Compositions and systems for conferring disease resistance in plants and methods of use thereof

Abstract

Compositions, systems and methods for conferring disease resistance in monocot plants to plant pathogens that express pathogen-specific proteases based on recognition of the pathogen specific proteases in a plant cell. The compositions, systems and methods are based upon modification of Receptor-Like Cytoplasmic Kinase Family VII (RLCK VII) sequences that have been modified such that the endogenous sequence has been replaced with a heterologous protease recognition sequence for the protease secreted by a plant pathogen.

Description

COMPOSITIONS AND SYSTEMS FOR CONFERRING DISEASE RESISTANCE IN PLANTS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Application 63 / 504,070, filed May 24, 2023, and entitled “COMPOSITIONS AND SYSTEMS FOR CONFERRING DISEASE RESISTANCE IN PLANTS AND METHODS OF USE THEREOF,” the entire disclosure of which is expressly incorporated by reference herein.STATEMENT OF GOVERNMENTAL RIGHTS

[0002] This invention was made with government support under 58-5020-0-013 awarded by the Agricultural Research Service, and 2022-67013-38265 awarded by the National Institute of Food and Agriculture. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present disclosure relates generally to plant genetics and plant molecular biology, and more particularly relates to compositions, systems and methods of conferring disease resistance to plant pathogens that express pathogen-specific proteases based on recognition of the pathogen-specific proteases in a plant cell.BACKGROUND

[0004] Plant diseases are a serious limitation on agricultural productivity and influence the development and history of agricultural practices. A variety of plant pathogens are responsible for plant diseases including bacteria, fungi, insects, nematodes and viruses.

[0005] Incidence of plant diseases can be controlled by agronomic practices that include conventional breeding techniques, crop rotation and use of synthetic agrochemicals. Conventional breeding methods, however, are time-consuming and require continuous effort to maintain diseaseresistance as plant pathogens evolve. See, Grover & Gowthaman (2003) Curr. Sci. 84:330-340. Likewise, agrochemicals increase costs to farmers and cause harmful effects on the ecosystem. Because of such concerns, regulators have banned or limited the use of some of the most harmful agrochemicals.

[0006] In light of the significant impact of plant pathogens on the yield and quality of plants, additional compositions, systems and methods are needed for protecting plants from plant pathogens. Compositions, systems and methods for conferring disease resistance to plant pathogens that express pathogen-specific proteases are provided herein to address this need.SUMMARY OF THE INVENTION

[0007] In a first aspect of the invention, a method for generating disease resistance in monocot plants by conferring disease resistance to plant pathogens that express pathogen-specific proteases.

[0008] In a second aspect of the invention, the method for introducing disease resistance into a monocot plant cell, by introducing at least one nucleic acid molecule, construct, expression cassette or vector as described herein to confer disease resistance to plant pathogens that express pathogenspecific proteases.

[0009] In a third aspect of the invention, the method for conferring disease resistance into monocot plants also includes the use of genome editing to modify an endogenous Receptor Like Cytoplasmic Kinase (RLCK) family VII member belonging to groups II or III (as defined in FIG. 1) in the monocot plant to replace the endogenous AvrPphB recognition sequence (SEQ ID NO: 22) with the recognition sequence for a different pathogen protease (for example, SEQ ID NOS: 23-25).

[0010] In a fourth aspect of the invention, compositions for conferring disease resistance into monocot plants include recombinant nucleic acid molecules having a nucleotide sequence that encodesa modified substrate protein of a pathogen specific protease, where the modified substrate protein has a heterologous protease recognition sequence.

[0011] In a fifth aspect of the invention, the compositions for conferring disease resistance into monocot plants also include nucleic acid constructs, such as expression cassettes and vectors, having a nucleotide sequence that encodes a modified substrate protein of a pathogen-specific protease as described herein operably linked to a promoter that drives expression in a plant cell, plant part or plant. Such a nucleic acid construct can be used to provide a modified substrate protein to a plant cell, plant part or plant that natively expresses the corresponding nucleotide binding leucine-rich repeat (NLR) protein. The modified substrate protein can associate with, and can activate, the NLR protein.

[0012] In a sixth aspect of the invention, transformed plant cells, plant parts and plants having a nucleotide sequence that encodes at least one modified substrate protein of a pathogen-specific protease as described herein operably linked to a promoter that drives expression in a plant cell, plant part or plant. Optionally, the plant cells, plant parts and plants are transformed to include a nucleotide sequence that encodes an NLR protein operably linked to a promoter that drives expression in the plant cell, plant part or plant. The NLR protein can associate with, and can be activated by, the modified substrate protein of the pathogen-specific protease.

[0013] In a seventh aspect of the invention, a system for conferring disease resistance in monocot plants, the system including a nucleic acid construct having a nucleotide sequence for a first promoter that drives expression in a plant cell, plant part or plant operably linked to a nucleotide sequence that encodes a modified substrate protein of a pathogen-specific protease as described herein and a nucleotide sequence for a second promoter that drives expression in a plant cell, plant part or plant operably linked to a nucleotide sequence that encodes a NLR protein. The NLR protein can associate with, and can be activated by, the modified substrate protein. Such systems can be used to provide theprotein pair to a plant cell, plant part or plant that does not natively express both members of the protein pair.

[0014] In an eighth aspect of the invention, the compositions, systems and methods therefore find use in conferring disease resistance to plant pathogens by transferring to plant cells, plant parts or plants, nucleotide sequences that encode at least one modified substrate protein of a pathogen-specific protease. Optionally, the nucleotide sequence encodes an NLR protein when such NLR protein is not native to the plant cell, plant part or plant. Alternatively, the nucleotide sequence may provide an additional copy of the nucleotide sequence encoding an NLR protein to the native plant cell, plant part or plant. The pair is thus engineered to be specific for a plant pathogen-specific protease by including in the modified substrate protein a heterologous protease recognition sequence for that plant pathogenspecific protease. When activated by the plant pathogen-specific protease, the pair initiates host defense responses thereto, including programmed cell death.

[0015] A first embodiment is a composition that includes recombinant nucleic acid molecules having a nucleotide sequence that encodes a modified substrate protein of a pathogen specific protease, where the modified substrate protein has a heterologous protease recognition sequence.

[0016] In a second embodiment, the heterologous protease recognition sequence of the recombinant nucleic acid can be within, for example, an exposed loop of the modified substrate protein.

[0017] In a third embodiment, the recombinant nucleic acid molecule of the composition also has a nucleotide sequence that encodes an NLR protein so that the nucleic acid molecule encodes the protein pair.

[0018] In a fourth embodiment, the recombinant nucleic acid molecule of the composition having a nucleotide sequence that encodes the NLR protein can be co-transformed with the recombinant nucleic acid molecules having a nucleotide sequence that encodes a modified substrate protein of a pathogen-specific protease so that the modified substrate protein and the NLR protein are co-expressed.

[0019] In a fifth embodiment, the composition wherein the NLR protein can be associate with, and can be activated by, the modified substrate protein of the pathogen-specific protease.

[0020] In a sixth embodiment, a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleotide sequence that encodes at least one member of the Receptor-Like Cytoplasmic Kinase Family VII (RLCK VII) that has been modified such that the endogenous sequence GDKSHVS (SEQ ID NO: 22) has been replaced with a heterologous protease recognition sequence for a protease secreted by a plant pathogen.

[0021] In a seventh embodiment, the recombinant nucleic acid molecule of the invention wherein the heterologous protease recognition sequences is selected from the group consisting of GGCSHQS (SEQ ID NO: 23), QYCVYES (SEQ ID NO: 24), and HKYVFES (SEQ ID NO: 25).

[0022] In an eighth embodiment, a transformed plant containing the recombinant nucleic acid molecule according to the invention.

[0023] In a ninth embodiment, a transgenic seed of the transformed plant of the invention.

[0024] In a tenth embodiment, a method of protecting a plant against infection by a plant pathogen that secretes at least one specific protease, the method comprising the steps of introducing to the plant a nucleotide sequence that encodes at least one member of RLCK Family VII wherein the endogenous sequence GDKSHVS (SEQ ID NO: 22) has been replaced by a pathogen-specific protease recognition sequence, including, but not limited to, GGCSHQS (SEQ ID NO: 23), QYCVYES (SEQ ID NO: 24), and HKYVFES (SEQ ID NO: 25).

[0025] In an eleventh embodiment, a method of protecting a plant against infection by a plant pathogen that secretes at least one specific protease, the method comprising the steps of modifying an endogenous RLCK Family VII gene in a monocot crop species using genome editing wherein the endogenous sequence GDKSHVS (SEQ ID NO: 22) has been replaced by a pathogen-specificprotease recognition sequence, including, but not limited to, GGCSHQS (SEQ ID NO: 23), QYCVYES (SEQ ID NO: 24), and HKYVFES (SEQ ID NO: 25).

[0026] In a sixth aspect of the invention, the nucleic acid constructs, including expression cassettes and vectors, can optionally include a nucleotide sequence that encodes an NLR protein operably linked to a promoter that drives expression in a plant cell, plant part or plant. The nucleic acid constructs having a nucleotide sequence that encodes a modified substrate protein of a pathogen-specific protease and the nucleic acid constructs having a nucleotide sequence that encodes a NLR protein can be coexpressed in a plant cell, plant part or plant. The NLR protein can associate with, and can be activated by, the modified substrate protein of the pathogen-specific protease. Such a nucleic acid construct can be used to provide the protein pair to a plant cell, plant part or plant that does not natively express both members of the protein pair.BRIEF DESCRIPTION OF THE FIGURES

[0027] The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments, and together with the description, serve to explain the principles of the disclosure.

[0028] FIG. 1 shows a phylogenetic tree of Receptor-Like Cytoplasmic Kinase VII (RLCK VII) Family members from Arabidopsis thaliana and Hordeum vulgare (barley). Protein names that start with ‘HORVU’ are all from barley (variety Morex). Protein names that start with PBL or PBS are all from Arabidopsis. Proteins Hv-PBSl-2 and Hv-PBSl-1 are encoded by barley genes that are the most similar to Arabidopsis PBS1 based on amino acid sequence identity. The bolded black lines in the tree indicate branches in which all members lack a cleavage site for AvrPphB, thus are unlikely to participate in activation of the NLR protein PBR1 by AvrPphB. Triangles indicate RLCK Family VII members that activate PBR1 following cleavage of their activation loop, while boxes indicate members that do not activate PBR1. Members without designation have not been tested.

[0029] FIG. 2A and 2B show that proteolytic cleavage of the barley PBS1 proteins does not activate PBR1 -dependent immune signaling in Nicotiana benthamiana. The indicated proteins were transiently expressed in N. benthamiana leaves using Agrobacterium tumefaciens . FIG. 2A: Immunoblots showing detection of the indicated PBS1 proteins (anti-HA) and the NIa protease (anti- myc) from turnip mosaic virus (TuMV). AtPBSlTuMVis Arabidopsis PBS1 modified to contain a cleavage site for the TuMV NIa protease (GGCSHQ / S). HvPBS 1 - lTuMVand HvPBSl-2TuMVare barley PBS1 orthologs that were also modified to contain the same TuMV NIa protease cleavage site. Cleavage in the presence of the protease is shown by the band at ~30 kDa. FIG. 2B: Images of N. benthamiana leaves. Co-expression of Arabidopsis AtPBSlTuMVwith TuMV NIa and Arabidopsis RPS5 induces a strong cell death response (far left leaf). In contrast, co-expression of barley HvPBS lTuMVproteins with barley PBRl.c and TuMV NIa does not trigger cell death (right two leaves), which indicates that neither HvPBSl-1 nor HvPBSl-2 can activate PBRl.c. The second leaf from the left shows that co-expression of AvrPphB and PBRl.c induces strong cell death even in the absence of an added barley PBS1 protein, which suggests that cleavage of an endogenous N. benthamiana PB SI -like protein can activate PBR1.

[0030] FIG. 3A, B, and C show that proteolytic cleavage of multiple barely PBSl-like (PBL) proteins (RLCK Family VII) can activate PBRl.c-dependent immune signaling in N. benthamiana. The indicated proteins were transiently expressed in N. benthamiana leaves using A. tumefaciens. FIG 3A shows immunoblots that detect the indicated PBS and PBL proteins (anti-HA) and the indicated proteases (anti-myc). Bands at 20-35 kDa in the anti-HA blot indicate cleavage products. FIG3B shows cell death responses in N. benthamiana leaves expressing the indicated protein combinations. All leaves expressed TuMV NIa protease. FIG 3C shows electrolyte leakage data, which provides a quantitative measurement of cell death. Cleavage of HORVU5890 and HORVU 3870 induced strongcell death, while cleavage of HORVU1140, HORVUR8280 and HORVU1570 did not. Activation of cell death required the presence of PBRl.c.

[0031] FIG 4A and B further show that proteolytic cleavage of Hv3870 and Hv5890 activates PBRl.c-dependent immune signaling in N. benthamiana. FIG 4A. Images of N. benthamiana leaves transiently expressing the indicated proteins using A. tumefaciens. All leaves are expressing TuMV NIa protease. Cell death is observed when PBRl.c is co-expressed with Hv3870TuMVor Hv5890TuMV. No cell death is observed if either PBRl.c or an appropriate NIa substrate is left out, indicating that all three proteins are required. The leaf on the left shows a positive control expressing Arabidopsis RPS5 and Arabidopsis AtPBSlTuMV. FIG 4B shows electrolyte leakage data from leaf disks expressing the indicated proteins, which confirms the visible results shown in the leaves above.

[0032] FIG 5 shows that additional barley RLCK Family VII proteins activate PBRl.c-dependent immune signaling in N. benthamiana. The indicated proteins were transiently expressed in N. benthamiana leaves using A. tumefaciens. The numbers below each leaf indicate the number of leaves injected in the denominator and the number of leaves showing cell death in the numerator. Hv3050TuMV, Hv4200TuMV, and Hv7770TuMVall activated PBRl.c upon cleavage by TuMV NIa protease. The immunoblots confirm that all three proteins are being cleaved by TuMV NIa protease.

[0033] FIG 6 shows that additional barley RLCK Family VII proteins activate PBRl.c-dependent immune signaling in N. benthamiana. The indicated proteins were transiently expressed in N. benthamiana leaves using A. tumefaciens. Hv2240TuMV, Hv3640l ll''lv, and Hv4670TuMVall activated PBRl.c upon cleavage by TuMV NIa protease.

[0034] FIG 7 shows that proteolytic cleavage of RLCK family VII members from rice, sorghum, and maize activate PBR1 c-mediated cell death in N. benthamiana. The indicated proteins were transiently expressed in N. benthamiana leaves using A. tumefaciens. The graph shows electrolyte leakage from leaf disks expressing the indicated proteins. OsPBLl 1 (SEQ ID NO: 19), SbPBLl 1 (SEQID NO: 20) and ZmPBLl l (SEQ ID NO: 21) are orthologs of barley HvPBLl l (HORVU3870; SEQ ID NO: 5) from rice, sorghum and maize, respectively.

[0035] FIG 8 shows that the replacement of the AvrPphB cleavage sequence (SEQ ID NO:22) within AtPBSl with the cleavage sequence for WSMV NIa protease (SEQ ID NO:24) enables it to be cleaved by WSMV NIa protease.

[0036] FIG 9 show that proteolytic cleavage of wheat PBL11 induces cell death in wheat protoplasts. Co-transfection of protoplasts with plasmid DNA encoding TaPBLl 1WSMV(SEQ ID NO: 26), HvPBRl.c (SEQ ID NO: 1) and firefly luciferase resulted in significantly lower expression of luciferase resulted in significantly lower expression of luciferase when a plasmid encoding WSMV NIa protease was included, indicating that cleavage of TaPBLl 1WSMVtriggers cell death in wheat cells. AvrPphB / PBSl / RPS5 is a positive control for induction of cell death, whereas C98S / PBS1 / RPS5 is a negative control (C98S) is a protease inactive version of AvrPphB.DETAILED DESCRIPTION

[0037] Many plant pathogens employ proteases as virulence factors, including bacteria, fungi and viruses. As used herein, "plant pathogen" or "pathogen" means an organism that interferes with or is harmful to plant development and / or growth. Examples of plant pathogens include, but are not limited to, bacteria (e.g., Xanthomonas spp. and Pseudomonas spp.), fungi (e.g., members in the phylum Ascomycetes orBasidiomycetes), and Oomycetes, which are fungal-like organisms (e.g., Pythium spp. and Phytophthora spp.), insects, nematodes (e.g., soil transmitted nematodes including Clonorchis spp., Fasciola spp., Heterodera spp., Globodera spp., Opisthorchis spp. and Paragonimus spp.), protozoans (e.g., Phytomonas spp.), and viruses (e.g., Comovirus spp., Cucumovirus spp., Cytorhabdovirus spp., Luteovirus spp., Nepovirus spp., Potyvirus spp., Tobamovirus spp.,Tombusvirus spp. and Tospovirus spp.)

[0038] Plants, however, contain innate disease resistance against a majority of plant pathogens. Natural variation for resistance to plant pathogens has been identified by plant breeders and pathologists and can be bred into many plants. These natural disease resistance genes provide high levels of resistance (or immunity) to plant pathogens and represent an economical and environmentally friendly form of plant protection.

[0039] Innate disease resistance in plants to plant pathogens typically is governed by the presence of dominant or semidominant resistance (R) genes in the plant and dominant avirulence (avr) genes in the pathogen. The largest group of R genes encodes proteins characterized by the presence of a nucleotide binding site and leucine-rich repeats (NLR). This form of innate disease resistance typically initiates programmed cell death in infected plant cells / tissues.

[0040] A. thaliana, for example, uses R genes to confer resistance to P. syringae strains that express the avr gene, AvrPphB. Specific recognition of AvrPphB requires at least two genes, RPS5 and PBSL RPS5 encodes an NLR disease resistance protein, and PBS1 encodes a serine / threonine protein kinase belonging to RLCK Family VII. AvrPphB functions as a protease to cleave the PBS1 protein, along with many other members for the RLCK Family VII. See Zhang et al. (2010) Cell Host Microbe 7:290-301. Significantly, AvrPphB also activates cell death responses in monocots such as barley and wheat, but this response is mediated by an NLR protein distantly related to RPS5, which we have named PBR1. See Carter et al. (2019) Mol. Plant-Microbe Interact. 32:550-565.

[0041] The work described herein is the first to show that activation of PBR1 by AvrPphB is mediated by cleavage of multiple RLCK VII Family members, but not by cleavage of the family members most closely related to Arabidopsis AtPBSl (FIG. 1). This finding confirms that that ability to recognize AvrPphB evolved independently in monocot and dicot plant species, with the underlying molecular mechanism differing between monocots and dicots.

[0042] The work described herein also shows that the AvrPphB protease recognition sequence found within RLCK Family VII members (GDKSHVS; SEQ ID NO: 22) can be replaced with amino acid sequences that enable cleavage by proteases from other pathogens. For example, substitution with the sequence GGCSHQS (SEQ ID NO: 23) enables cleavage by the NIa protease from turnip mosaic virus (TuMV), as shown in FIG. 2.

[0043] Activation of the barley HvPBRl.c protein by cleavage of an RLCK Family VII member can be tested by transiently expressing these proteins in leaves of Nicotiana benthamiana using Agrobacterium tumefaciens. FIG. 2 shows that proteolytic cleavage of barley PBS1 proteins does not activate PBR1 -dependent immune signaling in N. benthamiana. Given our prior work on soybean and Arabidopsis PBS1 proteins, this was an unexpected result. See Helm el al. (2019) Mol. Plant Microbe Interact. 32: 760-769.

[0044] The failure of barley PBS1 proteins to activate PBRl.c suggested that HvPBRl.c might be activated by cleavage of a different member of the RLCK VII family that contained the AvrPphB recognition sequence (GDKSHVS; SEQ ID NO: 22). We therefore tested multiple members of the barley RLCK VII family using the same assay. In brief, the GDKSHVS (SEQ ID NO: 22) motif in each tested family member was replaced with the amino acid sequence GGCSHQS (SEQ ID NO: 23), which enabled it to be cleaved by TuMV NIa protease. The modified protein was then transiently coexpressed in N. benthamiana with barley HvPBRl.c and TuMV NIa protease. Activation of HvPBRl c was assessed by monitoring visible collapse of the injected leaf tissue and by quantifying electrolyte leakage from the injected tissue. We also used immunoblots to confirm cleavage of the modified RLCK protein. As shown in FIG. 3 -FIG. 6, these assays revealed that multiple RLCK VII family members can activate HvPBRl.c upon cleavage. These findings are summarized in FIG. 1, with triangles marking family members that can activate HvPBRl.c and boxes marking family members that cannot activate HvPBRl c.

[0045] FIG. 1 shows a subset of barley RLCK VII family members that were tested for the ability to activate HvPBRl .c. Based on their positions in the phylogenetic tree, we can conclude that many other RLCK VII family members will also activate HvPBRl.c upon cleavage. Indeed, only RLCK VII family members closely related to HvPBSl-1 and HvPBSl-2 are unlikely to activate HvPBRl.c (Group 1 in FIG. 1). Embodiments of this invention thus include modifications of any RLCK VII family member in Groups II and III shown in FIG. 1 such that the GDKSHVS motif is replaced with a heterologous protease recognition sequence.

[0046] As used herein, the term “heterologous protease recognition sequence” refers to an amino acid sequence that can be cleaved by a pathogen-specific protease and which differs from GDKSHVS (SEQ ID NO: 22). The heterologous protease recognition sequence can differ from the GDKSHVS sequence (SEQ ID NO: 22) at anywhere from one position to all seven positions.

[0047] This approach described herein builds upon our prior invention described in US Patent 9,816,102 B2 issued November 14, 2017, incorporated by reference herein. However, the present disclosure differs from US Patent 9,816,102 B2 in that the approach described herein employs protease substrate proteins that were not previously described and are not obvious, even to those skilled in the art of molecular plant-microbe interactions. In the previously granted patent, the protease substrate protein described was the PBS1 protein from a di cot plant, Arabidopsis thaliana (NCBI gene ID 831155). Cleavage of this protein by a pathogen protease activates the Arabidopsis NLR protein RPS5 (NCBI accession number 064973).

[0048] Notably, A. thaliana is a dicot plant. As disclosed herein, monocot plants lack an ortholog of RPS5 and that cleavage of the monocot ortholog of Arabidopsis PBS1 does not activate resistance. Thus, the previously described method of conferring disease resistance in dicot plants does not work in monocot plants such as wheat, barley, rice, maize and sorghum. Instead, monocots have evolved an independent mechanism for detecting pathogen proteases. Specifically, monocot plants, includingbarley, wheat, rice, and sorghum, have evolved a novel NLR protein that we have named PBR1 that fulfills this function (SEQ ID NOS: 1-4). See Carter etal. (2019) Mol. Plant-Microbe Interact. 32:550- 565. In this application we show that PBR1 is activated by cleavage of any of multiple different protein kinases that are phylogenetically distinct from the Arabidopsis PBS1 (SEQ ID NOS: 5-14). The examples set out below focus on protein kinases from the monocot plant species barley, but these kinases are also found in other monocot species. Our data disclosed herein shows that these kinases would function equivalently with regards to activating PBR1 when cleaved in other monocot species. Embodiments of the invention include commercially farmed monocot species such as wheat (Triticum spp), rice (Oryza spp.), sorghum (Sorghum spp), and maize (Zea spp).

[0049] The compositions in a first aspect of the invention include recombinant nucleic acid molecules having a nucleotide sequence that encodes a modified substrate protein of a pathogen specific protease, where the modified substrate protein has a heterologous protease recognition sequence.

[0050] As used herein, the term “recombinant nucleic acid molecule” means a nucleic acid molecule that has been created or modified through deliberate human intervention such as by genetic engineering. This includes, but is not limited to, nucleic acid sequences that have been modified to include an artificial nucleotide sequence or to include some other nucleotide sequence that is not present within its native (non-recombinant) form.

[0051] The heterologous protease recognition sequence can be within, for example, an exposed loop of the modified substrate protein. Optionally, the recombinant nucleic acid molecule can have a nucleotide sequence that encodes an NLR protein so that the nucleic acid molecule encodes the protein pair. For example, in one embodiment, a recombinant nucleic acid molecule having a nucleotide sequence that encodes the NLR protein can be co-transformed with the recombinant nucleic acid molecules having a nucleotide sequence that encodes a modified substrate protein of a pathogen-specific protease so that the modified substrate protein and the NLR protein are co-expressed. The NLR protein can associate with, and can be activated by, the modified substrate protein of the pathogen-specific protease.

[0052] Compositions of the invention also include nucleic acid constructs, such as expression cassettes and vectors, having a nucleotide sequence that encodes a modified substrate protein of a pathogen-specific protease as described herein operably linked to a promoter that drives expression in a plant cell, plant part or plant. Such a nucleic acid construct can be used to provide a modified substrate protein to a plant cell, plant part or plant that natively expresses the corresponding NLR protein. The modified substrate protein can associate with, and can activate, the NLR protein.

[0053] The constructs of the invention, including expression cassettes and vectors, can optionally include a nucleotide sequence that encodes an NLR protein operably linked to a promoter that drives expression in a plant cell, plant part or plant. The nucleic acid constructs having a nucleotide sequence that encodes a modified substrate protein of a pathogen-specific protease and the nucleic acid constructs having a nucleotide sequence that encodes a NLR protein can be co-expressed in a plant cell, plant part or plant. The NLR protein can associate with, and can be activated by, the modified substrate protein of the pathogen-specific protease. Such a nucleic acid construct can be used to provide the protein pair to a plant cell, plant part or plant that does not natively express both members of the protein pair.

[0054] Compositions of the invention described herein also include transformed plant cells, plant parts and plants having a nucleotide sequence that encodes at least one modified substrate protein of a pathogen-specific protease as described herein operably linked to a promoter that drives expression in a plant cell, plant part or plant. Optionally, the plant cells, plant parts and plants are transformed to include a nucleotide sequence that encodes an NLR protein operably linked to a promoter that drivesexpression in the plant cell, plant part or plant. The NLR protein can associate with, and can be activated by, the modified substrate protein of the pathogen-specific protease.

[0055] Compositions of the invention also include isolated, modified substrate proteins of pathogen-specific proteases as described herein.

[0056] As disclosed herein, systems of the invention include nucleic acid constructs having a nucleotide sequence for a first promoter that drives expression in a plant cell, plant part or plant operably linked to a nucleotide sequence that encodes a modified substrate protein of a pathogenspecific protease as described herein and a nucleotide sequence for a second promoter that drives expression in a plant cell, plant part or plant operably linked to a nucleotide sequence that encodes a NLR protein. The NLR protein can associate with, and can be activated by, the modified substrate protein. Such systems can be used to provide the protein pair to a plant cell, plant part or plant that does not natively express both members of the protein pair. Expression of the protein pair will confer pathogen and disease resistance to the plants based upon the recognition of the pathogen-specific proteases in the cells of the plant.

[0057] The systems enclosed herein also include a first nucleic acid construct having nucleotide sequence for a promoter that drives expression in a plant cell, plant part or plant operably linked to a nucleotide sequence that encodes a modified substrate protein of a pathogen-specific protease as described herein, and a second nucleic acid construct having a nucleotide sequence for a promoter that drives expression in a plant cell, plant part or plant operably linked to a nucleotide sequence that encodes an NLR protein. Additional nucleic acid constructs also can be included in the system, where each construct has a nucleotide sequence that encodes a distinct modified substrate protein, each having a heterologous recognition sequence for a separate pathogen-specific protease. Although each modified substrate protein has a heterologous recognition sequence distinct from one another, each can associate with, and can activate, the NLR protein. Alternatively, the first nucleic acid construct can encode morethan one modified substrate protein, where each modified substrate protein has a heterologous recognition sequence distinct from one another and where each can associate with, and can activate, the NLR protein. Alternatively, the second nucleic acid construct can encode one or more modified substrate proteins, where each modified substrate protein has a heterologous recognition sequence distinct from one another and where each can associate with, and can activate, the NLR protein. Such systems can be used to provide the protein pair to a plant cell, plant part or plant that does not natively express the protein pair or can be used to provide more than one modified substrate protein to a plant cell, plant part or plant. Expression of the modified substrate proteins will confer pathogen and disease resistance to the plants based upon the recognition of the pathogen-specific proteases in the cells of the plant.

[0058] By way of example, the substrate protein of the pathogen-specific protease can be an RLCK family VII member from barley belonging to groups II or III (SEQ IDS NOS: 5-17) or their orthologo us sequences from other monocot crop species such as wheat, rice, sorghum and / or maize. As a specific example, it can be wheat TaPBLl 1 (SEQ ID NO: 18). The NLR protein can be wheat TaPBRl (SEQ ID NO: 2), where the TaPBLl 1 is modified to include a heterologous protease recognition sequence (for example, SEQ ID NO: 26).

[0059] In view of the foregoing, the methods described herein include introducing into a plant cell, plant part or plant at least one nucleic acid molecule, construct, expression cassette or vector as described herein to confer disease resistance to plant pathogens that express pathogen-specific proteases.

[0060] The methods of conferring disease resistance in a plant described herein also include the use of genome editing to modify an endogenous RLCK family VII member belonging to groups II or III (as defined in FIG. 1) to replace the endogenous AvrPphB recognition sequence (SEQ ID NO: 22) with the recognition sequence for a different pathogen protease (for example, SEQ ID NOS: 23-25).

[0061] For example, in one embodiment, genome editing technology is utilized to modify an endogenous RLCK VII family member in a monocot crop plant such as wheat, rice, sorghum or com such that the endogenous GDKSHV motif (SEQ ID NO: 22) is replaced with a heterologous protease recognition sequence such as the recognition sequence for the NIa protease from wheat streak mosaic virus (QYCVYES; SEQ ID NO: 24) or the recognition sequence for the ORF1 protease from maize yellow dwarf virus (HKYVFE; SEQ ID NO: 25). These replacements will enable the modified RLCK proteins to be cleaved by the corresponding viral proteases, which will then activate the endogenous PBR1 protein and thus confer resistance to viral infection.

[0062] As an alternative to using genome editing technology, another embodiment of this invention is to use recombinant DNA technology to introduce a nucleic acid sequence encoding a full- length RLCK Family VII gene in which the endogenous GDKSHV motif (SEQ ID NO: 22) has been replaced with a heterologous protease recognition sequence. Such nucleic acid constructs would include a promoter sequence that drives expression of the modified RLCK Family VII gene in the tissues of interest (e.g., roots and / or leaves) and a terminator to mark the end of transcription. Introduction of the recombinant nucleic acid, construct, expression cassette or vector may be performed through standard monocot plant transformation techniques known to those skilled in the art, including but not limited to Agrobacterium tumefaciens-m ate transformation of cultured tissue or immature embryos.

[0063] An example of a modified RLCK VII family member that is a substrate of a pathogenspecific protease includes SEQ ID NO:26 (wheat TaPBLl l containing a WSMV NIa protease recognition sequence). Another example of a modified RLCK VII family member that is a substrate of a pathogen-specific protease includes SEQ ID NO:27 (maize ZmPBLl l containing a MYDV ORF1 protease recognition sequence). These examples are provided just to clarify the invention and are not meant to limit the invention in anyway. As obvious to anyone with skill in the art, this system can beused to engineer recognition of any pathogen that employs a protease that has a defined recognition sequence of seven amino acids or less and which is translocated into host cells during infection. The invention thus includes the use of any monocot RLCK VII gene that contains a heterologous protease recognition sequence.

[0064] The compositions, systems and methods therefore find use in conferring disease resistance to plant pathogens by transferring to plant cells, plant parts or plants nucleotide sequences that encode at least one modified substrate protein of a pathogen-specific protease. Optionally, the nucleotide sequence encodes an NLR protein when such NLR protein is not native to the plant cell, plant part or plant. Alternatively, the nucleotide sequence may provide an additional copy of the nucleotide sequence encoding an NLR protein to the native plant cell, plant part or plant. The pair is thus engineered to be specific for a plant pathogen-specific protease by including in the modified substrate protein a heterologous protease recognition sequence for that plant pathogen-specific protease. When activated by the plant pathogen-specific protease, the pair initiates host defense responses thereto, including programmed cell death.EXAMPLES

[0065] Example 1: Phylogenetic Analysis of Arabidopsis and Barley PBSl-like (PBL) genes.

[0066] FIG. 1 shows a phylogenetic tree depicting the relationships between protein kinases of Arabidopsis and barley belonging to the subfamily of protein kinases that includes Arabidopsis PBS1. This subfamily is known as Receptor Like Cytoplasmic Kinase VII (RLCK VII). See Rao et al. (2018) Plant Physiol. T1 , 1679-1690. Members of this family have been shown to interact with cell surface immune receptors to activate downstream immune responses upon detection of pathogen-derived signals such as chitin or flagellin. Pathogens often translocate ‘effector proteins’ into host plant cells that modify this family of kinases and thereby block immune signalling from cell surface immunereceptors. As an example of this, the AvrPphB protein, which is a cysteine protease, is translocated from the bacterial pathogen Pseudomonas syringae into host cells, where it cleaves multiple members of the RLCK VII family, including PBS1, BIK1, PBL1 and PBL11. See Zhang et al. (2010) Cell Host Microbe 7:290-301. By doing so, AvrPphB suppresses immune signalling.

[0067] As illustrated in the phylogenetic tree (FIG. 1), protein names that start with ‘HORVU’ are all from barley (variety Morex). Protein names that start with PBL or PBS are all from Arabidopsis. The barley genome contains two genes that are co-orthologous to Arabidopsis PBS1. The barley genome also contains multiple genes that are co-orthologous to Arabidopsis genes BIK1, PBL1, PBL9, PBL10, and PBL11 (e g., HORVU.MOREX.r2.1HG0003870 (HvPBLl l), HORVU.MOREX.r2.5HG0403300, HORVU. MOREX. r2.5HG0403310,HORVU.MOREX.r2.5HG0437770, HORVU. MOREX. r2. 1HG0003870,HORVU.MOREX.r2.4HG0323050, HORVU.MOREX.r2.3HG0274200). This latter set belongs to a group (designated Group III in FIG. 1) that is phylogenetically distinct from the group that includes PBS1 (designated Group I in FIG. 1). In addition, the barley genome also contains multiple genes that are co-orthologous to Arabidopsis genes PBL34, PBL35 and PBL36 (e.g., HORVU.MOREX.r2.3HG0222240, HORVU.MOREX.r2.5HG0393640,HORVU.MOREX.r2.7HG0544670). This group of genes (designated Group II in FIG. 1) are also distinct from the PBS1 group. Below we show that cleavage of any member of the Group II or Group III kinases, but not Group I kinases, is sufficient to activate barley PBRl.c. Thus, in monocot plant species, any kinase from RLCK VII groups II or III (as defined in FIG. 1) can be used to create decoy substances for recognition of pathogen proteases. Furthermore, it does not matter what plant species the kinase originates from.

[0068] The work described herein is the first to show that activation of PBR1 by AvrPphB is mediated by cleavage of multiple RLCK VII Family members, but not by cleavage of the familymembers most closely related to Arabidopsis AtPBSl. This phylogenetic analysis confirms that that ability to recognize AvrPphB evolved independently in monocot and dicot plant species, with the underlying molecular mechanism differing between monocots and dicots.

[0069] However, based on their positions in the phylogenetic tree (FIG. 1), we can also conclude that many other RLCK VII family members will also activate HvPBRl.c upon cleavage. Indeed, only RLCK VII family members closely related to HvPBSl-1 and HvPBSl-2 are unlikely to activate HvPBRl.c (Group I of FIG I). Thus, in monocot plant species, any kinase from RLCK VII groups II or III (as defined in FIG. 1) can be used to create decoy substrates for recognition of pathogen proteases. Furthermore, it does not matter what plant species the kinase originates from. Embodiments of this invention thus include modification of any RLCK VII family member in Groups II and III of FIG 1 , such that the GDKSHVS motif is replaced with a heterologous protease recognition sequence.

[0070] Example 2: Proteolytic cleavage of the barley PBS1 proteins does not activate PBRl-dependent immune signaling in N. benthamiana.

[0071] In order to evaluate whether or not modification of the proteolytic cleavage site of the barley PBS1 proteins activate PBRl-dependent immune signalling, we created a model system in Nicotiana benthamiana. Three modified proteins were transiently expressed in N. benthamiana leaves using Agrobacterium tumefaciens following the methods described in Helm et al. (2019) Mol. Plant Microbe Interact. 32: 760-769: (1) AtPBSlTuMVis Arabidopsis PBS1 modified to contain a cleavage site for the TuMV NIa protein (GGCSHQ / S); (2) HVPBS1-1TUMVis a barley PBS1 ortholog also modified to contain a cleavage site for the TuMV NIa protein (GGCSHQ / S); and (3) HVPBS1-2TUMVwhich is another barley PBS1 ortholog modified to contain the same TuMV NIa protease cleavage site.

[0072] As shown in FIG. 2A, detection of PBS1 proteins (anti-HA) and the NIa protease (anti- myc) from turnip mosaic virus (TuMV) was done using an immunoblotting assay. Cleavage in the presence of the protease is shown by the band at ~30 kDa.

[0073] Phenotypic changes in N. benthamiana leaves transiently expressing the modified proteins were also noted. Co-expression of Arabidopsis AtPBSlluMVwith TuMV NIa and Arabidopsis RPS5 induces a strong cell death response (far left leaf in FIG. 2B). In contrast, co-expression of barley HVPBS1TUMVproteins with barley PBRl.c and TuMV NIa did not trigger cell death (right two leaves in FIG 2B), which indicates that neither HvPBSl-1 nor HvPBSl-2 can activate PBRl.c. The second leaf from the left shows that co-expression of AvrPphB and PBRl.c induces strong cell death even in the absence of an added barley PBS1 protein, which suggests that cleavage of an endogenous N. benthamiana PBS 1 -like protein can activate PBR1.

[0074] Example 3: Proteolytic cleavage of multiple barley PBSl-like (PBL) proteins activate PBRl.c-dependent immune signaling in N. benthamiana.

[0075] Using our model system described above, we tested barley PBSl-like (PBL) proteins to determine if they activated PBRl.c- dependent immune signalling. As shown in FIG. 3, proteolytic cleavage of multiple barely PBSl-like (PBL) proteins (RLCK Family VII) can activate PBRl.c- dependent immune signalling in N. benthamiana. In this example, five different barley PBL proteins were transiently expressed in / V. benthamiana ea QS using A. tumefaciens'. Hv3870TuMV, Hv5890l u IV, Hvl 570TuMV, Hvl 140Tu V, and Hv8280TuMV. Immunoblots detect the indicated PBS and PBL proteins (anti-HA) and the indicated proteases (anti-myc). (FIG 3 A) Bands at 20-35 kDa in the anti-HA blot indicate cleavage products. Phenotypic changes indicative of cell death responses in N. benthamiana leaves expressing the indicated protein combinations were also observed. (FIG 3B). All leaves expressed TuMV NIa protease. Electrolyte leakage data, which provides a quantitative measurement of cell death, was also measured. (FIG. 3C)

[0076] Cleavage of HORVU5890 and HORVU3870 induced strong cell death. Cleavage of H0RVU1 140, HORVUR8280 and H0RVU1570 did not induce cell death.

[0077] Further analysis of Hv3870TuMVand Hv5890TuMVwere conducted to determine if the activation of cell death required the presence of PBRl.c. Hv3870 and Hv5890 were transiently expressed in N. benthamiana leaves using A. tumefaciens with all leaves co-expressing TuMV NIa protease. Cell death is observed when PBRl.c is co-expressed with Hv3870TuMVor Hv5890TuMV. No cell death is observed if either PRBl.c or an appropriate NIa substrate is left out, indicating that all three proteins are required. (FIG. 4A) Electrolyte leakage data from leaf disks expressing the indicated proteins confirmed the visible results observed. (FIG. 4B) Activation of cell death required the presence of PBRl.c.

[0078] Example 4: Proteolytic cleavage of barley RLCK Family VII proteins activate PBRl.c-dependent immune signaling in N. benthamiana.

[0079] Additional barley RLCK Family VII proteins were tested for their ability to activate PBRl.c-dependent immune signaling in N. benthamiana. Hv3050TuMV, Hv4200TuMV, Hv7770TuMV, HV2240TUMV, HV3640TUMV, and HV4670TL IVwere transiently expressed in N. benthamiana leaves using A. tumefaciens. Hv3O5OTuMV, Hv4200Tu IV, and Hv7770TuMV(FIG. 5) as well as Hv2240TuMV, HV3640TUMV, and Hv4670TuMV(FIG. 6) all activated PBRl.c upon cleavage by TuMV NIa protease. These results were consistently observed both visually and through immunoblot assays (FIG 5).

[0080] Example 5: Proteolytic cleavage of RLCK family VII members from rice, sorghum, and maize activate PBRl-mediated cell death

[0081] FIG. 2-6 show that many barley RLCK VII family members can activate HvPBRl.c. RLCK VII genes are highly conserved across flowering plants (angiosperms), thus it is expected that RLCK VII family members from other plant species will also be able to activate HvPBRl.c. To test this, we obtained RLCK VII family members from rice (SEQ ID NO: 19), sorghum (SEQ ID NO: 20), and maize (SEQ ID NO: 21) and replaced their GDKSHVS motif (SEQ ID NO: 22) with the TuMV NIa protease recognition sequence GGCSHQS (SEQ ID NO: 23). These proteins were then transientlyexpressed in N. benthamiana along with HvPBRl c and TuMV Nia protease. RLCK proteins from all three species activated HvPBRl. c. (FIG. 7).

[0082] Embodiments of this invention thus include use of RLCK VII family members from any monocot plant species in which the GDKSHVS motif has been replaced with a heterologous protease recognition sequence. FIG. 8 shows that replacement of SEQ ID NO: 22 with SEQ ID NO: 24 in Arabidopsis PBS1 enables it to be cleaved by WSMV NIa protease. FIG. 9 shows that replacement of SEQ ID NO: 22 with SEQ ID NO: 24 in wheat PBL11 (SEQ ID NO: 26) enables activation of cell death in wheat protoplasts when co-expressed with WSMV Nia protease, which will confer resistance to infection by WSMV.EQUIVALENTS AND SCOPE

[0083] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present invention is not intended to be limited to the above, but rather is as set forth in the appended claims.

[0084] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0085] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses and descriptive terms, from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0086] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention is / are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps.

[0087] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the method of the invention can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0088] Each of the foregoing patents, patent applications and references is hereby incorporated by reference, particularly for the teaching referenced herein.INCORPORATION OF SEQUENCE LISTING

[0089] Information regarding SEQ ID NOs:l -27 are set out below. The instant application contains a Sequence Listing XML which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML copy, created on May 22, 2024 is named "IU202106102WOST26" and is 34,827 bytes in size.

[0090] SEQ ID NO : 1Number of amino acids: 939Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: AZI15803Common name: HvPBRl.cSEQUENCE: !1 MVSALAGVMT SVIGKLTALL GEEYAKLKGV HREVEFMKDE LSSMNALLQR LAEADRDLDV 61 QTKEWRDQVR EMSYDIEDCM DDFMKSLGQT DSAQTAGLVQ SWQQLKALR ARHQISSKIQ 121 GLKARVEDAS KRRMRYKLDE RTFEPSISRA INPRLPSLYA EPDGLVGIDK PRDELIKCLM 181 EGMGASVQQQ KVLSIVGPGG LGKTTLANEV YRKLEGQFQC RAFVSLSQQP DVNKILRNIL 241 SQVCQQELPS TSVQDEGKLI DAIREVLKNK RYLWIDDIW STQAWKIIKC SLFLNDLGSR 301 IMTTTRSIDI AKSCCSRRHD RVYEIMPLTT ANSKGLFFKR IFGSEDICPP QLEEISSEIL361 KKCGGSPLAI LTIASLLANK DSTNEEWKWV YNSIGSTLGK DPGVEEMRRI LSLSYDDLPH421 HLKTCLLYLS IFPEDYEIER DRLIRRWIAE GFIDTDGGRD LEEIGECYFN DLINRSMLEP481 VKIQYDGQW SCRVHDMILD LLASKSIEEN FATFSGNQNE ILVLRHKIRR LSLNYYAQEH541 TMLPSTAIIS HCRSLSIVGY AEKMPSLSKF RVLRVLDIEN GEEMESNCFE HLRTLFQLRY601 LRLHVRSISA LPEQLGELQH LRTLDMGWTK ITKMPKSIVQ LQHLTCLRVS NLELPEGIGN661 LQALQELSDI KVNRHSTASC LLELGSLTKL KILGLRWSIV STHGNEDTFV DNLVSSLRKL721 GRSSLRSICI RSYHGYTMEF LLDSWFPSPH LMQKFQMGTY YNFPRIPPWI ASLDKLTYLD781 INIDPVEEEA LEILGELPSL LFLWLTSKSA APKQRLWSS SMFVRLKELH FTCWSNGQGL841 MFEAGAMPRL EKLWVPFDAG SGLDSGIQHL SSLTHLAVEI ICVGATARD V EALEEAIRGA901 ARLLPNRPAV EFRTWDDEKM WEEEEGQGV PEEEIHASG

[0091] SEQ ID NO 2Number of amino acids: 967Sequence type: ProteinOrganism Source: wheat (Triticum aestivum)NCBI Accession Number: KAF7033218.1Common name: TaPBRl-B3

[0092] SEQUENCE: 21 MCLLCRAEAW GHFLQNWALI LVATLIEHPQ MVSALAGVMT SVIAKLTALL GEEYAKLKGV61 HREVEFMKDE LSSMNALLQR LAEVDRDLDV QTKEWRDQVR EMSYDIEDCI DDFMKSLGQT121 DSAKAAGLVQ SVLQQLKALR ARHQISSQIQ GLKAR VEDAS KRRMRYKLDE RTFEPSISRA181 IDPRLPSLYA EPDGLVGIDK PRSELIECLM EGMGASVQQQ KVISIVGPGG LGKTTLANEV241 FRKLEGQFQC RAFVSLSQQP DVSKILRNIL SQVCQQELPS TDIQDEGKLI DTIREVLKNK301 RYLWIDDIW STQAWKIIKC SLFLNDLGSR IMTTTRSIDI AKSCCSRRHD RVYEIMPLTA361 ANSKSLFFKR IFGSEDICPP QLEEVSSEIL KKCGGSPLAI LTIASLLANK DCTNEEWEWV421 YNSIGSTLGK DPGVEEMRRI LSLSYDDLPH HLKTCLLYLS IFPEDYEIER DRLVRRWIAE481 GFIDTNGGRD LEEIGERYFN DLINRSMLQP AEIQYDGQW SCRVHDMILD LLTSKSMEEN541 FATFFRNQNE ILVLQHKIRR LSLSYYDQEH IMLPSTAIIS HCRSLSIVGY AEKMPSLSKF601 RVLRVLDIEN GEEMESNCFE HLRKLFQLKY LRLHVRSISA LPEQLGELQH LKTLDMGWTK661 ITKMPKSIVQ LQHLTCLRVS NLELPEGIGN LQALQELSDI KVNRYSMASC LLELGSLTKL721 KILGLRWYIV STHSNKDTFVDNLVSSLRKL GRFSLRSICI RSYHGYSMEF LLDSWFPSPY781 LMQKFQMGTY YNFPRVPPWI VSLDKLTYLD INIDPVDEET LEILGELPAL LFLWLMSKSA841 APKQRLIISS SMFVCLREFH FTCWSNGEGL MFEAGAMPRL EKLWVPFDAG SGLDFGIQHL901 SSLRHLAVEI ICVGATARDV EALEEAIRDA AHLLPNRPAV EFRTWDDEKM AGEEGQGVTE961 EEIHASG

[0093] SEQ ID NO 3Number of amino acids: 938Sequence type: ProteinOrganism Source: rice (Oryza sativa)NCBI Accession Number: XP_015620262Common name: OsPBRlSEQUENCE: 31 MASALTGAMT SVIIKLSALL GEEYAKLKGL QREVEFMKDE LSSMNALLHR LAEVDSDLDV61 QTEEWRNQVR EMSYDIEDCI DGFTHRLGHI GIAEAAGPVQ RVAQQLKVLK VRRQIASQIQ121 ELKGR VEDAS KRRMRYKLDD RIFEPSIARA IDPRLPSLYA ESDGLVGIET PRAVLVKLIM181 EGDDASFQQL KVISIVGPGG LGKTTL ANEV YRRLEGQFQC RAFVSLSQQP DVKRILRNIF241 CQVSQQVYDS TSVWDEENLI DAIRGFLKDK RYFIVIDDIW SIQAWKTIKC ALLMNNLGSR301 IITTTRSVTI AKSCCSPQHD HVYEIMPLST ANAMSLFLKR IFGTEDICPP QLEEISCKIL361 KKCSGSPLAI ITIASLLTNK ASTKEEWERV HNSIGSTLEK DPSVEEMQRI LSLSYDDLPH421 HLKTCLLYLC IFPEDCEIER DQLVKRWIAE GFINTGSGQD LEKIGESYLN DLISRSMIQP481 VKVRYDGQVD SCRIHDMILD LLMSKSIKEN FATFLGEQNQ KLVLQGKVRR LSLSYYSQEN541 VMVPSTAIIS SCRSLSIFGY AEEMPSLSEF RVLRVLDIEH GEDMDSNYLE HVRRLSQLKY601 LRLNVRSIDA LPEQLGELQH LQTLDLVSTK LRKSPKSIVR LQNLTCLRIN NLELPEGIGC661 MRALQEVSEI KISRNSSASS LQELGNLTKL KILGLCWCIS DIHGGTKTLV NNLVSSLRKL721 GRLNLRSLCI QSSFKYSIDF LLDSWLPTPH LLQKFQMGMC YYFPRIPVWI ASLENLTYLD781 INLNPVKEEV LEILGNLPAL LFLWLTSKSA DPKQRLIINS NMFMCLKELY FTCWSIESGL841 MFQEGCMAKL EKLHLPFHAA TALEFGIHHL SSLRLLWEI ICSGATIRQV ESLEETIRKT901 ADLLPYRPTV EIRTWDEENM VEEQKEKDMG EEGTQTSC

[0094] SEQ ID NO: 4Number of amino acids: 903Sequence type: ProteinOrganism Source: sorghum (Sorghum bicolor')NCBI Accession Number: EES 16173Common name: sorghum SbPBRlSEQUENCE: 41 MVS VLTGVMT SV1DKLTALL GEEYTKETGV QREVNFMKDE LSSMNALLQR LAEADSDLDL61 QTKEWRRQVQ EMSYDIEDCV DEFMHRVGNS STTDSGGLVH GVVQQLKVLW ARYQIGSKIQ121 DLKARVEDAN KRRVRYKVDE LAFQSSTKSA IDPRLPSLYA DPDGLVG1GR PRDDLTRMLM181 EGEGTSVQQL KVISIVGPGG LGKTTLANEV YHRLEGQFQC RAFVSLSQQP DVKKILRNIL241 YQVSHQEYAN METWDEEMYF IVIDDIWSTQ AWKTIRCALY VNNCASRIMT TTRIVSIAKS301 CCSPHHDHVY EITPLSTDNS KCLFFKRIFG SEHICPPHLE DISSEILEKC SGSPLAIVTM361 ASLLANKACT KQEWDRVCNS IGSTLEKDPD VEEMRRILSL SFDDLPHHLK TCLLYLSIFP421 EDYEIERDQL VKRWIAEGFI NMEGGQDLEE IGENYFNDLI NRSMIQPMKI KCDGRVASCQ481 VHDMILDLLI SKSVEENFAT FISGKNKTLL LQHKVRRLSL NYYSQDHTMF PSAAIISQCR541 SLSIFGYSEQ MPSLSKFRVL RVLDIENGEE MEHKYFEHIR RLLQLKYLRL HVRSISALPE601 QLGELWQLRT LDLGGTKITK LPKSIVQLQN LTCLRVCNME LPEEIGNLHA LQELSEIKIN661 RNSMASSLLG LGSLTKLRIL RLRWCISNTD TDNRTFIDNF LSSLRKLGRL NLRSLCIQSY721 CGYSIDFMLD SWFPTPYLLQ KFQMNLEYYF PRIPSWIASL GNLTYLDINV DPLEEETLEI781 LGNLPSLMCL WVSSKAAAPK ERLWSNGMF GFLKEFQFMC WRNKVGLVFE AGSMPRLEKL841 RIPFNAGTGL NFGIEHLSSL RHLIVEIICS GASVQEVEAL EEAMRSAADL LPNRLFLEVR901 TWD

[0095] SEQ ID NO 5Number of amino acids: 406Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044974206Common name: HvPBLl l; HORVU.MOREX.r2.1HG0003870SEQUENCER1 MGNCWGAKIS SDTSSSSSSS PSGANSRYAS RNGAALSSSS SYASAASVQR SEGEILESAN61 VKAFTFNELR TATRNFRPDS VLGEGGFGSV FKGWIDEKTL APTKPGTGMV IAVKKLNQES121 YQGHREWLAE VNYLGQLSHP NLVKLVGYCV EDEQRLLVYE FMPRGSLENH LFRRSTHFQP181 LSWNLRMKIA HGAAKGLAFL HSDKAKVIYR DFKTSNILLD ANYDAKLSDF GLAKDGPTGD241 KSHVSTRVMG TYGYAAPEYL ATGHLTTKSD VYSFGWLLE MLSGRRAVDK NQPTGEHNLV301 EWARPYLTSK RRIFRVLDPR LGGQYSLAKA QKAASLALQC LSADSRHRPS MEQVWALEQ361 LHDAKEGGNS PHPQLQRKPS SSRSMTGSRP SSTKGSNNRP ASLRPA

[0096] SEQ ID NO 6Number of amino acids: 372Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044982017Common name: HORVU.MOREX.r2.4HG0323050SEQUENCE: 61 MGNCAGVQGN AEINPTFSAP NTSGNNSKSS SSNATDTSTF GKSSSSSVPP TPRSEKEILQ61 SSNLRKFTFS ELKSCTRNFR TDSLLGEGGF GSVFKGW1DE RTFTPVKPGT GMIVAVKKLK121 LDSFQGHKEW LAEVNYLGQL SHPNLVKLIG YCLEDEQRLL VYEFMPRGSL EHHLFRRAPH181 FQPLSWNLRM KVALEAARGL AFLHSDEAKV IYRDFKTSNV LLDSEYNAKL SDFGLAKDGP241 SGDKSHVSTR VMGTQGYAAP EYLATGHLTT KSDVYTYGW LLELLTGQRA LDKNRPPGQH301 NLVEWARPYI NSKRRVIHVL DPRLGSQYSL PAAQKAAALA MQCLSMDARC RPDMDQWTV361 LQKLPEVKKT YK

[0097] SEQ ID NO: 7Number of amino acids: 423Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044978667Common name: HORVU.MOREX.r2.3HG0274200SEQUENCE: 71 MGNCFGSRIS SDSPYKTTSG SASPSSSSGW AWRRKGKGGV SVSSSRVSSS PSTTVPPTPR61 SEGEILQSAN VKSFAFTELK TATRNFRPDS VLGEGGFGSV FKGWVDETTF APARPGTGMV121 IAVKKLNQEG FQGHREWLAE VNYLGQLSHP NLVRLVGYCL EDEQRLLVYE FMPRGSLENH181 LFRRGSHFQP LSWNLRMKVA LGAAKGLAFL HSDNAKVIYR DFKTSNVLLD SSYNAKLSDF241 GLAKDGPTGD KSHVSTRVMG THGYAAPEYL ATGHLTAKSD VYSFGVVLLE MLSGRRALDK301 NRPAGEHNLV EWARPYLTSK RRVFRILDAR LGGQYSLPGA QKTAALAMQC LSGDARARPG361 MAQWTALEQ LQDAKETAAG AGQGKAFVRM RGGGGSGAGR QQRQPEPMAV RRLPAAPLRS421 HPE

[0098] SEQ ID NO: 8Number of amino acids: 425Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044985743Common name: HvPbll; HORVU.MOREX.r2.5HG0437770SEQUENCE: 81 MGNCWGTRIK DGSTHPGASG MFSRGSGKDG SRLSACSSRA SSASMPPSAK TECEILQSAN61 VKVFSYNDLR LATRNFRPDS VLGEGGFGS V YKGWIDEHTL SACKPGTGIP VAVKRLNLEG121 LQGHREWLAE VNYLGQFCHQ NLVKLIGYCL EDEYRLLVYE CMPRGSLENH LFRRGSHFQP181 LSWNLRMKVA LGAAKGLAYL HSAEAKVIYR DFKTSNILLD TDYTAKLSDF GLAKDGPVGE241 KSHVSTRVMG TYGYAAPEYL STGHLTAKSD IYSFGWLLE MLSGRRAIDK NRPQGEHNLV301 EWARPYLTHK RKIFRVLDTR LEGQYSLNGA QTIAALAVEC LSFEAKMRPS MEAWSILEG361 IQDSSDPTRR PADPARRPAA ERPQDPKSGS KTAPGASNSG KGRRKSSGDL LKEPGRDPKP421 SAYSS

[0099] SEQ ID NO: 9Number of amino acids: 417Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044982756Common name: HvPBL2; HORVU.MOREX.r2.4HG0335890SEQUENCE: 91 MGNCMKSTAR VDHSMNTSAA YPSKVTSKTS LSSATSASKT NSTRSTFTLP SIRDRSEPPR61 TEGEILSSSN LKAFLFNDLK NATKNFRPDS LLGEGGFGHV FKGWIDEHTL APSKPGSGMV121 VAVKKLKPEG FQGHKEWLTE VNYLGQLHHA NLVKLIGYCS DGDNRLLVYE FMPKGSLENH181 LFRRGADPLS WGIRLKVAIG AAKGLSFLHH AENQVIYRDF KASNILLDSE FNAKLSDFGL241 AKAGPTGDKT HVSTQVMGTH GYAAPEYIAT GRLSAKADVY SFGWLLELL TGRRALDKSK301 PGIEQNLVDW AKPHLRDKRR LYRVMDTKLG GQYPKKGAHA VANLALQCIC NDAKMRPQIS361 EVLEELEQLQ DSKSNLTSPQ VDIRRTSNTVPKSPMRGQPS PRRSLGASPA YRTAQVH

[0100] SEQ ID NO: 10Number of amino acids: 415Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044969390Common name: HORVU.MOREX.r2.2HG0090920SEQUENCE: 101 MGNFCVCMGG SAKCASASTP FESKVNPRSS TSNSNSNSKA SRRGSSGPEK PQVAEVQAAA61 AGEEVAEVPA SLKSFSMADL RAATKNFGST SYLGEGGFGC VYKGWIDEAT LAPARPGATN121 AMMVAIKKLK KESFQGHREW LTE;VTYLGDL HHDNLVKLVG YCSDSDSNKL LVYEYMPRGS181 LENHLFRRGS QPPLPWSTRV AVAVDVARGI AFLHSRDVIF RDLKSSNVLL GPDHRAKLSD241 FGLARAGPTG GKSHVSTRW GTRGYAAPEY VATGHLSAKS DVYGFGWLL ELMTGRRALD301 ESRGLASELL VDWAMPMLQG ERRKVIRVMD TRLGGQYPKR QAQDMAALAL RCLQNDPKSR361 PSMADDVLPS LQLLLQPTTA AKSSSSSSSL TTTSSRSPAT TMTTPVQRGH RRHHR

[0101] SEQ ID NO: 11Number of amino acids: 477Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044956557Common name: HvPBL17; HORVU.MOREX.r2.7HG0612990SEQUENCE: 111 MGGCFSLEEQ RLQSRTGTSA EAGGPDGLRK CKSDSKAISS VLAPPKDVED LQTEGYGNVN61 1FTYNELRAA TKNFRPDQ1L GEGGFGVVYK GVIDENVR1G FKSTQVAVKE LNPEGFQGDK121 EWLAEVNYLG QLSHPNLVEL IGYCCEGSHR LLVYEYMACG SLEKHLFRRV CLNMPWSTRM181 KIALGAARGL EYLHGAERSI IYRDFKTSNI LLDADYNAKL SDFGLARTGP SGDQTHVSTR241 VMGTYGYAAP EYVMTGHLTA RSDVYGFGW LLEMIIGRRA VDKSRPSREH NLVEWARPLL301 VHNRKLFRII DPRMEGQYST KAAIEVASLC YRCLSQNPKG RPTMSQWET FEEVQSMPEC361 QDILLQNCMT GSVTLYEVPK EPVEHVETEK AKQEPTAKTD AVSVAVPPTN GKPVPQSRRT421 RPANGRSKSE PPLECKLYIP SPDSDGHQLG LETLASPPSR NSSRDDPPPV DEDLYKI

[0102] SEQ ID NO 12Number of amino acids: 435Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044955417Common name: HvPBL15; HORVU.MOREX.r2.6HG0451040SEQUENCE: 121 MPRPWRQVLA SATKCWSAED DDVEDEAAAY HRPANSEFSR RLASFRRLSA MANGPATLTE61 DKEEEYDNDA AAAAASGEIP MQLHSFSLSE LRGVTHDFST GYLLGEGGFG AVHKGFVDAG121 MRPGLEPQPV AVKQLNIAGH QGHREWLAEV IFLGQFRHQH LLKLLGYCCQ DEERLLVYEF181 MPRGSLDNHL FKRISATLPW CTRLKVAIGA AKGVAFLHGG KQPVIYRDLK ASNILLDSDY241 TAKLSDFGLA KMGPEGEETH VSTRVMGTHG YAAPEYVQTG HLQVKSDVYS FGWLLELLT301 GRRAMEHVPG RTARAEQTIK LVEWTRPYLA SSRRLRCIMD AKLSGHYSVK GARAMAHLAV361 QCTSPQPRDR PTMAAWEAL EQLEGLKDMA VSMGLFWPTA PAAGRNALSA KFRAEMKSAG421 TSAVPQRKAA SDKLS

[0103] SEQ ID NO: 13Number of amino acids: 458Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP 044953024Common name: HORVU.MOREX.r2.6HG0514460SEQUENCE: 131 MRAFLMGCFH PRGGATDPAA VADGDEAATA TATAKATAPP TRKGKKKSMR RAPSATARLR61 SLSFDDLSRT LASSGMHAFT VAELRAATRN FSGSHFIGEG GFGPVYKGFL DDKWAGMQP121 QHVAVKYLDA EGPQGHREWL AEVVYLGMQL SHPHLVKLVG YCYQEHHRML VYEYMARGSL181 EHHLFKNLLA SLPWATRLKI AVGAAKGLAF LHEAETPVIY RDFKASNILL ESDYTAKLSD241 FGLAKEGPSG DDTHVSTRVM GTHGYAAPEY ILTGHLTARS DVYSFGWLL ELLTGRRSVD301 KRRRGREQNL VDWARPYLRR PDKLHRVMDP SLEGSYSDQA AAKAAAVAYS CLHSVPKNRP361 TMREWDSLE PLMRLCGDVP AGLFVYTAPE AKLVDDEPDK AAGEEAKDGG ASVSAAAAAR421 KKCQRSAVHA ESAAPKYASS VAGKESRGSP RQRRDRGA

[0104] SEQ ID NO: 14Number of amino acids: 433Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044949565Common name: HORVU.MOREX.r2.5HG0397880SEQUENCE: 141 MAAKSWNPFS CCVGGARVAD DGDDCKRRIR RSAKGCPRSS SRMSFKSLSS SGTLSPEDLS61 ITLSGSNLHA FTYAELRAAT GSFSRANYLG CGGFGPVYKG AVDDKLRPGL AAQAVAVKYL121 DLDCGTQGHK EWLAEVFFLG QLRHKNLVKL IGYCYEDKHR MLVYEFMSGE SLEKHLFKSI181 NGSLPWMTRM KIAVGAAKGL AFLHDADPPV IYRDFKASNI LLDSDYNTKL SDFGLAKDGP241 QGDATHVTTC VMGTHGYAAP EYIMTGHLTA KSDVYSFGW LLELLSGLRS VDRSRRLREQ301 NLVDWARPYL KRSDRLYKVM DLALECQYSC KGAEVAALVA YKCLSQNPKS RPTMREWKA361 LEPVLGMEDF FPVGPFVFTV IVEEDKWNM KVEVEEKHQQ HRQNHQDRHR KKYPDSAIHA421 GIVLRRRDGI AQR

[0105] SEQ ID NO: 15Number of amino acids: 416Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044960398Common name: HvPBL36; HORVU.MOREX.r2.7HG0544670SEQUENCE: 151 MPPPPPPSPR HRQERRGCGC WAVLARGLRG SCFRPAAATA AAAPAGAAVK GGHVYDAAEM61 RYLNSSNRDL ADHFQRKLGD ENGVDTSIEN KISPKLLEFT FQELKSATVN FRPDSILGEG121 GFGYVFKGWI EPNSTAPAKP GTGLTVAVKS LKENALQGHR EWVAEVDFLG QLHHKHLVKL181 IGYCIEDDQR LLVYEFMARG SLENHLFRRT LPLPWPCRMK WLGAAKGLA FLHVGPKPVI241 YRDFKTSNIL IDAEYNSKLS DFGLAKAGPQ GDKTHVSTRV LGTYGYAAPE YVMTGHLTTK301 SDVYSFGWL LEVLTGRRSVDKKRPPGEQN LVAWARPYLS DRRRLYQLVD PRLGLNYSVR361 GVQKVAQICH HCLNRDSKSR PMMDEWKHL TPLQDLNDMA AASYRPRSSP RGKARR

[0106] SEQ ID NO: 16Number of amino acids: 477Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044976238Common name: HvPBL34; HORVU.MOREX.r2.3HG0222240SEQUENCE: 161 MGKEGTRRGQ DVRRRKGKEA AMDEEDAAPP TGCWIRLPRL GGGCMSSGSK VDSSTSGACA61 NASESKKVNH SCRDQSAAPA ASGSTTSSNI GSISPSSIVG EELKLAAQLR RFTFNELKCA121 TRNFRPESLL GEGGFGCVFK GWIEENGTAP MKPGTGLTVA VKTLNHDGLQ GHKEWVAEVD181 FLGNLQHPHL VKLVGYCIED DQRLLVYEFM PRGSLENHLF RRSFPLPWAI RMKIALGAAK241 GLAFLHEEAE RPVIYRDFKT SNILLDAEYN AKLSDFGLAK DGPEGDKTHV STRVMGTYGY301 AAPEYVMTGH LTSKSDVYSF GVVLLEMMSG RRSMDKNRPN GEHNLVEWAR PYLGERRRFY361 RLVDPRLEGN FSIKGAQKTA QLAHACLSRD PKARPLMSQV VEVLKPLPNL KDMASSSYFF421 QSMRQERAAS LNNPNGSQSM KAQSTFARNG VQPMRSLSYG PHASPYRQSP RPNGKQS

[0107] SEQ ID NO: 17Number of amino acids: 497Sequence type: ProteinOrganism Source: barley (Hordeum vulgare)NCBI Accession Number: XP_044985690Common name: HvPBL34-like; HORVU.MOREX.r2.5HG0393640SEQUENCE: 171 MGLAPPELGQ FDGWESSGEE ERERWGWCRR SSRRGRRLPR KGGGEDASVA TGCCIRLWPM61 GNCPPQPRSK VDTSTSSAST HGGEKSAENG SRNQPWSW SGSTSTSNAE SSSS ASKAGE121 DIKVSSKLRK FGFSDLKCAT RNFRPESLLG EGGFGCVFKG WIEENGTAPV KPGTGLTVAV181 KTLNHDGLQG HKEWVAEVDF LGNLHHPNLV KLIGYCVEDD QRLLVYEFMP RGSLDNHLFR241 RSLPLPWSIR MKVALGAAQG LSFLHEEAER PVIYRDFKTS NILLDSEYNA KLSDFGLAKD301 GPVGDKTHVS TRVMGTYGYA APEYVMTGHL TSKSDVYSFG WLLEMMSGR RSMDKNRPNG361 EHNLVEWARP LLGERQRFYK LVDPRLEGNF SVKGAQKAAQ LARACLSRDP KARPLMSQW421 EALKPLLNLK DMASSSYFYQ TMQAERMAHS SSMNGRNSHS LKVHGSFARA NGQQPMRSMS481 DGPRASPFRY SPKPNVK

[0108] SEQ ID NO: 18Number of amino acids: 403Sequence type: ProteinOrganism Source: wheat (Triticum aestivuni)NCBI Accession Number: KAF6981921Common name: TaPBLl 1SEQUENCE: 181 MGNCWGAKIS SDSSSSSPSG TNSKYASRNG AALSSSSSYA SAASVPRSEG EILESANVKA61 FSFNELRTAT RNFRPDSVLG EGGFGSVFKG WIDEKTLTPT KPGTGMVIAV KKLNQESYQG121 HREWLAEVNY LGQLSHPNLV KLVGYCVEDE QRLLVYEFMP RGSLENHLFR RSTHFQPLSW181 NLRMKIAHGA AKGLAFLHSD KAKVIYRDFK TSNILLDANY D AKLSDFGLA KDGPTGDKSH241 VSTRVMGTYG YAAPEYLATG HLTTKSDVYS FGWLLEMLS GRRAVDKNRP TGEHNLVEWA301 RPYLTSKRRI FRVLDPRLGG QYSLAKAQKA ASLALQCLSV DSRNRPSMEQ VWALEQLHD361 AKEGGNSPRP QLQRKPSSNR SLTGSRPSST KGNNNRPASS RPV

[0109] SEQ ID NO: 19Number of amino acids: 395Sequence type: ProteinOrganism Source: rice Oryza sativa)NCBI Accession Number: XP 015638719Common name: OsPBLl 1SEQUENCE: 191 MGNCWGAKIS SESPCRSASS PSGGTSKYAS NSSVSAASVP PTPRSEDEIL EAANVKAFAF61 NELRTATRNF RPDSVLGEGG FGSVFKGWID EKTLAPTKPG TGMVIAVKKL NQEGHQGHRE121 WLAEVNYLGQ LSHPYLVRLV GYCVEDEQRL LVYEFMPRGS LENHLFRRST HFQPLSWNLR181 MKIALGAAKG LAFLHSDKVK VIYRDFKTSN VLLDANYDAK LSDFGLAKDG PTGDKSHVST241 RVMGTYGYAA PEYLATGHLT TKSDVYSFGV VLLEMLSGRR ALDKNRPTGE HNLVEWARPY301 LMSKRRIFRI LDARLGGQYS LAKAQKAATL ALQCISVEAK NRPNMEQWA VLEQLQDSKE361 TGANPQLQKK SSSKNAGSNG SKPSSKGKPA NARLV

[0110] SEQ ID NO: 20Number of amino acids: 405Sequence type: ProteinOrganism Source: sorghum (Sorghum bicolor)NCBI Accession Number: XP_002440455Common name: SbPBLl lSEQUENCE: 201 MGNCWGAKIS SDSPSRGAIS PSGATSKFTS RNGAAALSGC SSHASSASML PTPRSEDEIL61 ESANVKAFTF NELRTATRNF RPDSVLGEGG FGSVFKGWID EKTLAPTRPG TGMVIAVKKL121 NQEGYQGHKE WLTEVNYLGT LSHPYLVKLV GYCLEDEQRL LVYEFMPRGS LENHLFRRSS181 YFQPLSWNLR MKIALGAAKG LAYLHSDEAK VIYRDFKTSN VLLDANFNAK LSDFGLAKDG241 PTGDKSHVST RVMGTHGYAA PEYLATGHLT TKSDVYSFGV VLLEMLSGRR ALDKNRPNGE301 HNLVEWARPY LRSKRRIFRI LDPRLGGQYS LARAQKAAAL ALQCLSVESR HRPSMDEWT361 ALEQLQDTKE GGNHHLQKRP SSRSMDNNGV KAAVKGKPAP SVKPV

[0111] SEQ ID NO: 21Number of amino acids: 406Sequence type: ProteinOrganism Source: maize (Zea mays)'NCBI Accession Number: NP_001151952Common name: ZmPBLl l 211 MGNCWGAKIS SDSPSRGASS PSGTASKLAS RNGAAAALSG CSSHASSASM LPTPRSEDEI61 LESANVRAFS FNELRTATRN FRPDSVLGEG GFGSVFKGWV DEKTLAPARP GTGMVIAVKK121 LNQDGYQGHK EWLTEVNYLG TLSHPYLVKL VGYCLEDEQR LLVYEFMPRG SLENHLFRRS181 SYFQPLSWNL RMKIALGAAK GLAYLHSDEA KVIYRDFKTS NVLLDANFNA KLSDFGLAKD241 GPTGDKSHVS TRVMGTHGYA APEYLATGHL TTKSDVYSFG WLLEMLSGR RALDKNRPNG 301 EHNLVEWARP YLRSKRRIFR ILDPRLGGQY SLARAQKAAA LALQCLSVES RHRPSMDEVV361 TALEQLQDTK EGGNHHLQKR PGSRSLDNNG GKAAGKGKPA PHVKPV

[0112] SEQ ID NO: 22Number of amino acids: 7Sequence type: ProteinOrganism Source: Arabidopsis thaliana, but found in many dicot and monocot plant speciesNCBI Accession Number: not applicableCommon name: AvrPphB Recognition SequenceSEQUENCE: 221 GDKSHVS 7

[0113] SEQ ID NO 23Number of amino acids: 7Sequence type: ProteinOrganism Source: syntheticNCBI Accession Number: not applicableCommon name: TuMV NIa protease recognition sequenceSEQUENCE: 231 GGCSHQS 7

[0114] SEQ ID NO: 24Number of amino acids: 7Sequence type: ProteinOrganism Source: syntheticNCBI Accession Number: not applicableCommon name: WSMV NIa protease recognition sequenceSEQUENCE: 241 QYCVYES 7

[0115] SEQ ID NO: 25Number of amino acids: 7Sequence type: ProteinOrganism Source: syntheticNCBI Accession Number: not applicableCommon name: MYDV ORF1 protease recognition sequenceSEQUENCE: 251 HKYVFES 7

[0116] SEQ ID NO 26Number of amino acids: 403Sequence type: ProteinOrganism Source: syntheticNCBI Accession Number: not applicableCommon name: TaPbll 1 containing a WSMV NIa protease recognition sequenceSEQUENCE: 261 MGNCWGAKIS SDSSSSSPSG TNSKYASRNG AALSSSSSYA SAASVPRSEG EILESANVKA61 FSFNELRTAT RNFRPDSVLG EGGFGSVFKG WLDEKTLTPT KPGTGMV1AV KKLNQESYQG121 HREWLAEVNY LGQLSHPNLV KLVGYCVEDE QRLLVYEFMP RGSLENHLFR RSTHFQPLSW181 NLRMKIAHGA AKGLAFLHSD KAKVIYRDFK TSNILLDANY DAKLSDFGLA KDGPTQYCVY241 ESTRVMGTYG YAAPEYLATG HLTTKSDVYS FGVVLLEMLS GRRAVDKNRP TGEHNLVEWA301 RPYLTSKRRI FRVLDPRLGG QYSLAKAQKA ASLALQCLSV DSRNRPSMEQ VWALEQLHD 361 AKEGGNSPRP QLQRKPSSNR SLTGSRPSST KGNNNRPASS RPV

[0117] SEQ ID NO: 27Number of amino acids: 406Sequence type: ProteinOrganism Source: syntheticNCBI Accession Number: NP_001151952Common name: ZmPBLl 1 containing aMYDV ORF1 protease recognition sequenceSEQUENCE: 271 MGNCWGAKIS SDSPSRGASS PSGTASKLAS RNGAAAALSG CSSHASSASM LPTPRSEDEI61 LESANVRAFS FNELRTATRN FRPDSVLGEG GFGSVFKGWV DEKTLAPARP GTGMVIAVKK121 LNQDGYQGHK EWLTEVNYLG TLSHPYLVKL VGYCLEDEQR LLVYEFMPRG SLENHLFRRS181 SYFQPLSWNL RMKIALGAAK GLAYLHSDEA KVIYRDFKTS NVLLDANFNA KLSDFGLAKD241 GPTHKYVFES TRVMGTHGYA APEYLATGHL TTKSDVYSFG WLLEMLSGR RALDKNRPNG301 EHNLVEWARP YLRSKRRIFR ILDPRLGGQY SLARAQKAAA LALQCLSVES RHRPSMDEVV 361 TALEQLQDTK EGGNHHLQKR PGSRSLDNNG GKAAGKGKPA PHVKPV

Claims

CLAIMSWe claim1. A recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleotide sequence that encodes least one gene of the Receptor-Like Cytoplasmic Kinase Family VII (RLCK VII) that has been modified such that the endogenous sequence GDKSHVS (SEQ ID NO: 22) has been replaced with a heterologous protease recognition sequence for a protease secreted by a plant pathogen.

2. The recombinant nucleic acid molecule of claim 1 wherein the heterologous protease recognition sequences is selected from the group consisting of GGCSHQS (SEQ ID NO: 23), QYCVYES (SEQ ID NO: 24), and HKYVFES (SEQ ID NO: 25).

3. The recombinant nucleic acid molecule of claim 1 wherein the RLCK VII gene is co- orthologous to an Arabidopsis PBL gene.

4. A transformed monocot plant containing the recombinant nucleic acid molecule according to claim 1.

5. The transformed monocot plant of claim 4 wherein the monocot plant is selected from the group consisting of barley, rice, wheat, sorghum, and maize.

6. The transformed monocot plant of claim 5 wherein the plant is a species of barley and RLCK VII gene is a Group III RLCK gene selected from the group consisting of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NOTO.

7. The transformed monocot plant of claim 5 wherein the plant is a species of barley and RLCK VII gene is a Group II RLCK gene selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16 and SEQID NO: 17.

8. The transformed monocot plant of claim 5 wherein the plant is rice and the RLCK VII gene is SEQ ID NO: 19, wherein the endogenous sequence GDKSHVS (SEQ ID NO: 22) has been replaced.

9. The transformed monocot plant of claim 5 wherein the plant is sorghum and the RLCK VII gene is SEQ ID NO:20, wherein the endogenous sequence GDKSHVS (SEQ ID NO: 22) has been replaced.

10. The transformed monocot plant of claim 5 wherein the plant is maize and the RLCK VII gene is SEQ ID NO:21, wherein the endogenous sequence GDKSHVS (SEQ ID NO: 22) has been replaced.

11. A transgenic seed of the transformed plant according to claim 4.

12. A method of protecting a monocot plant against infection by a plant pathogen that secretes at least one specific protease, the method comprising the steps of: introducing to the monocot plant a nucleotide sequence that encodes at least one gene of RLCK Family VII wherein the endogenous sequence GDKSHVS (SEQ ID NO: 22) has been replaced by a pathogen-specific protease recognition sequence, including, but not limited to, GGCSHQS (SEQ ID NO: 23), QYCVYES (SEQ ID NO: 24), and HKYVFES (SEQ ID NO: 25).

13. A method of protecting a monocot plant against infection by a plant pathogen that secretes at least one specific protease, the method comprising the steps of: modifying an endogenous RLCK Family VII gene in a monocot crop species using genome editing wherein the endogenous sequence GDKSHVS (SEQ ID NO: 22) has been replaced by a pathogen-specific protease recognition sequence, including, but not limited to, GGCSHQS (SEQ ID NO: 23), QYCVYES (SEQ ID NO: 24), and HKYVFES (SEQ ID NO: 25).

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