positions of DNA (upper panel) and protein markers (middle and lower panels) are indicated.

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[0060] Figure 6 shows HA-PCSPVY-aTm-22confers resistance against PVY-GFP.

[0061] a, Inoculated leaves under normal light (left panel) and ultra-violet light (right panel) at 7-dpi by PVY-GFP. Leaves of WT, #3-T1 and #5-T1 came from the same plants showed in Figure 1g. HR lesions are indicated (arrows). The bar is 2 cm. b, c, Inoculated leaves showed HR lesions at 7-dpi (b), but whole plant showed systemic hypersensitive response (SHR) at 21-dpi (c) for a few T1 transgenic HA-PCSPVY-aTm-22plant infected by PVY-GFP. Wild-type (WT) plant was used as control. The bar is 2 cm and 5 cm in pannels b and c, respectively.

[0062] Figure 7 shows HA-PCSPVY-aTm-22confers resistance against potyviruses.

[0063] a-d, Leaves of T1 transgenic HA-PCSPVY-aT m-22plants were inoculated with T uMV-GFP (a), PPV-GFP (b), PepMoV (c) and ChiVMV (d), respectively, and photographed at 7 days post infection. HR lesions are indicated (arrows) in T1 transgenic HA-PCSPVY-aTm-22plants inoculated with PepMoV (c).

[0064] Figure 8 shows Confirmation of expression in transgenic plants of HA-PCSPVY-aAtNRG1.1. a, PVY Nla-Myc caused cell death in the leaf of TO transgenic HA-PCSPVY-aAtNRG1.1 plants, b, T1 transgenic plants of line #1 grew and developed normally, c, Transgene and HA-PCSPVY-aAtNRG1.1 protein were detected by genomic PCR and immunoblotting in three independent individuals of T1 plants of transgenic line #1.

[0065] Figure 9 shows HA-PCSPVY-aAtNRG1.1 confers resistance against multiple potyviruses. a, Inoculated leaves of wild-type and T1 transgenic HA-PCSPVY-aAtNRG1.1 plants challenged with PVY-GFP. Photographs were taken under normal light and UV light at 7- dpi. HR lesions are indicated (arrows), b, Systemic tissues of PPV-inoculated WT and transgenic plants at 7 dpi. Bar is 5cm. c, Inoculated leaves of T1 transgenic HA-PCSPVY-aAtNRG1.1 plants co-infected with TuMV-GFP, PPV-GFP, PepMoV and ChiVMV and wild-type (WT) plants infected with each of the four individual potyviruses. Two halves of one leaf of transgenic plants were infected with TuMV-GFP and PPV-GFP, respectively. Two halves of another leaf of the same transgenic plant were infected with PepMoV and ChiVMV, respectively. One half leaf from different wild-type plants was infected with TuMV-GFP, PPV-GFP, PepMoV and ChiVMV, separately. Leaf photographs were taken at 7-dpi. HR lesions are indicated (arrows). Bar is 2 cm.

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[0068] Figure 10 shows HA-PCSPVY-aNLR failed to confer resistance against TEV. a, Coexpression of HA-PCSPVY-aNLR with PVY Nla-Myc, but not TEV Nla-Myc, caused cell death. The photograph was taken at 3 days post agroinfiltration, b, TEV-GFP spread over WT and T1 transgenic HA-PCSPVY-aAtNRG1.1 plants of line #1 at7-dpi. c, RT-PCR detected the presence of TEV RNA in WT and transgenic HA-PCSPVY-aAtNRG1.1 plants at 7-dpi by TEV-GFP.

[0069] Figure 11 shows confirmation of expression and resistance in transgenic plants of HA-PCSTEV-PCSPVY-aAtNRG1.1. a, T1 transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants from line #3 showed no abnormal development, b, Transgene and its protein expression were detected by genomic PCR and immunoblotting in three independent individual T1 plants of line #3. c, d, The inoculated leaves of T1 transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants were photographed under normal light and UV light at 7 days post infection with PVY-GFP (c) and TEV-GFP (d).

[0070] Figure 12 shows HA-PCSTEV-PCSPVY-aAtNRG1.1 confers resistance against multiple potyviruses. a, The inoculated leaves of T1 transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants infected with TuMV-GFP, PPV-GFP, PepMoV and ChiVMV. HR lesions are indicated with arrows. Photographs were taken at 7 days post infection, b, T1 transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants showed resistance against co-infection of TuMV-GFP, PPV-GFP, PepMoV and ChiVMV. T1 transgenic plants were co-infected with all four potyviruses TuMV-GFP, PPV-GFP, PepMoV and ChiVMV. Two halves of one leaf of transgenic plants were infected with TuMV-GFP and PPV-GFP, respectively. Two halves of another leaf of the same transgenic plants were infected with PepMoV and ChiVMV, respectively. One half leaf from different wild-type plants was infected with TuMV-GFP, PPV-GFP, PepMoV and ChiVMV, separately. Photographs were taken at 21-dpi. The bar is 5 cm. c, RT-PCR showed that no viral RNA was detected in the systemic leaves of T1 transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants co-infected by TuMV-GFP, PPV-GFP, PepMoV and ChiVMV.

[0071] Figure 13 shows some transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants showed systemic hypersensitive response (SHR) upon TEV-GFP infection, a, The inoculated leaves and whole plants were photographed under normal light and UV light at 7 and 21-dpi respectively, b, The enlarged view of top tissues accompanied with SHR at 21-dpi.

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[0074] c, RT-PCR showed that small amount of TEV RNA was detected in the systemic leaves of SHR plants.

[0075] Figure 14 shows HA-PCSAvrRpt2-aAtNRG1.1 recognized AvrRpt2. a, Co-expression of AvrRpt2-Flag and HA-PCSAvrRpt2-aAtNRG1.1 caused much severer cell death than individual expression of AvrRpt2-Flag. The photograph was taken at 28 hours post agroinfiltration, b, HA-PCSAvrRpt2-aAtNRG1.1 was cleaved by AvrRpt2-Flag. c, HA-PCSAvrRpt2-aAtNRG1.1-HA was cleaved by AvrRpt2-Flag. Engineered NLRs were coexpressed with wild type AvrRpt2-Flag and its protease-inactive C122A mutant at the left or right half of same leaves. Leaf tissues without agroinfiltration (NA) and expressing empty vector (EV) were used as negative controls. Total protein was isolated and analyzed. The NLR proteins and proteases were detected by anti-HA and anti-Flag antibody, respectively. Back arrow indicates position of the HA-PCSAvrRpt2-aAtNRG1.1-HA, and the red one indicates that of C-terminal cleavage product aAtNRG1.1-HA.

[0076] Figure 15 shows co-expression of AvrPphB and HA-PCSAvrPphB-aAtNRG1.1 caused cell death. The photograph was taken at 3 days post agroinfiltration.

[0077] Figure 16 shows that when fused to a PM localization signal named Rop, Tm-22CC domain alone can induce cell death. We fused YFP and TEV Nla protease cleavage site to the N-terminus of CC-Myc-Rop, and found thatYFP-PCSTEV-CC-Myc-Rop caused cell death when co-expressed with HA-TEV Nla-Pro. The photograph was taken at 3 days post agroinfiltration.

[0078] Figure 17 shows that an extra six-amino acid sequence (YEVHHQ) is sufficient to block the autoimmune activity of aTm-22, whereas PCSPVY-aTm-22can induces cell death when co-expressed with and Myc-Nla-ProPVY. The photograph was taken at 2 days post agroinfiltration. PCSPVY(YEVHHQA) consists of the additional six amino acids (YEVHHQ) followed by the alanine corresponding to the first residue of Tm-22after removal of the initiating methionine.

[0079] Figure 18 shows that co-expression of PAT-PCSPVY-CCRNbNRG1and Myc-Nla-ProPVYcaused cell death. The photograph was taken at 2 days post agroinfiltration. PAT (phosphinothricin acetyltransferase) confers resistance to phosphinothricin-based

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[0082] herbicides. CCRNbNRG1denotes the N-terminal RPW8-like coiled-coil domain of NbNRGI. PAT-PCSPVY-CCRNbNRG1is therefore expected to confer resistance to both viruses and herbicides.

[0083] Figure 19 shows that co-expression of PAT-PCSPVY-CCRA‘ADR1and Myc-Nla-ProPVYcaused cell death. The photograph was taken at 2 days post agroinfiltration. PAT-PCSPVY-CCRA‘ADR1is therefore expected to confer resistance to both viruses and herbicides.

[0084] Figure 20 shows that co-expression of HA-PCSPVY-CCMLA10(F99E) and Myc-Nla-ProPVYcaused cell death. The photograph was taken at 2 days post agroinfiltration. CCMLA1° denotes the N-terminal coiled-coil domain of MLA10. F99E mutation was reported to enhance the cell death induced by CCMLA1° (Bai S, et al. Structure-function analysis of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in cell death and disease resistance, PLoS Pathogens 8(6): e1002752, 2012;).

[0085] DETAILED DESCRIPTION

[0086] The following embodiments apply to all aspects of the invention.

[0087] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0088] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, which are within the skill of the art. Such techniques are explained fully in the literature.

[0089] As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide

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[0092] analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or “gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and / or may include cDNAs in combination with regulatory sequences.

[0093] The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

[0094] For the purposes of the invention, a “genetically altered plant” is a plant that has been genetically altered compared to the naturally occurring wild type (WT) plant. In one embodiment, a genetically altered plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant using a mutagenesis method, such as targeted genome modification or genome editing. In one embodiment, the plant genome expresses the chimeric protein, preferably a chimeric NLR protein or vectors expressing the chimeric NLR proteins. Such plants have an altered phenotype as described herein, such as increased immunity to a pathogen. Therefore, in this example, these phenotypes are conferred by the presence of an altered plant genome, for example the mutation of at least one gene encoding an NLR gene. In particular, the aspects of the invention involve recombination DNA technology and exclude embodiments that are solely based on generating plants by traditional breeding methods.

[0095] NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins are modular proteins that consist of an N-terminal domain (NTD), a central NB-ARC domain, and a C-terminal region containing leucine-rich repeats (LRR) or other superstructureforming repetitive elements. The NB-ARC domain functions as a switch that controls the “on / off” state of the receptor, whereas the variable NTD defines different NLR classes. In plants, there are three types of NLRs distinguished by their N-terminal domain: CNLs - CC (coiled-coil) domain NLRs, TNLs - the TIR (Toll / interleukin- 1 receptor) domain NLRs and the RPW8 (resistance to powdery mildew 8)-like coiled-coil domain NLR. NLR NTDs are the “executioner” or “signalling” domain encoding the biochemical activities that lead

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[0098] to immunity. Indeed, ectopic expression of NTDs in planta, either alone or translationally fused to fluorescent proteins like yellow fluorescent protein (YFP), is often sufficient to promote domain self-association and activate plant immune responses. Typical outputs downstream of NLR NTD activity include defensive hormone accumulation / signalling, transcriptional reprogramming, reactive oxygen species (ROS) accumulation, and in many cases a localized form of programmed cell death known as the hypersensitive response (HR).

[0099] NLRs have been investigated over decades with the aim to use them for improving plant disease resistance. Here, we describe a simple and effective strategy to redesign NLRs, such as the CNL / RNL-type of NLR immune receptors to expand their recognition specificities for engineering new broad-spectrum or individual disease resistance in plants. This strategy is based on the realisation that a N-terminal peptide such as HA and YFP can inactivate an autoactive NLR (referred to herein as a aNLRs) or an autoactive N-terminal fragment of NLR, and that a PCS-containing polypeptide can be used to switch on or off the autoactivity of aNLR or autoactive N-terminal fragment of NLR in the presence versus absence of pathogen-originated proteases. Using Tm-22(CNL) and NRG1 (RNL) coupled with the potyviral Nla protease-targeted PCSs, we provide compelling “proof of principle” evidence that this strategy can confer complete plant resistance, such as to individual or mixed potyviral infection (as shown in Figs. 1-16). Given that many DNA and RNA viruses, bacteria, oomycetes, fungi, and nematodes as well as pests express and secrete proteases, this strategy is applicable to control across-kingdom pathogens and pests in plants.

[0100] Indeed, using the same experimental design, we found that co-expression of the Pseudomonas syr / ngae-originated cysteine protease AvrRpt2 and HA-PCSAvrRpt2-aAtNRG1.1 caused more severe cell death than individual expression of AvrRpt2 did in N. benthamiana leaf (Figure 14). Further, transgenic Arabidopsis RPS2 knockout plants expressing HA-PCSAvrRpt2-aAtNRG1.1 showed resistance to Pseudomonas syringae DC3000 expressing avrRPT2. (Table 6). Interestingly, tandem PCSs can even further expand the spectrum of disease resistance, and an aNLR bearing an 8-aa short peptide at its N-terminus remains functional to elicit immunity in plant cells (Figure 3).

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[0103] Moreover, modifying a R-gene by our method is easy and fast. Indeed, a short DNA sequence can be added to the 5’ end of autoactive NLR gene to construct a chimeric gene that encodes a fusion protein consisting of a N-terminal short peptide, one or two pathogen-derived PCSs, and an aNLR or an autoactive N-terminal fragment of NLR. Upon infection, pathogens produce protease(s) that will cleave the fusion protein and release free aNLR or an autoactive N-terminal fragment of NLR to trigger immunity. It should be noted that aNLR can also be simply created by site-directed mutagenesis. For instance, D-to-V mutation in the MHD motif is reported to cause NLR activation for many NLRs. Further, aNLR can be replaced with CC / CCR domain or very short MADA motifs which have been reported to induce HR cell death in plants.

[0104] The reported engineered or native NLRs usually confer resistance to single pathogens. By contrast, single NLRs engineered by our strategy can confer extremely broadspectrum resistance against multiple potyviruses, and is expected to be resistant to at least 110 potyviruses when using single conserved PCSPVY, even to more pathogens when using tandem PCSs. In addition, the classic NLR-mediated resistance often endures an average effective 3.5-years life span of a single all- stage R-gene. Similarly, resistance engineered by the reported approaches for artificially expanding the recognition specificity of NLRs mainly depend on direct interactions between engineered R-protein and pathogen effector protein, that can be easily overcome during the plantpathogen arms race. By contrast, the resistance engineered by our strategy is expected to be durable because breakdown of resistance will require simultaneous loss-of-function mutations in both pathogen-originated proteases and their conserved PCSs within pathogenic polyproteins which are essential for pathogens to survive. This seems to be unlikely to occur since such double mutations will be lethal to pathogens.

[0105] Lastly, we have also envisaged that our innovative strategy when coupled with the genome editing technology such as CRISPR / Cas can be directly employed to engineer endogenous R-gene and condition autoactivity of endogenous NLR for improving plant immunity. For example, we can use CRISPR to add in a PCS and a blocking peptide to the endogenous gene. In one embodiment, base editing or prime editing can be used to introduce the point mutation to create the activated NLR, and gene knock-in technologies can be used to insert the short DNA fragment encoding the polypeptide including the cleavage site into the genome of crops.

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[0108] Accordingly, in one aspect of the invention, there is provided a fusion protein comprising a nucleotide-binding domain and leucine-rich repeat-containing (NLR) protein or the N-terminal fragment of NLR thereof and at least one pathogen-originated protease cleavage (PCS) site. Preferably the PCS is at the N-terminal of the NLR protein as shown in Figure 1a.

[0109] As used herein “fusion” refers to the NLR protein of the invention that comprises both a NLR protein or a N-terminal fragment of NLR thereof and at least one PCS site, as described in further detail below. As used here “chimeric” may refer to the nucleic acid sequence that encodes the fusion protein of the invention. However, the terms “fusion protein” and “chimeric protein” for the purposes of the present invention may be used interchangeably.

[0110] The NLR may be selected from a CNL or a RNL-type of NLR immune receptor. In one embodiment, the NLR is Tm-22, which is a plasma membrane (PM)-localized coiled coil (CC)-type NLR.

[0111] It is known that the CC / CCR domains or N-terminal MADA motifs alone are sufficient to activate plant defence and HR cell death. As such, in one embodiment of the invention, the fusion protein comprises a NLR fragment and at least one pathogen cleavage site. For example, as we show in Figure 16, fusion of YFP and a TEV Nla protease cleavage site to the N-terminus of Tm-22CC-Myc-Rop leads to cell death when co-expressed with the pathogen, TEV Nla-Pro.

[0112] By “fragment” is meant a functionally active series of consecutive nucleic acids from a longer nucleic acid sequence. The fragment may be at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10% or at least 1% the length of the full NLR sequence and is able to elicit a hypersensitive response (HR). A number of standard techniques in the art can be used to determine if a hypersensitive response has been elicited, and as such, whether a given fragment is capable of eliciting a hypersensitive response (and therefore, resistance). For example, Agrobacterium-mediated transient expression in N.

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[0115] benthamiana can be used to test whether a given fragment is capable of eliciting a hypersensitive response.

[0116] In one embodiment, the N-terminal fragment of an NLR comprises or consists of a CC or CCR domain. In a further embodiment, the N-terminal domain comprises or consists of a MADA motif.

[0117] By a “Coiled coil (CC) domain” is meant the coiled coil domain found at the N-terminus of an NLR. Many CC domains contain a conserved EDVID motif, which was which first described in Rx, a potato CC domain-containing NRL conferring resistance to Potato virus X (PVX) via recognition of the PVX coat protein. According to their sequence features, the CC domains can be classified into five major groups.

[0118] The SD-CC type of coiled coil domains is exemplified in the NRLs Sw-5, Prf and Mi-1.2 found in tomato.

[0119] The CCEDviotype of coiled coil domain is exemplified in the NRLs Rx (potato), MLA10 (Barley), Sr33 (Wheat), Sr50 (Rye), Rp1-D21 (Maize), RPM1 (Arabidopsis), PM21 (Wheat) and NRC4 ( / V. bethamiana).

[0120] The I2-Iike type of coiled coil domain is exemplified in the NRLs I2 (Tomato), N’ (Nicotiana sylvestris), R3a (Potato). These I2-Iike type of CC domains lack the EDVID motif.

[0121] The CCR type of coiled coil domains is exemplified in the NRLs ADR1, and NRG1. These CCR type of CC domains lack the EDVID motif.

[0122] The CCNO-EDVID type of coiled coil domains is exemplified in the NRLs RPS2 (Arabidopsis), RPS5 (Arabidopsis) and Pvr4 (Pepper). These CCNO-EDVID type of CC domains lack the EDVID motif.

[0123] While the specific structure of the N-terminal domain or fragment may vary between NLR proteins, all N-terminal domains contain at least one alpha helix, which is responsible for inserting into the plasma membrane and forming a pore to initiate the HR response.

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[0126] Thus, in one embodiment, the N-terminal domain or fragment of the NLR comprises or consists of at least one alpha helix.

[0127] By a “TIR (Toll / interleukin-1 receptor / resistance protein) domain” is meant a domain found at the N-terminus of an NLR, typically consisting of 135-160 residues and displaying a five-stranded parallel p-sheet (strands A- E) surrounded by five a-helices (aA-aE).

[0128] Plant Tl R domains are enzymatic Tl R-Domains, that undergo self-association to facilitate NAD+ activity and eventually cell death. Single TIR domains are characterised by a BB-loop found at the BE interface and BCD, EE and AE surfaces. Self-association between TIR domains forms assemblies of TIR domains in a head-to-tail arrangement, mediated by interactions between the AE surface and BB-loop-containing BE interface. The interface corresponds to a symmetric “AE interface”, involving the aA and aE helices. Crystal structures of numerous plant TIR domains (including those from RPS4 and RPS4-RRS1 heterodimer, AtTIR, SNC1, RPP1 and RPV1) feature a symmetrical AE interface. The AE interface often features conserved residues, such as the SH (serinehistidine) motif in the aA helices of the TIR domains, which allows stacking and hydrogen-bonding interactions across the interface. Charged residues surrounding the SH motif further stabilize the AE interface.

[0129] By a “MADA motif” is meant a sequence with the consensus sequence of MADAxVSFxVxKLxxLLxxEx.

[0130] Accordingly, one embodiment, the NLR may be selected from a CC-NLR (or CC-NBS-LRR), which contains a coiled-coil (CC) domain at the N-terminus) and a CCr-NLR (or RPW8-NBS-LRR, which contain a Resistance to Powdery Mildew 8 (RPW8) domain at the N-terminus).

[0131] In one (non-limiting) example, the NLR may be selected from one of the following NLR proteins: ADR1-like, Adnrl, Adnr1-RGA4, AtNRG1.1, BnRPRI, CHS3, DAR5 CcRppI, GhDSCI, MLA8, NRC1, NRC2a, NRC2b, NbNRC4b, NbPRFb, NiattPtrl, OsRPRI, Pi1-5, Pi1-6, Pi5-3, Pi7-J-1, Pi7-J-2, Pia-2 (RGA5), Pii-2, Pik-1, Pik-2, PIK5-NP, PIK6-NP, Pik-e1, Pik-e2, Pik-h1, Pik-h2, Pik-m1, Pik-m2, Pik-p1, Pik-p2, Piks-1, PiPR1, Pik-s1m Pi-ta, R1A-3, R1B-14, R1B-16, R1B-23, RGA2a, RGA4-like, RGH2, RLM3_Col, RPG1,

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[0134] RPG5, Rph15, Rpp1-R1, Rpp1-R3, Rpp1-R4, Rpp1-R5, RPP2A, RRS1B, RRS1-R, RRS1-S, RRSI-Ws, SLH1, Ta4ANPR1, Ta7ANPR1 (RGA5), Ta7DNPR1, TRIDC5AG050380, Tsn1, WRKY19, Xa1, Xo1, Yr5, Yr7, YrSP, YrU1, Sw-5, Prf, Mri-1.2, Rx, MLA10, Sr33, Sr50, Rp1-D21, RPM1, PM21, NRC4, I2, R3a, ADR1, NRG1, RPS2, RPS4, RPS1, RPP1, RPP4, SNC1, N’, L6, RLM3, RLM10, DM1, DM2, CHS1, RPS5 and Pvr4.

[0135] In one embodiment the NLR may be selected from Sw-5, Prf, Mri-1.2, Rx, MLA10, Sr33, Sr50, Rp1-D21, RPM1, PM21, NRC4, I2, R3a, ADR1, NRG1, RPS2, RPS4, RPS1, RPP1, RPP4, SNC1, N’, L6, RLM3, RLM10, DM1, DM2, CHS1, RPS5 and Pvr4.

[0136] In one embodiment, the NLR protein may be a NRC protein (NLR-required for cell death). In an embodiment, the NLR protein is selected from NRC2, NRC3 and NRC4. In this embodiment, the plant is a Solanaceous plant.

[0137] In an embodiment, the NLR protein or N-terminal fragment thereof is selected from Xa1 and Xa1-like nucleotide-binding leucine-rich repeat (NLR) proteins from rice, Pi-family NLR proteins including Pi54, Pia, Pik, and Pita from rice, RB (Rpi-blb1) from Solanum bulbocastanum, Rpi-vnt1 and Rpi-blb2 from potato, R3a from potato, MI-1.2 from tomato, Bs2 from pepper, N NLR protein or Nfrom tobacco, Rsv1 and Rsv3 from soybean, Rpp-family NLR proteins from soybean, stem rust resistance proteins selected from Sr33, Sr35, and Sr45 from wheat.

[0138] Pathogen originating proteases are proteases produced by pathogens, including but not limited to viruses, bacteria, fungi and insects. Non-limiting examples of pathogens that express proteases are provided in Tables 1, 2 and 3. Pathogen originating proteases have a critical role in the pathogen infection of numerous plants, mediated by cleaving pathogen and / or host proteins. The sites at which such cleavages take place are referred to as pathogen-originated protease cleavage sites. Accordingly, by “pathogen-originated protease cleavage site” or “PCS” is meant a sequence of amino acids in a protein that are targeted and cleaved by at least one protease produced by at least one pathogen.

[0139] In one embodiment, the PCS is selected from one of the cleavage sites described in Table 1, 2 or 3.

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[0142] In one embodiment the cleavage site comprises the consensus sequence: xxVxHQj.A(G / S) orxxVxxQj.A(G / S), where x is any amino acid. In one embodiment, the cleavage site comprises the sequence: PCSPVY(YEVHHQJ.A). In another embodiment, the protease cleavage site is GDK / SHVS or VPKFG / DW, and the pathogen in Pseudomonas syringae.

[0143] In one embodiment, the cleavage site comprises or consists of at least one sequence selected from: resiudes 68 to 74 of SEQ ID NO: 3 to 8.

[0144] That is, in one embodiment, the cleavage site comprises or consists of the sequence at least one of:

[0145] • YEVHHQA (corresponding to the PVY Nla protease cleavage site, and residues 68 to 74 of SEQ ID NO: 3, 4 and 7);

[0146] • ESVSLQS (corresponding to the SMV Nla protease cleavage site, and residues 68 to 74 of SEQ ID NO: 5);

[0147] • ENLYFQG (corresponding to the TEV Nla protease cleavage site, and residues 68 to 74 of SEQ ID NO: 6);

[0148] • VPKFGDW (corresponding to the AvrRpt2 protease cleavage site and residues 68 to 74 of SEQ ID NO: 8)

[0149] As can be seen from the notes of SEQ ID NO: 6, it is possible to select more than one cleavage site from the list above.

[0150] Table 1: Pathogen-originated protease cleavage (PCS) sites of exemplary viruses form the virus family Potyviridae.

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[0203] Table 2: Exemplary viruses and their Pathogen-originated protease cleavage (PCS) sites (outside of the family Potyviridae).

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[0265] Table 3: Pathogen-originated protease cleavage (PCS) sites of exemplary bacteria and insects.

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[0269] By “at least one” pathogen-originated protease cleavage (PCS) site is meant one or more PCS site. In one embodiment, the fusion protein comprises one PCS site. In another embodiment, the fusion protein comprises two or more PCS sites. In a further embodiment, the fusion protein comprises three, four, five, six, seven, eight, nine or ten PCS sites. The PCS sites may be the same, but preferably are different. That is, the sequence of the PCS site differs, although in some embodiments the different PCS sites originate from the same pathogen, while in other embodiment, the different PCS sites originate from different pathogens. For example, as shown in Figure 3, using tandem PCS (i.e. two or more consecutive PCSs) - specifically HA-PCSTEV-PCSPVY-aAtNRG1.1 - impressively led to extreme resistance or classic complete resistance against both TEV and co-infection with TuMV, PepMoV, ChiVMV and PPV.

[0270] As used herein “aNLRs” refers to an autoactive NLR. NLRs act as molecular switches; under basal conditions they exhibit an inactive resting state characterised by binding of ADP to the nucleotide-binding domain and autoinhibitory contacts within their structure. However, upon recognition of a specific ligand (such as a pathogen) via the sensor domain, a conformational change occurs allowing nucleotide exchange and binding of ATP, which in turn is accompanied by relaxation of the autoinhibitory contacts, and an active state of the NLR. Once activated, the NLR is able to mediate a HR response.

[0271] An NLR is said to be ‘autoactive’ or an ‘autoactive variant’ if it is constitutively active independent of any ligand. In other words, this means that the NLR does not require prior pathogen invasion for activation. In particular, according to the invention, an autoactive NLR may comprise one or more mutation that results in stimulus-independent NLR activation through altering the intramolecular peptide contacts that promote ATP binding. In other words, an autoactive NLR exhibits an increase in ATP binding. An “increase” may mean at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the amount of ATP bound to an NLR that is not autoactive. The amount of ATP bound can be measured by any technique in the art, for example using a malachite green phosphate detection kit.

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[0274] Within the nucleotide-binding domain of a NLR there are typically at least four highly conserved regions or domains. These are the hhGRExE motif (two hydrophobic residues followed by glycine-arginine-glutamic acid then any amino acid then another glutamic acid), RNBS-B (Resistance Nucleotide Binding Site B), GLPL (glycine-leucine-proline-leucine) and the MHD (Methionine-Histidine-Aspartate motif). Accordingly, in one embodiment, the autoactive NLR comprises at least one mutation in at least one nucleic acid sequence encoding at least one of the CO domain, hhGRExE, RNBS-B, GLPL and the MHD motif, wherein the at least one mutation leads to autoactivity.

[0275] In one embodiment, an autoactive NLR (aNLR) can be constructed by introducing a “D-to-V” mutation in the methionine-histidine- aspartate (MHD) motif. Accordingly, in one embodiment, the aNLR comprises a D to V mutation at position 481 of Tm-22, as shown in SEQ ID NO: 3 or position 485 of AtNRG1.1, as shown in SEQ ID NO: 4 or 5 or 6 or 8, or position 519 of NbNRGI, as shown in SEQ ID NO: 7. Such D to V mutation positions correspond to the Tm-22(D481V), AtNRG1.1 (D485V) and NbNRGI (D519V) autoactive NLR respectively. Another example of an autoactive mutation is the F99E mutation in the CO -domain of the NLR protein, MLA10 (shown in SEQ ID NO: 12).

[0276] In a further embodiment, the NLR or N-terminal fragment thereof comprises at least one mutation that enhances the cytotoxicity of the NLR.

[0277] In one embodiment, the fusion NLR of the invention further comprises at least one blocking peptide. A free N-terminus is crucial for CNLs, RNLs or their defence-activating domains to function, and an extra polypeptide tagged at the N-termini of NLRs can inactivate their functionality to trigger plant immunity. Such excisable polypeptide blocks autoactivation of the chimeric aNLR proteins. As shown in Figure 1a, upon invasion, pathogens express proteases. The latter cleaves the chimeric NLR and release free aNLR to trigger cellular innate immunity. Alternatively, a blocking peptide is not needed, as the presence of the PCS alone is sufficient to prevent the free NLR or N-terminal fragment of NLR thereof from eliciting a hypersensitive response (HR) (as shown in Figure 17).

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[0280] The blocking peptide is preferably at the N-terminus of a chimeric aNLR, and even more preferably, at the N-terminus of the at least one PCS site as shown in Figure 1- Figure 4.

[0281] The blocking peptide can be any sequence or length of amino acids provided it is sufficient to block activation of the NLR. That is any peptide sufficient to block or significantly reduce an NLR-mediated HR response. In one embodiment, the blocking peptide is at least six, seven or eight amino acids in length.

[0282] In one embodiment, the blocking peptide is selected from residues 1 to 67 of SEQ ID NO: 3 to 8 (for the avoidance of doubt, the same blocking sequence is included in each of SEQ ID NO: 3 to 8).

[0283] Alternatively, the blocking peptide may be selected from FLAG (for example, DYKDDDDK, Myc (for example, EQKLISEEDL), HA (for example, YPYDVPDYA), a fluorescent protein (such as, but not limited to GFP, GFP-like, YFP, RFP and so on), His (for example, HHHHHH), V5 (for example, GKPIPNPLLGLDST), Xpress (for example, DLDDDDK or DLYDDDDK), Thrombin (for example, LVPRGS), BAD (Biotin Acceptor Domain) (for example, GLNDIFEAQKIEWHE), Factor Xa (for example, IEGR or IDGR), VSVG (for example, YTDIEMNRLGK), SV40 NLS (for example, PKKKRKV or PKKKRKVG), Protein C (for example, EDQVDPRLIDGK), S Tag (for example, KETAAAKFERQHMDS), OneStrap (for example, SAWSHPQFEK2GGSAWSHPQFEK), SB1 (for example, PRPSNKRLQQ), ALFA-tag (for example, SRLEEELRRRLTE), AviTag (for example, GLNDIFEAQKIEWHE), C-tag, Calmodulin-tag (for example, KRRWKKNFIAVSAANRFKKISSSGAL), E-tag (for example, GAPVPYPDPLEPR), NE-tag (for example, TKENPRSNQEESYDDNES), Rho1D4-tag (for example, TETSQVAPA), SBP-tag (for example, MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), Softag 1(for example, SLAELLNAGLGGS), Softag 3(for example, TQDPSRVG), T7-tag (for example, MASMTGGQQMG), TC tag (for example, CCPGCC), Ty tag (for example, EVHTNQDPLD), V5 tag (for example, GKPIPNPLLGLDST) and VSV-tag (for example, YTDIEMNRLGK) - or fragments thereof.

[0284] In a further embodiment, the fusion NLR of the invention further comprises at least one pathogen effector. The pathogen effector may be at N-terminus of the NLR, and optionally at the N-terminus of the at least one PCS. As used herein by “pathogen

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[0287] effector” is meant any molcecule, typically a protein, secreted by a pathogen into the extracellular and intracellular spaces of host plants, which are capable of eliciting effector-triggered plant immunity. Pathogen effectors are produced by a wide range of pathogens, including bacteria, virsues, insects and parasites to assist with the infection of a plant.

[0288] Pathogen effectors are well described in the literature. Non-limiting examples include, PC1, MyMSP32, AI6, MeTCTP, XopL, VdAMP3, BLN08, PWL3, PWL2, PWL1 and AVR2 etc.

[0289] Typical effector are approximately 50-300 amino acid residues in length, contain an N-terminal signal peptide with a highly specific sequence, have no transmembrane structural domain, no anchor site for glycosylphosphatidylinositol (GPI), no subcellular localization signal for mitochondria or other intracellular organelles, and are rich in cysteine residues.

[0290] In a further embodiment, the fusion NLR of the invention further comprises at least one protein conferring abiotic stress resistance or tolerance. In an embodiment, the protein confers herbicide resistance or tolerance. As shown in Figure 18, the at least one protein conferring herbicide resistance or tolerance may be at the N-terminus of the NLR, and optionally at the N-terminus of the at least one PCS. In one embodiment, the protein conferring herbicide resistance or tolerance may be selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (including CP4 EPSPS and herbicide-tolerant EPSPS variants), bialaphos resistance (bar), phosphinothricin acetyltransferase (pat), acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS), herbicide-tolerant alleles, acetyl-CoA carboxylase (ACCase) herbicide-tolerant alleles, protoporphyrinogen oxidase (PPO) herbicide-tolerant alleles including protoporphyrinogen oxidase 1 (PPX1) and protoporphyrinogen oxidase 2 (PPX2), photosystem II reaction center protein D1 (psbA) herbicide-tolerant alleles, aryloxyalkanoate dioxygenase genes selected from aryloxyalkanoate dioxygenase- 1 (aad-1) and aryloxyalkanoate dioxygenase- 12 (aad-12), 4-hydroxyphenylpyruvate dioxygenase (HPPD) herbicide-tolerant alleles, glutathione S-transferase (GST) genes, cytochrome P450 monooxygenase (CYP) genes, ATP-binding cassette (ABC) transporter genes associated with herbicide tolerance, and combinations thereof. In an

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[0293] embodiment, the protein conferring herbicide resistance or tolerance is phosphinothricin acetyltransferase (pat).

[0294] Accordingly, in another aspect of the invention there is provided a method of producing a plant with improved abiotic and biotic stress resistance or tolerance, the method comprising introducing and expressing the chimeric protein of the invention, the nucleic acid molecule of the invention or the vector of the invention.

[0295] In plants, NLRs function at varying levels of connectivity, ranging from standalone “singleton” receptors sufficient to induce NLR-mediated immunity, paired NLRs that distribute perception (sensor) and transduction (helper) activities, and in fortified networks where a minimal set of helper NLRs function alongside an extensive repertoire of sensors. Accordingly, in one embodiment, the NLR of the invention may act as a “singleton” standalone receptor that is sufficient to induce NLR-mediated immunity without relying on additional NLRs. Alternatively, the NLR of the invention may act in a pair. In a paired NLR system one NLR receptor acts is a sensor that senses a pathogen effector. The other NLR acts as a helper to activate downstream immune signalling. The NLR of the present invention may be a helper or a sensor NLR.

[0296] In one embodiment, the NLR fusion protein of the invention comprises or consists of a sequence defined in SEQ ID NO: 3 to 8, or a functional variant or homolog thereof.

[0297] The term “variant” or “functional variant” as used herein with reference to any of the sequences defined herein refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full non-variant sequence. Accordingly, in the context of an NLR protein (or fragment thereof), a functional variant may be a variant that is able to mediate an immune response, such as the hypersensitive (HR) response.

[0298] A functional variant also comprises a variant of the gene of interest, which has sequence alterations that do not affect function, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active (e.g. is able to oligomerise and cause cell

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[0301] death). Alterations in a nucleic acid sequence that result in the production of a different amino acid at a given site that does not affect the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

[0302] As used in any aspect of the invention described herein a “variant” or a “functional variant” has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the non-variant nucleic acid or amino acid sequence.

[0303] Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognised that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for

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[0306] other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Non-limiting examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms. The overall sequence identity of a variant can be determined using any number of sequence alignment programs known in the art. As an example, Emboss Stretcher from the EMBL-EBI may be used: https: / / www.ebi.ac.uk / Tools / psa / emboss stretcher / (using default parameters: pair output format, Matrix = BLOSUM62, Gap open = 1, Gap extend = 1 for proteins; pair

[0307]

[0308] = 16, Gap extend = 4 for

[0309]

[0310] In a further embodiment, a variant as used herein can comprise a nucleic acid sequence encoding an NLR polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to a nucleic acid sequence as defined herein.

[0311] In another aspect of the invention there is provided a nucleic acid molecule that comprises a nucleic acid sequence that encodes at least the chimeric protein of the invention, and preferably a chimeric NLR protein or the chimeric N-terminal fragment of NLR thereof of the invention.

[0312] In another aspect of the invention, there is provided a nucleic acid construct or vector (such terms may be used interchangeably), wherein the nucleic acid construct or vector comprises a nucleic acid sequence that encodes the chimeric protein of the invention, and preferably the chimeric NLR of the invention. Alternatively, there is provided a vector that comprises a nucleic acid sequence that encodes the chimeric N-terminal fragment

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[0315] of NLR of the invention. The nucleic acid sequence may also be operably linked to a regulatory sequence. In some embodiments, the nucleic acid sequence encodes a chimeric NLR as defined in any of SEQ ID NO: 1 to 12 or a variant thereof.

[0316] According to all aspects of the invention, including the method below and including the plants, methods and uses as described below, the term "regulatory sequence" is used interchangeably herein with "promoter" and all terms are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term "regulatory sequence" also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.

[0317] In one embodiment, the promoter may be a constitutive or a strong promoter. Alternatively, the promoter may be a tissue-specific promoter.

[0318] A "constitutive promoter" refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Examples of constitutive promoters include the cauliflower mosaic virus promoter (CaMV35S or 19S), rice actin promoter, maize ubiquitin promoter, rubisco small subunit, maize or alfalfa H3 histone, OCS, SAD1 or 2, GOS2 or any promoter that gives enhanced expression.

[0319] A "strong promoter" refers to a promoter that leads to increased or overexpression of the gene. Examples of strong promoters include, but are not limited to, CaMV-35S, Arabidopsis ubiquitin LIBQ1, rice ubiquitin, actin, or Maize alcohol dehydrogenase 1 promoter (Adh-1).

[0320] The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.

[0321] In one embodiment, the progeny plant is stably transformed with the nucleic acid construct described herein and comprises the exogenous polynucleotide, which is heritably maintained in the plant cell. The method may include steps to verify that the

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[0324] construct is stably integrated. The method may also comprise the additional step of collecting seeds from the selected progeny plant.

[0325] In an alternative aspect of the invention, there is provided a genetically altered plant, plant part thereof or plant cell, wherein the plant, part thereof or plant cell expresses a nucleic acid construct comprising a nucleic acid sequence encoding a chimeric protein of the invention, and preferably a chimeric NLR or chimeric N-terminal fragment of NLR as described herein.

[0326] In another aspect of the present disclosure, there is provided a genetically altered plant, plant part thereof or plant cell, wherein the plant, part thereof or plant cell comprises at least one mutation in at least one nucleic acid sequence encoding a nucleotide-binding domain and leucine-rich repeat-containing (NLR) protein, wherein the mutation is the addition of one or more copies of the fusion protein of the invention.

[0327] In another aspect of the present disclosure, there is provided a method of producing a plant with improved pathogen resistance, the method comprising introducing at least one mutation into the plant genome, wherein the mutation is the addition of one or more nucleic acid sequences encoding a pathogen-originated protease cleavage site (PCS) and / or a blocking peptide, as described herein. Preferably, the PCS and / or blocking peptide are added at the N-terminus of the NLR protein, as described herein.

[0328] In one embodiment, the mutation is introduced using targeted genome editing. That is, in one embodiment, the invention relates to a method and plant that has been generated by genetic engineering methods as described above, and does not encompass naturally occurring varieties or generating plants by traditional breeding methods.

[0329] Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events. In a preferred embodiment, the genome editing method that is used according to the various aspects of the invention is CRISPR.

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[0332] In another aspect of the invention, there is provided a method of providing or improving pathogen resistance in a plant, the method comprising providing the plant with the fusion protein of the invention, a nucleic molecule of the invention or a vector of the invention. Alternatively, the method may comprise introducing at least one of the above-described mutations into the plant genome. In one embodiment, the method of the invention does not require a second protein, specifically a non-NLR protein, such as PBS1, to mediate pathogen resistance.

[0333] The method may comprise directly administering the NLR protein to the plant - for example, by injection into a cell - or may comprise introducing nucleic acids as described herein and allowing the nucleic acids to be expressed. Said introduction may be transient (as, for example, mRNA), or may be more permanent (as, for example, introduction and integration into the genome).

[0334] In another aspect of the invention, there is also provided a method of producing a plant with improved pathogen resistance, wherein the method comprises introducing into a plant cell, or at least one plant cell of a plant part or plant, a chimeric protein, a nucleic acid molecule, or a vector, as described herein. The method may further comprise breeding the modified plant, or cell, or part, to produce offspring.

[0335] In another aspect of the invention, there is also provided a method of producing a plant with improved pathogen resistance, wherein the method comprises introducing into a plant cell, or at least one plant cell of a plant part or plant, a nucleic acid molecule encoding the chimeric NLR or chimeric N-terminal fragment of NLR thereof, as described above. The method may further comprise breeding the modified plant, or cell, or part, to produce offspring.

[0336] By “providing or improving pathogen resistance” is meant a reduction in at least one of pathogen / pest growth, fitness or an improvement of the fecundity on the plant carrying a fusion NLR protein as described above, compared to a wild-type or control plant. An improvement as used herein may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% compared to the level of pathogen / pest growth or fitness in a control or wild-type plant or compared to the fecundity of a control or wild-type plant. The method of providing or

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[0339] improving pathogen resistance may also comprise providing or improving pathogen evasion. The method of providing or improving pathogen resistance can also be seen as a method of providing or improving plant immunity.

[0340] Pathogen (or plant disease) resistance (or immunity) can be broadly classified into (i) complete resistance with visible hypersensitive response (HR) (classic complete resistance) or without visible HR (ER, extreme resistance), and (ii) partial resistance. A hypersensitive response (HR) is characterized by pathogen containment in the initially infected tissues and programmed cell death (PCD) of the infected cells. In one embodiment, there is provided a method of providing or improving classic complete resistance or extreme resistance in a plant, wherein the method comprises introducing into a plant cell, or at least one plant cell of a plant part or plant, a nucleic acid molecule encoding the chimeric protein, preferably a chimeric NLR or chimeric N-terminal fragment of NLR thereof, as described above.

[0341] An improvement in plant immunity can be measured by any technique in the art. In one embodiment, an improvement in immunity can be measured by measuring cell death in the presence of a pathogen or pathogen effector compared to cell death in a wild-type or control plant. An example of a cell death assay that may be carried out is described in Example 7.

[0342] By “improvement” is meant an improvement of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% in immunity compared to a wild-type or control plant.

[0343] A “pathogen” may include any disease causing agent, particularly a disease causing organism such as bacteria, viruses, nematodes and fungi. Pathogens infect plants and cause disease, and may, for example, reduce yield or otherwise damage the plant, for example through the action of toxins. “Pests” may include organisms such as animals, insects or nematodes, and may infect plants with pathogens or cause damage to the plant directly (e.g. through sap-feeding, boring into stems and fruits, or and cutting the root stem and leaves).

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[0346] The pathogen may be a virus, bacteria, oomycete, fungi, nematode or a proteaseexpressing pest, such as piercing-sucking pests.

[0347] In one embodiment, when the pathogen is a virus, the virus is in the family Potyviridae, preferably the virus is in the genus potyvirus, more preferably selected from any one of Potyvirus achyranthis, Potyvirus adiuris, Potyvirus algeriaense, Potyvirus alliumagrestis, Potyvirus almeidi, Potyvirus alstromeriae, Potyvirus amaranthi, Potyvirus ampeloprasi, Potyvirus anemones, Potyvirus angelicae, Potyvirus apii, Potyvirus apiumtessellati, Potyvirus arachidis, Potyvirus araujiae, Potyvirus arracachae, Potyvirus artichokis, Potyvirus ascaloniae, Potyvirus ascaloniavirgae, Potyvirus ashitabae, Potyvirus asparagi, Potyvirus atuberosi, Potyvirus barbacenense, Potyvirus basellae, Potyvirus batatalatentis, Potyvirus batatamaculae, Potyvirus batataplumei, Potyvirus begonia, Potyvirus betaceum, Potyvirus betaci, Potyvirus bidensia, Potyvirus bidenstessellati, Potyvirus brugmansiae, Potyvirus calistephi, Potyvirus callanthis, Potyvirus cannae, Potyvirus capsianuli, Potyvirus capsiflavi, Potyvirus capsimaculae, Potyvirus capsiseverum, Potyvirus capsivenae, Potyvirus capsivenamaculae, Potyvirus caricae, Potyvirus carotae, Potyvirus carotatenuifoli, Potyvirus caryae, Potyvirus catharantessellati, Potyvirus cebatatae, Potyvirus cepae, Potyvirus ceratobii, Potyvirus chamaescillae, Potyvirus chichorii, Potyvirus citrulli, Potyvirus citrullimoroccense, Potyvirus citrullufolimaculae, Potyvirus clitoriae, Potyvirus cliviaflavilineae, Potyvirus colchici, Potyvirus commelinae, Potyvirus cordophani, Potyvirus costus, Potyvirus croci, Potyvirus cucurbitae, Potyvirus cucurbitaflavitesselati, Potyvirus cynanchi, Potyvirus cypripedii, Potyvirus cyrtanthi, Potyvirus dactylis, Potyvirus daphnis, Potyvirus dasheenis, Potyvirus dendrobii, Potyvirus dianthi, Potyvirus dioscoreae, Potyvirus duobatatae, Potyvirus esculentinecrosis, Potyvirus eucharae, Potyvirus euphorbiae, Potyvirus fountaingrassi, Potyvirus freesia, Potyvirus fritillariae, Potyvirus gebatatae, Potyvirus gladioli, Potyvirus gloriosae, Potyvirus glycitessellati, Potyvirus gomphocarphi, Potyvirus habenariae, Potyvirus halapensis, Potyvirus hardenbergiae, Potyvirus heliannulabis, Potyvirus helichloromaculae, Potyvirus helitenuitessellati, Potyvirus helitessellati, Potyvirus henbanis, Potyvirus hibbertiae, Potyvirus hiemalisdaphnis, Potyvirus hippeastri, Potyvirus hyacinthi, Potyvirus impatiensis, Potyvirus iriseverum, Potyvirus iristenuis, Potyvirus jasmine, Potyvirus kalanchoes, Potyvirus konjac, Potyvirus lactucae, Potyvirus lactucaitalicense, Potyvirus lilimaculae, Potyvirus lupines, Potyvirus lycorsis, Potyvirus malvae, Potyvirus melongenae, Potyvirus melozonati,

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[0350] Potyvirus mirabilis, Potyvirus miscanthi, Potyvirus morindatessellati, Potyvirus muricati, Potyvirus musae, Potyvirus narcissus, Potyvirus narcissusdegeneris, Potyvirus narcissuslineae, Potyvirus nerinis, Potyvirus nicotianainsculpentis, Potyvirus nicotianamaculae, Potyvirus nicotianavenamaculae, Potyvirus nicotianavenaobscurum, Potyvirus nippodioscoreae, Potyvirus orionspassiflorae, Potyvirus ornithogali, Potyvirus ornithogalitessellati, Potyvirus pachyrhizus, Potyvirus panax, Potyvirus papayanuli, Potyvirus paris, Potyvirus parisnecrosis, Potyvirus passifloradistorti, Potyvirus passiflorae, Potyvirus passifloraflavi, Potyvirus passiflorafricanse, Potyvirus passifloramaculae, Potyvirus passiflory, Potyvirus pennisseti, Potyvirus pepo, Potyvirus peporesticulae, Potyvirus pepotigris, Potyvirus perulycopersici, Potyvirus pfaffiae, Potyvirus phaseoli, Potyvirus phaseoluteum, Potyvirus phaseovulgaris, Potyvirus phytolaccae, Potyvirus pisumsemenportati, Potyvirus platycodonis, Potyvirus pleioblasti, Potyvirus pleionis, Potyvirus plumpoxi. Potyvirus polianthis, Potyvirus polianthismaculae, Potyvirus polygonati, Potyvirus ranunculi, Potyvirus ranunculideformationis, Potyvirus ranunculitenuis, Potyvirus rapae, Potyvirus rhopalanthi, Potyvirus rutae, Potyvirus sacchari, Potyvirus sarcochili, Potyvirus schizanthi, Potyvirus scorzaureum, Potyvirus sorghitessellati, Potyvirus spiranthesis, Potyvirus streptopi, Potyvirus suaveolens, Potyvirus tagetis, Potyvirus telosmae, Potyvirus tetraparis, Potyvirus thevetiae, Potyvirus thladiatessellati, Potyvirus thurnbergii, Potyvirus torvi, Potyvirus tradescantiae, Potyvirus trifolii, Potyvirus trompetae, Potyvirus tropaeoli, Potyvirus tuberosiflavi, Potyvirus tulipadefractum, Potyvirus tulipatessellati, Potyvirus vallotae, Potyvirus vanilla, Potyvirus verbenae, Potyvirus vetuberosi, Potyvirus vignae, Potyvirus wisteriae, Potyvirus yamplacidum, Potyvirus yamtesselati, Potyvirus yidiuris, Potyvirus yililii, Potyvirus yiornithogali, Potyvirus yipleionis, Potyvirus yituberosi, Potyvirus zantedeschiae, Potyvirus zantedeschiatenuis, Potyvirus zeananus or Potyvirus zeatessellati.

[0351] In another embodiment, when the pathogen is a virus said the virus is in the family Potyviridae, preferably the virus is in the genus potyvirus and said potyvirus is unclassified. An unclassified potyvirus is a member of the potyvirus genus that is yet to be classified into a known species. In an embodiment, the pathogen is an unclassified potyvirus selected from any one of Ecuadorian rocoto virus, Celery yellow mosaic virus, Ornamental onion stripe mosaic virus, Commelina mild mosaic virus, Trillium crinkled leaf virus, Cotyledon virus Y, Ammi majus latent virus, Arisaema potyvirus 1, Bermuda grass mosaic virus, Bermuda grass southern mosaic virus, Commelina mild mosaic

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[0354] virus, Delphinium vein-clearing virus, Lily virus A, melon vein-banding mosaic virus, Muscari mosaic virus, Omphalodes virus Y, Ornithogalum virus 4, Passiflora foetida virus Y, siratro 1 virus Y, siratro 2 virus Y, snowdrop virus Y, Stenomesson mosaic virus, Tricyrtis virus Y, Triteleia mosaic virus, Veltheimia mosaic virus or Veltheimia virus Y.

[0355] In one embodiment, when the pathogen is a virus, the family Secoviridaem, preferably from the genus comovirus, and more preferably is Comovirus siliquae (bean pod mottle virus).

[0356] In another embodiment, when the pathogen is a bacteria, the bacteria is from the family Pseudomonadaceae, preferably from the genus Pseudomonas and more preferably is selected from the species Pseudomonas syringae or Pseudomonas aeruginosa.

[0357] In another embodiment, when the pathogen is a bacteria, the bacteria is from the family Burkholderiaceae, preferably from genus Ralstonia, and preferably the species Ralstonia solanacearum.

[0358] In another embodiment, when the pathogen is a bacteria the bacteria is from the family Xanthomonadaceae, preferably from the genus Xanthomonas, and preferably the species is selected from any one of Xanthomonas campestris, Xanthomonas oryzae, or Xanthomonas campestris.

[0359] In another embodiment, when the pathogen is a bacteria the bacteria is from the family, Erwiniaceae preferably from the genus Erwinia, and preferably the species is selected from any one of Erwinia amylovora or Erwinia chrysanthemi.

[0360] In another embodiment, when the pathogen is a bacteria the bacteria is from the family, Enterobacteriaceae, preferably from the genus Proteus, and preferably the species Proteus mirabilis.

[0361] In another embodiment, when the pathogen is a bacteria the bacteria is from the family, Yersiniaceae, preferably from the genus Serratia, and preferably the species Serratia marcescens.

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[0364] In another embodiment, when the pathogen is a bacteria the bacteria is from the family, Caulobacteraceae, preferably from the genus Caulobacter, and preferably the species Caulobacter crecentus.

[0365] In another embodiment, when the pathogen is a bacteria the bacteria is from the family, Rhizobiaceae, preferably from the genus Liberibacter, and preferably the species Candidatus Liberibacter asiaticus.

[0366] In another embodiment, when the pathogen is a bacteria the bacteria is from the family, Pectobacteriaceae, preferably from the genus Pectobacterium, and preferably the species Pectobacterium carotovorum.

[0367] In another embodiment, when the pathogen is a bacteria the bacteria is from the family, Pectobacteriaceae preferably from the genus Dickeya, and preferably the species is selected from any one of Dickeya dadantii or Dickeya chrysanthemii.

[0368] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Nectriaceae, preferably from the genus Fusarium, and preferably the species is selected from any one of Fusarium verticillioides, Fusarium oxysporum, Fusarium oxysporum f.sp. Lycopersici, Fusarium solani, Fusarium proliferatum, Fusarium graminearum or Fusarium culmorum.

[0369] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Peronosporaceae, preferably from the genus Phytophthora, and preferably the species is selected from any one of Phytophthora infestans, Phytophthora mirabilis and Phytophthora parasitica, Phytophthora sojae, Phytophthora ramorum, Phytophthora pseudosyringae, Phytophthora chamydospora or Phytophthora gonapodyides.

[0370] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Plectosphaerellaceae, preferably from the genus Verticullium, and preferably the species is Verticillium dahliae.

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[0373] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Magnaporthaceae, preferably from the genus Magnaporthe, and preferably the species is Magnaporthiopsis poae.

[0374] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Mycosphaerellaceae, preferably from the genus Zymoseptoria, and preferably the species is Zymoseptoria tritici.

[0375] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Cryphonectriaceae, preferably from the genus Cryphonectria, and preferably the species is Cryphonectria parasitica.

[0376] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Aspergillaceae, preferably from the genus Penicillium, and preferably the species is Penicillium expansum.

[0377] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Glomerellaceae, preferably from the genus Colletotrichum, and preferably the species is Colletotricum acutatum.

[0378] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Glomerellaceae, preferably from the genus Gonapodya, and preferably the species is Gonapodya prolifera.

[0379] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Clavicipitaceae, preferably from the genus Torrubiella, and preferably the species is Torrubiella hemipterigena.

[0380] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Spizellomycetaceae, preferably from the genus Spizellomyces, and preferably the species is Spizellomyces punctatus.

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[0383] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Ustilaginaceae, preferably from the genus Mycosarcoma, and preferably the species is Mycosarcoma maydis.

[0384] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Ustilaginaceae, preferably from the genus Ustilago, and preferably the species is Ustilago hordei.

[0385] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Pleosporaceae, preferably from the genus Bipolaris, and preferably the species is Bipolaris maydis or Bipolaris zeicola.

[0386] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Pleosporaceae, preferably from the genus Bipolaris, and preferably the species is Bipolaris maydis or Bipolaris zeicola.

[0387] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Diaporthaceae, preferably from the genus Stenocarpella, and preferably the species is Stenocarpella maydis.

[0388] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Sclerotiniaceae, preferably from the genus Botryris, and preferably the species is Botrytis cinerea.

[0389] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Hypocreaceae, preferably from the genus Acremonium, and preferably the species is Acremonium strictum.

[0390] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Hypocreaceae, preferably from the genus Acremonium, and preferably the species is Acremonium strictum.

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[0393] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Saprolegniaceae, preferably from the genus Saprolegnia, and preferably the species is Saprolegnia parasitica.

[0394] In another embodiment, when the pathogen is a fungi, the fungi is selected from the family Leptolegniaceae, preferably from the genus Aphanomyces, and preferably the species is Aphanomyces invadans.

[0395] In another embodiment, when the pathogen is an insect, the insect is selected from the family Aphididae, preferably from the genus Myzus, and preferably the species Myzus persicae.

[0396] In another embodiment, when the pathogen is an insect, the insect is selected from the family Aphididae, preferably from the genus rhopalosiphum, and preferably the species rhopalosiphum padi.

[0397] In another embodiment, when the pathogen is an insect, the insect is selected from the family Aphididae, preferably from the genus Acyrthosiphon, and preferably the species Acyrthosiphon pisum.

[0398] In another embodiment, when the pathogen is an insect, the insect is selected from the family Aphididae, preferably from the genus Macrosiphum, and preferably the species Macrosiphum euphorbiae.

[0399] In another embodiment, when the pathogen is an insect, the insect is selected from the family Aphididae, preferably from the genus Sitobion, and preferably the species Sitobion avenae.

[0400] In another embodiment, when the pathogen is an insect, the insect is selected from the family Aphididae, preferably from the genus Metopolophium, and preferably the species Metopolophium dirhodum.

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[0403] In another embodiment, when the pathogen is an insect, the insect is selected from the family Aphididae, preferably from the genus Aphis, and preferably the species Aphis glycines.

[0404] In another embodiment, when the pathogen is an insect, the insect is selected from the family Aphididae, preferably from the genus Diuraphis, and preferably the species Diuraphis noxia.

[0405] In another embodiment, when the pathogen is an insect, the insect is selected from the family Delphacidae, preferably from the genus Nilaparvata, and preferably the species Nilaparvata lugens.

[0406] In another embodiment, when the pathogen is an insect, the insect is selected from the family Delphacidae, preferably from the genus Sogatella, and preferably the species Sog atelia furcifera.

[0407] In another embodiment, when the pathogen is an insect, the insect is selected from the family Delphacidae, preferably from the genus Laodelphax, and preferably the species Laodelphax striatellus.

[0408] In another embodiment, when the pathogen is an insect, the insect is selected from the family Trichostrongylidae, preferably from the genus Haemonchus, and preferably the species Haemonchus contortus.

[0409] In another embodiment, when the pathogen is an insect, the insect is selected from the family Heteroderidae, preferably from the genus Meloidogyne, and preferably the species is selected from any one of Meloidogyne incognita or Meloidogyne hapla.

[0410] In another embodiment, when the pathogen is an insect, the insect is selected from the family Heteroderidae, preferably from the genus Heterodera, and preferably the species is selected from any one of Heterodera glycines or Heterodera schachtii.

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[0413] In another embodiment, when the pathogen is an insect, the insect is selected from the family Heteroderidae, preferably from the genus Globodera, and preferably the species is selected from any one of Globodera pallida or Globodera rostochiensis.

[0414] In another embodiment, when the pathogen is an insect, the insect is selected from the family Liviidae, preferably from the genus Diaphorina, and preferably the species Diaphorina citri, commonly known as the Asian citrus psyllid (AGP) or oriental citrus psyllid.

[0415] Alternatively, the provision of or an improvement in pathogen resistance can lead to an increase in yield or seed yield. As such, the provision of or an improvement in pathogen resistance can be measured by measuring an increase in yield or seed yield.

[0416] The term "yield" in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and / or weight. The actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square metres.

[0417] Preferably, increased yield comprises at least one of an increased number and / or weight of seeds, increased number of pods per plant (where the plant contains pods), increased thousand kernel weight (TKW), increased biomass, increased fresh weight and increased growth, preferably root growth. Yield is increased relative to a control or wildtype plant. For example, the yield is increased by may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to a wild-type or control plant.

[0418] In a further aspect of the invention there is provided a plant obtained or obtainable by the above-described methods. In a further aspect, there is provided a seed or other reproductive material obtained or obtainable from the plant. Also included in the scope of the invention is progeny plants obtained from the seed or other reproductive material and as well as seed or other reproductive material obtained from the progeny plants.

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[0421] As described above, the plant (in which immunity is provided or improved) may be a dicot or a monocot. Alternatively, the plant may be a gymnosperm.

[0422] A dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (eg Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, watermelon, squash, cabbage, tomato, potato, yam, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine or citrus species. In one embodiment, the plant is oilseed rape.

[0423] Also included are biofuel and bioenergy crops such as rape / canola, sugar cane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).

[0424] A monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as wheat, rice, barley, maize, oat, sorghum, rye, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species, or a crop such as onion, leek, yam or banana.

[0425] In one embodiment, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In another embodiment the plant is Arabidopsis, Nicotiana benthamiana, Nicotiana tabacum, Medicago truncatula, or other suitable model organisms used in plant research.

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[0428] By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. Preferred plants are maize, wheat, rice, oilseed rape, sorghum, soybean, potato, tomato, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.

[0429] The term "plant" as used herein encompasses whole plants and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned mutations of the invention. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores. The invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs.

[0430] The term “reproductive material” encompasses seeds and other vegetative propagation material, such as tubers.

[0431] In a most preferred embodiment, the plant part or harvestable product is a seed or grain. Therefore, in a further aspect of the invention, there is provided a seed or grain produced from a genetically altered plant as described herein. Accordingly, in one aspect of the invention there is provided seed, wherein the seed comprises a nucleic acid molecule or vector encoding a fusion protein of the invention. Also provided is a progeny plant obtained from the seed as well as seed obtained from that progeny.

[0432] A control plant as used herein according to all of the aspects of the invention is a plant, which has not been modified according to the methods of the invention. Accordingly, in one embodiment the control plant does not comprise a nucleic acid molecule or vector encoding a fusion protein of the invention. In one embodiment, the control plant is a wild type plant. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.

[0433] Further provided is a plant protection product (e.g. pesticide) comprising a fusion protein, nucleic acid sequence, or vector, as described herein.

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[0436] While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0437] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments, which are described.

[0438] The invention is now described in the following non-limiting examples.

[0439] EXAMPLES

[0440] Example 1 : Engineering CNL Tm-22confers PVY resistance

[0441] We first tested whether our remodeling aNLRs approach (Figure 1a) can be used to generate a new NLR for conferring resistance against potato virus Y (PVY), a typical potyvirus. We used the PVY Nla PCS YEVHHQ> A (abbreviated as PCSPVY), one of the conserved amino acid (aa) sequences of the Nla PCS between Nib and CP (Figure 1b), and generated HA-PCSPVY-aTm-22, a NLR chimeric protein. HA-PCSPVY-aTm-22comprises a flexible 2xHA-containing polypeptide, a 7-aa PVY Nla PCSPVYand an autoactive CNL Tm-22(aTm-22) with D481V mutation in the MHD motif (Figure 1c). Neither HA-PCSPVY-aTm-22nor NlaPVY-Myc alone was able to trigger cell death, by contrast, cell death was induced by co-expression of HA-PCSPVY-aTm-22and NlaPVY-Myc (Figure 1 d). These data suggest that the flexible polypeptide fused to the N-terminus of aTm-22inactivated the immune function of HA-PCSPVY-aTm-22and such engineered NLR, once cleaved by PVY Nla, led to release of free aTm-22and the latter induced plant cell death. To confirm that HA-PCSPVY-aTm-22was cleaved by PVY Nla (or Nla-Pro), we performed immunoblotting assays using anti-HA antibody. The accumulation of HA-PCSPVY-aTm-22protein was reduced when co-expressed with Myc-Nla-ProPVY,

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[0444] consistent with the occurrence of in planta cleavage of the N-terminal HA tag in the chimeric protein, although we could not detect the released HA tag (Figure 1e), perhaps due to the degradation of this small protein (7.8 kD). To further confirm this cleavage, we generated HA-PCSPVY-aTm-22-HA comprising an extra 3xHA tag at the C-terminus of HA-PCSPVY-aTm-22. When co-expressed with Myc-Nla-ProPVY, both HA-PCSPVY-aTm-22-HA and a smaller aTm-22-HA were readily detectible (Figure 1f). Taken together, these data suggest that PVY both Nla and Nla-Pro are able to cleave HA-PCSPVY-aTm-22and the resultant free aTm-22is autoactive to trigger plant cell death.

[0445] We next generated transgenic Nicotiana benthamiana plants expressing HA-PCSPVY-aTm-22. Transient expression of NlaPVY-Myc induced severe cell death on TO transgenic plant leaves (Figure 5a, b). T1 plants from independent transgenic lines grew and developed normally with traits (height, biomass production, seed setting, and seed germination rate) similar to wild-type plants (Figure 5c, d). Transgene and its protein expression in T1 plants were confirmed by PCR and immunoblotting (Figure 5e). To test their viral resistance, wild-type (WT) and transgenic plants were mechanically inoculated with GFP-tagged PVY (PVY-GFP). At 7 days post inoculation (dpi), PVY-GFP was observed on the local leaves of WT, but not HA-PCSPVY-aTm-22plants, although visible HR lesions appeared on the inoculated leaves of all tested transgenic plants (Figure 6a, b). At 21 -dpi, neither viral symptom nor GFP fluorescence was noticeable in most HA-PCSPVY-aTm-22plants (Figure 1g, Table 4); consistently, no viral RNA was detected by RT-PCR (Figure 1h). On rare occasions, we noticed a few transgenic plants showing SHR (Figure 6 c), a phenomenon known as partial resistance46. Taken together, these data demonstrate that the engineered CNL HA-PCSPVY-aTm-22can confer classic complete resistance (with visible HR lesions) against PVY infection in plants.

[0446] Table 4. Resistance to potyviruses in T1 transgenic HA-PCSPVY-aTm-22plants

[0447] X. Plants HA-PCSPVY-aTm-22HA-PCSPVY-aTm-22WT

[0448] line #3 line #5

[0449] Viru

[0450]

[0451] PVY-GFP 18 / 21 (86%) 41 / 45 (91%) 0 / 18 (0%)

[0452] TuMV-GFP 16 / 17 (94%) 26 / 29 (90%) 0 / 17 (0%)

[0453] PPV-GFP 10 / 14 (71%) 21 / 30 (70%) 0 / 13 (0%)

[0454] PepMoV 14 / 16 (88%) 15 / 18 (83%) 0 / 10 (0%)

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[0457] ChiVMV 17 / 19 (89%) 25 / 26 (96%) 0 / 9 (0%)

[0458] Example 2: HA-PCSPVY-aTm-22enables plants to resist a different potyviral infection.

[0459] The cleavage site PCSPVY(YEVHHQ> A), a derivative of the consensus motif xxVxHQ> A(G / S) or xxVxxQ> A(G / S), is expected to be the target for cleavage by Nla proteases encoded by more than 110 of the 199 potyviruses (Table 1). Thus, HA-PCSPVY-aTm-22is likely to detect and confer resistance against multiple viruses in the Potyvirus genus. We tested this assumption with four additional potyviruses turnip mosaic virus (TuMV), pepper mottle virus (PepMoV), chilli veinal mottle virus (ChiVMV) or plum pox virus (PPV). Based on their native cleavage sites (Table 1), Nla and Nla-Pro of the four potyviruses are expected to target PCSPVY, an isoform of XXVXHCUA(G / S), and cleave HA-PCSPVY-aTm-22to release aTm-22. Indeed, in transgenic HA-PCSPVY-aTm-22N. benthamian plants (TO), transient expression of TuMV Nla-Pro, but not its loss-of-function mutant (C151A) in which a cysteine was mutated to alanine within the key protease active site, caused cell death (Figure 5a). T1 transgenic HA-PCSPVY-aTm-22plants were further tested for their resistance against TuMV. At 7-dpi, HR lesions appeared on inoculated leaves of transgenic plants challenged with GFP-tagged TuMV (TuMV-GFP) (Figure 7a). At 21 -dpi, most HA-PCSPVY-aTm-22plants infected with TuMV-GFP remained healthy and symptomless, and displayed no GFP fluorescence (Figure 1 i, Table 4). No TuMV RNA was detected in systemic leaves of the H A-PCSPVY-aTm-22resistant plants (Figure 1j). By contrast, all wild-type N. benthamian plants developed severe dwarf viral symptom with obvious GFP fluorescence (Figure 1i). In similar experiments, we also found that HA-PCSPVY-aTm-22plants developed local HR on inoculated leaves and showed complete resistance to GFP-tagged PPV (PPV-GFP), PepMoV or ChiVMV (Figure 1 k-p, Figure 7b-d, Table 4). These results demonstrate that the remodeled CNL protein HA-PCSPVY-aTm-22can detect various “effector” proteases and confer plant broad-spectrum and complete resistance against multiple potyviruses.

[0460] Example 3: Redesigning autoactive RNL NRG1 convenes extreme resistance. In addition to modifying canonical CNL Tm-22, we also examined if autoactive RNLs can be similarly restructured to confer plant immunity. We generated HA-PCSPVY-aAtNRG1.1 and HA-PCSPVY-aNbNRG1 by replacing aTm-22in HA-PCSPVY-aTm-22with autoactive

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[0462] RNL AtNRG1.1 (D485V) and NbNRGI (D519V), respectively (Figure 2a). These RNL-redesigned proteins induced cell death when PVY NlaPVYwas co-expressed in N. benthamiana (Figure 2b). We then focused on HA-PCSPVY-aAtNRG1.1 for further research. Through similar assays done for HA-PCSPVY-aTm-22(Figure 1e, f), we showed that HA-PCSPVY-aAtNRG1.1 cleaved by Nla-ProPVYin plants (Figure 2c, d).

[0463] We then generated transgenic N. benthamiana lines expressing HA-PCSPVY-aAtNRG1.1. Transient expression of NlaPVY-Myc caused strong cell death in TO transgenic HA-PCSPVY-aAtNRG1.1 plants (Figure 8a). We then chose T1 plants from representative line #1 expressing HA-PCSPVY-aAtNRG1.1 for further disease resistance test. These HA-PCSPVY-aAtNRG1.1 T1 plants underwent normal growth and development (Figure 8b). Moreover, transgene and the HA-PCSPVY-aAtNRG1.1 chimeric protein were detected by genomic PCR and immunoblotting in T1 plants (Figure 8c). After PVY-GFP infection, no or some HR lesions appeared on the inoculated leaves of T 1 plants (Figure 9a), and all tested T 1 plants showed complete resistance against PVY-GFP (Table 6), evidenced by the fact that no GFP fluorescence occurred at 21-dpi (Figure 2e) and no viral RNAs were detected in the systemic leaves on T1 plants challenged with PVY-GFP at 23-dpi (Figure 2f). No visible HR lesion on the local leaves challenged with PVY-GFP, and undetectable PVY RNA on systemic leaves suggest that HA-PCSPVY-aAtNRG1.1 conferred extreme resistance against PVY-GFP. Further, we challenged HA-PCSPVY-aAtNRG1.1 transgenic plants through inoculation of TuMV-GFP, PPV-GFP, PepMoV and ChiVMV on different half-leaf of each tested transgenic plant. Astoundingly, these T1 transgenic HA-PCSPVY-aAtNRG1.1 plants showed extreme resistance or classic complete resistance against co-infection of these four potyviruses (Figure 2g, h; Fig 9 b and Table 6). HR lesions hardly appeared on the inoculated leaves infected with PPV-GFP and PepMoV, but were often observed on the inoculated leaves challenged with TuMV-GFP, and sometimes observed on the inoculated leaves infected with ChiVMV (Figure 9c). These results suggest that the remodeled RNL can confer plant extreme resistance against multiple potyviruses.

[0464] Table 5. Resistance to multiple virus infection in T1 transgenic HA- PCSPVY-aAtNRG1.1 and HA-PCSTEV-PCSPVY-aAtNRG1.1 plants

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[0468]

[0469] Example 4: Using tandem PCSs to expand aNLR recognition specificities.

[0470] Co-expression of HA-PCSPVY-aNLR (aTm-22, aAtNRG1.1) and Nla from tobacco etch potyvirus (TEV) failed to elicit cell death (Figure 10a), and transgenic HA-PCSPVY-aAtNRG1.1 plants were susceptible to GFP-tagged TEV (TEV-GFP) infection (Figure 10b, c; Table 6). Consistent with these data, we noticed that the TEV Nla cleavage site PCSTEV(ENLYFQ> G, a derivative of the consensus motif ExxxxQ> A(G / S) differs from the consensus motif XXVXXCHA(G / S) or PCSPVY(YEVHHQ> A) used in the engineered aNLR proteins (Table 1). This prompted us to investigate if a second PCS can be utilized to expand aNLR recognition specificity. To test this idea, we generated HA-PCSTEV-PCSPVY-aAtNRG1.1 by incorporating the PCSTEVinto HA-PCSPVY-aAtNRG1.1 (Figure3a). Strikingly, co-expression of HA-PCSTEV-PCSPVY-aAtNRG1.1 with either NlaPVY-Myc or NlaTEV-Myc provoked strong cell death (Figure 3b), suggesting that the engineered aRNL fusion protein with tandem PCSTEVand PCSPVYgained the ability to recognize both PVY and TEV Nla proteases. Moreover, the cleavages of HA-PCSTEV-PCSPVY-aAtNRG1.1 and HA-PCSTEV-PCSPVY-aAtNRG1.1-HA by Myc-Nla-ProPVYand MycNla-ProTEVwere confirmed (Figure 3c, d). Further, we generated transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 lines (Figure 11a, b). All tested T1 plants in the representative line #3 showed extreme resistance or classic complete resistance against PVY-GFP (Figure 3e, f; Figure 11c; Table 6). When TuMV-GFP, PepMoV, ChiVMV and PPV-GFP were inoculated on four half leaf of same transgenic plant, HR lesions were hardly seen on the leaves inoculated with PPV-GFP and PepMoV, but often observed on the inoculated leaves infected with TuMV-GFP and ChiVMV (Figure 12a). All tested T1 plants exhibited extreme resistance or classic complete resistance against coinfection of TuMV-GFP, PepMoV, ChiVMV and PPV-GFP without detectable viral RNAs in the systemic leaves (Figure 12; Table 6). When infected with TEV-GFP, HR loci always

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[0473] appeared on the inoculated leaves (Figure 11d, 13a). Seventeen of thirty-one tested transgenic plants showed complete resistance against TEV-GFP, evidenced by the fact that no GFP fluorescence occurred and no viral RNAs were detected in systemic leaves (Figure 3g, h; Table 6). However, the remaining 14 tested plants showed partial resistance accompanied by viral RNA accumulation, leaf curl symptom, and SHR (Figure 13). It is possible that when the second cleavage site (located distal to the N-terminus of NRG1.1) in HA-PCSTEV-PCSPVY-aAtNRG1.1 is targeted by a protease from one specific pathogen, the "PCSPVY" peptide is not removed. This peptide, however, may interfere with the total immune activity of NRG1.1. Nevertheless, our results imply that aAtNRG1.1 can tolerate a short peptide of up to 8 amino acids at its N-terminus without severely affecting its capability to elicit immunity in plant cells. Taken together, our data suggest that using tandem PCSs can expand the recognition specificities of an aNLR for conferring a broader-spectrum of disease resistance in plants.

[0474] Example 5: HA-PCSSMV-aAtNRG1.1 confers soybean complete resistance against soybean mosaic virus.

[0475] To test whether our resistance engineering works in crop, we generated HA-PCSSMV-aAtNRG1.1 by replacing PCSPVYin HA-PCSPVY-aAtNRG1.1 with soybean mosaic virus (SMV) Nla cleavage site PCSSMV(ESVSLQ> S) (Figure 4a). Co-expression of HA-PCSSMV-aAtNRG1.1 with NlaSMV-Myc, but not NlaPVY-Myc, induced cell death in N. benthamiana (Figure 4b). Through similar assays done for HA-PCSPVY-aTm-22(Figure 1e, f), we showed that HA-PCSSMV-aAtNRG1.1 was cleaved by NlaSMV-Myc in plants (Figure 4 cd). Further, we generated transgenic soybean lines expressing HA-PCSSMV-aAtNRG1.1 and challenged these transgenic plants with eGFP-tagged SMV (SMV-eGFP). At 25 dpi, all infected T1 soybean plants from representative soybean line #2 underwent proper growth and development and have 5 expanding trifoliate leaves like the non-infected wild-type soybean, as indicated by their fifth trifoliate leaves (Figure 4e). These T 1 soybean plants showed complete resistance to SMV-eGFP without any visible eGFP fluorescence and detectable viral RNAs in systemic leaves (Figure 4e, f). However, all eight SMV-eGFP infected wild-type soybean plants exhibited severe viral symptom (Figure 4e) and only developed four trifoliate leaves. We did not observe any eGFP fluorescence and obvious HR lesions on the inoculated leaves of these T1 soybean plants except the damage caused by mechanical inoculation (Figure 4g). These

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[0478] data demonstrate that our method can be used to engineer disease resistance in the important crops.

[0479] Example 6: Resistance is conferred by HA-PCSAvrRpt2-aAtNRG1.1

[0480] Given that many DNA and RNA viruses, bacteria, oomycetes, fungi, and nematodes pests express and secrete proteases, the present strategy could be applicable to control across-kingdom pathogens and pests in plants. Indeed, using the same experimental design, we found that co-expression of the Pseudomonas syr / ngae-originated cysteine protease AvrRpt2 and HA-PCSAvrRpt2-aAtNRG1.1 caused much more severe cell death (which is a good indicator of ETI) than individual expression of AvrRpt2 did in N. benthamiana, and HA-PCSAvrRpt2-aAtNRG1.1 or HA-PCSAvrRpt2-aAtNRG1.1-HA was successfully cleaved by AvrRpt2 (Figure 14). To further confirm the resistance conferred by HA-PCSAvrRpt2-aAtNRG1.1, we generated transgenic Arabidopsis thaliana rps2 knockout plants expressing HA-PCSAvrRpt2-aAtNRG1.1, on the basis of rps2 plants in which the RPS2 gene was loss-of-function and was unable to confer resistance against P. syringae pv tomato strain DC3000 (Pst) expressing AvrRpt2. These transgenic rps2 Arabidopsis TO plants expressing HA-PCSAvrRpt2-aAtNRG1.1 were inoculated with Pst (avrRpt2) bacteria. Under same condition and treatment, fewer numbers of Pst (avrRpt2) were detected in transgenic rps2 knockout lines expressing HA-PCSAvrRpt2-aAtNRG1.1 compared with rps2 knockout plants as shown in Table 6.

[0481] Table 6. Numbers of Pst (avrRpt2) colonies from diluted plant extract

[0482]

[0483] Example 7: Materials and Methods

[0484] Plant materials and growth conditions - N. benthamiana plants were grown under a 16 h light / 8 h dark photoperiod at 25 ± 2°C in insect-free greenhouses.

[0485] Plasmid construction - To generate the engineered novel NLRs expression vector, HA-PCSPVY-aTm-22, HA-PCSPVY-aAtNRG1.1, HA-PCSPVY-aNbNRG1, HA- PCSTEV-PCSPVY-

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[0488] aAtNRGI.1 and HA-PCSAvrRpt2-aAtNRG1.1 DNA fragments, which contain an N-terminal 2xHA-containing blocking polypeptide, a 7-aa protease cleavage site (or 14-aa tandem sites), and an autoactive Tm-22(D481V), AtNRG1.1 (D485V) or NbNRGI (D519V) mutant, were cloned into pWJZ576, a binary T-DNA vector which contains 2xCaMV 35S promoter, 5'- and 3'-untranslated region from Cowpea mosaic virus RNA-2 and the NOS terminator. Binary T-DNA vectors pLIC-Myc and pLIC-HA were used to generate fusion protein with a C-terminal 4xMyc and 3xHA tag by LIC method, respectively. Binary T-DNA vector pMyc-LIC was used to generate fusion protein with a N-terminal 4xMyc tag by LIC method. Potyviral Nla and Nla-Pro DNA fragments were PCR amplified and cloned into sites between CaMV 35S promoter and 4xMyc tag of pLIC-Myc or between 4xMyc tag and rbcS-E9 terminator of pMyc-LIC vector to generate potyviral Nla or Nla-Pro expression constructs via ligation-independent cloning (LIC) method as described previously. Similarly, to be fused with C-terminal 3xHA, the above engineered novel NLRs were cloned into pLIC-HA by LIC method.

[0489] Transient expression mediated by agroinfiltration - A. tumefaciens GV3101 strain containing gene expression constructs of interest was grown, harvested, re-suspended in the infiltration buffer (10 mM MgCI2, 10 mM MES, and 0.2 mM acetosyringone). GD600 was adjusted to 0.1 -0.5 using the infiltration buffer. Agrobacterium suspension was kept at room temperature for 2-6 hours, then infiltrated into leaves of N. benthamiana plants using a needleless syringe.

[0490] Immunoblotting - Target proteins were expressed for 22-48 h in N. benthamiana leaves after agroinfiltration and extracted with Laemmli buffer. Proteins were then separated by SDS-PAGE for western blotting. For analyzing the cleavage of HA-PCSPVY-aTm-22, total proteins were separated through 12% SDS-PAGE gels. Larger proteins were then transferred onto 0.45 pm PVDF membrane under the condition of 300 mA current for 1 hour. Smaller proteins were transferred onto 0.2 pm PVDF membrane using 240 mA electricity for 16 min. For analyzing the cleavage of HA-PCS-aAtNRG1.1, total proteins were also separated by 12% SDS-PAGE gels. For analyzing the cleavage of HA-PCS-aNLR-HA, total proteins were separated by 8% SDS-PAGE gels for better distinguishing full-length HA-PCS-aNLR-HA and its expected C-terminal product aNLR-HA. The engineered NLRs with HA tags and proteases with Myc / Flag tags were detected by anti-HA (Roche) and anti-Myc / anti-Flag (Abmart) antibodies, respectively. For detecting the

[0491] 15301539-1M&C PC933865LU

[0492] 88

[0493] cleavage of HA-PCS-aNLR or HA-PCS-aNLR-HA, the PVDF membrane was firstly detected with anti-HA antibody, then was washed with stripping buffer to get rid of anti-HA antibody. Lastly, this PVDF membrane was detected with anti-Myc / anti-Flag (Abmart) antibodies.

[0494] Plant transformation - Leaf disc transformation-mediated by Agrobacterium tumefaciens GV3101 was performed to generate transgenic N. benthamiana plants. Positive transgenic lines were screened by kanamycin (100 mg / L). T1 transgenic N. benthamiana plants from TO seeds were geminated and grown on MS medium containing kanamycin (50 mg / L) while the wild-type plants were cultivated without any antibiotic before being transferred to compost. Furthermore, agrobacterium-mediated explant transformation was performed to generate transgenic soybean plants. Seeds of soybean (Nannong 1138-2) were chlorinated overnight in a fume hood. After being rinsed in sterile distilled water four times, the seeds were soaked in 200 mL of sterile MSB culture medium for imbibition at 28°C for 18-24 h. Subsequently, the seed coat was excised with a scalpel, and the explants for transformation were generated by removing one of two cotyledons of the soybean seed under a dissecting microscope to expose the embryo tip. T1 transgenic soybean plants from TO seeds were grown in the growth chamber.

[0495] Potyvirus infection - N. benthamiana plants were initially infected with individual potyvirus through A. tumefaciens-mebi\a\.e infection (agro-infection). Systemically infected leaf tissues were collected and homogenized in ddH2O through mortar, pestle and silica sand. Saps were then gently rubbed on the leave of tested plants, and the residual sap and silica sand were washed off by water. For virus resistance test for single potyvirus, tested plants were inoculated with individual virus onto one whole leave. For testing virus resistance against co-infection of four potyviruses, T1 transgenic plants were inoculated with the four potyviruses on 4 different halves of leaves, and each virus on one half leaf of same plants. Wild-type plants were only inoculated with individual virus on one half leaf.

[0496] RT-PCR - Total RNA from the systemic leave was extracted with Trizol reagent (Transgen Biotech). Then cDNA was synthesized from 1 pg of RNA using TransScript

[0497] 15301539-1M&C PC933865LU

[0498] 89

[0499] One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen Biotech) with oligo dT primers. A pair of specific primers was used to amplify viral coat protein fragment.

[0500] SEQUENCE LISTING PVY Nla protease cleavage sites are (underlined)

[0501] SEQ ID NO: 1 (Figure 1). N-terminal amino acid (aa) sequence of HA-PCSPVY-aTm- 22MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVYEHVHHQAEILLTSVINKSVEIAG

[0502] SEQ ID NO: 2 (Figure 4). N-terminal amino acid sequences of HA-PCSTEV-PCSPVY-aAtNRG1.1 MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVYENLYFQGYEVHHQANDWASLGIG

[0503] SEQ ID NO: 3. Protein sequence of HA-PCSPVY-aTm-22:

[0504] MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVYEVHHQAEILLTSVINKSVEIAGNLLIQEGKRLYWLKEDIDWLQREMR HIRSYVDNAKAKEAGGDSRVKNLLKDIQELAGDVEDLLDDFLPKIQQSNKFNYCLKRS SFADEFAMEIEKIKRRWDIDRIRKTYNIIDTDNNNDDCVLLDRRRLFLHADETEIIGLDD DFNMLQAKLLNQDLHYGWSIVGMPGLGKTTLAKKLYRLIRDQFECSGLVYVSQQPR ASEILLDIAKQIGLTEQKMKENLEDNLRSLLKIKRYVILLDDIWDVEIWDDLKLVLPECDS KVGSRMIITSRNSNVGRYIGGESSLHALQPLESEKSFELFTKKIFNFDDNNSWANASP DLVNIGRNIVGRCGGIPLAIWTAGMLRARERTEHAWNRVLESMGHKVQDGCAKVLA LSYNDLPIASRPCFLYFGLYPEDHEIRAFDLINMWIAEKFIVVNSGNRREAEDLAEDVLN DLVSRNLIQLAKRTYNGRISSCRIHVLLHSLCVDLAKESNFFHTAHDAFGDPGNVARLR RITFYSDNVMIEFFRSNPKLEKLRVLFCFAKDPSIFSHMAYFDFKLLHTLVVVMSQSFQ AYVTIPSKFGNMTCLRYLRLEGNICGKLPNSIVKLTRLETIDIDRRSLIQPPSGVWESKH LRHLCYRDYGQACNSCFSISSFYPNIYSLHPNNLQTLMWIPDKFFEPRLLHRLINLRKL GILGVSNSTVKMLSIFSPVLKALEVLKLSFSSDPSEQIKLSSYPHIAKLHLNVNRTMALN SQSFPPNLIKLTLAYFSVDRYILAVLKTFPKLRKLKMFICKYNEEKMDLSGEANGYSFP QLEVLHIHSPNGLSEVTCTDDVSMPKLKKLLLTGFHCRISLSERLKKLSK*

[0505] 15301539-1M&C PC933865LU

[0506] 90

[0507] Note for SEQ ID NO: 3. 1-67 aa: N-terminal 2xHA-containing block peptide; 68-74 aa: PVY Nla protease cleavage site (underlined); 75-933 aa: Tm-22(D481V)

[0508] SEQ ID NO: 4 Protein sequence of HA-PCSPVY-aAtNRG1.1 :

[0509] MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVYEVHHQANDWASLGIGSIGEAVFSKLLKVVIDEAKKFKAFKPLSKDL VSTMEILFPLTQKIDSMQKELDFGVKELKELRDTIERADVAVRKFPRVKWYEKSKYTR KIERINKDMLKFCQIDLQLLQHRNQLTLLGLTGNLVNSVDGLSKRMDLLSVPAPVFRDL CSVPKLDKVIVGLDWPLGELKKRLLDDSWTLVVSAPPGCGKTTLVSRLCDDPDIKGK FKHIFFNVVSNTPNFRVIVQNLLQHNGYNALTFENDSQAEVGLRKLLEELKENGPILLV LDDVWRGADSFLQKFQIKLPNYKILVTSRFDFPSFDSNYRLKPLEDDDARALLIHWAS RPCNTSPDEYEDLLQKILKRCNGFPMEVVGVSLKGRSLNTWKGQVESWSEGEKILG KPYPTVLECLQPSFDALDPNLKECFLDMGSFLEDQKIRASVIIDMWVELYGKGSSILYM YLEDLASQNLLKLVPLGTNEHEDGFYNDFLVTQHVILRELAICQSEFKENLERKRLNLEI LENTFPDWCLNTINASLLSISTDDLFSSKWLEMDCPNVEALVLNLSSSDYALPSFISGM KKLKVLTITNHGFYPARLSNFSCLSSLPNLKRIRLEKVSITLLDIPQLQLSSLKKLSLVMC SFGEVFYDTEDI VVSNALSKLQEI DI DYCYDLDELPYWISEI VSLKTLSITNCN KLSQLPE AIGNLSRLEVLRLCSSMNLSELPEATEGLSNLRFLDISHCLGLRKLPQEIGKLQNLKKIS MRKCSGCELPESVTNLENLEVKCDEETGLLWERLKPKMRNLRVQEEEIEHNLNLLQM

[0510] F*

[0511] Note for SEQ ID NO: 4. 1-67 aa: N-terminal 2xHA-containing block peptide. 68-74 aa: PVY Nla protease cleavage site (underlined). 75-882 aa: AtNRG1.1 (D485V)

[0512] SEQ ID NO: 5. Protein sequence of HA-PCSSMV-aAtNRG1.1 :

[0513] MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVESVSLQSNDWASLGIGSIGEAVFSKLLKVVIDEAKKFKAFKPLSKDLV STMEILFPLTQKIDSMQKELDFGVKELKELRDTIERADVAVRKFPRVKWYEKSKYTRKI ERINKDMLKFCQIDLQLLQHRNQLTLLGLTGNLVNSVDGLSKRMDLLSVPAPVFRDLC SVPKLDKVIVGLDWPLGELKKRLLDDSVVTLVVSAPPGCGKTTLVSRLCDDPDIKGKF KHIFFNVVSNTPNFRVIVQNLLQHNGYNALTFENDSQAEVGLRKLLEELKENGPILLVL DDVWRGADSFLQKFQIKLPNYKILVTSRFDFPSFDSNYRLKPLEDDDARALLIHWASR PCNTSPDEYEDLLQKILKRCNGFPIVIEVVGVSLKGRSLNTWKGQVESWSEGEKILGK PYPTVLECLQPSFDALDPNLKECFLDMGSFLEDQKIRASVIIDMWVELYGKGSSILYMY LEDLASQNLLKLVPLGTNEHEDGFYNDFLVTQHVILRELAICQSEFKENLERKRLNLEIL

[0514] 15301539-1M&C PC933865LU

[0515] 91

[0516] ENTFPDWCLNTINASLLSISTDDLFSSKWLEMDCPNVEALVLNLSSSDYALPSFISGMK KLKVLTITN HGFYPARLSN FSCLSSLPN LKRI RLEKVSITLLDI PQLQLSSLKKLSLVMCS FGEVFYDTEDIVVSNALSKLQEIDIDYCYDLDELPYWISEIVSLKTLSITNCNKLSQLPEA IGNLSRLEVLRLCSSMNLSELPEATEGLSNLRFLDISHCLGLRKLPQEIGKLQNLKKISM RKCSGCELPESVTNLENLEVKCDEETGLLWERLKPKMRNLRVQEEEIEHNLNLLQMF* Note for SEQ ID NO: 5. 1-67 aa: N-terminal 2xHA-containing block peptide; 68-74 aa: SMV Nla protease cleavage site (underlined); 75-882 aa: AtNRG1.1 (D485V)

[0517] SEQ ID NO: 6. Protein sequence of HAS-PCSTEV-PCSPVY-aAtNRG1.1 :

[0518] MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVENLYFQGYEVHHQANDWASLGIGSIGEAVFSKLLKWIDEAKKFKAF KPLSKDLVSTMEILFPLTQKIDSMQKELDFGVKELKELRDTIERADVAVRKFPRVKWYE KSKYTRKIERINKDMLKFCQIDLQLLQHRNQLTLLGLTGNLVNSVDGLSKRMDLLSVPA PVFRDLCSVPKLDKVIVGLDWPLGELKKRLLDDSVVTLVVSAPPGCGKTTLVSRLCDD PDIKGKFKHIFFNWSNTPNFRVIVQNLLQHNGYNALTFENDSQAEVGLRKLLEELKEN GPILLVLDDVWRGADSFLQKFQIKLPNYKILVTSRFDFPSFDSNYRLKPLEDDDARALLI HWASRPCNTSPDEYEDLLQKILKRCNGFPIVIEWGVSLKGRSLNTWKGQVESWSEG EKILGKPYPTVLECLQPSFDALDPNLKECFLDMGSFLEDQKIRASVIIDMWVELYGKGS SILYMYLEDLASQNLLKLVPLGTNEHEDGFYNDFLVTQHVILRELAICQSEFKENLERK RLNLEILENTFPDWCLNTINASLLSISTDDLFSSKWLEMDCPNVEALVLNLSSSDYALP SFISGMKKLKVLTITNHGFYPARLSNFSCLSSLPNLKRIRLEKVSITLLDIPQLQLSSLKK LSLVMCSFGEVFYDTEDIVVSNALSKLQEIDIDYCYDLDELPYWISEIVSLKTLSITNCNK LSQLPEAIGNLSRLEVLRLCSSMNLSELPEATEGLSNLRFLDISHCLGLRKLPQEIGKLQ NLKKISMRKCSGCELPESVTNLENLEVKCDEETGLLWERLKPKMRNLRVQEEEIEHNL NLLQMF*

[0519] Note for SEQ ID NO: 6. 1-67 aa: N-terminal 2xHA-containing block peptide; 68-74 aa: TEV Nla protease cleavage site (underlined); 75-81 aa: PVY Nla protease cleavage site (italicised); 82-890 aa: AtNRG1.1 (D485V)

[0520] SEQ ID NO: 7. Protein sequence of HA-PCSPVY-aNbNRG1:

[0521] MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVYEVHHQAATLLGGAALGPVFDILLKAVLDVGIKIATFRSKFQSLIKTLN DIKPVFDDIERLNKALDGRDYEIEMFKKQLFAGEELVRKCSKTKCYDALKKWNYSRKL TKLENSLVRFCQVHGFIQVCRDSKIILVNVIEHGKKLDQITSMLRGISLRNGSSIGFTNS

[0522] 15301539-1M&C PC933865LU

[0523] 92

[0524] NGSSGWMNGNSFGSTNGSGFSGWSDVPQFSDSVVGFDLPLQELKVKLLEEKEKW VLSAPAGCGKTTLAAMLCQEDDIKDKYRDIFFVTVSKKANIKRIVGEIFEMKGYKGPDF ASEHAAVCQLNNLLRRSTSQPVLLVLDDVWSESDFVIESFIFQIPGFKILVTSRSVFPKF DTYKLNLLSEKDAKALFYSSAFKDSIPYVQLDLVHKAVRSCCGFPLALKVVGRSLCGQ PELIWFNRVMLQSKRQILFPTENDLLRTLRASIDALDEIDLYSSEATTLRDCYLDLGSFP EDHRIHAAAILDMWVERYNLDEDGMKAMAIFFQLSSQNLVNLALARKDAPAVLGLHNL HYIQQHVLLRELVIHQCDEKTVEERKRLYINIKGNDFPKWWSQQRLQPLQAEVLSIFTD EHFESVWYDVRFPKVEVLVLNFETKTYNFPPFVEQMSQLKTLIVANNYFFPTKLNNFQ LCSLLNLKRISLERISVTSIFTANLQLPNLRKISFIMCEIGEAFENYAANMSYMWPKLVE MNIEYCSDLVEVPAETCDLVGLKKLSICYCHELVALPEELGKLSNLEVLRLHSCTNVSK LPESVVKLNRLGFLDVYDCVELDFLPREMDQLCSLRTICMGSRLGFTELPDSVLRLVK LEDVVCDEETASLWEYYKEHLRNLRITVIKEDINLNLLHKSLFI*

[0525] Note for SEQ ID NO: 7. 1-67 aa: N-terminal 2xHA-containing block peptide; 68-74 aa: PVY Nla protease cleavage site (underlined); 75-922 aa: NbNRG1.1 (D485V)

[0526] SEQ ID NO: 8. Protein sequence of HA-PCSAvrRpt2-aAtNRG1.1 :

[0527] MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVVPKFGDWNDWASLGIGSIGEAVFSKLLKVVIDEAKKFKAFKPLSKDL VSTMEILFPLTQKIDSMQKELDFGVKELKELRDTIERADVAVRKFPRVKWYEKSKYTR KIERINKDMLKFCQIDLQLLQHRNQLTLLGLTGNLVNSVDGLSKRMDLLSVPAPVFRDL CSVPKLDKVIVGLDWPLGELKKRLLDDSWTLVVSAPPGCGKTTLVSRLCDDPDIKGK FKHIFFNVVSNTPNFRVIVQNLLQHNGYNALTFENDSQAEVGLRKLLEELKENGPILLV LDDVWRGADSFLQKFQIKLPNYKILVTSRFDFPSFDSNYRLKPLEDDDARALLIHWAS RPCNTSPDEYEDLLQKILKRCNGFPMEVVGVSLKGRSLNTWKGQVESWSEGEKILG KPYPTVLECLQPSFDALDPNLKECFLDMGSFLEDQKIRASVIIDMWVELYGKGSSILYM YLEDLASQNLLKLVPLGTNEHEDGFYNDFLVTQHVILRELAICQSEFKENLERKRLNLEI LENTFPDWCLNTINASLLSISTDDLFSSKWLEMDCPNVEALVLNLSSSDYALPSFISGM KKLKVLTITNHGFYPARLSNFSCLSSLPNLKRIRLEKVSITLLDIPQLQLSSLKKLSLVMC SFGEVFYDTEDI VVSNALSKLQEI DI DYCYDLDELPYWISEI VSLKTLSITNCN KLSQLPE AIGNLSRLEVLRLCSSMNLSELPEATEGLSNLRFLDISHCLGLRKLPQEIGKLQNLKKIS MRKCSGCELPESVTNLENLEVKCDEETGLLWERLKPKMRNLRVQEEEIEHNLNLLQM

[0528] F*

[0529] Note for SEQ ID NO: 8. 1-67 aa: N-terminal 2xHA-containing block peptide. 68-74 aa: avrRPT2 protease cleavage site (underlined). 75-882 aa: AtNRG1.1 (D485V)

[0530] 15301539-1M&C PC933865LU

[0531] 93

[0532] SEQ ID NO: 9: Protein sequence of PCSPVY-aTm-22 MYEVHHQAEILLTSVINKSVEIAGNLLIQEGKRLYWLKEDIDWLQREMRHIRSYVDNAK AKEAGGDSRVKNLLKDIQELAGDVEDLLDDFLPKIQQSNKFNYCLKRSSFADEFAMEI EKIKRRWDIDRIRKTYNIIDTDNNNDDCVLLDRRRLFLHADETEIIGLDDDFNMLQAKLL NQDLHYGVVSIVGMPGLGKTTLAKKLYRLIRDQFECSGLVYVSQQPRASEILLDIAKQI GLTEQKMKENLEDNLRSLLKIKRYVILLDDIWDVEIWDDLKLVLPECDSKVGSRMIITSR NSNVGRYIGGESSLHALQPLESEKSFELFTKKIFNFDDNNSWANASPDLVNIGRNIVG RCGGIPLAIVVTAGMLRARERTEHAWNRVLESMGHKVQDGCAKVLALSYNDLPIASR PCFLYFGLYPEDHEIRAFDLINMWIAEKFIWNSGNRREAEDLAEDVLNDLVSRNLIQLA KRTYNGRISSCRIHVLLHSLCVDLAKESNFFHTAHDAFGDPGNVARLRRITFYSDNVMI EFFRSNPKLEKLRVLFCFAKDPSIFSHMAYFDFKLLHTLWVMSQSFQAYVTIPSKFGN MTCLRYLRLEGNICGKLPNSIVKLTRLETIDIDRRSLIQPPSGVWESKHLRHLCYRDYG QACNSCFSISSFYPNIYSLHPNNLQTLMWIPDKFFEPRLLHRLINLRKLGILGVSNSTVK MLSIFSPVLKALEVLKLSFSSDPSEQIKLSSYPHIAKLHLNVNRTMALNSQSFPPNLIKLT I KYFSVDRYI LAVLKTFPKLRKLKM FICKYN EEKM DLSGEANGYSFPQLEVLH I HSPNG LSEVTCTDDVSMPKLKKLLLTGFHCRISLSERLKKLSK

[0533] SEQ ID NO: 10 Protein sequence of PAT-PCSPVY-CCRNbNRG1 MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQEWTDDLVRLRERYP WLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGSTLYTHLLKSLE AQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGFWQLDFSL PVPPRPVLPVTEIYEVHHQAATLLGGAALGPVFDILLKAVLDVGIKIATFRSKFQSLIKTL NDIKPVFDDIERLNKALDGRDYEIEMFKKQLFAGEELVRKCSKTKCYDALKKWNYSRK LTKLENSLVRFCQVHGFIQVCRDSKIILVNVIEHGKKLDQITSMLRGISLRNGSSIGFTN SNGSSGWMNGNSFGSTNGSGFSGWSDVPQFSDSWGFDLPLQELKVKLLEEKEKV VVLSAPAGC

[0534] SEQ ID NO: 11 Protein sequence of PAT-PCSPVY-CCRAtADR1 MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQEWTDDLVRLRERYP WLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGSTLYTHLLKSLE AQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGFWQLDFSL PVPPRPVLPVTEIYEVHHQASFIDLFAGDITTQLLKLLALVANTVYSCKGIAERLITMIRD VQPTIREIQYSGAELSNHHQTQLGVFYEILEKARKLCEKVLRCNRWNLKHVYHANKMK

[0535] 15301539-1M&C PC933865LU

[0536] 94

[0537] DLEKQISRFLNSQILLFVLAEVCHLRVNGDRIERNMDRLLTERNDSLSFPETMMEIETV SDPEIQTVLELGKKKVKEMMFKFTDTHLFGISGMSGS

[0538] SEQ ID NO: 12 Protein sequence of HA-PCSPVY-CCMLA10(F99E) MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVYEVHHQADIVTGAISNLIPKLGELLTEEFKLHKGVKKNIEDLGKELDS MNAALIKIGEVPREQLDSQDKLWADEVRELSYVIEDWDKFLVQVDGIKSDDNNNKEK GLMKRTTELLKKVKHKHGIAHAIKDIQEQLQKVADRRDRNKVFVPHPTRTIAIDPCLRA L

[0539] SEQ ID NO: 13AtNRG1.1 in Figure 2 MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVYENLYFQGYEVHHQANDWASLGIGEA

[0540] SEQ ID NO: 14 NbNRGI in Figure 2 MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVYENLYFQGYEVHHQAATLLGGAALGPVFD

[0541] SEQ ID NO: 15 Figure 1c MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAGSYPYDVPDYAAQCSSRGG GSSGGGQISYASRGGTFDDKTVYEVHHQAEILLTSVINKSVEIAGNLLIQEGKR

[0542] SEQ ID NO: 16. Figure 4a MGLINIFYGGDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCSSRGGGSSGGGQISYA SRGGTFDDKTVESVSLQSNDWASLGIGSIGEAVFS

[0543] 15301539-1

Claims

M&C PC933865LU95CLAIMS:

1. A fusion protein comprising an immune receptor protein or fragment thereof, and at least one pathogen-originated protease cleavage site (PCS).

2. The fusion protein of claim 1 , wherein the immune receptor protein is a plant receptor protein.

3. The fusion protein of claim 1 or 2, wherein the immune receptor protein is a nucleotide-binding domain and leucine-rich repeat-containing (NLR) protein or N- terminal fragment thereof.

4. The fusion protein of claim 3, wherein the NLR protein or N-terminal fragment thereof comprises or consists of a coiled-coil (CC) domain, a RESISTANCE TO POWDERY MILDEW 8-like CC (CCR ) domain, or a MADA motif.

5. The fusion protein of claim 3 or 4, wherein the NLR or fragment thereof is an autoactive variant.

6. The fusion protein of any of claims 1 to 5, wherein the NLR protein or N-terminal fragment thereof comprises at least one blocking peptide, wherein the at least one blocking peptide is added at the N-terminus of the PCS, and wherein the blocking peptide and PCS together prevent the NLR or N-terminal fragment thereof from eliciting a hypersensitive response (HR).

7. The fusion protein of any of claims 1 to 6, wherein the at least one PCS and optionally the at least one blocking peptide are at the N-terminus of the NLR protein or N- terminal fragment thereof.

8. The fusion protein of claim 6 or 7, wherein the blocking peptide comprises at least one amino acid residue, wherein more preferably the blocking peptide is selected from FLAG, GFP, YFP, HA and Myc or fragments thereof.15301539-1M&C PC933865LU969. The fusion protein of any of claims 1 to 8, wherein the pathogen of the pathogen- originated protease is selected from at least one of a virus, bacteria, oomycete, insect or fungus.

10. A nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein of any of claims 1 to 9.

11. A vector comprising the nucleic acid molecule of claim 10, operably linked to at least one regulatory sequence.

12. A genetically altered plant, part thereof or plant cell comprising the fusion protein of any of claims 1 to 9, the nucleic acid molecule of claim 10 or the vector of claim 11.

13. A method of providing or improving pathogen resistance in a plant, the method comprising providing a plant with the fusion protein of any of claims 1 to 9, the nucleic acid molecule of claim 10 or the vector of claim 11.

14. A method of producing a plant with improved pathogen resistance, the method comprising introducing and expressing the fusion protein of any of claims 1 to 9, the nucleic acid molecule of claim 10 or the vector of claim 11.

15. A method of producing a plant with improved pathogen resistance, the method comprising introducing at least one mutation in at least one nucleic acid sequence encoding a nucleotide-binding domain and leucine-rich repeat-containing (NLR) protein, wherein the mutation is the addition of one or more nucleic acid sequences encoding a pathogen-originated protease cleavage site (PCS) and / or a blocking peptide.

16. The method of claim 15, wherein the mutation is introduced using targeted genome editing.

17. A plant obtained or obtainable by the method of any of claims 13 to 16.15301539-1M&C PC933865LU9718. A plant protection product comprising the fusion protein of any of claims 1 to 9, the nucleic acid molecule of claim 10 or the vector of claim 11.15301539-1

Images