WO2020219879A1 - Use of pbs1 genes from plant species to engineer disease resistance - Google Patents

Abstract

Engineering of novel pathogen disease resistance traits into crops such as soybeans, barley, wheat, rice, sorghum, corn, tomato, canola and citrus is accomplished by modifying endogenous PBS1 genes in these crops so that the proteins they encode are cleaved by proteases from pathogens of interest.

Description

USE OF PBS1 GENES FROM PLANT

SPECIES TO ENGINEER DISEASE RESISTANCE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) U.S. Provisional

Patent Application Serial Nos. 62/838,786 filed April 25, 2019, and 62/844,310, filed May 7, 2019. The disclosures set forth in the referenced applications are incorporated herein by reference in their entireties.

[0002] This invention was made with government support under IOS1551452 awarded by

National Science Foundation. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted

electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 23, 2020, is named 312830_SEQ.txt and is 270,906 bytes in size.

BACKGROUND

[0004] Engineering resistance to some of the most devastating crop diseases facing humanity is of worldwide benefit. For example, crop losses due to infection by rust pathogens in wheat costs billions of dollars. Genetic disease resistance to rust diseases is rapidly overcome in the field, but control by fungicides is expensive and bad for the environment. The methods and compositions disclosed herein have the potential to reduce environmental impact of farming, while increasing global food security and decreasing toxins in food from both fungi and agrichemicals.

[0005] Plant disease resistance may be mediated by intracellular innate immune receptors known as nucleotide-binding leucine-rich repeat proteins (NLRs). The primary function of NLRs is to detect the presence of pathogen- secreted effector proteins, sometimes indirectly through effector-induced modification of other host proteins. Recognition of effectors by NLRs usually activates a programmed cell death response known as the hypersensitive reaction (HR). A well-studied example of an NLR that indirectly detects its cognate effector is RPS5 (RESISTANCE TO PSEUDOMONAS SYRINGAE 5) from Arabidopsis. RPS5 detects the Pseudomonas syringae pv. phaseolicola effector protease AvrPphB by monitoring the conformational status of an Arabidopsis substrate of AvrPphB, the serine/threonine protein kinase PBS1 (AvrPphB SUSCEPTIBLE 1). RPS5 forms a“pre-activation complex” with PBS1, and when PBS1 is cleaved by AvrPphB, the resulting conformational change is sensed by RPS5, culminating in activation of the NLR and subsequent induction of HR.

[0006] The gene PBS1 is one of the most well conserved, defense-related genes in flowering plants. The products of PBS1 orthologs in wheat, soybean, and Arabidopsis can be cleaved by AvrPphB. PBS1 belongs to receptor- like cytoplasmic kinase (RLCK) family VII, which has many members with demonstrated roles in pattern-triggered immunity (RTI). For example, family VII RLCKs BIK1 and PBL1 physically associate with the flagellin-detecting receptor FLS2. Of the 45 Arabidopsis proteins within the RLCK family VII, 9 are PBS 1 -like (PBL) kinases cleaved by AvrPphB. Importantly, AvrPphB inhibits FLS2-dependent PTI, as well as defense responses triggered by ElTu and chitin. However, only AvrPphB’s cleavage of PBS1 activates RPS5.

[0007] Because of their role in PTI, RLCKs and other kinases are commonly targeted by

pathogen effectors. Examples beyond the AvrPphB-PBSl interaction include the RLCK BIK1, which is uridylylated by AvrAC from Xanthomonas campestris pv. campestris, and the receptor-like kinase BAK1 which is bound by the Pseudomonas syringae pv. tomato effectors AvrPto and AvrPtoB to inhibit signaling. Some kinases targeted by effectors appear to play little or no primary role in immunity, but function as decoys, guarded by NLRs to detect effector activity. An example is the RLCK PBL2 in

Arabidopsis, which is uridylylated by AvrAC like BIK1, and is guarded by the NLR ZAR1.

[0008] Determining whether PBS1 orthologs are guarded in diverse plant species is of particular interest because it provides insight into the evolution of disease resistance gene specificity and could enable engineering of new disease resistance specificities in crop plants. The AvrPphB cleavage site sequence within PBS1 could be substituted with a sequence recognized by an effector protease of another pathogen, thereby generating a synthetic PBS1 decoy. Cleavage of PBS 1 decoys in planta activates RPS5-dependent HR, effectively broadening the recognition specificity of RPS5. Thus, in plant species in which a PBS1 ortholog is guarded, engineering these orthologs to serve as substrates of other pathogen proteases offers an attractive approach for generating resistance tailored to pathogens of those species. Given that plant pathogenic viruses, bacteria, fungi, oomycetes, and nematodes express proteases during infection, engineering the RPS5/PBS1 surveillance system may be an effective strategy for developing resistance to many important plant diseases.

[0009] Although it was recently reported that bread wheat ( Triticum aestivum subsp. aestivum ) encodes a homolog of Arabidopsis PBS1, TaPBSl, that can be cleaved by AvrPphB, it was previously unknown whether TaPBSl is guarded, i.e., whether wheat or other cereals can recognize and respond to AvrPphB. Unlike the PBL kinases, NLR genes are under intense selection pressure to diversify, and they vary greatly in number and structure across plant genomes. However, it is possible that proteins functionally analogous to RPS5 guard AvrPphB-cleavable PBS1 homologs in the grasses, especially given the central role that RLCK proteins play in immunity. This was confirmed using diploid barley ( Hordeum vulgare subsp. vulgare) as a model because of its rich genetic resources, including a high-quality genome sequence and large nested association mapping (NAM) populations.

SUMMARY

[00010] The present invention is applicable to a wide variety of plants, in particular crop plants, and to the pathogens that threaten the plants. Materials and methods disclosed herein teach persons of skill in the art to apply this general approach to any pathogen of any crop falling within the limitations of the steps below:

1. the plant to be protected has a PBS1 gene or its equivalent;

2. the PBS1 gene or equivalent can be modified by inserting a cleavage site that can be cleaved by a protease of a pathogen of interest;

3. transcriptome data to identify proteases expressed by a pathogen of interest during infection is available;

4. the proteases identified must be able to enter the plant host cells;

5. genomic data is available to identify a subset of the proteases that are

conserved among related species of the pathogen of interest;

6. the amino acid sequences that are cleaved by the conserved proteases of interest can be determined;

7. the cleavage site sequences are able to be inserted into a PBS1 gene or the equivalent in the crop plant to produce a modified PBS1 gene; (using gene editing or by overexpressing the modified PBS1 gene); 8. expression of the modified PBS1 gene confers resistance to infection by pathogens expressing matching proteases.

[00011] There are several examples of the general approach disclosed herein. The Arabidopsis resistance protein RPS5 is activated by proteolytic cleavage of the protein kinase PBS1 by the Pseudomonas syringae effector protease AvrPphB. Replacing seven amino acids at the cleavage site of PBS 1 with a motif cleaved by the NIa protease of turnip mosaic virus (TuMV) enables RPS5 activation upon TuMV infection. However, this engineered resistance conferred a trailing necrosis phenotype indicative of a cell death response too slow to contain the vims. This problem was elimated by over-expressing the PBS1^TuMV decoy protein, which conferred complete resistance to TuMV when delivered by either Agrobacterium or by aphid transmission, showing that RPS5-mediated defense responses are effective against bacterial and viral pathogens.

[00012] A PBS1 decoy approach was extended to soybeans by modifying a soybean PBS1

ortholog to be cleaved by the NIa protease of soybean mosaic virus (SMV). Transgenic overexpression of this soybean PBS1 decoy conferred immunity to SMV, demonstrating that endogenous PBS1 proteins are useful in crop plants to engineer economically relevant disease resistant traits.

[00013] The present disclosure demonstrates that a PBS1 protein from a monocot crop e.g. barley was modified so that it is cleaved by the NIa protease of Wheat streak mosaic vims. Disease resistance was activated in many barley varieties in response to proteolytic cleavage of a barley PBS1 protein. This resistance response is mediated by a barley disease resistance protein identified and named AvrPphB Recognition 1 (PBR1). Thus, engineered recognition of diverse pathogen proteases in barley, and other monocot crops such as rice, wheat and corn is effected by modifying the native PBS1 genes in these crops. To support this assertion, the Pbrl gene is conserved in wheat, and cleavage of wheat PBS1 proteins activates resistance in wheat. This disclosure thus teaches modification of PBS1 genes in all monocot plant species by enabling cleavage of their corresponding protein products by pathogen proteases and activation of disease resistance.

[00014] Multiple barley varieties recognize and respond to AvrPphB protease activity, and barley also contains PBLs that are cleaved by AvrPphB. Using newly developed NAM resources, the AvrPphB response was mapped to a single segregating locus on chromosome 3HS and an NLR gene was identified that designated AvrPphB Response 1 ( Pbrl ). PBR1 mediates AvrPphB recognition. Using transient expression assays in Nicotiana benthamiana, it was confirmed that PBR1 associates with PBS1 homologs in planta. Phylogenetic analyses indicated that Pbrl and RPS5 are not orthologous, hence the ability to recognize AvrPphB protease activity has evolved independently in monocots and dicots. Wheat varieties also recognize AvrPphB protease activity and harbor an ortholog of Pbrl in a syntenic position on chromosome 3B, suggesting that the PBSl-decoy system can be deployed in barley and in wheat.

[00015] The method disclosed herein has wide applicability to, for example, tomatoes, canola, and citrus.

BRIEF DESCRIPTION OF THE DRAWINGS

[00016] FIG. 1. AvrPphB protease activity elicits a range of responses in barley lines.

Representative barley leaves from 12 lines after infiltration with strains of Pseudomonas syringae pv. tomato DC3000(D36E) expressing AvrPphB or a catalytically inactive mutant, AvrPphB(C98S). Primary leaves of ten day old plants were infiltrated using needleless syringe with a bacterial suspension at an OD600=0.5 and photographed at 5 dpi. Phenotypes were scored as: N - no response; LC - low chlorosis; C - chlorosis;

HC - high chlorosis; HR - hypersensitive reaction. At least six plants were infiltrated with both strains per line over two repeats. Asterisks (*) indicate parental lines of the mapping population families used for GWAS. Responses of all lines tested are recorded in Table 1.

[00017] FIG. 2A-2C. Barley contains two PBS1 homologs that are cleaved by AvrPphB.

(FIG. 2A) HORVU2HrlG070690.2 (HvPBSl-1) and HORVU3HrlG035810.1 (HvPBSl-2) are co-orthologous to Arabidopsis PBS1. Shown is a Bayesian phylogenetic tree generated from the amino acid sequences of Arabidopsis PBS1 (AtPBSl) and closely related barley homologs of AtPBSl. This tree is a subset of (FIG. 9) displaying the proteins most similar to AtPBSl. Branch annotations represent Bayesian posterior probabilities as a percentage. (FIG. 2B) Alignment of the activation segment sequences of AtPBSl and the barley PBS1 homologs (SEQ ID NOS 1-3, respectively, in order of appearance). The AvrPphB cleavage site is indicated by the arrow. Numbers indicate amino acid positions. (FIG. 2C) Cleavage of HvPBSl-1 and HvPBSl-2 by AvrPphB. HA-tagged barley PBS1 homologs or AtPBSl were transiently co-expressed with or without myc-tagged AvrPphB, or a protease inactive derivative [AvrPphB(C98S)] in N. benthamiana. Six hours post-transgene induction, total protein was extracted and immunoblotted with the indicated antibodies. Two independent experiments were performed with similar results.

[00018] FIG. 3A-3C. Genome wide association study identifies a single locus in the barley genome significantly associated with AvrPphB response. Manhattan plots of the association between SNPs and AvrPphB response of NAM barley lines for (FIG. 3A) all 175 lines and (FIG. 3B) the lines from each HR subpopulation individually. The X-axis shows SNPs in the region graphed, either the whole genome or the interval containing the significant locus in the short arm of Chromosome 3H (3HS). The Y-axis shows the negative logarithm of the p- value for the association. The locations of genes encoding NLRs predicted by NLR-Parser are indicated by open triangles; the blue triangle points to Pbrl, the orange to Goi2 ( Gene of interest 2). The dotted horizontal line indicates a false discovery rate of 0.05 with Bonferroni correction. (FIG. 3C) Graphical representation of 18 recombinant lines from four additional families used to fine map the AvrPphB response determinant. Green indicates regions containing SNPs matching the Rasmusson genotype. Blue indicates regions matching the other parental genotype. Uncolored regions represent the intervals in which it can be concluded the recombination took place, based on the nearest flanking SNPs. Lines labeled in green font display the Rasmusson HR phenotype (1), in blue the other parent phenotype (0).

[00019] FIG 4. RPS5 and PBR1 are phylogenetically distant. Neighbor-Joining phylogenetic tree of the amino acid sequence of the NB-ARC domains from 304 NLRs predicted to be encoded in the barley genome (HORVU), and 15 known Coiled Coil NLRs from Arabidopsis thaliana (At). For simplicity, clades containing >5 predicted NLRs from barley were collapsed, with the number of sequences represented in the adjacent parentheses. Node labels indicate confidence probabilities (shown for those above 50%) from an interior-branch test with 1000 replicates. Triangles indicate predicted protein products encoded within the GWAS interval, and RPS5 is marked with a circle.

[00020] FIG. 5A-5D. Sequence and expression polymorphism in Pbrl across barley lines correlate to AvrPphB response. (FIG. 5A) Schematic illustration of Pbrl.b, the allele in the AvrPphB -responding line Rasmusson, showing the approximate location of a C nucleotide deletion that disrupts the open reading frame in Pbrl. a, the allele in the non responding line Morex. Below, the Pbrl.b protein product is represented, with the amino acid positions of the CC domain, NB-ARC domain, and LRR domain indicated. (FIG. 5B) PCR amplification from cDNA and genomic DNA (gDNA) of 12 representative lines that differ in their response to AvrPphB, showing expression and primer compatibility, respectively, for Pbrl and Goi2. cDNA was generated from RNA extracted from 10-day old plants, the same age used for phenotyping in FIG. 1. See FIG. 11 for data from additional lines. (FIG. 5C) A neighbor joining tree showing the sequence relationships of Pbrl alleles from the barely lines represented in (FIG. 5B) and the (at right) the responses of those lines to AvrPphB. The tree is based on aligned genomic DNA sequence from start codon to stop codon. Nodes are labeled with bootstrap values and the scale bar represents number of base substitutions per site. N, no response; LC, low chlorosis; C, chlorosis; HC, high chlorosis; HR, hypersensitive reaction. FIG. 5D Schematic illustration of the PBRl.b protein annotated with the approximate location of amino acid substitutions present in the predicted protein products of other Pbrl alleles and the responses of the corresponding barley lines. *C includes all 3 chlorotic responses.

[00021] FIG. 6A-6E. Transient co-expression of PBRl.c with AvrPphB induces cell death in

N. benthamiana. (FIG. 6A) Schematic representation of the PBRl.b protein product from Rasmusson, an HR line, showing the approximate locations of amino acid substitutions between the PBRl.b protein product and the PBRl.c protein product from Cl 16151, a low-chlorosis line. (FIG. 6B) Induction of cell death by PBRl.b:sYFP, but not PBRl.c:sYFP, independent of AvrPphB expression when transiently expressed in N. benthamiana. PBRl.b:sYFP or PBRl.c:sYFP were agroinfiltrated into 3-week old N. benthamiana. All transgenes were under the control of a dexamethasone-inducible promoter. A representative leaf was photographed 24 hours post-transgene induction under white light and UV light. Three independent experiments were performed with similar results. (FIG. 6C) Activation of HR by transient co-expression of PBRl.c:sYFP and AvrPphB :myc in N. benthamiana. Agroinfiltrations were used to transiently express combinations of PBRl.c:sYFP, empty vector (e.v.), AvrPphB :myc, and a protease inactive derivative, AvrPphB (C98 S):myc. HA-tagged Arabidopsis PBS1 co-expressed with RPS5:sYFP and AvrPphB :myc was used as a positive control. All trans genes were under the control of a dexamethasone-inducible promoter. A representative leaf was photographed 24 hours post- transgene induction under white light and UV light. Three independent experiments were performed with similar results. (FIG. 6D) Electrolyte leakage as a measure of cell death resulting from co-expression of PBRl.c :sYFP with AvrPphB:myc relative to PBRl.c:sYFP with e.v. or AvrPphB(C98S):myc in N.

benthamiana leaf discs. The assay was performed using N. benthamiana leaf discs transiently expressing the indicated combinations of constructs. Conductivity is shown as mean ± S.D. (n = 4). Three independent experiments were performed with similar results. (FIG. 6E) Cleavage of N. benthamiana PBS1 (NbPBSl) by AvrPphB. HA- tagged N. benthamiana PBS1 or AtPBSl was transiently co-expressed with or without myc-tagged AvrPphB or AvrPphB(C98S) in N. benthamiana. Total protein was extracted six hours post-transgene induction and immunoblotted with the indicated antibodies. Two independent experiments were performed with similar results.

[00022] FIG. 7. PBS1 proteins immunoprecipitate with PBRl.c when transiently coexpressed in N. benthamiana. The indicated construct combinations were transiently co-expressed in leaves of 3-week old N. benthamiana plants using agroinfiltration. All transgenes were under the control of a dexamethasone-inducible promoter. Total protein was extracted six hours post-transgene induction. HA-tagged Arabidopsis PBS1 co expressed with RPS5:sYFP was used as a positive control. The sYFP:LTI6b fusion protein, which is targeted to the plasma membrane (Cutler et al., 2000), was co expressed with the HA-tagged PBS1 proteins as a negative control. Results are representative of two independent experiments.

[00023] FIG. 8A-8C. Recognition of AvrPphB protease activity is conserved in wheat. (FIG.

8A) Responses of wheat cultivars Fielder and Centana infiltrated with (top to bottom) 10 mM MgCl₂ (mock), P. syringae DC3000(D36E) expressing empty vector (e.v.), AvrPphB(C98S), or AvrPphB three days post-infiltration (dpi), photographed under white and UV light. Bacteria (OD600=0.5) were infiltrated into the adaxial surface of the second leaf of two-week old seedlings. Three independent experiments were performed with similar results. Responses of all lines tested are recorded in Table 3. (FIG. 8B) Hydrogen peroxide accumulation. Cultivars and treatments assayed were as in panel A. Three dpi, leaf segments were excised from the infiltrated regions, stained with DAB solution, cleared with 70% ethanol, and photographed under white light. This experiment was repeated twice with similar results. (FIG. 8C) Full-length amino acid sequence alignment between barley PBRl.c (SEQ ID NO: 5) and the most closely related homolog in wheat, TRIAE_CS42_3B_TGACvl_226949_AA0820360 (TaPBRl) (SEQ ID NO:

4). Conserved residues and conservative substitutions are highlighted with black and grey backgrounds, respectively. The predicted coiled-coil (CC), nucleotide binding (NB- ARC), and leucine-rich repeat (LRR) domains of TaPBRl are indicated by pink, green, and cyan bars, respectively. The predicted palmitoylation site is indicated by a purple box.

[00024] FIG. 9. Bayesian phylogenetic tree based on amino acid alignment of full-length products of Arabidopsis PBS1 ( AtPBSl ), all characterized Arabidopsis PBS1- like (. AtPBL ) genes, and barley PBS1- like ( HvPBL ) genes homologous to Arabidopsis PBS1. AtPBSl and AtPBL sequences were obtained from The Arabidopsis Information Resource (TAIR10) website (arabidopsis.org). Homology searches were performed using BLASTp to identify barley amino acid sequences homologous to Arabidopsis PBS1 and PBS 1-like proteins. Thirty-two barley protein sequences were identified as homologous to the 29 Arabidopsis sequences used in the analysis. Bayesian phylogenetic trees were generated for the collected sequences using the program MrBayes under a mixed amino acid model. Scale bars indicate amino acid substitutions per site and nodes are labeled with Bayesian posterior probabilities as a percentage. The gray box highlights the clade presented in FIG. 2.

[00025] FIG. 10. Full-length amino acid sequence alignment between Arabidopsis PBS1 and the barley PBS1 homologs (SEQ ID NOS 6-8, respectively, in order of appearance).

Conserved residues and conservative substitutions are highlighted with black and grey backgrounds, respectively. Predicted myristoylation and palmitoylation sites are indicated with red and blue boxes, respectively. The activation segment is indicated with a green box and the AvrPphB cleavage site with a black arrow.

[00026] FIG. 11A-11B. Expression of Pbrl and Goi2 in additional representative barley lines. PCR amplification from cDNA showing expression and from gDNA showing primer compatibility for FIG. 11A two recombinant inbred lines each from each of the three NAM subpopulations used for GWAS (HR620, HR656, and HR658), exhibiting the parental phenotypes FIG. 11B. Additional lines bringing the total lines tested (excluding RILS) to 12 responding and 12 non-responding when considered with FIG. 5. cDNA was generated from RNA extracted from 10-day old plants, the same age used for AvrPphB response assays. The AvrPphB response for each line is indicated: N, no response; LC, low chlorosis; C, chlorosis; HC, high chlorosis.

[00027] FIG. 12A-12B. NIa protease-mediated cleavage of the Arabidopsis PBS1‘decoy’ protein in Nicotiana benthamiana. (FIG. 12A) The Wheat streak mosaic virus (WSMV) PBS1 decoy protein contains the WSMV NIa protease cleavage motif QYCVYES (SEQ ID NO: 37), while the Soybean mosaic virus (SMV) PBS1 decoy protein contains the SMV NIa protease cleavage motif ESVLSQS (SEQ ID NO: 38). FIG. 12A also discloses "GDKSHVS" as SEQ ID NO: 39. (FIG. 12B) The indicated proteins were transiently co-expressed in N. benthamiana leaves and then protein extracted and analyzed by immunoblot. Bands marked with an * indicate cleavage products of PBS 1, demonstrating cleavage of the decoy PBS1 proteins only by the matching proteases.

[00028] FIG. 13A-13B shows that transgenic soybean plants with GmPBSl^SMV are resistant to SMV. FIG. 13A are photographs of transgenic soybeans (Family A, Family B, Family C) wherein the pairs of photographs show plants with GmPBSl^SMV (left) and non-transgenic plant (right), wherein the plant photograph to the left of each pair is resistant. (FIG. 13B) shows results of gel analysis corresponding to Family A, Family B, Family C photograph pairs.

[00029] FIG. 14A-14B. Overexpression of PBS l^TuMV confers complete immunity to TuMV.

FIG. 14A, White light and UV photographs showing the spread of TuMV infection in homozygous 35S:PBSl^TuMV transformants in Col-0, pbsl and rps5 backgrounds and native promoter pbsl::PBSl^TuMV in Col-0 at three weeks post infection by

Agrobacterium-mediated delivery of viral RNAs. FIG. 14B, Immunoblot showing PBS1^TuMV and virally produced 6K2-GFP expression levels in the lines pictured in FIG. 14A.

[00030] FIG. 15A-15B. Arabidopsis expressing both WT PBS1 and PBS1^TuMV retain recognition of AvrPphB. Bar charts showing the number of leaves expressing an HR phenotype in response to infiltration with Pseudomonas syringae expressing either AvrPphB or an empty vector. Bar height represents the number of leaves infiltrated. The black bar represents the number of leaves expressing cell death. HR tests were done in four week old homozygous 35S:PBSl^TuMV transformants in Col-0, pbsl and rps5 backgrounds and pbsl:PBSl^TuMV in Col-0.

[00031] FIG. 16A-16B. Aphid transmission of TuMV is blocked in PBSl^TuMV overexpressing lines. FIG. 16A, Table showing the percentage of Arabidopsis plants expressing GFP as a result of TuMV infection in Col-0, pbsl and rps5 backgrounds at 15 days post aphid inoculation. FIG. 16B, Immunoblot showing PBS1^TuMV and TuMV Coat Protein expression levels in the lines represented in 16A. [00032] FIG. 17A-17C. Box plots showing above ground dry weight and seed weight at senescence for each of the three 35S::PBSl^TuMV-HA transgenic lines in Col-0, pbsl SALK and rps5 SALK backgrounds (FIGS. 17A, 17B, 17C). Each set of transgenic lines is compared to WT Col-0 plants grown in the same tray. Analysis was done in R using a one-way ANOV A. * = p<0.05, ** = p<0.01, *** = p<0.001

DETAILED DESCRIPTION

[00033] Persons skilled in the art can easily envision applying the general approach disclosed herein is applicable to any pathogen of any crop using the following steps, which can be summarized as follows: 1) use transcriptome data to identify proteases expressed by the pathogen and whose expression increases during the infection process; 2) use genomic data to identify a subset of these proteases that are conserved among related species of the pathogen; 3) determine the amino acid sequences of the host plant cleaved by the conserved proteases of interest; 4) insert these cleavage site sequences into a PBS1 gene in the crop plant using genome editing, or alternatively, overexpressing the modified PBS1 gene using traditional transformation methods to generate a transgenic plant that expresses the modified PBS I gene as a newly introduced gene. Expression of the modified PBS1 gene is expected to then confer resistance to infection by pathogens expressing matching proteases.

[00034] The PBS1 gene is highly conserved across all angiosperms, including barley, wheat, rice and corn. AvrPphB protease from Pseudomonas syringae cleaves PBS1 proteins from all crops tested, including barley and soybean. Furthermore, AvrPphB protease activity induces resistance in barley, wheat and soybean.

[00035] By way of example, the substrate protein of a pathogen- specific protease can be a PBS1 homolog from Glycine max (soybean) (e.g., PBS1 homolog GmPBSla (SEQ ID NO:65). GmPBSlb (SEQ ID NO:67), and GmPBSlc (SEQ ID NO:69)). The PBS1 homolog is modified to include a heterologous protease recognition sequence. As understood by those skilled in the art, "PBS1" refers to avrPphB susceptible 1. As understood by those skilled in the art, "avrPphB" refers to the bacterial avirulence from Pseudomonas syringae that encodes the "AvrPphB " polypeptide having a role in plant-P syringae interactions.

[00036] Many examples support this general approach. The Pseudomonas syringae cysteine protease AvrPphB activates the Arabidopsis resistance protein RPS5 by cleaving a second host protein, PBS1. AvrPphB induces defense responses in other plant species, but the genes and mechanisms mediating AvrPphB recognition in those species have not been previously defined. This disclosure shows that AvrPphB induces defense responses in diverse barley cultivars. Barley contains two PBS1 orthologs. Their products are cleaved by AvrPphB, and barley AvrPphB response maps to a single locus containing a nucleotide-binding leucine- rich repeat (NLR) gene, which is termed AvrPphB Response 1 ( Pbrl ).

[00037] AvrPphB, and not a catalytically inactive derivative, triggers defense responses in barley.

[00038] To test whether barley can detect AvrPphB protease activity, AvrPphB was delivered to barley leaves using P. syringae pathovar tomato strain D36E, which is a derivative of strain DC3000 lacking all type III secretion system effectors. Seedlings were infiltrated with D36E expressing AvrPphB or the catalytically inactive mutant AvrPphB(C98S) and scored for visible responses at 2 and 5 days post infiltration·

[00039] A diverse set of barley lines were tested and a variety of responses were observed.

Representative examples are shown in FIG. 1, and the complete list of cultivars and their responses are provided in Table 1. Based on the range of responses, the phenotypes were scored as no response (N) or one of 4 responses: low chlorosis (LC) indicates a weak, but noticeable response, chlorosis (C) for strong yellow, high chlorosis (HC) for a chlorotic response that gives way to cell death, and hypersensitive reaction (HR) for cell collapse and browning visible by day 2.

[00040] Of the 150 barley genotypes screened, 29 were scored as LC, 17 as C, 13 as HC and 6 as

HR (Table 1). Both chlorotic and cell death responses were considered defense responses, as both have been documented as such for grasses.

[00041] PBS1 homologs in barley contain the AvrPphB recognition site and are cleaved by

AvrPphB.

[00042] Having found that many barley lines recognize AvrPphB, a question was whether barley contains a recognition system functionally analogous to the Arabidopsis RPS5-PBS1 pathway. Because PBS1 is one of the most well conserved defense genes in flowering plants, with orthologs present in monocot and dicot crop species, an initial question was whether barley contains a PBS1 homolog cleavable by AvrPphB.

[00043] Amino acid sequences were used from all characterized Arabidopsis PBS 1 -like (AtPBL) proteins, Arabidopsis PBS1 (AtPBSl), and twenty barley PBSl-like (HvPBL) protein sequences homologous to AtPBSl and AtPBL proteins to identify the barley proteins most closely related to Arabidopsis PBS1. Bayesian phylogenetic analyses showed that HORVU2HrlG070690.2 (MLOC_13277) was the closest homolog to AtPBSl, whereas HORVU3HrlG035810.1 (MLOC_12866) was the second most closely related (FIG. 2A; FIG. 9). Both proteins are more similar to AtPBSl than to other AtPBL and HvPBL proteins, indicating that the two barley genes are co-orthologous to AtPBSl. Full-length amino acid alignments showed that HORVU2HrlG070690.2 and

HORVU3HrlG035810.1 are 66% and 64% identical to Arabidopsis PBS1, respectively (FIG. 10). Alignment of the two barley gene products and Arabidopsis PBS1 across the kinase domain showed 86% and 79% identity, respectively. Further characterization of the HvPBSl orthologs showed that each contains several domains that are conserved in AtPBSl, including putative N-terminal palmitoylation and myristoylation sites required for plasma membrane localization and the protease cleavage site sequence recognized by AvrPphB (FIG. 2B; FIG. 10). HORVU2HrlG070690.2 (MLOC_13277) was designated as HvPbsl-1 and HORVU3HrlG035810.1 (MLOC_12866) was designated as HvPbsl- 2.

[00044] Conservation of the AvrPphB cleavage site sequences within the barley PBS1 homologs suggested that AvrPphB would cleave HvPBSl-1 and HvPBSl-2. To test this, HvPBSl- 1 and HvPBSl-2 were fused to a three-copy human influenza haemagglutinin (3xHA) epitope tag and transiently co-expressed with AvrPphB :myc in N. benthamiana. Western blot analysis indeed showed that HvPBSl-LHA and HvPBSl-2:HA are each cleaved by AvrPphB:myc (FIG. 2C). As a control, HvPBSl-LHA and HvPBSl-2:HA were co expressed with protease inactive AvrPphB(C98S):myc. This did not produce any cleavage products (FIG. 2C). Collectively, these data show that barley contains two PBS1 homologs whose protein products can be cleaved by AvrPphB and whose function may be analogous to AtPBSl.

[00045] A single NLR gene-rich region in the barley genome is associated with AvrPphB response.

[00046] Given the response to AvrPphB in some barley lines and the presence of conserved

AvrPphB- cleavable PBS1 homologs in barley, the possibility was that responding barley lines contain a PBS 1 -guarding NLR analogous to RPS5. To identify candidates, a genome wide association study was carried out (GWAS). The Rasmusson spring barley nested association mapping (NAM) population generated by the US Barley CAP (A. Ollhoff and K. Smith, University of Minnesota) contains 6,161 RILs derived from crosses between the elite malting line Rasmusson and 88 diverse donor parents, each of which has associated SNP marker data. Rasmusson, the common parent, displays an HR when infiltrated with P. syrinage strain D36E expressing AvrPphB whereas the other parents vary in their responses (FIG. 1). For the GWAS, three NAM sub- populations (families) were chosen: two derived from non-responding parents, PI329000 (family HR656) and PI366207 (HR658), and one from a low-chlorosis response parent, CIhol5600 (HR620) (FIG. 1).

[00047] As expected for a qualitative, single gene trait, the responses segregated ~ 1 : 1 within each family of RILs; 39 of 73 HR656 lines, 19 of 36 HR658 lines, and 29 of 66 HR620 lines displayed an HR following infiltration with D36E expressing AvrPphB (a total of 87 out of 175 RILs tested; Table 2). Co-segregation of AvrPphB response with SNPs was analyzed using the R/NAM package, which included 13,981 SNPs in the analysis of the 175 lines (see Methods). The GWAS identified a 22.65 Mb region between positions 660,376,398 (SNP 3H2_266065765) and 683,030,529 (SNP 3H2_288719896) on the short arm of chromosome 3H associated with AvrPphB response (FIG. 3A). Neither HvPbsl-1 nor HvPbsl-2 are in this region, supporting the hypothesis that an NLR, and not a PBS1 homolog, is the determinant of AvrPphB response. Notably, analysis of each of the three families individually identified the same locus on chromosome 3H as the only significant association (FIG. 3B). The most significant SNP and the number of SNPs used in the analysis varied by population due to the SNP variation between Rasmusson and each of the other parents.

[00048] Within the GWAS interval, there are 13 predicted NLR genes, as called by NLR-parser

(FIG. 3A and 3B). In the reference genome, only four encode putative full length NLRs; the rest are fragments, mostly LRR domains and some partial NB-ARC domains. The most significant SNP in the analysis of all lines was S3H2_279293442 (3H:673604075; - log(p)=25.48). The nearest predicted NLR to this SNP, HORVU3HrlG107310 (3H: 672,928,614-672,932,121), was selected as the top candidate for the determinant of the response to AvrPphB and tentatively named Pbrl ( AvrPphB Response 1). Despite its association with the most significant SNP, the protein product PBR1 is less closely related to RPS5 in both the encoded CC (FIG. 4A) and NB-ARC (FIG. 4B) domains than the protein products of the other NLR genes within the 22.65 Mb region. Of these, the one encoding the NLR most closely related to RPS5, HORVU3HrlG109680 (3H: 679064240-679072712), on the edge of the GWAS interval, was selected as an additional gene of interest and referred to it hereafter as Goi2. PBR1 and RPS5 are 17% identical to each other across the CC domain and 29% identical across the NB- ARC domain, while GOI2 and RPS5 are 24% and 45% identical to each other across those domains, respectively. For comparison, PBR1 and GOI2 are 23% identical to each other across the CC domain and 20% across the NB-ARC domain.

[00049] As a next step to identify the determinant of the AvrPphB response, SNP data were used for the entire NAM population to find additional recombinants within the 22.65 Mb GWAS interval. Based on haplotype data within the region, eighteen apparent recombinants were selected from four additional families with non- AvrPphB -responding parents and phenotyped (Table 2). Adding the genotype and phenotype data of these new lines to the GWAS increased the significance of many of the SNPs, but did not narrow the interval. However, using the estimated recombination breakpoints and the phenotypes of the individual RILs to fine map the determinant of the response resulted in a 3.04 Mb region within the GWAS peak that contains Pbrl and no other NLR gene (FIG. 3C), supporting Pbrl rather than Goi2 as the candidate determinant.

[00050] Pbrl is expressed in lines responding to AvrPphB and allelic variation correlates with phenotype.

[00051] The reference genome used in the GWAS is from the barley line Morex, an AvrPphB - non-responding line (FIG. 1). Therefore, the reference genome is likely to have a nonfunctional copy of, or lack completely, the NLR hypothesized to detect activity of AvrPphB. In the Morex genome, Pbrl is annotated as containing just a truncated NB- ARC domain and LRR domain, missing an N-terminal domain. In contrast, Goi2 encodes a full length NLR (965 aa) with an RPS5-like CC domain (aa 27-66), NB-ARC domain (aa 156-439), and LRR (aa 537-864). To see if either gene sequence varies in the responding line Rasmusson, Pbrl and Goi2 from that line were sequenced. The Rasmusson allele of Goi2 is highly similar to the Morex allele, with only 3

nonsynonymous mutations between them (N860I, R808H, and V282L). Among the differences between the two Pbrl sequences, a single nucleotide insertion in Rasmusson was found that restores a larger open reading frame (FIG. 5A), resulting in a predicted full-length NLR (939 aa) with an intact CC_EDVID ("EDVID" disclosed as SEQ ID NO:

21) domain (aa 7-131), NB-ARC domain (aa 174-454), and an LRR domain containing 12 repeats (aa 474-886). For Pbrl, the allele in Morex is referred to as Pbrl.a (GenBank: MH595617) and the allele in Rasmusson as Pbrl.b (GenBank:MH595618).

[00052] Reverse transcriptase PCR (RT-PCR) was used to test the expression of Pbrl alleles in

Morex and Rasmusson, as well as a variety of other barley lines ranging in AvrPphB - induced responses. Pbrl was expressed in lines that respond to AvrPphB either with HR or chlorosis (Rasmusson, Haruna Nijo, PI061533, Gorak, PI584977, PI163409, CIhol5600, and Cl 16151), but not in non-responding lines (PI329000, PI386650, PI362207, and Morex) (FIG. 5B). The primers used for RT-PCR were compatible with all genotypes tested, as shown by amplification from genomic DNA, and spanned an intron to differentiate cDNA from any genomic DNA contamination· 30 total lines were investigated: 12 responders, 12 non-responders, and 6 RILS from the 3 NAM

subpopulations used for GWAS. Pbrl was expressed in all responding lines, but not expressed in 9 out of 12 non-responding lines (FIG. 5B and FIG. 11). For comparison, Goi2 expression was assayed in these lines as well and varying levels of expression were found that did not correspond to AvrPphB response (FIG. 5B).

[00053] Because point mutations within an NLR can lead to changes in observable HR in planta, to see if the responses to AvrPphB observed across different barley lines corresponded with sequence polymorphism at Pbrl, Pbrl alleles of 10 additional barley lines selected at random from among the different response phenotypes (1 HR, 2 HC, 2 C, 2 LC, and 3 non- responding lines) were sequenced and compared to Pbrl.a and Pbrl.b from Morex and Rasmusson, respectively. The nucleotide sequences cluster by phenotype (FIG. 5C) (SEQ ID NOS: 9-20), respectively, in order of appearance) when analyzed from start codon to stop codon using the Neighbor-Joining method. The non-responding lines, PI329000, PI386650, PI362207, and Morex have unique but similar alleles. The HR line Haruna Nijo, like Rasmusson, has the Pbrl.b allele. The amino acid sequences of PBR1 in the two lines each from the three chlorosis response groups (LC lines Cl 16151 and CIhol5600, C lines PI584977 and PI163409, and HC lines Gorak and PI061533) are identical within and different across the groups; all contain 3 common substitutions compared to the Rasmusson allele Pbrl.b, including an L538Q substitution in the LRR. Together these observations suggest that sequence polymorphism in Pbrl determines response to AvrPphB.

[00054] The product of Pbrl allele Pbrl.c recognizes AvrPphB protease activity in N.

benthamiana. [00055] To directly test whether PBR1 mediates recognition of AvrPphB, a transient expression assay was developed in N. benthamiana. Pbrl.b from cultivar Rasmusson was cloned into a dexamethasone-inducible vector along with a C-terminal fusion to super yellow fluorescent protein (PBRl.b:sYFP). Unfortunately, transient expression of PBRl.b:sYFP alone resulted in HR with complete tissue collapse within 24 hours of transgene induction (FIG. 6B), indicating that PBRl.b is auto-active when overexpressed in N. benthamiana.

[00056] To circumvent the problem posed by auto-activity of the PBRl.b protein, a Pbrl allele from the LC line Cl 16151 was tested (FIG. 1). This allele was designed Pbrl.c (GenBank: MH595619). PBRl.b and PBRl.c differ by five amino acid substitutions, of which three are located within the leucine-rich repeat domain (FIG. 6A). Transient expression of a PBRl.c:sYFP fusion protein in the absence of AvrPphB consistently produced a weaker HR than PBRl.b (FIG. 6B). This result allowed testing whether the HR was enhanced in the presence of active AvrPphB.

[00057] PBRl.c:sYFP and AvrPphB:myc were transiently co-expressed in A. benthamiana and assessed cell death. As a control, AtPBSUHA and RPS5:sYFP with AvrPphB:myc, a combination that activates cell death in N. benthamiana were co-expressed. Transient co expression of PBRl.c:sYFP with AvrPphB:myc resulted in observable tissue collapse 24 hours post-transgene induction, whereas co-expression of PBRl.c:sYFP with either empty vector (e.v.) or AvrPphB(C98S):myc resulted in a much weaker cell death response (FIG. 6C). Further, transient expression of AvrPphB :myc in the absence of PBRl.c:sYFP did not trigger HR, indicating that the cell death response requires PBRl.c (FIG. 6C). An electrolyte leakage analysis was performed to better quantify PBRl.c- mediated cell death. Transient co-expression of PBRl.c:sYFP with AvrPphB-myc induced greater ion leakage than PBRl.c:sYFP co-expressed with either empty vector or AvrPphB(C98S):myc between 9 and 16 hours after transgene induction, confirming that PBRl.c:sYFP recognizes and mediates a response to AvrPphB protease activity (FIG. 6D). By 26 hours post transgene induction, PBRl.c:sYFP expressed with

AvrPphB(C98S) or empty vector induced ion leakage similar to that observed with co expression of PBRl.c:sYFP and wild-type AvrPphB, indicating that PBRl.c:sYFP is weakly auto-active, consistent with the HR assays (FIG. 6D).

[00058] The observation that AvrPphB, but not AvrPphB(C98S), activates PBRl.c-mediated cell death in N. benthamiana even in the absence of a barley PBS1 protein suggested that AvrPphB might be cleaving an N. benthamiana ortholog of PBS 1 and that PBRl.c is recognizing that cleavage. Using a reciprocal BLAST and the amino acid sequence of Arabidopsis PBS 1, an ortholog of PBS 1 was identified in the N. benthamiana genome, Nibenl01Scf02996g03008.1, which was designated NbPBSl. Importantly, NbPBSl contains the AvrPphB cleavage site sequence and is thus predicted to be cleaved by AvrPphB. To determine whether in the transient assay PBRl.c is guarding an endogenous PBS1 ortholog, NbPBSLHA was co-expressed with either AvrPphB:myc or AvrPphB(C98S):myc. Co-expression with AvrPphB:myc, but not the protease inactive mutant, resulted in cleavage of NbPBSLHA within 6 hours post-transgene expression, showing that NbPBSLHA is a substrate for AvrPphB (FIG. 6E) and that its cleavage could be the trigger for PBRl.c.

[00059] PBS1 proteins immunoprecipitate with barley PBRl.c when transiently coexpressed in N. benthamiana.

[00060] To determine whether PBRl.c is activated by sensing cleavage of PBS1 proteins, co- immunoprecipitation (co-IP) analyses of PBRl.c with HvPBSl-LHA, HvPBSl-2:HA, AtPBSLHA, or NbPBSLHA was performed. As a positive control, AtPBSLHA was co expressed with RPS5:sYFP, which forms a pre- activation complex in the absence of AvrPphB. As a negative control, the plasma membrane-localized fusion protein sYFP:LTI6b was co-expressed with each of the PBS1 proteins (Cutler et al., 2000). Consistent with the hypothesis being tested, HvPBSl-LHA, HvPBSl-2:HA, and AtPBSLHA immunoprecipitated with PBRl.c:sYFP and not with sYFP:LTI6b, demonstrating that PBRl.c forms a complex with PBS1 proteins from barley and Arabidopsis in the absence of AvrPphB (FIG. 7). NbPBSLHA also immunoprecipitated with PBRl.c:sYFP (and not with sYFP:LTI6b) supporting the notion that AvrPphB - mediated cleavage of NbPBSl activates PBRl.c-dependent HR in N. benthamiana. Though all of the PBS1 proteins immunoprecipitated with PBRl.c:sYFP, PBRl.c:sYFP preferentially interacted with HvPBSl-2:HA and AtPBSLHA (FIG. 7). Collectively, these data suggest that PBR1 forms a pre-activation complex with one or more barley PBS1 orthologs, providing further evidence that PBR1 is the guard that recognizes AvrPphB activity. Importantly, CSS-PALM 4.0 predicts that PBRl.b and PBRl.c are palmitoylated at Cys314, suggesting co-localization with AvrPphB and barley PBS1 orthologs at the plasma membrane. [00061] Wheat ( Triticum aestivum subsp. aestivum ) also recognizes AvrPphB protease activity.

[00062] Recently an ortholog of Arabidopsis PBS1 was identified in wheat, TaPBSl, that

localizes to the plasma membrane when transiently expressed in N. benthamiana and is cleaved by AvrPphB. However, it remained unclear whether wheat recognizes AvrPphB protease activity and would thus likely contain a functional analog of RPS5, such as Pbrl. 34 wheat varieties obtained from the U.S. Department of Agriculture Wheat Germplasm Collection were screened for their response to P. syringae strain D36E expressing AvrPphB (FIG. 8A; Table 3). Twenty-nine responded with chlorosis, while five showed no visible response by three days post- inoculation (FIG. 8A; Table 3). No line responded to the protease inactive mutant AvrPphB(C98S).

[00063] To further characterize the chlorotic response in wheat, 3,3’-diaminobenzidine (DAB) staining was used to examine hydrogen peroxide accumulation following leaf infiltration with D36E expressing either AvrPphB or AvrPphB(C98S). Consistent with the chlorotic phenotype, wheat cv. Fielder accumulated detectable hydrogen peroxide within the infiltrated area when inoculated with D36E expressing AvrPphB, whereas the mock and AvrPphB(C98S) treatments resulted in minimal hydrogen peroxide accumulation (FIG. 8B). In contrast, there was no significant hydrogen peroxide accumulation in wheat cv. Centana inoculated with either strain (or mock), consistent with the lack of chlorotic response of this line to AvrPphB. The correlation of chlorosis and hydrogen peroxide accumulation specifically in response to active AvrPphB is consistent with recognition in wheat associated with defense.

[00064] To examine whether this recognition in wheat might be mediated by a Pbrl ortholog, the

T aestivum subsp. aestivum genome was searched using the Ensembl genome browser (release TGACvl). TRIAE_CS42_3B_TGACvl_226949_AA0820360 was found to be an ortholog, and was designated TaPbrl. TaPbrl is located on wheat chromosome 3B in a position syntenic with barley Pbrl and encodes an NLR consisting of a predicted Rx- like coiled-coil domain (aa 7-131), a nucleotide -binding domain (aa 174-454), and a leucine-rich repeat domain (aa 474-895) (FIG. 8C). Full-length amino acid sequence alignment of barley PBRl.c and TaPBRl shows 93% amino acid identity (FIG. 8C). Further, TaPBRl, like barley PBR1, is predicted to be palmitoylated at Cys314, suggesting co-localization with AvrPphB and wheat PBS1. It thus seems likely that TaPBRl functions as the cognate NLR protein that mediates recognition of AvrPphB protease activity in wheat.

[00065] Transient co-expression of Pbrl.c with wild- type AvrPphB, but not a protease inactive mutant, triggers defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 co-immunoprecipitates with barley and N. benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. The disclosed wheat cultivars also recognize AvrPphB protease activity and contain a Pbrl ortholog. These results indicate that PBS 1 -based decoys can be used to engineer protease effector recognition-based resistance in barley and wheat, including protease effectors from fungal pathogens.

[00066] Use of Monocot PBS1 Decoy Proteins to Engineer Disease Resistance in Crops.

[00067] The PBS1 protein of Arabidopsis is a protein kinase that functions to regulate defense signaling in response to perception of pathogen-derived molecules by pattern recognition receptors, which are transmembrane proteins localized on the cell surface. PBS1 was identified in 2001. Subsequently, PBS1 was shown to be among the most highly conserved proteins in flowering plants, with clear orthologs present in both monocot and dicot plant species. As a central regulator of plant defense responses, PBS1 proteins from diverse plant species have been shown to be targeted by effector proteins from pathogens in order to suppress immune responses. Most plant species, however, have evolved a second layer of defense involving intracellular receptors that can detect effector-induced modification of PBS1. For example, in Arabidopsis, the intracellular receptor RPS5 monitors the status of PBS1 and activates a strong defense response upon proteolytic cleavage of PBS1 by the effector protease AvrPphB, which is injected into plant cells by the bacterial pathogen Pseudomonas syringae. AvrPphB also cleaves PBS1 proteins from soybean, barley and wheat, which strongly indicates that AvrPphB can cleave PBS1 proteins from all flowering plants. Importantly, AvrPphB induces a strong defense response in soybean, barley and wheat, which indicates that these crop species all contain intracellular immune receptors that are activated by PBS1 cleavage. In barley, the intracellular receptor PBR1 mediates recognition of AvrPphB protease activity. Collectively, these data indicate that most, if not all, crop plants contain intracellular immune receptors that are activated by cleavage of PBS 1, or close homologs. [00068] The above summary focused on cleavage of PBS 1 by AvrPphB. This protease cleaves PBS1 at a single site located in the activation loop of PBS1, which is an exposed loop located between the conserved kinase motifs‘DFG’ and‘APE’ (between amino acids 231 and 261 of Arabidopsis PBS1). A seven amino sequence within this loop can be replaced with alternative sequences that then enable cleavage of PBS1 by proteases from other pathogens. Importantly, cleavage of these modified PBS1 proteins, which are referred to as‘decoy PBS1’ proteins, still activates RPS5, and thus a strong immune response. Such decoy PBS1 proteins thus enable RPS5 to recognize proteases from most any pathogen and confer resistance to these pathogens. For example, RPS5 confers resistance to infection by Turnip mosaic virus (TuMV) when a cleavage site for the NIa protease from TuMV is inserted into the PBS1 activation loop (Kim et al., 2016).

[00069] The PBS1 decoy approach has been extended to engineering novel disease resistance traits in crop plants using endogenous PBS1 genes of these species. (FIGS. 13A-13B)

For example, insertion of a cleavage sequence for the NIa protease from Soybean mosaic virus (SMV) into a soybean PBS1 protein confers resistance to infection by SMV. FIG. 13 shows that transgenic soybean plants that express GmPBSl^SMV are resistant to infection by SMV. The present disclosure shows that PBS1 decoy engineering will also be effective in monocot crop species such as barley, wheat, rice, com, and sorghum. The genomes of all of these crop species encode one or more copies of PBS1. The proteins encoded by these PBS1 genes are highly similar to each other (>80% amino acid sequence identity across the full length of the protein, and >95% identical within the kinase domain). The AvrPphB cleavage sites are conserved in all of them, and this disclosure shows that the PBS1 proteins from wheat and barley are cleaved by PBS 1, and that AvrPphB induces immune responses in these species. Insertion of a protease cleavage site for the NIa protease from Wheat streak mosaic virus (WSMV) enables cleavage of PBS1 by this protease. Thus, it is straight forward to produce PBS1 decoy proteins from PBS1 proteins of any plant species, including monocot crops such as barley (SEQ ID NOS: 28-30), wheat (SEQ ID NOS: 25-27), rice (SEQ ID NOS: 31-33), corn (SEQ ID NOS: 22-24) and sorghum (SEQ ID NOS: 34-36). FIGS. 19A-19B

Generalization and Summary

[00070] Recognition of the P. syringae AvrPphB protease by the Arabidopsis RPS5 NLR protein is a well characterized example of indirect effector recognition. Though AvrPphB is recognized by other plant species such as soybean and common bean, the disease resistance genes responsible for recognition outside of Arabidopsis have not been cloned, and the underlying molecular mechanisms are unknown. The evidence herein supports the conclusion that barley and Arabidopsis have convergently evolved NLRs able to detect effectors that structurally modify PBSl-like kinases: barley cultivars respond to AvrPphB but not to a protease inactive mutant of AvrPphB, barley contains an NLR gene evolutionarily distinct from RPS5 that mediates a strong HR when co-expressed with avrPphB in N. benthamiana, and AvrPphB associates with and cleaves PBS1 orthologs from monocots and dicots.

[00071] While AvrPphB is not known to be present in any pathogens of barley, it is a member of a family of proteases present in many phytopathogenic bacteria. More generally, proteases that target host proteins are found in many, diverse types of pathogens, and conserved kinases that are involved in PTI are expected to be common effector targets. Though the functional roles of HvPBSl-1 and HvPBSl-2 as well as other barley PBSl- like proteins are unknown, given their conservation in many flowering plant families, they may have a role in PTI signaling as observed in Arabidopsis. Results herein however, support that barley deploys an effector protease recognition mechanism similar to that of recognition of AvrPphB by Arabidopsis RPS5, wherein barley PBR1 guards RLCKs such as HvPBSl-1 and HvPBSl-2 such that it is activated upon their cleavage. Within Arabidopsis populations, RPS5 is maintained as a balanced presence/absence polymorphism despite inconsistent interaction with Pseudomonas strains expressing AvrPphB homologs, suggesting other effectors are also imposing selection pressure.

How many and which effectors from barley pathogens target RLCKs is unknown.

[00072] The convergent evolution of the shared ability of PBR1 and RPS5 to recognize AvrPphB aligns with the prediction that RLCKs that function in plant immunity are common targets of pathogen effectors and that selection to guard these proteins is ancient and widespread. A similar example of convergent evolution of NLR specificity has been described for the RPM1 and Rpglb/Rpglr proteins of Arabidopsis and soybean, all three of which detect effector induced modifications of RIN4 proteins. Like PBL proteins, RIN4 is targeted by multiple effectors, consistent with these proteins serving critical functions in plant immunity. PBR1 and RPS5 independently evolved to detect PBL cleavage instead of directly interacting with AvrPphB or integrating a PBL decoy. Direct interaction limits the number of effectors a single NLR can detect, while guarding a commonly targeted host protein expands the response spectrum, thus allowing the NLR to detect multiple pathogen effectors. The guarding strategy might impose purifying selection on RLCKs themselves or selection to integrate an RLCK decoy into an NLR: either would reduce the risk of any guard-guardee genetic mismatch that might lead to hybrid necrosis.

[00073] When assessing the functional role of PBRl.c in AvrPphB recognition, it was found its co-expression with AvrPphB elicited cell death in N. benthamiana even in the absence of barley PBS1 expression. Given that PBRl.c does not contain the AvrPphB cleavage site sequence, it is likely PBRl.c is sensing AvrPphB -mediated cleavage of an endogenous N. benthamiana PBL protein. PBS1 ortholog NbPBSl indeed associates with PBR1 in the absence of AvrPphB and is cleaved by AvrPphB. This was true also of AtPBSl and the barley PBS orthologs HvPBSl-1 and HvPBSl-2. Taken together, these data strongly suggest that in barley, PBRl.c detects AvrPphB protease activity by sensing cleavage of a PBS1 protein, analogous to AvrPphB -detection by RPS5.

[00074] Pbrl is expressed in the 12 tested barley lines that respond to AvrPphB, and in only 3 of

12 lines that do not respond. The sequence polymorphisms found in Pbrl alleles across the 12 responding barley lines correlate with the presence and severity of the AvrPphB response (i.e. chlorosis versus strong HR). These data support that mutations within the Pbrl coding sequence impact the macroscopic phenotype observed when AvrPphB is present. Mutagenesis screens of specific NLRs have been shown to modify the severity of phenotype and specificity of interaction. Natural examples of the effect of single or few mutations impacting NLR function include the Pi-ta NLR in Oryzae spp., in which a single amino acid is highly correlated to resistance, and the barley Mia locus, which encodes alleles with over 90% amino acid sequence identity that recognize different effector proteins. In wheat, alleles of the Pm3 gene have very little sequence diversity, but just two amino acid mutations expand the effector recognition capacity of Pm3f and increase its activity. The polymorphisms in PBR1 will be characterized to determine which, if any, modify the response to AvrPphB response or if any impact specificity. However, the difference in auto-activity between PBRl.b and PBRl.c when expressed in N. benthamiana is further evidence that the allelic sequence polymorphism contributes to phenotype.

[00075] The evidence that PBR1 is activated by cleavage of a PBS1 or PBSl-like protein

supports that PBS1- based decoys can be used to expand protease effector recognition in barley. Barley powdery mildew ( Blumeria graminis f. sp. border, Bgh) and Wheat streak mosaic virus (WSMV) are two barley pathogens known to deploy proteases as part of the infection process. BEC1019 is a putative metalloprotease secreted by Bgh and is conserved among ascomycete fungi. Notably, silencing of BEC1019 by both Barley stripe mosaic virus- and single cell RNAi-based methods reduces Bgh virulence, suggesting BEC1019 is required for Bgh pathogenicity. Similar to other Potyviruses, WSMV expresses a protease, designated the nuclear inclusion antigen (NIa), that is essential for viral replication and for proper temporal expression of potyviral genes in planta. The cleavage site sequence recognized by the NIa protease has been identified. Insertion of the BEC1019 or NIa protease cleavage site sequence into the barley PBS1 proteins should enable recognition of these proteases by PBR1. This approach could also be extended into wheat given that PBR1 and PBS1 are conserved.

[00076] Example 1: Optimizing the PBS1 Decoy System to Confer Resistance to Potyvirus

Infection in Arabidopsis and Soybean

[00077] Results of these investigations are as follows:

[00078] 1. Overexpression of PBS1^TuMV in Arabidopsis confers complete immunity to

TuMV

[00079] To test whether overexpressing plasma membrane (PM)-localized PBS1^TuMV in

Arabidopsis would enhance resistance and ameliorate the trailing necrosis phenotype (a correlation was observed previously between TuMV resistance and the level of PBS1^TuMV expression), three independent homozygous transgenic Arabidopsis lines expressing HA tagged PBS1^TuMV under a 35S promoter were generated in wild-type Col- 0, pbsl and rps5 backgrounds. The rps5 background was used to confirm dependency on RPS5, and the pbsl line was used to ensure there would be no loss of signaling efficiency through competition with the WT PBS1 protein for association with RPS5.

The stable overexpression of PBS1^TuMV in these lines appeared to have no fitness consequences, because plants grew similarly to wild type and, with the exception that the pbsl lines 1-5 and 2-1, produced an equivalent amount of seed (FIG. 17).

[00080] Transgenic lines were inoculated with TuMV (6K2-GFP) using Agrobacterium-mediated delivery and assessed for TuMV accumulation in the non-inoculated, systemic leaves via ultra-violet light imaging at three weeks post infection (FIG. 14A). Wild type Col-0 and pbsl genotypes over-expressing PBS1^TuMV displayed complete resistance to TuMV. In contrast, the rps5 lines over-expressing PBS1^TuMV showed systemic GFP fluorescence derived from virally produced 6K2-GFP, indicative of successful TuMV infection (FIG. 14A). The lines expressing PBS1^TuMV under a native promoter showed the trailing necrosis phenotype previously published by Kim et al. 2016. These data establish that TuMV resistance in the wild-type and pbsl transgenic lines is dependent on RPS5 expression and the expression level of PBSl^TuMV. These results were verified using immunoblot analyses (FIG. 14).

[00081] A conclusion is that the trailing necrosis phenotype originally observed in the

PBS1/RPS5 decoy system can be overcome through overexpression of the decoy protein. Furthermore, expression of PBS1^TuMV in a Col-0 background does not compromise the recognition of AvrPphB. It can be seen in (FIG. 15) that both the 35S and native promoter PBS1^TuMV lines in a Col-0 background were capable of AvrPphB recognition, whereas the lines in the rps5 background showed a response to AvrPphB comparable to that of the empty vector. Interestingly, lines in the pbsl background showed a variable level of HR, suggesting that PBS1^TuMV may still be able to be cleaved by AvrPphB at a low level. These data indicate that the pool of RPS5 available for partnering with decoy PBS1 proteins is not severely limiting. Therefore expand the recognition specificity of RPS5 may be expanded through the addition of multiple different decoy PBS1 proteins.

[00082] To confirm that overexpression of PBS1^TuMV confers resistance to TuMV, infection

assays using aphid transmission, which is the natural vector for TuMV infection, were conducted. Homozygous lines expressing 35S:PBSl^TuMV were inoculated with aphids that had recently been feeding on TuMV (6K2:GFP)-infected plants. The pbsl lines over-expressing PBS1^TuMV displayed no observable viral symptoms two weeks post infection (FIG. 16A). In Col-0 lines, viral symptoms were only observed in the final of three replicates, in 2 of the 10 plants tested. Results from the final replicate are shown. The rps5 lines, however, showed a strong GFP fluorescence caused by TuMV produced 6K2-GFP. These results were verified using immunoblot analysis (FIG. 16B). Here PBSl^TuMV expression levels are shown to be more variable, possibly due to increased protein turnover during aphid infection. However, these data still demonstrate that the PBS1^TuMV decoy is capable of conferring strong resistance to TuMV in a natural context.

[00083] 2. Overexpression of a soybean PBS1 decoy protein, GmPBSl-l^SMV, in soybean confers immunity to soybean mosaic virus. [00084] PBS1 proteins from soybean can also be engineered to confer recognition of the NIa protease from SMV, with cleavage of these decoy proteins activating cell death in soybean protoplasts, suggesting the decoy engineering approach can be extended to a crop plant (Helm et al., 2019). It was unclear, however, whether transgenic soybean expressing a GmPBS 1 - 1 ^SMV decoy protein would confer resistance to SMV. Therefore, transgenic soybean lines constitutively expressing HA-tagged G'/r/PBS 1 - 1 ^SMV under an enhanced 35S promoter were generated. Three independent TO soybean plants were obtained. Screening of T1 progeny from each line for expression of b-glucuronidase, a marker on the T-DNA vector used for transformation, revealed that only one T1 family segregated the T-DNA in a 3:1 ratio. Seed was harvested from three sibling T1 progeny of this line to generate three T2 families, two of which contained the T-DNA (families A and B) and one of which did not (family C), based on staining for GUS expression and genotyping by PCR. To test for resistance to SMV infection, unifoliate leaves of ~ 12-day old T2 soybean plants were rub inoculated with SMV-Nv::GFP virions. Systemic spread of SMV was assessed by scoring the third trifoliate leaves for virus presence three weeks after inoculation. No observable mosaic symptoms or SMV coat protein (CP) accumulation was observed in families A or B (10 plants each), while 7 of 10 family C plants showed vims accumulation (FIG. 13). Consistent with these results, immunoblot analysis revealed that all plants in families A and B expressed PBS1-1^smv, while none of the family C plants showed PBS1^SMV expression (FIG. 13). These data indicate that transgenic overexpression of the GmPBSl-l^SMV decoy protein confers resistance to systemic infection by SMV without a trailing necrosis phenotype, nor any obvious deleterious effects on plant growth.

[00085] 3. Discussion of Results

[00086] The above experiments show that the PBS1 decoy system is capable of conferring

complete resistance to a viral pathogen in both Arabidopsis and soybean, provided that the PBS1 decoy protein is overexpressed.

[00087] Kim et al., 2016 reported correlation between the expression levels of PBS1^TuMV and the corresponding severity of trailing necrosis. These observations indicate that the trailing necrosis phenotype was due to suboptimal activation of RPS5, and that this can be overcome by increasing the amount of PBS 1 available for cleavage, and hence increasing the amount of RPS5 activated above a threshold required to kill host cells prior to virus spread. It is noteworthy that overexpression of PBS1^TuMV or soybean PBS1^SMV did not cause observable changes in plant growth, which suggests that overexpression does not cause constitutive activation of defense responses, or other deleterious effects. Furthermore, it may be possible to express multiple PBS1 decoys in a single plant, further expanding the recognition specificity of RPS5.

[00088] The PBS1/RPS5 decoy system should be capable of conveying immunity to any

biotrophic pathogen that requires a protease as part of its infection or replication machinery, providing that the protease accumulates inside the host cell and recognizes a cleavage sequence of seven, or fewer, amino acids. This has the potential to be broadly applicable as bacterial, viral and fungal pathogens, as well as oomycetes and nematodes, are known to employ proteases as part of their infection cycle (Alfano and Collmer 2004; Lim et al. 2011; Raffaele et al. 2010; Gardner et al. 2018).

[00089] This decoy system also has the potential to be transferred to crop plants without the need to transfer AtRPS5, as many crop species are already resistant to Pseudomonas syringae through the recognition of AvrPphB, suggesting that they possess a functional analogue of RPS5 (Russell et al. 2015; Carter et al. 2019). Indeed, soybean, barley and wheat all respond to AvrPphB protease activity (Carter et al. 2019; Helm et al. 2019), and this response in barley is mediated by a disease resistance protein designated PBR1 (Carter et al. 2019).

[00090] PBS1 is highly conserved in both dicots and monocots (Caldwell and Michelmore 2009;

Swiderski and Innes 2001), including soybean, which contains three PBS1 co-orthologs (Helm et al. 2019). Similar to the above work with Arabidopsis PBS1, insertion of an NIa cleavage site in the soybean PBS1 proteins enables their cleavage by the NIa protease from SMV (Helm et al. 2019). Significantly, co-expression of a decoy soybean PBS1 protein with the SMV NIa protease triggers cell death in soybean protoplasts (Helm et al. 2019). That transgenic overexpression of a soybean PBS1 decoy can confer complete resistance to SMV confirms that the PBS1 decoy approach can be deployed in crops using only endogenous genes. Furthermore, in crops such as soybean and wheat, which have three or more PBS1 co-orthologs, it should be possible to use genome editing to create multiple different PBS1 decoys that target different pathogens, or that target multiple proteases in a single pathogen to enhance durability. MATERIALS AND METHODS FOR EXAMPLE 1

[00091] Plant growth

[00092] Nicotiana benthamiana and Arabidopsis thaliana plants were grown under a 12h

light/12h dark cycle at 24°C in Pro-Mix PGX Biofungicide plug and germination mix supplemented with Osmocote 14-14-14 slow release fertilizer (Scotts).

[00093] Seeds of soybean [Glycine max (L.) Merr.] cultivars‘Williams82’ and Williams82

PBS1-1^SMV transgenic lines were sown in clay pots containing greenhouse soil (generated by composting plants and previously used potting mix, and then

supplementing with small amounts of sand, perlite and vermiculite) supplemented with Osmocote slow-release fertilizer (14-14-14) and grown in a growth chamber under a 16 hr light/8 hr dark photoperiod at 23°C with average light intensities of 300 pEinsteins nr² s^-1 (at soil height).

[00094] Generation of plant expression constructs

[00095] To generate the SSSiPBS1^TUMV construct, PBS 1 ^TUMV in pBSDONR P1P4 and a C- terminal 3X HA tag were recombined into the destination vector pEGlOO (contains a cauliflower mosaic vims 35S promoter to drive transgene expression; Earley et al. 2006) using LR Clonase. The TuMV (6K2:GFP) construct was previously described by Wan et al. 2015.

[00096] To generate the pWI-1000:E35S::GmPBSl-l^SMV-HA construct for soybean

transformation, GmPBSl-l^SMV-HA was PCR- amplified from the pBAV154:GmPBSl- 1^SMV-HA template (Helm et al., 2019) using primers designed to introduce Xbal restriction sites at each end. The resulting PCR products were gel-purified using the QIAquick gel extraction kit (Qiagen) and cloned into the Xbal site of pWI-1000 (M. Peterson; Wisconsin Crop Innovation Center). The resulting constructs were sequence- verified to check for proper sequence and reading frame.

[00097] Generation of transgenic Arabidopsis lines

[00098] pEGlOO-based constructs were transformed into Agrobacterium tumefaciens strain

GV3101 (pMP90). This strain was then used to prepare floral dips of Arabidopsis wild- type Col-0, rps5 and pbsl knockout lines (SALK lines 127201 and 062464 respectively) (Clough and Bent 1998). Progeny were selected via BASTA resistance in T1 and T2 generations, and then homozygous T3 families identified. The pbsl::PBS1^TuMV-3xHA lines were described previously (Kim et al. 2016).

[00099] Generation of transgenic soybean lines and infection with SMV

[000100] Transgenic Williams 82 soybean lines were generated by the Wisconsin Crop Innovation

Center using an Agrobacterium- mediated transformation protocol. T1 seed from three independent TO plants were obtained from the Wisconsin Crop Innovation Center. T1 plants were assessed for expression of a GUS (beta-glucuronidase) marker gene present on the T-DNA of the pWI-1000 vector using X-gluc staining of unifoliate leaves (Jefferson et al. 1987). T1 plants were selfed to obtain T2 seed. T2 plants were grown in a growth chamber at 22°C under a 16-hour day (300 mEinsteins n^-2 s ). Unifoliate leaves of 12-14 day old plants were rub-inoculated plasmid DNA encoding full-length SMV-NV::GFP (T1 plants), or SMV virions prepared from infected soybean plants (T2 plants), as described in Helm et al. 2019. In brief, virions were obtained from previously infected wild-type Williams 82 plants that were rub-inoculated with infectious pSMV- Nv:GFP plasmid DNA and maintained after infection for 4 weeks in a growth chamber. Heavily infected trifoliate leaves were harvested in IX phosphate-buffered saline (137 mM NaCl, 2.7 mM KC1, 10 mM Na2HP04, 1.8mM KH2P04, pH 7.4), with cell debris cleared by centrifugation, and the supernatant containing virions stored at -80°C until use. Soybean plants were grown in a growth chamber until unifoliate leaves were fully expanded (approximately 12-14 days after planting). One of the two unifoliate leaves was wounded by rubbing carborundum on the abaxial side of the leaf. Using a clean cotton swab, virion suspension was rubbed on the abaxial side, and then plants were returned to the growth chamber for an additional three weeks. Third trifoliate leaves were harvested when completely expanded (approximately 21-days post inoculation), and photographed under white light and ultraviolet light. Tissue was flash frozen in liquid nitrogen and stored at -80°C.

[000101] Transient protein expression

[000102] Agrobacterium cells were taken from LB plates and suspended in 10 mM MgC12 with

100 mM acetosyringone (Sigma- Aldrich). After a two-hour incubation at room temperature, bacterial cultures were infiltrated into the leaves of three to four week old N. benthamiana with a needleless syringe. For microscopy and HR, the final concentration of infiltrated bacteria, once mixed, was OD600 of 0.3 +/- 0.01, and for immunoblot analyses 0.15 +/- 0.01.

[000103] TuMV Infection

[000104] Agrobacterium cells containing pCAMBIA carrying TuMV(6K2-GFP) (Wan et al. 2015) were taken from plates and suspended to an ODeoo of 0.1 in 10 mM MgCh with 100 mM acetosyringone (Sigma- Aldrich). After a two-hour incubation at room temperature, bacterial cultures were infiltrated into three-week-old Arabidopsis leaves with a needleless syringe. Infection was allowed to progress for three weeks before being photographed under white and UV light.

[000105] Immunoblots

[000106] For transient assays in N. benthamiana, 48 hours after infiltration, plants were sprayed with 50 mM dexamethasone. Six hours post-transgene induction, samples were taken for protein extraction. For assays in Arabidopsis, whole plant samples were taken immediately after TuMV symptoms were photographed. For N. benthamiana samples, inoculated leaves were pooled from two to three plants. Leaf tissue was ground in extraction buffer (150 mM NaCl, 50 mM Tris [pH 7.5], 0.2 % Nonidet P-40 [Sigma- Aldrich], 1% plant protease inhibitor cocktail [Sigma- Aldrich], 2 mM 2,2'-dipyridyl disulfide). Cell debris was pelleted at 8,000 x g, 4°C for 10 min, and the collected supernatants were separated on a 4-20% Tris-glycine Stain Free polyacrylamide gel (BioRad) at 185V for 1 hour in IX Tris/Glycine/SDS running buffer. Total proteins were transferred to nitrocellulose membrane (GE Water and Process Technologies). Membranes were blocked overnight at 4°C in 5% milk. Proteins were subsequently detected with HRP-conjugated anti-HA antibody (rat monoclonal, Roche, catalog number 12013819001) or mouse monoclonal anti-GFP antibody (Novus Biologicals, Littleton, catalog number NB600-597). Membranes were washed 3 times for 15 mins in IX Tris-buffered saline (TBS) with 0.1% Tween20 (TBST) and incubated with HRP- conjugated goat anti-mouse antibody (abeam, catalog number ab6789). All antibodies were used at a concentration on 1:5000. The nitrocellulose membranes were washed three times for 15 minutes in TBST and imaged using Supersignal® West Femto Maximum Sensitivity Substrates (Thermo Scientific) and X-ray film. The experiment was repeated three times. [000107] For protein extraction from soybean, trifoliate leaves were ground to a powder under liquid nitrogen with mortar and pestle. Three times the volume of ice cold IP buffer (50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 1 mM diothioreitol, 1% NP-40, 0.1% Triton X-100, 1% Plant Protease Inhibitor cocktail, 1% DPDS, 1 mM EDTA) was added to powder and ground until homogenous. Leaf extracts were centrifuged 2x at 12,500xg at 4°C and supernatants free of plant tissue debris were collected. 500 pi of cleared supernatant was added to 10 mΐ anti-HA magnetic beads (MedChemExpress catalog number HY-K0201) and incubated on a rotator at 4°C for 3 hours. Immunoprecipitation samples were collected on a magnetic stand for 5 minutes and unbound protein lysate was removed and saved for SMV-CP analysis. Magnetic beads were washed 5 times in ice cold IP buffer. Protein was eluted in 40 mΐ of IP buffer and 10 mΐ of 5x SDS-PAGE buffer. Samples were boiled at 95°C for 5 minutes and returned to magnetic stand to separate sample from magnetic beads. A volume of 10 mΐ was loaded onto a 4-20% SDS- PAGE gel. 40 mΐ of unbound protein lysate was added to 10 mΐ of 5x SDS-PAGE loading buffer and boiled at 95°C for 5 minutes. Samples were briefly centrifuged and 10 mΐ of sample was loaded onto A 4-20% SDS-PAGE gel. Gels were ran for approximately 1 hour at 160V and transferred to nitrocellulose membranes at 300 amps for 1 hour. Membranes with unbound protein lysates were stained with Ponceau for 3 minutes, rinsed with deionized water, and photographed for RUBISCO loading control.

[000108] SMV coat protein immunoblots were blocked overnight with 5% dry milk in lx Tris- buffered saline (TBS; 50mM Tris-HCl, 150 mM NaCl, pH 7.5) solution containing 0.1% Tween 20 (TBST) and then anti-SMV coat protein antisera (rabbit polyclonal; a gift from Sue Tolin) added at a dilution of 1:20,000 and incubated at room temperature with gentle shaking for 1 hour. Membranes were washed three times with TBS-T, after which secondary goat anti-rabbit antibody conjugated to horse radish peroxidase (HRP)

(Abeam, catalog number ab205718, Cambridge, MA) was added at a dilution of 1:5000 and incubated at room temperature for 1 hr, followed by washing three times with TBS-T. Anti-HA immunoblots were prepared in a similar fashion, but using an anti-HA-HRP conjugated antibody (rat monoclonal, Roche, catalog number 12013819001) at 1:5000, with incubation at room temperature for 1 hr.

[000109] Immunblots were developed using equal parts of Clarity TM Western ECL substrate peroxide solution and luminol/enhancer solution (BioRad) with incubation at room temperature for 5 minutes. Immunoblots were imaged using the chemiluminescent setting on the KwikQuant Imager (Kindle Biosciences, LLC).

[000110] Cell death assays in N. benthamiana

[000111] At 48 hours after Agrobacterium infiltration into N. benthamiana leaves, plants were sprayed with 50 mM dexamethasone. Twenty-four hours later the infiltrated leaves were assessed for cell death and photographed under white light. Ten leaves over four to six plants were tested for every treatment in the experiment and the experiment was replicated three times.

[000112] Cell death assays in Arabidopsis

[000113] Pseudomonas syringae strain DC3000 expressing either AvrPphB or an empty vector

(Simonich and Innes 1995) were sub-cultured from plates into 10 mM MgCh and syringe infiltrated into leaves of four week old Arabidopsis. Twenty-four hours later the infiltrated leaves were assessed for cell death and photographed under white light.

[000114] Aphid vector assays

[000115] Non-viruliferous aphid clones of a tobacco-adapted strain of Myzus persicae were reared under controlled conditions (23 °C with a photoperiod of 12/12 h day/night) on tobacco ( Nicotiana tabacum). Adult aphids were transferred to Col-0 leaves infected with TuMV (6k2-GFP) for a 10 min acquisition period. After virus acquisition, 2 aphids were transferred to a 20-day-old plantlet for each treatment for a 24 h inoculation period. After inoculation, the aphids were removed from the plants. Two weeks later, the number of infected plants was recorded for each line using a handheld UV light to visualize GFP, and tissue was collected for immunoblot confirmation. Seven to ten plants were inoculated per plant line.

[000116] For immunoblots of aphid- treated plants, 10 mg of leaf tissue was collected from each plant and pooled for each line. Leaf tissue was immediately frozen in liquid nitrogen and ground to a fine powder in a 1.5 mL tube using steel beads and a paint shaker. Lysis buffer (1 mL 0.5M Sodium citrate, 0.5 g SDS powder, 0.2 mL Beta-meracptoethanol, 1 mL 1.5M NaCl, 7.8 mL water, 1 tablet of EDTA-free Complete protease inhibitor cocktail) was added directly to the frozen tissue and mixed until homogeneous at room temperature (1 mg tissue:2 mL buffer). After mixing, samples were boiled for 10 minutes and cell debris pelleted in a microfuge at 15,000 RPM for 10 minutes at room temperature. Supernatants were mixed with loading buffer in a 1 : 1 ratio before separation at 70V for 15 minutes then 90V for 1.5 hours on a 10% Tris-glycine gel (Biorad) in IX Tris/Glycine/SDS running buffer. Total proteins were transferred to a nitrocellulose membrane and blocked overnight at 4°C. Proteins were detected with anti- TuMV coat protein antibody at a concentration of 1: 1500 and Goat anti-Rabbit IgG (H+L)-HRP conjugate (Miltenyi Biotec) at a concentration of 1: 10,000, or using HRP- conjugated anti-HA antibody (Miltenyi Biotec) at a concentration of 1:2000. The nitrocellulose membranes were washed three times for 5, 15, and 10 minutes in TBST (IX TBS, 0.3% Tween) and imaged using a 1:4 ratio of Supersignal® West Femto Maximum Sensitivity Substrates and Supersignal® West Pico Plus (Thermo Scientific) and a ChemiDoc Imaging System (Biorad). Gels were stained with Coommassie blue (0.5g Coommassie R250, 200uL Methanol, 50uL Acetic Acid, 250uL Water) overnight at room temperature. Destaining solution (500mL water, 400mL methanol, lOOmL Acetic Acid) was used for 2 hours. Coommassie blue stained gels were visualized using Gel Doc EZ Imager.

[000117] Example 2: Application of PBS1 Decoy Technology in Monocot Crops

[000118] (a) As an example of how PBS1 decoy technology may be deployed to control diseases in monocot crops, this technology can be used to confer resistance to infection by wheat stripe rust fungus ( Puccinia striiformisf sp. tritici (Pst)). Wheat stripe rust is one of the most prevalent and destructive diseases in wheat, responsible for an estimated 5 million ton yield loss annually. In addition, the prevalence and severity of losses to wheat stripe rust have been increasing in the last decade within the United States, primarily attributed to geographical adaptation of the pathogen to warmer and dryer climates. Current strategies to control wheat stripe rust include using resistant wheat cultivars and fungicide application. Genetic -based resistance to wheat stripe rust is achieved by Yellow rust ( Yr ) resistance genes; however, most resistant varieties expressing single Yr genes provide transient protection due to emergence of virulent Pst isolates. Although fungicide application is effective, emerging evidence suggests wheat stripe rust is evolving resistance to several commercially available fungicides. There is thus an urgent need to develop durable, genetic -based resistance to wheat stripe rust.

[000119] To develop PBS1 decoys that will confer resistance to Pst, proteases that are expressed during infection of wheat, and which are predicted to be secreted, were identified. These are listed in Table 4, and are provided as SEQ ID NOS: 40-47. Importantly, these proteases are conserved in other species of Puccinia, which suggests they play a fundamental role in pathogenesis, and cannot be easily eliminated by the fungus. This latter property is important for developing disease resistance traits that are durable in a field setting (i.e. not easily overcome by mutations in the fungus).

[000120] To generate PBS1 decoys that can be cleaved by these proteases, the amino acids

sequences to which these proteases bind and cleave are identified using a yeast-based genetic screen (Kang et al, 2001). Cleavage sequences (typically seven amino acids or less) are then inserted into the activation loop of a wheat PBS I gene, replacing the equivalent number of amino acids at the AvrPphB cleavage site. Introduction of the modified PBS1 sequence into the wheat genome is accomplished using CRISPR-Prime genome editing (Lin et al, 2020). Expression of the resulting wheat PBS I gene is expected to produce a wheat PBS1 decoy protein that can be cleaved by the matching Pst protease, and thus activate resistance, preventing further growth of the fungus.

[000121] (b) In a second example, the PBS1 decoy system may be used to engineer resistance to infection by Fusarium graminearum, causal agent of Fusarium head blight (FHB), also known as wheat scab. FHB causes billions of dollars in economic losses worldwide each year and leads to contamination of wheat with mycotoxins such as deoxynivalenol, which inhibits protein biosynthesis, and zearalenone, an estrogenic mycotoxin. No completely resistant wheat varieties are currently available. Using published transcriptome data (Brown et al, 2017; Dilks et al, 2019), a set of 10 secreted F.

graminearum proteases that are expressed during infection of wheat heads are identified (Table 5 and SEQ ID NOS: 48-57).

[000122] As described in the first example, cleavage site sequences for each of these proteases are identified using a yeast genetic system, and then these sequences are inserted into a wheat PBS1 gene. These modifications are then inserted into a wheat genome using CRIPSR- Prime genome editing. Wheat varieties that then express these wheat PBS1 decoy proteins are expected to be resistant to infection by Fusarium because strong resistance responses are activated as soon as the protease gains entry into host wheat cells.

[000123] Example 3: Application of PBS 1 Decoy Technology in Dicot Crops

[000124] (a) Example 1 above described successful application of PBS1 decoy technology in a dicot crop (soybean), in which a soybean PBS1 gene was modified to introduce a cleavage site for the NIa protease from soybean mosaic vims. Transgenic soybean plants expressing this modified PBS1 gene were completely resistant to SMV infection. (FIG.

13)

[000125] Three genes with the Glycine max genome encode proteins with significant amino acid homology to Arabidopsis (SEQ ID NOS: 64, 66, and 68).

[000126] A recombinant nucleic acid molecule (SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID

NO: 74) encodes a Glycine max GmPBSl substrate protein and a heterologous pathogen- specific protease recognition sequence (SEQ ID NO: 71, SEQ ID NO: 73 and SEQ ID NO: 75).

[000127] (b) As an additional example, modification of a PBS1 gene in tomato encoding (SEQ ID

NO: 58) is presented. Replacement of the the AvrPphB cleavage site (GDKSHVS) with a site that can be cleaved by the AvrRpt2 protease from Pseudomonas syringae pathovar tomato (VPKFGDW) produces (SEQ ID NO: 59) and should confer resistance to bacterial speck disease in tomato. An identical substitution in the Arabidopsis PBS1 gene renders it resistant to this disease (Kim et al., 2016).

[000128] (c) As an additional example, modification of a PBS1 gene from rapeseed (Canola;

Brassica rapa)\ encoding SEQ ID NO: 60 is presented. This plant species is closely related to Arabidopsis and is also infected by turnip mosaic virus (TuMV). Modification of Canola PBS1 to include a cleavage site for the NIa protease of TuMV (GGCSHQS) (SEQ ID NO: 61) should render rapeseed fully resistant to TuMV infection.

[000129] (d) As yet another example, modification of a PBS1 gene from Valencia orange ( Citrus sinensis, SEQ ID NO: 62) to confer resistance to citrus greening disease (also known as Huanglongbing, or HLB), which is devastating the Florida citrus industry. HLB is caused by the bacterium Candidatus Liberibacter asiaticus (Las). Las is known to secrete proteases when it colonizes citrus trees (see Table 3 in Prasad et al. 2016).

Modification of citrus PBS I to contain a cleavage site for one or more of these proteases is expected to render citrus resistant to HLB disease. As an example of a protease from Las that can be targeted, SEQ ID NO: 63 is provided. GENERAL MATERIALS AND METHODS

[000130] U.S. Pat. 9,816,102 (incorporated by reference) provides materials and methods modified herein; the modification is that the present application uses barley and wheat PBS1 genes rather than Arabidopsis PBS1, and introduces cleavage sites into PBS1 that enable cleavage by fungal proteases.

[000131] Plant Material and Growth Conditions

[000132] Barley seeds were planted in Cornell mix soil (1.2 cubic yards of mix contains 10.6 cubic feet of compressed peat moss, 20 lb of dolomitric limestone, 6 lb of 11-5-11 fertilizer, 12 cubic ft of vermiculite) in plastic pots. Barley plants were grown in a growth room on a

16 hr light/8 hr dark cycle with cool white fluorescent lights (85 to 112 pmol/m² /s at soil level) at 22°C. Plants were watered as needed to keep soil damp.

[000133] N. benthamiana seeds were sown in plastic pots containing Pro-Mix B Biofungicide potting mix supplemented with Osmocote slow-release fertilizer (14-14-14) and grown under a 12 hr photoperiod at 22°C in growth rooms with average light intensities at plant height of 150 pEinsteins/m² /s.

[000134] Seed for wheat ( Triticum aestivum subsp. aestivum ) cultivars were ordered from the U.S.

Department of Agriculture Wheat Germplasm Collection via the National Plant Germplasm System Web portal or provided by S. Hulbert (Washington State University). Wheat plants were grown in clay pots containing Pro-Mix B Biofungicide potting mix supplemented with Osmocote slow-release fertilizer (14-14-14) and grown under a 12 hr photoperiod at 22°C in growth rooms with average light intensities at plant height of 150 pEinsteins/m² /s.

[000135] P. syringae strain DC3000(D36E) in planta assays

[000136] Previously generated plasmids pVSP61-AvrPphB and pVSP61-AvrPphB(C98S) (a

catalytically inactive mutant) (Simonich and Innes, 1995; Shao et al., 2003) were each transformed into D36E, a strain of Pseudomonas syringae pv. tomato DC3000 with 36 effectors removed (Wei et al., 2015). Bacteria were grown on King’s media B (KB), supplemented with 50 pg of kanamycin per milliliter, for two days at 28 °C, then suspended in 10 mM MgC1₂ to an OD600 of 0.5. Suspensions were infiltrated into the underside of the primary leaf of 10-day old barley seedlings by needleless syringe. Each leaf was infiltrated with bacteria expressing AvrPphB and bacteria expressing AvrPphB(C98S), and the infiltrated areas were marked with permanent marker.

Infiltrated leaves were checked for cell collapse two days post infiltrations, then photographed and phenotyped for chlorosis and necrosis five days post infiltrations·

[000137] For wheat inoculations, bacteria were grown and prepared in the same way, but the adaxial side of the second leaf of 14-day old wheat seedlings was infiltrated at three spots with one of the strains of bacteria per leaf. Responses were photographed three days after infiltration using a high intensity long-wave (365 nm) ultraviolet lamp (Black- Ray B-100AP, UVP, Upland, CA).

[000138] Phylogenetic Analyses

[000139] Homology searches were performed using BLASTp to gather barley amino acid

sequences homologous to Arabidopsis PBS1 and PBS 1 -like proteins. First, AtPBL (1 to 27), BIK1, and other PBS1- homologous sequences were gathered by searching the Arabidopsis genome (TAIR10, GCA_000001735.1) with the AtPBSl (0A091748.1) amino acid sequence and by name search. Potential barley PBLs were collected by searching the barley protein database (assembly Hv_IBSC_PGSB_v2) with each Arabidopsis homologue and taking the top five hits derived from distinct genes.

[000140] For NLR phylogenetic analysis, the NB-ARC domain was extracted by NLR-parser

(Steuernagel et ah, 2015). For genes where no NB-ARC domain was automatically found, the upstream nucleotide sequence in the genome was inspected using BLASTx to look for fragments encoding an NB-ARC domain or CC domain. CC domains were identified by analyzing each predicted NLR with the BLAST Conserved Domain Search or by comparison to the CC domain in RPS5 for domains lacking the EDVID motif (SEQ ID NO: 21) (Marchler-Bauer and Bryant, 2004).

[000141] Nucleotide or amino acid sequences were aligned with Clustal Omega (Sievers et ah,

2011). Bayesian phylogenetic trees were generated for the collected sequences using the program MrBayes under a mixed amino acid model (Ronquist et al., 2012). Parameters for the Markov chain Monte Carlo method were; nruns = 2, nchains = 2, diagnfreq = 1000, diagnstat = maxstddev. The number of generations (ngen) was initially set at 200,000 and increased by 100,000 until the max standard deviation of split frequencies was below 0.01, or until it was below 0.05 after 1,000,000 generations. Phylogenetic trees were visualized.

[000142] For the analysis of Pbrl alleles, nucleotide sequences were selected from each sequenced allele that spanned from the start codon to the stop codon of the Rasmusson allele, including the intron. Sequences were aligned with Clustal Omega and then used to construct Neighbor-Joining trees in MEGA7 (Kumar et al., 2016). A bootstrap test of 1000 replicates was applied.

[000143] Genome Wide Association Study

[000144] The University of Minnesota Spring Barley Nested Association Mapping (NAM)

population comprises 6,161 RILs generated from the variety Rasmusson crossed to 88 diverse parents that represent 99.7% of captured SNP diversity. In total, ~24,000 SNPs were generated through use of genotyping by sequencing and the barley iSelect 9K SNP chip. The 89 parental lines were assayed for AvrPphB response as part of the initial survey of barley lines. Because the common parent, Rasmusson, displayed a strong hypersensitive response, NAM families derived from Rasmusson and a parent showing no response were chosen for GWAS.

[000145] Plants were assayed as described above using infiltrations of two Pseudomonas strains expressing either AvrPphB or AvrPphB(C98S). Phenotypes for at least six plants of each recombinant inbred line (RIL) were recorded as 0 (no response/low chlorosis) or 1 (hypersensitive reaction) depending on the parental phenotype they exhibited. Lines that showed phenotypic segregation between individuals were not included in the analysis.

[000146] Genome wide association analysis was performed with the gwas2 function from the

R/NAM (Nested Association Mapping) package, which uses an empirical Bayesian framework to determine likelihood ratios for each marker (Xavier et al., 2015). Lines from each family were identified within a family vector to account for population stratification. Markers with a minor allele frequency below 0.05 or missing data of more than 20% were removed using the snpQC function prior to analysis. A threshold of 0.05 for the false discovery rate was used to identify significant associations. NLR-encoding gene prediction was generated using NLR-parser (Steuernagel et al., 2015) and the high confidence Morex barley genome protein predictions (Mascher et al., 2017).

[000147] Lor genetic fine mapping, eighteen additional RILs with recombination events in the

GWAS interval were selected from other families that also had an AvrPphB-non- responding parent. To determine which RILs to select, we subset the master SNP file by family and removed SNPs that were not variable between Rasmusson and the other parent. Lor visualization, SNPs that did not match neighboring markers across RILs were assumed to be miscalls and were also removed; while these could indicate double recombination events, the probability for a double recombination occurring within the 22.65 Mb interval is 0.001, and would be even less between two or three SNPs.

[000148] Construction of Transgene Expression Plasmids

[000149] The AvrPphB:myc, AvrPphB(C98S):myc, RPS5:sYFP, and AtPBSl:HA constructs have been described previously (Shao et al., 2003; Ade et al., 2007; DeYoung et al., 2012). HORVU2HrlG070690 ( HvPbsl-1 ) and HORVU3HrlG035810 ( HvPbsl-2 ) were PCR amplified from barley accession Cl 16151 (Manchuria background) and Rasmusson cDNA, respectively. The resulting fragments were gel-purified, using the QIAquick gel extraction kit (Qiagen), and cloned into the Gateway entry vector pCR8/GW/TOPO (Invitrogen) to generate pCR8/GW/TOPO:HORVU2HrlG070690 and

pCR8/GW/TOPO:HORVU3HrlG035810, which we then designated

pCRS/GW/TOPO.HvPbs1-1 and pC R 8/GW/TO PO : HvPbs 1-2, respectively.

[000150] The following genes were PCR amplified with attB-containing primers from the

corresponding templates: HvPbsl-1 Fro m pC R 8/G W /TO PO : vPbs 1-1 , HvPbsl-2 from pCR8/GW/TOPO:HvPbs1-2, Pbrl.b (HORVU3HrlG107310) and Goi2

(HORVU3HrlG109680) from Rasmusson cDNA, Pbrl.c from Cl 16151 gDNA, LT16b from Arabidopsis thaliana gDNA (Col-0), and NbPbsl (Nibenl01Scf02996g03008.1) from Nicotiana benthamiana cDNA. The resulting PCR products were gel-purified, using the QIAquick gel extraction kit (Qiagen) or the Monarch DNA gel extraction kit (NEB), and recombined into the Gateway donor vectors pBSDONR(Pl-P4) or pBSDONR(P4r-P2) using the BP Clonase II kit (Invitrogen) (Qi et al., 2012). The resulting constructs were sequence-verified to check for proper sequence and reading frame.

[000151] To generate protein fusions with the desired C-terminal epitope tags, pBSDONR(Pl- P4):HvPbs1-1, pBSDONR(Pl -P4):HvPbs1-2, and pBSDONR(Pl -P4):M/PAs1 were mixed with the pBSDONR(P4r-P2):Jx77A construct and the Gateway-compatible expression vector pBAV154 in a 2:2:1 molar ratio. A derivative of the destination vector pTA7001, pBAV154, carries the dexamethasone inducible promoter (Aoyama and Chua, 1997; Vinatzer et al., 2006). The pBSDONR(Pl -P4):Pbrl.b and pBSDONR(Pl- P4 y.Pbrl.c constructs were mixed with the pBSDONR(P4r-P2):.sYFP construct and pBAV154 in a 2:2:1 molar ratio. The pBSDONR(P4r- P2 ):sYFP and pBSDONR(P4r- P2):3xHA constructs have been described previously (Qi et al., 2012). To generate the sYFP:LTI6b fusion protein, the pBSDONR(P4r-P2):LTI6b construct was mixed with the pBSDONR(P1 -P4):.sYFP construct and pB AV154 in a 2:2: 1 molar ratio. Plasmids were recombined by the addition of LR Clonase II (Invitrogen) and incubated overnight at 25 °C following the manufactures instructions. Constructs were sequence verified and subsequently used for transient expression assays in N. benthamiana.

[000152] Transient Expression Assays in N. benthamiana

[000153] For transient expression assays in N. benthamiana, we followed the protocol described by DeYoung et al. (2012) and Kim et al. (2016). Briefly, the dexamethasone-inducible constructs were transformed into Agrobacterium tumefaciens GV3101 (pMP90) strains and were streaked onto Luria-Bertani (LB) plates containing 30 mg of gentamicin sulfate per milliliter and 50 mg of kanamycin per milliliter. Cultures were prepared in liquid LB media (5 ml) supplemented with 30 pg of gentamicin per milliliter and 50 pg of kanamycin per milliliter and shaken overnight at 30 °C and 250 rpm on a New Brunswick orbital shaker. After overnight culture, the bacterial cells were pelleted by centrifuging at 3000 x g for 3 minutes and resuspended in 10 mM MgC1₂ supplemented with 100 pM acetosyringone (Sigma- Aldrich). The bacterial suspensions were adjusted to an OD600 of 0.9 for HR and electrolyte leakage assays and an OD₆₀₀ of 0.3 for

immunoprecipitation and immunoblotting assays, and incubated for 3 hours at room temperature. For co-expression of multiple constructs, suspensions were mixed in equal ratios. Bacterial suspension mixtures were infiltrated by needleless syringe into expanding leaves of 3-week-old N. benthamiana. Leaves were sprayed with 50 pM dexamethasone 45 hours after injection to induce transgene expression. Samples were harvested 6 hours after dexamethasone application for protein extraction, flash- frozen in liquid nitrogen, and stored at -80°C. HR was evaluated and leaves photographed 24 hours after dexamethasone application using a high intensity long- wave (365 nm) ultraviolet lamp (Black-Ray B-100AP, UVP, Upland, CA).

[000154] Immunoblot Analysis

[000155] Frozen N. benthamiana leaf tissue (0.5 g) was ground in two volumes of protein

extraction buffer (150 mM NaCl, 50 mM Tris [pH 7.5], 0.1% Nonidet P-40 [Sigma- Aldrich], 1% plant protease inhibitor cocktail [Sigma- Aldrich], and 1% 2,2’-dipyridyl disulfide [Chem-Impex]) using a ceramic mortar and pestle and centrifuged at 10,000 x g for 10 minutes at 4°C to pellet debris. Eighty microliters of total protein lysate were combined with 20 pi of 5X SDS loading buffer, and the mixture was boiled at 95°C for 10 minutes. All samples were loaded on a 4-20% gradient Precise™ Protein Gels (Thermo Fisher Scientific, Waltham, MA) and separated at 185 V for 1 hour in IX Tris/Glycine/SDS running buffer. Total proteins were transferred to a nitrocellulose membrane (GE Water and Process Technologies, Trevose, PA). Ponceau staining was used to confirm equal loading of protein samples and successful transfer. Membranes were washed with IX Tris- buffered saline (TBS; 50 mM Tris-HCl, 150 mM NaCl, pH 7.5) solution containing 0.1% Tween 20 (TBST) and blocked with 5% Difco™ Skim Milk (BD, Franklin Lakes, NJ) overnight at 4°C. Proteins were detected with 1:5,000 diluted peroxidase-conjugated anti-HA antibody (rat monoclonal, Roche, catalog number 12013819001) and a 1:5,000 diluted peroxidase-conjugated anti-c-Myc antibody (mouse monoclonal, Thermo Fisher Scientific, catalog number MAI-81357) for 1 hour and washed three times for 10 minutes in TBST solution. Protein bands were imaged using an Immuno-Star™ Reagents (Bio-Rad, Hercules, CA) and X-ray film.

[000156] Allele Sequencing and Expression Analysis

[000157] DNA was isolated from ground frozen leaf tissue using the GeneJET Plant Genomic

DNA Purification Kit (Thermo Scientific™). Primers were designed throughout the genes of interest and fragments were amplified from genomic DNA using Q5 2X Master Mix (NEB), then Sanger sequenced at the Cornell Biotechnology Resource Center. RNA was isolated from the primary leaf of a 10-day old plant using the RNeasy Plant Mini Kit (QIAGEN) after freezing and grinding. RNA samples were quantified using a NanoDrop™ spectrophotometer (Thermo Scientific™) and 500 ng of RNA from each sample were used to make cDNA with Superscript III Reverse Transcriptase (Invitrogen) and oligo dT primers. DreamTaq™ DNA Polymerase (Thermo Scientific™) was used for 30-cycle PCRs of 1 pi of cDNA or 50 ng of gDNA template. Eight microliters of the PCR products were then visualized in a 1% agarose gel. Samples chosen for expression and sequence analysis were done so based on NAM population parent lines and to encompass two or more lines for all phenotypes.

[000158] Electrolyte leakage assays in N. benthamiana

[000159] Electrolyte leakage assays were performed as described previously (Kim et al., 2016). In brief, after infiltration of Agrobacterium strains into N. benthamiana, leaf discs were collected from the infiltrated area using a cork borer (5 mm diameter) 2 h post dexamethasone application. Four leaf discs from four individual leaves of four different plants were included for each replication. The leaf discs were washed three times with distilled water and floated in 5 ml of distilled water supplemented with 0.001% Tween 20 (Sigma- Aldrich). Conductivity was monitored using a Traceable Pen Conductivity Meter (VWR) at the indicated time points after dexamethasone induction.

[000160] Immunoprecipitation assay in N. benthamiana

[000161] Frozen N. benthamiana leaf tissue (four leaves) was ground in 1 ml of IP buffer (50 mM

Tris-HCl [pH 7.5], 150 mM NaCl, 10% Glycerol, 1 mM DTT, 1 mM EDTA, 1% NP40, 0.1% Triton X-100, 1% plant protease inhibitor cocktail [Sigma- Aldrich], and 1% 2,2’- dipyridyl disulfide [Chem-Impex]) using a ceramic mortar and pestle and gently rotated for 1 hour at 4°C. The samples were centrifuged at 10,000 x g for 10 minutes at 4°C twice to remove plant debris. Five hundred microliters of the clarified extract were then incubated with 10 pi of GFP-Trap A (Chromotek) a-GFP bead slurry overnight at 4°C with constant end-over-end rotation. After overnight incubation, the a-GFP beads were pelleted by centrifugation at 4000 x g for 1 minute at 4°C and washed five times with 500 mΐ of IP wash buffer. Eighty microliters of the immunocomplexes were resuspended in 20 mΐ of 5X SDS loading buffer, and the mixture was boiled at 95 °C for 10 minutes. All protein samples were resolved on a 4-20% gradient Precise™ Protein Gels (Thermo Scientific, Waltham, MA) and separated at 185 V for 1 hour in IX Tris/Glycine/SDS running buffer. Total proteins were transferred to a nitrocellulose membrane (GE Water and Process Technologies, Trevose, PA). Membranes were blocked with 5% Difco™ Skim Milk (BD, Franklin Lakes, NJ) overnight at 4°C. Proteins were detected with 1:5,000 horseradish peroxidase conjugated anti-HA antibody (rat monoclonal, Roche, catalog number 12013819001) or 1:5,000 monoclonal mouse anti-GFP antibody (Novus Biologicals, Littleton, CO, catalog number NB600-597), washed in X Tris- buffered saline (TBS; 50 mM Tris-HCl, 150 mM NaCl, pH 7.5) solution containing 0.1% Tween 20 (TBST) overnight and incubated with 1:5,000 horseradish peroxidase-conjugated goat anti-mouse antibody (abeam, Cambridge, MA catalog number ab6789). The nitrocellulose membranes were washed three times for 15 minutes in TBST solution and protein bands were imaged using an Immuno-Star™ Reagents (Bio-Rad, Hercules, CA) or Supersignal® West Femto Maximum Sensitivity Substrates (Thermo Scientific, Waltham, MA) and X-ray film.

[000162] DAB assay for hydrogen peroxide accumulation in wheat

[000163] Hydrogen peroxide accumulation was detected following the protocol described by Liu et al. (2012) and Thordal-Christensen et al. (1997). In brief, 0.01 g of DAB powder (Sigma-Aldrich) was dissolved in 10 ml of distilled water (pH 3.6) and incubated at 37°C for 1 hour on a New Brunswick orbital shaker to dissolve the DAB powder. Wheat leaf segments were harvested from the infiltrated leaves 3 days post inoculation, (10 plants per treatment, experiment performed twice), immersed immediately in DAB solution and vacuum infiltrated for 10 seconds. The samples were wrapped in aluminum foil and incubated overnight in the dark. After overnight incubation, the stained leaf tissue was gently rinsed with distilled water, submerged in 70% ethanol and incubated at 70°C to clear the chlorophyll. The cleared leaves were rinsed and stored in a lactic

acid/glycerol/H20 solution (1:1:1, v/v/v) for photography. Wheat leaves inoculated with 10 mM MgC1₂ (mock) or P. syringae DC3000(D36E) expressing AvrPphB(C98S) were used as controls.

[000164] PBS1 Decoy Sequences

[000165] In one aspect, the present disclosure is directed to an isolated polypeptide encoded by the recombinant nucleic acid molecule comprising about 90% identity to an amino acid sequence selected from SEQ ID NO: 24, SEQ ID NO:27, and SEQ ID NO:30, SEQ ID NO: 33 and SEQ ID NO: 36 wherein the polypeptide is a substrate protein of a plant pathogen-specific protease. In another embodiment, the isolated polypeptide can comprise about 95% identity to an amino acid sequence selected from SEQ ID NO: 24, SEQ ID NO:27, and SEQ ID NO:30, SEQ ID NO: 33 and SEQ ID NO: 36, wherein the polypeptide is a substrate protein of a plant pathogen-specific protease. In other embodiments, the isolated polypeptide can comprise about 96% identity, about 97% identity, about 98% identity, about 99% identity, and even 100% identity to an amino acid sequence selected from SEQ ID NO: 24, SEQ ID NO:27, and SEQ ID NO:30, SEQ ID NO: 33 and SEQ ID NO: 36, wherein the polypeptide is a substrate protein of a plant pathogen-specific protease.

[000166] An example of a fusion protein (that is, a modified substrate protein of a pathogen- specific protease) therefore includes that of SEQ ID NO: 24, SEQ ID NO:27, and SEQ ID NO:30, SEQ ID NO: 33 and SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and including polypeptides comprising about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity and even 100% identity to an amino acid sequence selected from SEQ ID NO: 24, SEQ ID NO:27, and SEQ ID NO:30, SEQ ID NO: 33 and SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39. [000167] The present disclosure has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the present disclosure has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, one of skill in the art will realize that the present disclosure is intended to encompass all modifications and alternative arrangements of the compositions and methods as set forth in the appended claims.

[000168] For nucleotide sequences, "variant" means a substantially similar nucleotide sequence to a nucleotide sequence of a recombinant nucleic acid molecule as described herein, for example, a substantially similar nucleotide sequence encoding a modified substrate protein. For nucleotide sequences, a variant comprises a nucleotide sequence having deletions (/._<?., truncations) at the 5' and/or 3' end, deletions and/or additions of one or more nucleotides at one or more internal sites compared to the nucleotide sequence of the recombinant nucleic acid molecules as described herein; and/or substitution of one or more nucleotides at one or more sites compared to the nucleotide sequence of the recombinant nucleic acid molecules described herein. One of skill in the art understands that variants are constructed in a manner to maintain the open reading frame.

[000169] Conservative variants include those nucleotide sequences that, because of the degeneracy of the genetic code, result in a functionally active modified substrate protein as described herein. Naturally occurring allelic variants can be identified by using well-known molecular biology techniques such as, for example, polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also can include synthetically derived sequences, such as those generated, for example, by site-directed mutagenesis but which still provide a functionally active modified substrate protein. Generally, variants of a nucleotide sequence of the recombinant nucleic acid molecules as described herein will have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence of the recombinant nucleic acid molecules as determined by sequence alignment programs and parameters as described elsewhere herein.

[000170] Table 1. Responses of 150 barley lines when infiltrated with Pseudomonas syringae

DC3000(D36E) expressing AvrPphB. A catalytically inactive AvrPphB(C98S) mutant was used as a negative control and never elicited a response. Lines were scored as no response (N), low chlorosis (LC), chlorosis (C), high chlorosis (HC), and hypersensitive reaction (HR). [000171]

HR- hypersensitive reaction HC- high chlorosis C- chlorosis

LC- low chlorosis

N- no response

[000172] Table 2. Responses of 193 RILs from the UMN Spring Barley Nested Association

Mapping (NAM) population when infiltrated with Pseudomonas syringae DC3000(D36E) expressing AvrPphB. The inactive protease AvrPphB(C98S) never elicited a response. Response phenotypes were used in genome wide association analysis. Hypersensitive reaction = 1; no response or low chlorosis = 0. [000173]

1- HR; responds like Rasmusson

0- No response or low chlorosis; responds like other parent

* RIL chosen for recombination event in the GW AS interval

[000174] Table 3. Responses of wheat varieties to P. syringae DC3000(D36E) expressing

AvrPphB. The second leaves of 14-day old wheat seedlings were inoculated with P. syringae DC3000(D36E) carrying AvrPphB (OD₆₀₀= 0.5) by infiltration with a needleless syringe. Wheat responses were scored as no response, low chlorosis, or chlorosis three days post-inoculation. Wheat varieties were obtained from the U.S.

Department of Agriculture Wheat Germplasm Collection or generously provided by Scot Hulbert (Washington State University). [000175]

Table 4 Summary of candidate effector proteases from wheat stripe rust.

Table 5 Summary of candidate effector proteases from F. graminearum.

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Claims

1. A method to activate disease resistance in a plant, the method comprising:

(a) modifying a gene in the plant to produce a modified PBS1 protein, wherein modified means incorporating a pathogen protease cleavage site; and

(b) cleaving the modified protein with a pathogen protease to activate disease resistance in the plant.

2. The method of claim 1 wherein the plant is a monocot plant.

3. The monocot plant of claim 2 is selected from the group consisting of barley, wheat, rice, sorghum and corn.

4. The method of claim 1 wherein modifying the gene enables cleavage of the PBS1 protein by specific pathogen - derived proteases.

5. The method of claim 1, wherein the plant is Glycine max.

6. The method of claim 5, wherein the modified PBS1 protein is selected from the group consisting of a sequence SEQ ID NOS: 10, 12 and 14.

7. The method of claim 1, wherein the plant is tomato.

8. The method of claim 7, wherein a PBS1 protein is modified for cleaving by

AvrRpt2.

9. The method of claim 1, wherein the plant is Canola.

10. The method of claim 9, wherein the PBS1 protein is modified for cleavage with TuMV Nia protease.

11. The method of claim 1, wherein the plant is Valencia orange.

12. The method of claim 11, wherein a protease from a pathogen that is suitable for targeting is (SEQ ID NO: 63).

13. A monocot plant with a gene encoding a modified PBS1 protein.

14. The monocot plant of claim 13 is selected from the group consisting of barley, wheat, rice, sorghum and com.

15. The monocot plant of claim 14, wherein the modified PBS1 protein enables cleavage by specific pathogen-derived proteases that activate resistance to the pathogen.

16. A modified PBS1 protein, wherein the modification allows cleavage of the protein by a pathogen protease, and the modified PBS1 protein has an amino acid sequence selected from the group consisting of SEQ ID NOS: 24, 27, 30, 33 and 36.

17. A gene with a DNA sequence encoding a PBS1 protein of claim 16.

18. A synthetic PBS1 decoy protein with a protease cleavage motif.

19. The synthetic PBS1 decoy protein, of claim 18 wherein the protease cleavage motif is selected from the group consisting of SEQ ID NOS: 37, 38, 39.

20. A method to generate a PBS1 decoy protein in wheat that can be cleaved by proteases expressed during infection of wheat by a pathogen:

(a) identify the pathogen proteases;

(b) identify amino acids in wheat to which the proteases bind and cleave;

(c) insert cleavage sequences into the activation loop of a wheat PBS1 gene to replace the equivalent number of amino acids at the AvrPphB cleavage site.

21. The method of claim 20, wherein the pathogen is wheat rust fungus and wherein the proteases are selected from the group consisting of amino acid sequences designated SEQ ID NOS: 40-47.

22. The method of claim 20, wherein the pathogen is Fusarium and the proteases are selected from the group of amino acid sequences consisting of amino acid sequences designated SEQ ID NOS: 48-57.