Control of male fertility in plants by rhomboid-mediated cleavage of immune receptor

Abstract

Described are plants having reduced male fertility. Compositions and methods for making the plants are also described.

Description

Control of Male Fertility in Plants by Rhomboid-Mediated Cleavage of Immune Receptor CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 590,111, filed 13 October 2023, which is incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No.2114833 awarded by The National Science Foundation and Grant No.2015-67013-22910 awarded by The United States Department of Agriculture, National Institutes of Food & Agriculture. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The Sequence Listing written in file T19204WO001_SeqListing.xml is 119 kilobytes in size, was created October 10, 2024, and is hereby incorporated by reference. BACKGROUND

[0004] The production of F1 hybrid plants has great significance in agriculture due to the homogeneity, predictability, and higher performance of hybrid plants. The development of male sterile plant lines, which cannot undergo self-pollination due to the inability of the plant to generate functional anthers, pollen, or male gametes, has drastically improved the efficiency F1 hybrid seed production by replacing physical emasculation methods. However, the complex molecular mechanisms involved in male sterility are not well understood in many species and the development of male sterile plant lines remains a significant challenge for F1 hybrid plant production. Improved methods for controlling male sterility in plants are needed to advance plant breeding programs. SUMMARY

[0005] Described are compositions and methods for modifying male fertility in a plant. The method comprising modifying activity of a PVR1 rhomboid protease (i.e., an Oryza sativa PVR1 or an ortholog thereof in another plant species) in a plant cell. The compositions comprise CRISPR systems that target a PVR1 gene (i.e., an Oryza sativa PVR1 gene or an ortholog thereof in another plant). Also described are genetically modified plants having reduced male sterility, wherein the genetically modified plants contain a modified PVR1 gene. 1 LEGAL02 / 44913848v1

[0006] In some embodiments, modifying activity of a PVR1 rhomboid protease comprises reducing expression of a gene encoding the PVR1 rhomboid protease. In some embodiments, reducing expression of the PVR1 rhomboid protease expression comprises reducing mRNA encoding the PVR1 rhomboid protease (PVR1 mRNA). In some embodiments, reducing expression of the PVR1 rhomboid protease comprises introducing an RNA interference (RNAi) molecule or an antisense nucleotide targeting the PVR1 mRNA into the plant or expressing an RNAi nucleic acid sequence or an antisense nucleic acid sequence targeting the PVR1 mRNA in the plant. In some embodiments, reducing the PVR1 mRNA comprises modifying an endogenous gene encoding a PVR1 rhomboid protease to create a deletion, insertion, substitution, or frameshift mutation in the endogenous gene encoding the PVR1 rhomboid protease.

[0007] In some embodiments, modifying activity of the PVR1 rhomboid protease comprises introducing a mutation into an endogenous gene encoding the PVR1 rhomboid protease that results in reduced of loss of function of the encoded PVR1 rhomboid protease.

[0008] In some embodiments, modifying activity of the PVR1 rhomboid protease comprises introducing a CRISPR system into a plant cell, wherein the CRISPR system comprises an RNA-guided DNA endonuclease or a nucleic acid encoding the RNA-guided DNA endonuclease and a guide RNA or a nucleic acid encoding the guide RNA into the plant, wherein the RNA-guided DNA endonuclease and the guide RNA form a complex that targets a PVR1 genomic locus. In some embodiments, the CRISPR system is selected from the group consisting of: a CRISPR class 1 system, a CRISPR class 2 system, a CRISPR / Cas system, a CRISPR / Cas9 system, a CRISPR / zCas9 system and a CRISPR / Cas3 system. In some embodiments, the RNA-guided endonuclease comprises a Cas9 endonuclease.

[0009] In some embodiments, the guide RNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) as separate molecules or as a single chimeric guide RNA (sgRNA).

[0010] The described CRISPR systems can be introduced into plants using methods available in the art. In some embodiments, introducing a CRISPR system into a plant cell comprises electroporation, microprojectile bombardment, biolistic transformation, microinjection, protoplast transformation, an Agrobacterium tumefaciens vector transformation or an Agrobacterium rhizogenes vector transformation.

[0011] In some embodiments, the CRISPR system introduces a mutation into the genomic PVR1 locus that results in loss of enzymatic activity of the encoded PVR1 rhomboid protease 2 LEGAL02 / 44913848v1or reduces expression of the PVR1 gene. In some embodiments, the mutation comprises a deletion, insertion, substitution, or frameshift mutation in the encoded PVR1 rhomboid protease. In some embodiments, the mutation comprises a deletion, insertion, substitution, or frameshift mutation at a catalytic serine in the encoded PVR1 rhomboid protease. In some embodiments, the mutation comprises a deletion in 1 or more nucleotides or an insertion one or more nucleotides in the PVR1 genomic locus that results decreased expression of the PVR1 gene. In some embodiments, the mutation comprises a deletion in one or more nucleotides or an insertion one or more nucleotides in the PVR1 genomic locus that results in reduced of loss of activity of the encoded PVR1 rhomboid protease. In some embodiments, the mutation comprises a deletion of one or more nucleotides (e.g., four nucleotides) or an insertion of one or more nucleotides (e.g., one nucleotide) in the PVR1 genomic locus, wherein the insertion is upstream of the sequence encoding a catalytic serine, wherein the catalytic serine corresponds to position 187 of SEQ ID NO: 35.

[0012] In some embodiments, a CRISPR system targets introduces a mutation in the PVR1 gene at a site upstream of the catalytic serine and results in premature termination of translation of the PVR1 mRNA.

[0013] Also described are genetically modified plants made using the described methods, including progeny of such plants. In some embodiments, the plants have reduced male fertility.

[0014] In some embodiments, genetically modified plants have reduced anther dehiscence and / or pollen viability as compared to a similar wild-type plant that does not have a genetic modification of an endogenous PVR1 gene. In some embodiments, male fertility of the plants is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. In some embodiments, viability of pollen grains of the modified plant is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

[0015] The plant can be, but is not limited to, a rice plant, a maize plant, a sorghum plant, a rye plant, a barley plant, a wheat plant, an oat plant, a sugarcane plant, a soybean plant, a canola plant, an alfalfa plant, a sunflower plant, a cotton plant, a tobacco plant, a peanut plant, a tomato plant, or a potato plant. In some embodiments, the plant is rice (Oryza sativa).

[0016] In some embodiments, the PVR1 rhomboid protein comprises the amino acid sequence shown in FIG.2A (SEQ ID NO: 35) or an ortholog thereof. 3 LEGAL02 / 44913848v1BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1. CRISPR / Cas9-mediated mutagenesis of PVR1 in rice. (A) Diagram of the PVR1 locus and the CRISPR construct targeting PVR1 with the sgRNA target site and sequence indicated (atgggagcactggtatggg (SEQ ID NO:66)). Gray boxes represent coding regions, and solid lines indicate non-coding regions. Numbers above the boxes indicate distances in base pairs (bp). The conserved catalytic residue serine 187 (Ser-187) of PVR1, and the start and stop codons of PVR1 are shown. (B) Sequence analysis of pvr1 mutant alleles, showing the sgRNA target site (underlined), mutated nucleotides (red letters for insertion; red asterisks for deletions), and protospacer adjacent motif (PAM) sequences (italicized). The biallelic mutant pvr1-b is indicated with parenthesis. The Myc-Xa21Ltransgene was introduced into the pvr1-1 and pvr1-2 mutants by genetic crossing. SEQ ID NOs. for the sequences shown in FIG.1B are as follows: atgggagcactggtatgggacaagg (SEQ ID NO:67) atgggagcactggtaacaagg (SEQ ID NO:68) atgggagcactggtatgggaacaagg (SEQ ID NO:69); atgggagcactggtaacaagg (SEQ ID NO:68); atgggagcactggtatggggacaagg (SEQ ID NO:70); atgggagcactggtaacaagg (SEQ ID NO:68); and atgggagcactggtatgggaacaagg (SEQ ID NO:69).

[0018] FIG.2. Predicted amino acid sequence and transmembrane domains (TMDs) of PVR1 (SEQ ID NO:35). (A) The deduced TMDs are underlined. The L1 loop between TMD1 and TMD2 is also indicated. Conserved motifs shaded in gray include E / QXWRXXS / T (where X is any residue) in the L1 loop, GzSG (where z is one of several permitted residues) in TMD3 and TMD4, and AHXXGXXXG in TMD6. The putative catalytic residue Ser-187 (S) in TMD4 and His-239 (H) in TMD6 are highlighted (larger letter). (B) Prediction of seven TMDs by TMHMM v2.0. (C) Topology model of PVR1 predicted by TMHMM v2.0. The positions of Ser-187 (S) and His-239 (H) are indicated.

[0019] FIG.3. Phylogenetic tree of rice rhomboid-like (OsRBL) proteins and human RhoA2 members. The tree was generated in MEGA 6 using the neighbor joining method with 1,000 bootstrap trials. Bootstrap values are shown at the nodes of the tree. Scale bar refers to amino acid substitutions per site. Accession numbers for the proteins are OsRBL1 (Os11g0704800), OsRBL3a (Os03g0116400), OsRBL3b (Os10g0521900, PVR1, in red), OsRBL3c 4 LEGAL02 / 44913848v1(Os08g0546700), OsRBL3d (Os09g0525900), OsRBL3e (Os04g0569300), OsRBL4 (Os01g0147300), OsRBL10 (Os09g0454100), OsRBL11 (Os05g0220600), OsRBL13 (Os01g0895500), OsRBL14 (Os01g0283500), OsRBL15 (Os03g0651100), OsPARL (Os01g0763100), HsRHBDL1 (NP_003952), HsRHBDL2 (NM_017821), and HsRHBDL3 (XM_011524445). The rice RhoA1 and human RhoA2 subfamilies are indicated. The subcellular localizations of PVR1 and RHBDLs (60) are indicated. PM, plasma membrane.

[0020] FIG.4. Subcellular localization of PVR1 in rice protoplasts. Bright-field (Bright) and fluorescence (Green and Red) images of the same cell were captured 16 hours after transfection using a Zeiss LSM800 confocal laser scanning microscope under 40× magnification. Merge, a combination of green and red channel images. (A) Localization of GFP-PVR1 at the plasma membrane of a cell stained with FM4-64 dye. (B) Subcellular distribution of GFP as control. Scale bars, 20 μm.

[0021] FIG.5. Expression profiling of PVR1 and five additional RhoA1 members showing tissue-specific expression in rice. Microarray data are from RiceXpro.

[0022] FIG.6. Expression of PVR1 and accumulation of XA21 in rice spikelets. (A) PVR1 is preferentially expressed in spikelets. Levels of mRNA were quantified by RT-qPCR using the Myc-Xa21Lline. Results were normalized to expression of the housekeeping genes UBIQUITIN5 and ACTIN. Expression levels in diverse tissues were expressed relative to those of 2-month-old leaves, which were arbitrarily set to 1 unit. Values are means ± SD of three biological replicates, each with three technical replicates. Asterisks denote significant differences (*P < 0.05, **P < 0.01). DAF = days after flowering. (B) Accumulation of Myc- XA21 protein in spikelets. Myc-XA21 protein levels in indicated lines were determined by immunoblot analysis using an anti-Myc antibody, with ATPase levels as loading controls. WTsib is a negative control lacking XA21. (C) Quantification of Myc-XA21 from (B). The ratio value of Myc-XA21 to ATPase in the Myc-Xa21Lline was set as 1. Values are the means ± SD (n = 3). The asterisk denotes a significant difference (*P < 0.05). Statistical analyses were performed using Student's t test.

[0023] FIG. 7. Histochemical analysis of GUS activity from PVR1pro:GUS and Xa21pro:GUS reporter lines in spikelets. GUS activity was detectable in veins (V) of lemma and palea, rachillae (R), and anther filament (F) in the PVR1pro:GUS and Xa21pro:GUS lines. GUS staining was only detected in the style (S) and the central core (CC) of the anther in PVR1pro:GUS plants. Scale bars, 1 mm (red), 100 μm (black). Similar results were obtained from two (Xa21pro:GUS) or three (PVR1pro:GUS) independent lines. 5 LEGAL02 / 44913848v1

[0024] FIG. 8. PVR1 mediates cleavage of XA21 in N. benthamiana. (A) Diagram of the Xa21 construct used for the cleavage assay. LRR, leucine-rich repeat domain; TMD, transmembrane domain. c-Myc and 3xFLAG epitope tags were fused to XA21 for detection. The intracellular kinase domain (ICD) used to generate the α-XA21ICD antibody is indicated by the black horizontal line. (B) Cleavage of XA21 depends on active PVR1. Constructs encoding the indicated proteins were co-expressed in N. benthamiana. PVR1catis a catalytically dead variant. Total protein extracts were probed using the indicated antibodies. EV, empty vector. Full-length Myc-XA21-3xFLAG (~140 kD) and its N- and C-terminal cleavage products (Myc-XA21ncp1, ~110 kD; XA21-FLAGccp1, ~37 kD) are indicated. Asterisks indicate non-specific cleavage products. Ponceau S staining of Rubisco large subunit was used as loading control. (C) Mutations of Pro-652 and Pro-655 of XA21 to leucines abolished PVR1- mediated cleavage. (D) PVR1 specifically mediates XA21 cleavage.

[0025] FIG.9. Mutation of Pro-655 of XA21 to leucine markedly compromises, but does not abolish, PVR1-mediated cleavage of XA21 in N. benthamiana. Cleavage experiments were performed as described in Fig. 2. XA21-FLAGccp1was visible in the Myc-XA21P655L- FLAG / HA-PVR1 combination when immunoblots were exposed for a longer time.

[0026] FIG. 10A-E. pvr1 mutants decrease seed-set but retain resistance in an XA21- dependent manner. (A) Panicle phenotypes of control lines and pvr1 Myc-Xa21Lmutants with lower fertility grown in a temperature-controlled growth chamber. Scale bar, 1 cm. (B) Phenotypes of mature plants (upper) and florets (lower) of the indicated lines in (A). Scale bar, 1 mm. (C) Seed-setting rates of pvr1 Myc-Xa21Lmutants and their controls. Values are shown as the mean ± SD from 5 independent plants each with 2 panicles. Statistical analyses were performed using the Tukey-Kramer honestly significant difference test. Different letters indicate significant differences (P < 0.05). (D) Accumulation of Myc-XA21 protein in the spikelet of WTsib, Myc-Xa21L, and the pvr1 Myc-Xa21Lmutants. (E) Quantification of Myc- XA21 protein from (D). The ratio value of Myc-XA21 to ATPase in the Myc-Xa21Lline was set as 1. Values are the means ± SD (n = 3). Asterisks denote significant differences (*P < 0.05, **P < 0.01).

[0027] FIG 10F-I. (F) Relative Myc-Xa21 transcript levels, quantified by RT-qPCR, in spikelets of lines shown in (D). Results were normalized relative to levels of mRNAs for UBIQUITIN5 and ACTIN. Values are means ± SD of three biological replicates, each with three technical replicates. Transcript levels in the indicated lines containing Myc-Xa21 were not significantly different. (G) Myc-XA21 protein accumulation in leaves of WTsib, Myc-Xa21L, 6 LEGAL02 / 44913848v1and pvr1 Myc-Xa21L. Relative levels of Myc-XA21 protein in the indicated lines (D and G) were determined by immunoblot analysis using ATPase as a loading control. (H) XA21 confers normal resistance in the pvr1 mutant background. Indicated lines were inoculated with Xoo PXO99Aby the leaf-clipping method. Lesion length data were scored at 12 days post inoculation (dpi). Each data point represents 17 inoculated leaves. Values are means ± SD. (I) Inoculated leaves from the indicated lines showing lesion development at 12 dpi. Arrows indicate the inoculation sites. Statistical analyses in (E, F, and H) were performed using Student's t test. Lesion lengths in the indicated lines containing Myc-Xa21Lwere not significantly different.

[0028] FIG.11A-B. Phenotypes of mature plants, panicles and florets of lines WTsib, pvr1-1, pvr1-2, Myc-Xa21L, pvr1-1 Myc-Xa21L, and pvr1-2 Myc-Xa21L. (A) Mature plants of the indicated genotypes grown in the greenhouse during the summer season. (B) Floral structure of indicated lines. Scale bars, 1 mm.

[0029] FIG 11C-D (C) Panicles harvested from the plants shown in (A). (D) Seed-setting rates based on the numbers of filled and empty seeds of six representative panicles from three plants per line. Values are means ± SD. Statistical analysis was performed using the Tukey- Kramer HSD test. The differences among the indicated lines were not statistically significant.

[0030] FIG 12A-B. The pvr1-b Myc-Xa21Hmutant is sterile and accumulates a higher level of XA21 protein than does the Myc-Xa21Hcontrol. Materials used for the experiments described in this figure were harvested from the plants maintained by continuous ratooning in a greenhouse. (A) Panicle phenotypes of the control line Myc-Xa21Hand the sterile pvr1-b Myc-Xa21Hmutant. (B) Phenotypes of mature plants (Upper) and floral structure (Lower) of the indicated lines in (A). Scale bars, 1 mm.

[0031] FIG 12C-D. (C) Accumulation of Myc-XA21 protein in the spikelet of the pvr1-b Myc-Xa21Hmutant and in Myc-Xa21Hcontrol, determined by immunoblot analysis using an anti-Myc antibody and anti-ATPase as a loading control. (D) Quantification of Myc-XA21 protein from (C). The ratio value of Myc-XA21 to ATPase in the Myc-Xa21Hline was set as 1. Values are the means ± SD (n = 3). The asterisk denotes a significant difference (*P < 0.05). (E) Relative Myc-Xa21 transcript levels, quantified by RT-qPCR, in spikelets of the pvr1-b Myc-Xa21H and Myc-Xa21H lines. Results were normalized relative to levels of mRNAs for the housekeeping genes UBIQUITIN5 and ACTIN. Values are the means ± SD of three biological replicates, each with three technical replicates. The difference between the two Myc- Xa21 lines was not statistically significant. WTsib(lacking Xa21) was used as a negative control 7 LEGAL02 / 44913848v1for Myc-XA21 protein and Myc-Xa21 transcript abundance. Statistical analyses in (D and E) were performed using Student's t test.

[0032] FIG. 13. Rice seedlings carrying Xa21 survive Xoo infection regardless of the presence of a functional PVR1. Two-week-old seedlings of the indicated lines were inoculated with Xoo strain PXO99Aby the leaf-clipping method and kept under fluorescence light at 27°C. The photograph was taken 5 weeks post inoculation. Arrows indicate the inoculation sites. Similar results were obtained from 10 inoculated individuals.

[0033] FIG 14A-B. Mutation of PVR1 impairs pollen viability and anther dehiscence in an XA21-dependent manner. (A) pvr1 Myc-Xa21 mutants display lower starch accumulation in their pollen grains. (Left) I2-KI-stained pollen grains from pvr1 Myc-Xa21Lmutants and the indicated control lines grown in a temperature-controlled growth chamber before anthesis. (Right) Pollen viability was quantified based on the number of stained pollen grains (dark color) relative to the total pollen counted. Values are means ± SD, n = 3. Statistical analysis was performed using the Tukey-Kramer honestly significant difference test. Different letters indicate significant differences (P < 0.05). Values are means ± SD, n = 3. (B) pvr1 Myc-Xa21Lmutants display impaired anther dehiscence. Images show the distribution of dehiscent and indehiscent spikelets among more than 100 random samples chosen from five individual plants of each indicated line.

[0034] FIG 14C. Morphology of dissected spikelets from the indicated lines. (Top 2 rows) I2-KI staining of pollen grains in anthers of the indicated lines before anthesis and 2 h after excertion. (Third Row) Presence or absence of pollen grains on the stigmas of the indicated lines after anthesis. Both dehisced and indehiscent anthers were observed in the pvr1 Myc- Xa21Lmutants. (Bottom Row) Cross-sections of anthers from the indicated lines after anthesis. Scale bars, 500 μm (Rows 1, 2 and 4) or 50 μm (Row 3).

[0035] FIG 15. Mutation of PVR1 severely impairs pollen viability and causes anther indehiscence in the Myc-Xa21Hbackground. Materials used for the experiments described in this figure were harvested from the plants maintained by continuous ratooning in a greenhouse. (A) I2-KI-stained pollen grains from lines Myc-Xa21Hand pxv1-b Myc-Xa21H. (Right) Pollen viability was quantified based on the number of stained pollen grains (dark color) relative to the total pollen counted. Values are means ± SD, n = 3. Statistical analysis was performed using Student's t test. The asterisk indicates a statistically significant difference (**P < 0.01). (B) (Top 2 rows) ) I2-KI staining of pollen grains in anthers of the indicated lines before and after anthesis. (Third Row) Presence or absence of pollen grains on the stigmas of the indicated lines 8 LEGAL02 / 44913848v1after anthesis. (Bottom Row) Cross-sections of anthers from the indicated lines after anthesis. Scale bars in a and b, 500 μm (Rows 1, 2 and 4) or 50 μm (Row 3).

[0036] FIG 16. A simplified model for PVR1-mediated intramembrane proteolysis of XA21 in rice. In XA21 plants, PVR1 cleavage triggers the degradation of the immune receptor. The abundant PVR1 in the spikelet suppresses the vascular accumulation of XA21 over a threshold that would interfere with fertility (Left). When PVR1 is mutated, XA21 accumulates to a higher level, which leads to reduced or no seed set (Middle). Regardless of the presence of functional PVR1, XA21 accumulation in the leaf and immunity are not significantly affected owing to the low-level expression of PVR1 in this tissue (Right).

[0037] FIG. 17A-B. Upregulation of defense-related genes and down-regulation of JA- responsive / signaling genes in the indehiscent pvr1-b Myc-Xa21Hspikelets. (A) Volcano plot showing differentially expressed rice NLR genes detected in the indehiscent pvr1-b Myc-Xa21Hspikelets relative to the dehiscent Myc-Xa21H. (B) Volcano plot showing downregulation of a set of JA-responsive / signaling genes. Cut-off criteria for data in (A) and (B): log2[pvr1 Myc- Xa21H / Myc-Xa21H] ≥ 1 and ≤ -1 and FDR-value < 0.05.

[0038] FIG.17C. Graphs illustration relative transcript levels of the JA-responsive / signaling genes in (B) with RPKM (reads per kilo base per million mapped reads) > 1, quantified by RT- qPCR, in spikelets of lines Myc-Xa21Hand pvr1-b Myc-Xa21H. Results were normalized relative to levels of the mRNAs for UBIQUITIN 5 and ACTIN. Values are means ± SD of three biological replicates, each with three technical replicates. Statistical analyses were performed using Student's t-test. Asterisks denote significant differences (**P < 0.01).

[0039] FIG. 18. Graph illustration cleavage of the non-canonical TMDs of a subset Arabidopsis RLKs by PVR1. (C) Cleavage of the non-canonical TMDs of a set of plant RLKs by PVR1. Constructs encoding the indicated proteins were used to transfect HEK293 cells expressing PVR1 or PVR1S187A. Values are shown as means (released AP / total AP) ± SD (n = 3). Statistical analyses were performed using Student's t test. Asterisks denote significant differences (**P < 0.01). DETAILED DESCRIPTION

[0040] Unless otherwise defined, all terms of art, notations, and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. Some techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular 9 LEGAL02 / 44913848v1cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc.2001; Transgenic Plants: Methods and Protocols (Leandro Pena, ed., Humana Press, 1st edition, 2004); and, Agrobacterium Protocols (Wan, ed., Humana Press, 2nd edition, 2006). As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters unless otherwise noted.

[0041] The use of “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings. To the extent that any material incorporated by reference is inconsistent with the express content of this disclosure, the express content controls.

[0042] The term “about” or “approximately” indicates within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of 0 to 20%, 0 to 10%, 0 to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” means within an acceptable error range for the particular value should be assumed.

[0043] All ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions such as "not including the endpoints"; thus, for example, "within 10-15" includes the values 10 and 15. One skilled in the art will understand that the recited ranges include the end values, as well as whole numbers in between the end values, and where practical, rational numbers within the range (e.g., the range 5-10 includes 5, 6, 7, 8, 9, and 10, and where practical, values such as 6.8, 9.35, etc.). When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0044] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof ("polynucleotides") in either single- or double-stranded form. Unless specifically limited, the term polynucleotide encompasses nucleic acids containing known 10 LEGAL02 / 44913848v1analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited, the term polynucleotide encompasses nucleic acids having one or more modified nucleotides. Modified nucleotides can modify binding properties or alter in vitro or in vivo stability. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res.19: 5081; Ohtsuka et al., 1985 J. Biol. Chem.260: 2605-2608; and Cassol et al., 1992; Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0045] The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure.

[0046] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms, or by manual alignment and visual inspection.

[0047] Sequence identity can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspection, and the best alignment (i.e., resulting in the highest percentage of sequence similarity over a comparison window). Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the 11 LEGAL02 / 44913848v1number of matched positions, dividing the number of matched positions by the total number of matched and mismatched positions not counting gaps in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise indicated the window of comparison between two sequences is defined by the entire length of the shorter of the two sequences.

[0048] The term "plant" includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, reproductive organs, embryos, and parts thereof, etc.), seedlings, seeds and plant cells, and progeny thereof. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid (e.g., octoploid), diploid, haploid, and hemizygous.

[0049] The term "locus" refers to a position in the genome that corresponds to a measurable characteristic (e.g., a trait) or gene. A locus can be a genomic region or section of DNA (the locus) which correlates with a variation in a phenotype. A locus can comprise a single or multiple genes or other genetic information within a contiguous genomic region or linkage group.

[0050] A "marker" or "genetic marker" refers to a gene or nucleotide sequence that can be used to identify the presence or location of a trait determinant, locus, gene, and / or allele. A genetic marker may be described as a variation at a given genomic locus. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (for example as in a single nucleotide polymorphism (SNP)), or a longer DNA sequence, for example, a microsatellite / simple sequence repeat (SSR)). A "marker allele" refers to the version of the marker that is present in a particular individual. A genetic marker can be used to identify individuals, genes, or loci in an off-spring originating from an individual parent. An “PVR1 marker” is a genetic marker that is linked, closely linked, tightly linked, or extremely tightly linked to the OsRBL3b gene.

[0051] "Linked" refers to one or more genes or markers that are located within about 65 megabases (Mb) of one another on the same chromosome. Thus, two "linked" genes or markers may be separated, for example, by about 65 Mb, about 60 Mb, about 55 Mb, about 50 Mb, about 45 Mb, about 40 Mb, about 35 Mb, about 30 Mb, about 25 Mb, about 20 Mb, about 15 Mb, about 10 Mb, about 9.0. Mb, about 8.0 Mb, about 7.0 Mb, about 6.0 Mb, about 5.2 Mb, about 4.0 Mb, about 3.0 Mb, about 2.0 Mb, about 1.0 Mb, or fewer Mb. 12 LEGAL02 / 44913848v1

[0052] “Introgression” or “introgressing” of a PVR1 locus means introduction of a PVR1 locus from a donor plant into a recipient plant by standard breeding techniques, wherein selection can be done phenotypically or with the use of a genetic marker linked to the PVR1 locus markers through marker-assisted breeding, or combinations of these. The process of introgressing is often referred to as "backcrossing" when the process is repeated two or more times. In introgressing or backcrossing, the "donor" parent refers to the parental plant with the desired gene or locus to be introgressed. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. Selection is started in the F1 or any further generation from a cross between the recipient plant and the donor plant, suitably by using markers. The skilled person is however familiar with creating and using new molecular markers that can identify or are linked to the PVR1 locus.

[0053] A “homolog” or “homologous” sequence (e.g., nucleic acid sequence) includes a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Homologous sequences can include, for example, orthologs (orthologous sequences) and paralogs (paralogous sequences). Homologous genes, for example, typically descend from a common ancestral DNA sequence, either through a speciation event (orthologous genes) or a genetic duplication event (paralogous genes). "Orthologs" are genes and products thereof in different species that evolved from a common ancestral gene by speciation and retain the same or similar function. An ortholog is a gene that is related by vertical descent and is responsible for substantially the same or identical functions in different organisms. “Paralogous” genes include genes related by duplication within a genome. Paralogs can evolve new functions in the course of evolution.

[0054] A “heterologous” sequence is a sequence which is not normally present in a cell, genome, or gene in the genetic context in which the sequence is currently found. A heterologous sequence can be a sequence derived from the same gene and / or cell type, but introduced into the cell or a similar cell in a different context, such as on an expression vector or in a different chromosomal location or with a different promoter. A heterologous sequence can be a sequence derived from a different gene or species than a reference gene or species. A heterologous sequence can be from a homologous gene from a different species, from a 13 LEGAL02 / 44913848v1different gene in the same species, or from a different gene from a different species. For example, a regulatory sequence may be heterologous in that it is linked to a different coding sequence relative to the native regulatory sequence.

[0055] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a marker may contain the marker alone or in combination with other ingredients. The transitional phrase “consisting essentially of” means that the scope of a claim is to be interpreted to encompass the specified elements recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

[0056] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which it does not.

[0057] The term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). The term “or” refers to any one member of a particular list and also includes any combination of members of that list.

[0058] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a marker” or “at least one marker” can include a plurality of markers, including mixtures thereof.

[0059] An "RNA-guided DNA endonuclease" is an enzyme (endonuclease) that uses RNA- DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. An RNA-guided DNA endonuclease may be, but is not limited to, a zCas9 nuclease, a Cas9 nuclease, type II Cas nuclease, an nCas9 nuclease, a type V Cas nuclease, a Cas12a nuclease, a Cas12b nuclease, a Cas12c nuclease, a CasY nuclease, a CasX nuclease, a Cas12i nuclease, or an engineered RNA-guided DNA endonuclease.

[0060] A "guide RNA" (gRNA) comprises an RNA sequence (tracrRNA) bound by Cas and a spacer sequence (crRNA) that hybridizes to a target sequence and defines the genomic target to be modified. The tracrRNA and crRNA may be linked to form a "single chimeric guide RNA" (sgRNA).

[0061] The term "CRISPR RNA (crRNA)" has been described in the art (e.g., in Makarova et al. (2011) Nat Rev Microbiol 9:467-477; Makarova et al. (2011) Biol Direct 6:38; Bhaya et 14 LEGAL02 / 44913848v1al. (2011) Annu Rev Genet 45:273-297; Barrangou et al. (2012) Annu Rev Food Sci Technol 3:143-162; Jinek et al. (2012) Science 337:816-821; Cong et al. (2013) Science 339:819-823; Mali et al. (2013) Science 339: 823-826; and Hwang et al. (2013) Nature Biotechnol 31:227- 229). A crRNA contains a sequence (spacer sequence or guide sequence) that hybridizes to a target sequence in the genome. A target sequence can be any sequence that is unique compared to the rest of the genome and is adjacent to a protospacer-adjacent motif (PAM).

[0062] A "protospacer-adjacent motif" (PAM) is a short sequence recognized by the CRISPR complex. The precise sequence and length requirements for the PAM differ depending on the CRISPR system used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (i.e., target sequence). Non-limiting examples of PAMs include NGG, NNGRRT, NN[A / C / T]RRT, NGAN, NGCG, NGAG, NGNG, NGC, and NGA.

[0063] A "trans-activating CRISPR RNA" (tracrRNA) is an RNA species facilitates binding of the RNA-guided DNA endonuclease (e.g., Cas) to the guide RNA.

[0064] A "CRISPR system" comprises a guide RNA, either as a crRNA and a tracrRNA (dual guide RNA) or an sgRNA, and RNA-guided DNA endonuclease. The guide RNA directs sequence-specific binding of the RNA-guided DNA endonuclease to a target sequence. In some embodiments, the RNA-guided DNA endonuclease contains a nuclear localization sequence. In some embodiments, the CRISPR system further comprises one or more fluorescent proteins and / or one or more endosomal escape agents. In some embodiments, the gRNA and RNA- guided DNA endonuclease are provided in a complex. In some embodiments, the gRNA and RNA-guided DNA endonuclease are provided in one or more expression constructs (CRISPR constructs) encoding the gRNA and the RNA-guided DNA endonuclease. Delivery of the CRISPR construct(s) to a cell results in expression of the gRNA and RNA-guided DNA endonuclease in the cell. The CRISPR system can be, but is not limited to, a CRISPR class 1 system, a CRISPR class 2 system, a CRISPR / Cas system, a CRISPR / Cas9 system, a CRISPR / zCas9 system and a CRISPR / Cas3 system.

[0065] A “DNA donor template” is a nucleic acid, such as a single stranded DNA, linear double strand DNA, plasmid DNA or AAV sequence that provides the homology necessary for precise repair of a double-strand break. A DNA donor template contains two regions of homology, one region of homology to either side of the double strand break. A DNA donor template can also contain a heterologous sequence between the two regions of homology. Each region of homology can be about 30 to about 100 nucleotides in length. 15 LEGAL02 / 44913848v1

[0066] A "regenerant" is a plant produced from a plant tissue cell, such as a genetically modified plant tissue cell. II. Overview

[0067] To detect pathogen molecules and mount defense responses, plants rely on the wide expression of numerous genes encoding two large groups of receptors. The first of these are cell surface transmembrane domain (TMD) immune receptors and the second are intracellular immune receptors belonging to the nucleotide binding domain and leucine-rich repeat (NLR) family. Tight control of the abundance of these immune receptors and their function is crucial for hosts to maintain physiological homeostasis, avoid autoimmune diseases, and balance the trade-offs between growth and defense. Despite the importance, molecular mechanisms controlling immune receptors in specific cell types / tissues and the physiological significance of this regulation are not fully understood.

[0068] XA21 is a representative member of the cell surface TMD immune receptor group of plants and animals. The receptor kinase specifies rice (Oryza sativa) resistance to most strains of Xanthomonas oryzae pv. oryzae (Xoo) via recognition of the sulfated peptide RaXX-sY. The Gram-negative Xoo bacterium enters plants through hydathode water pores and / or wounds in leaves and roots before propagating and spreading in xylem vessels. The disease causes substantial yield loss in Asia and Africa, in particularly after the wide growing of high-yielding rice varieties in 1960s. Evidence increasingly indicates that XA21 can be proteolytically cleaved. However, neither the corresponding proteases nor the biological significance of these cleavages are known.

[0069] We show that the rhomboid protease OsRBL3b, named Protecting Physiologically Vulnerable Site from Cell Surface Immune Receptor 1 (PVR1) cleaves XA21 to regulate its levels and help maintain homeostasis. Plants carrying Xa21 but lacking a functional PVR1 displayed normal immunity but increased accumulation of XA21 in spikelets, as well as impaired anther dehiscence and pollen viability, which markedly reduced seed set. The findings reveal a conserved, widespread mechanism by which rhomboid-mediated proteolysis spatially prevents excessive accumulation of a transmembrane domain protein to avoid detrimental effects.

[0070] Described are methods and compositions for modifying male fertility in a plant comprising: modifying the activity of a PVR1 rhomboid protease. Methods disclosed herein can produce male sterile plants by reducing expression of an endogenous PVR1 gene or modifying activity of the encoded PVR1 rhomboid protease in plants. Also described are 16 LEGAL02 / 44913848v1methods of restoring male fertility in plants by providing a wild-type PVR1 gene, or repairing a loss of function mutation in a genomic PVR1 locus in a plant, in which endogenous PVR1 gene contains a mutation that decreases expression or decreases activity of the encoded PVR1 rhomboid protease. Fertility restoration plants can be generated by restoring PVR1 function of a male sterile plant for a two-line breeding system. As used herein a PVR1 rhomboid protease comprises an Oryza sativa PVR1 (SEQ ID NO: 35) or an ortholog thereof in another plant species. As used herein a PVR1 genomic locus comprises an Oryza sativa PVR1 genomic locus (SEQ ID NO: 37) or an ortholog thereof in another plant species (e.g., SEQ ID NOs: 91-95 and 98-99). As used herein a PVR1 gene comprises an Oryza sativa PVR1 gene (SEQ ID NO: 36) or an ortholog thereof in another plant species (e.g., SEQ ID NOs: 81-89).

[0071] A plant can be a diploid species, a tetraploid species, a hexaploid species, an octoploid species, or any other ploidy species or hybrid thereof. The plant can be, but is not limited to, a rice plant, a maize (corn) plant, a sorghum plant, a rye plant, a barley plant, a wheat plant, an oat plant, a sugarcane plant, a soybean plant, a canola plant, an alfalfa plant, a sunflower plant, a cotton plant, a tobacco plant, a peanut plant, a tomato plant, or a potato plant. In some embodiments, the plant is rice (Oryza sativa). An Oryza sativa plant can be any subspecies including, but not limited to Oryza sativa ssp. japonica.

[0072] “Male fertility” refers to the ability of a plant to generate functional anthers, pollen, or male gametes. A male sterile plant is incapable of fertilizing a female plant.

[0073] A “PVR1 rhomboid protease” or “PVR1” is a rhomboid protease encoded by the OsRLB3b gene or an ortholog of the OsRLB3b gene in another plant species. As used herein, OsRLB3b is also referred to as Protecting Physiologically Vulnerable Site from Cell Surface Immune Receptor 1 (PVR1). In Oryza sativa ssp. japonica, the amino acid sequence of PVR1 is SEQ ID NO: 35, and the nucleic acid sequence (PVR1 gene or PVR1) encoding the PVR1 comprises SEQ ID NO: 36. The Oryza sativa ssp. japonica PVR1 genomic sequence or locus is provided in SEQ ID NO: 37. A PVR1 rhomboid protease (PVR1) comprises Oryza sativa PVR1 (having the amino acid sequence of SEQ ID NO: 35) or an ortholog thereof in another plant species. In some embodiments, an ortholog of Oryza sativa PVR1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:35 and cleaves XA21. A PVR1 gene comprises Oryza sativa PVR1 (having the nucleic acid sequence of SEQ ID NO: 36) or an ortholog thereof in another plant species (e.g., SEQ ID NOs: 81-89). In some embodiments, an ortholog of the Oryza sativa PVR1 gene comprises a nucleic acid sequence encoding a protein having an amino acid sequence that is at least 80%, 17 LEGAL02 / 44913848v1at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:35 and cleaves XA21. A PVR1 genomic locus comprises the Oryza sativa PVR1 genomic locus (having the nucleic acid sequence of SEQ ID NO: 37) or an ortholog thereof in another plant species (e.g., SEQ ID NOs: 91-95 and 98-99). In some embodiments, an ortholog of the Oryza sativa PVR1 genomic locus comprises a nucleic acid sequence encoding a protein having an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:35 and cleaves XA21. Orthologs of Oryza sativa PVR1, the Oryza sativa PVR1 gene, or the Oryza sativa PVR1 gene locus in other plants are readily identified using methods available in the art for identifying orthologous genes.

[0074] In some embodiments, the modified rice PVR1 rhomboid protease gene has a coding sequence that has at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology, or at least 99.5% homology to SEQ ID NO: 36 and encodes a rice PVR1 rhomboid protease that cleaves XA21.

[0075] Described are constructs and systems for reducing expression of a PVR1 rhomboid protease in a plant to induce male sterility. Also described are constructs and systems for genetically modifying a PVR1 genomic locus in a plant to reduce expression of a PVR1 rhomboid protease or to decrease activity of the PVR1 rhomboid protease.

[0076] In some embodiments, genetically modifying a plant comprises creating a deletion, insertion, substitution, or frameshift mutation in the PVR1 gene wherein the mutant PVR1 encoded by the modified PVR1 gene has a reduced ability to cleave XA21.

[0077] In some embodiments, genetically modifying a plant comprises modifying an endogenous PVR1 genomic locus to reduce or knock our expression of the PVR1 gene.

[0078] In some embodiments, constructs and systems for CRISPR / Cas-mediated modification of a PVR1 genomic locus in a plant are described. The constructs and systems can be used to induce male sterility or restore male fertility in a plant.

[0079] In some embodiments, nucleic acids for modifying male fertility in a plant are described. The nucleic acids include, but are not limited to, nucleic acids comprising crRNAs or gRNAs and nucleic acids encoding crRNAs or gRNAs.

[0080] In some embodiments, nucleic acids for modifying male fertility in plants using CRISPR systems are described. The nucleic acids include, but are not limited to, nucleic acids comprising crRNAs or gRNAs, nucleic acids encoding crRNAs or gRNAs, and DNA donor template nucleic acids. 18 LEGAL02 / 44913848v1

[0081] The CRISPR systems can be used to modify an endogenous wild-type PVR1 rhomboid protease genomic locus or a mutant PVR1 rhomboid protease gene or genomic locus. The nucleic acids include, but are not limited to, nucleic acids comprising crRNAs or gRNAs, nucleic acids encoding crRNAs or gRNAs, and DNA donor template nucleic acids.

[0082] In some embodiments, the described CRISPR systems are used to modify an endogenous PVR1 genomic locus in a plant, and the resulting plants have a reduced ability to cleave XA21. In some embodiments, the modified plants have reduced male fertility. The CRISPS system can be used to create a deletion, insertion, substitution, or frameshift mutation in the PVR1 gene.

[0083] Also described are CRISPR constructs for repairing a mutant PVR1 gene or PVR1 genomic locus in a plant. In some embodiments, the CRISPR construct comprises a donor template comprising SEQ ID NO: 36 or 37 or a fragment thereof. In some embodiments, the CRISPR construct comprises a donor template comprising an ortholog of SEQ ID NO: 36 or 37 or a fragment thereof (e.g., SEQ ID NOs: 81-89, 91-95, and 98-99).

[0084] CRISPR systems for modifying an endogenous wild-type or mutant gene or gene locus are readily designed using the methods available in the art and the information described herein f.

[0085] In some embodiments, plants having a reduced male fertility phenotype produced using any one or more of the described CRISPR constructs are described. The described PVR1 genomic locus can be targeted to genetically modify plants to yield a reduced male fertility phenotype.

[0086] In some embodiments, fertility is restored to a plant having a reduced male fertility phenotype caused by loss of PVR1 function by introducing a heterologous PVR1 gene encoding a function PVR1(e.g., capable of cleaving its TMD immune receptor target in XA21). In some embodiments, fertility is restored to a plant having a reduced male fertility phenotype caused by loss of PVR1 function by repairing a mutation in an endogenous PVR1 gene, e.g., via CRISPR mediated gene repair. III. Targeted Genetic Modification

[0087] A targeted genetic modification can comprise a targeted alteration to a polynucleotide of interest including, for example, a PVR1 genomic locus (e.g., SEQ ID NO: 37 in rice or an orthologous gene in another plant), or insertion of a heterologous gene in a location suitable for expression of a heterologous gene in a plant. Such targeted modifications include, but are not limited to, additions of one or more nucleotides, deletions of one or more nucleotides, 19 LEGAL02 / 44913848v1substitutions of one or more nucleotides, a knockout of the polynucleotide of interest or a portion thereof, and a knock-in of a polynucleotide of interest or a portion thereof (e.g., a heterologous PVR1 gene, coding sequence, or fragment thereof). In certain embodiments, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more nucleotides are changed to form the targeted genomic modification.

[0088] Various methods can be used to generate the targeted modification in the polynucleotide or plant genome of interest. Methods of obtaining the targeted genetic modifications include, but are not limited to, unaided homologous recombination, recombinase-based insertion, and DNA repair-based insertion and are other methods known in the art (Dong et al., 2021, PNAS. 118(22) e2004834117). Recombinase-based insertion can comprise systems and constructs involving site-specific recombinases, including but not limited to, Cre:loxP systems, Flp:FRT systems, Dre:rox systems, VCre:loxV systems, Gin:gix systems, Bxb1:attP / attB systems, phiC31:attP / attB systems. DNA repair-based insertion methods rely upon the activity of a nuclease agent.

[0089] The term “recognition site for a nuclease agent” includes a DNA sequence at which a nick or double-strand break is induced by a nuclease agent. The recognition site for a nuclease agent can be endogenous (or native) to the cell or the recognition site can be exogenous to the cell.

[0090] The length of the recognition site can vary, and includes, for example, recognition sites that are about 30-36 bp for a zinc finger nuclease (ZFN) pair (i.e., about 15-18 bp for each ZFN), about 36 bp for a Transcription Activator-Like Effector Nuclease (TALEN), or about 20 bp for a CRISPR / Cas9 guide RNA.

[0091] In some embodiments, each monomer of the nuclease agent recognizes a recognition site of at least 9 nucleotides. In some embodiments, the recognition site is from about 9 to about 12 nucleotides in length, from about 12 to about 15 nucleotides in length, from about 15 to about 18 nucleotides in length, or from about 18 to about 21 nucleotides in length, and any combination of such subranges (e.g., 9-18 nucleotides). It is recognized that a given nuclease agent can bind the recognition site and cleave that binding site or alternatively, the nuclease agent can bind to a sequence that is different from the recognition site. Moreover, the term recognition site comprises both the nuclease agent binding site and the nick / cleavage site irrespective whether the nick / cleavage site is within or outside the nuclease agent binding site. In another variation, the cleavage by the nuclease agent can occur at nucleotide positions immediately opposite each other to produce a blunt end cut or, in other cases, the incisions can 20 LEGAL02 / 44913848v1be staggered to produce single-stranded overhangs, also called “sticky ends”, which can be either 5′ overhangs, or 3′ overhangs.

[0092] Any nuclease agent that induces a nick or double-strand break into a desired recognition site can be used in the methods and compositions disclosed herein. A naturally occurring or native nuclease agent can be employed so long as the nuclease agent induces a nick or double-strand break in a desired recognition site. Alternatively, a modified or engineered nuclease agent can be employed. An “engineered nuclease agent” includes a nuclease that is engineered (modified or derived) from its native form to specifically recognize and induce a nick or double-strand break in the desired recognition site. Producing a nick or double-strand break in a recognition site or other DNA can be referred to herein as “cutting” or “cleaving” the recognition site or other DNA. Assays to measure the double-strand break of a recognition site by a nuclease agent are known in the art (e.g., TaqMan® qPCR assay, Frendewey D. et al., Methods in Enzymology, 2010, 476:295-307, which is incorporated by reference herein in its entirety).

[0093] In some embodiments, the recognition site is positioned within a target genomic locus (e.g., a PVR1 genomic locus). Such a position can be located within a coding region of the target genomic locus, a regulatory region (e.g., a promoter or enhancer region), or an intron. In specific embodiments, a nick or double-strand break at the recognition site disrupts the activity of the target gene.

[0094] In some embodiments, the nuclease agent is a Transcription Activator-Like Effector Nuclease (TALEN). TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a prokaryotic or eukaryotic organism. TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, FokI. The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. See e.g., WO 2010 / 079430, which is herein incorporated by reference. Examples of suitable TAL nucleases, and methods for preparing suitable TAL nucleases, are disclosed, e.g., in US20110239315, US20110269234, US20110145940, US20030232410, US20050208489, US20050026157, US20050064474, US20060188987, and US20060063231, each of which is incorporated herein by reference. 21 LEGAL02 / 44913848v1

[0095] In some embodiments, the nuclease agent employed in the various methods and compositions disclosed herein comprise a zinc-finger nuclease (ZFN). Exemplary ZFNs are described in, e.g., US20060246567, US20080182332, US20020081614, US20030021776, WO2002057308, US20130123484, US20100291048, and WO2011017293, each of which is herein incorporated by reference.

[0096] In some embodiments, the nuclease agent comprises a meganuclease. Meganucleases include LAGLIDADG, GIY-YIG, H-N-H, and His-Cys box families. Exemplary meganuclease are described in, e.g., WO2005105989, WO2003078619, WO2006097854, WO2006097853, WO2006097784, and WO2004031346, each of which is herein incorporated by reference.

[0097] The nuclease agent employed in the various methods and compositions can also comprise a CRISPR / Cas system. Described are nucleic acids for producing AFR and / or ARN resistant plants using a CRISPR (e.g., CRISPR / Cas) system. The described nucleic acids can be used to target modification of the FaRCa1 locus and / or to insert or express one or more AFR and / or ARN resistance genes in a plant.

[0098] A CRISPR system comprises an RNA-guided DNA endonuclease enzyme and a CRISPR RNA. In some embodiments, a CRISPR RNA is part of a guide RNA. In some embodiments, the RNA-guided DNA endonuclease enzyme is a Cas9 protein. In some embodiments, a CRISPR system comprises one or more nucleic acids encoding an RNA- guided DNA endonuclease enzyme (such as, but not limited to a Cas9 protein) and a guide RNA. A guide RNA can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), either as separate molecules or a single chimeric guide RNA (sgRNA). The guide RNA contains a guide sequence having complementarity to a sequence in the target gene genomic region. The Cas protein can be introduced into the plant in the form of a protein or a nucleic acid (DNA or RNA) encoding the Cas protein (e.g., operably linked to a promoter expressible in the plant). The guide RNA can be introduced into the plant in the form of RNA or a DNA encoding the guide RNA (e.g., operably linked to a promoter expressible in the plant). In some embodiments, the CRISPR system further includes a DNA donor template. In some embodiments, the CRISPR system can be delivered to a plant or plant cell via a bacterium. The bacterium can be, but is not limited to, Agrobacterium tumefaciens.

[0099] In some embodiments, the CRISPR system is designed to knockout a PVR1 genomic locus, coding sequence, or fragment thereof, thereby reducing male fertility. In some embodiments, the CRISPR system is designed insert a heterologous PVR1 rhomboid protease 22 LEGAL02 / 44913848v1gene, coding sequence, or fragment thereof into the genome thereby resulting in restoration of male fertility.

[0100] In some embodiments, the CRISPR system is designed to introduce a nutation into a PVR1 genomic locus, wherein the mutation reduces or eliminates activity of the encoded PVR1 protein, thereby reducing male fertility. In some embodiments, the CRISPR system is designed introduce a mutation into a mutant PVR1 gene that encodes a PVR1 protein having reduced or no activity, wherein the mutation results in increased or restored activity of the encoded PVR1 protein, thereby resulting in restoration of male fertility.

[0101] The CRISPR / Cas system can be, but is not limited to, a CRISPR class 1 system, CRISPR class 2 system, CRISPR / Cas system, a CRISPR / Cas9 system, a CRISPR / zCas9 system or CRISPR / Cas3 system.

[0102] Guide sequences suitable for forming gRNAs or crRNAs for CRISPR system mediated genetic modification of a PVR1 rhomboid protease locus are described. Suitable guide sequences include 17-20 nucleotide sequences along the PVR1 rhomboid protease locus, that are unique compared to the rest of the genome and immediately adjacent (5′) to a protospacer-adjacent motif (PAM) site. For the RNA-guided DNA endonuclease enzyme zCas9, a PAM site is NGG. Thus, any unique 17-20 nucleotide sequence immediately 5′ of a 5′-NGG-3′ the PVR1 rhomboid protease locus can be used in forming a gRNA. In some embodiments, the guide sequence is 100% complementary to the target sequence. In some embodiments, the guide sequence is at least 90% or at least 95% complementary to the target sequence. In some embodiments, the guide sequence contains 0, 1, or 2 mismatches when hybridized to the target sequence. In some embodiments, a mismatch, if present, is located distal to the PAM, in the 5′ end of the guide sequence. In some embodiments, the gRNA comprises a sequence identical to or complementary to a sequence present in a PVR1 genomic locus. In some embodiments, the gRNA comprises a sequence identical to or complementary to a 17-20 nucleotide sequence present in a PVR1 genomic locus. In some embodiments, the gRNA comprises a sequence identical to or complementary to a sequence present in any of SEQ ID NOs: 36, 37, 81-89, 91-95, and 98-99. In some embodiments, the gRNA comprises a sequence identical to or complementary to a sequence present in SEQ ID NO: 36 or 37. In some embodiments, the gRNA comprises a sequence identical to or complementary to a 17-20 nucleotide sequence present in any of SEQ ID NOs: 36, 37, 81-89, 91-95, and 98-99. In some embodiments, the gRNA comprises a sequence identical to or complementary to a 17-20 nucleotide sequence present in SEQ ID NO: 36 or 37. 23 LEGAL02 / 44913848v1

[0103] Additional guide sequence suitable for forming gRNAs or crRNAs for CRISPR system mediated genetic modification of plants include 17-20 nucleotide sequences in the genome that are unique compared to the rest of the genome and immediately adjacent (5′) to a protospacer-adjacent motif (PAM) site and suitable for insertion of a heterologous gene. Such sites are term safe harbor sites.

[0104] The DNA donor template contains sequence to be inserted into the genome of the plant. In some embodiments, the DNA donor template to be used with a CRISPR system comprises coding sequences for a heterologous PVR1 gene. The donor template can further comprise one or more regulatory sequences operatively linked to the coding sequence that drives expression of the coding sequence in the plant cell. In some embodiments, the DNA donor template to be used with a CRISPR system comprises sequence for modifying the endogenous PVR1 genomic locus to reduce or knock out expression of the PVR1 gene or to modify the activity of the encoded PVR1. In some embodiments, the CRISPR system introduces a mutation into the genomic PVR1 rhomboid protease coding sequence that results in loss of enzymatic activity of the encoded PVR1 rhomboid protease. In some embodiments, the mutation comprises a deletion, insertion, substitution, or frameshift mutation. In some embodiments, the mutation comprises a deletion of four nucleotides or an insertion of one nucleotide in the PVR1 gene locus coding sequence at a site upstream of the catalytic serine, wherein the catalytic serine corresponds to position 187 of SEQ ID NO: 35.

[0105] DNA donor templates further comprise 5′ and 3′ homology regions located 5′ and 3′ to the sequence to be inserted into the genome. The homology regions comprise about 30 to about 100 nucleotides that are complementary to a corresponding number of nucleotides in the genome on either side of the double strand break created by the CRISPR nuclease.

[0106] The DNA donor template can be provided a single strand DNA, double strand DNA, plasmid DNA, or viral vector DNA (e.g., adeno-associated vector DNA).

[0107] It is understood that RNA equivalents of any listed DNA sequences, substituting uracils (U) for thymines (T), may be used. An "RNA equivalent" is an RNA molecule having essentially the same complementary base pair hybridization properties as the listed DNA sequence.

[0108] CRISPR modification of a PVR1 genomic locus or other genomic site is not limited to the CRISPR / zCas9 system. Other CRISPR systems using different nucleases and having different PAM sequence requirements are known in the art. PAM sequences vary by the species of RNA-guided DNA endonuclease. For example, Class 2 CRISPR-Cas type II endonuclease 24 LEGAL02 / 44913848v1derived from S. pyogenes utilizes an NGG PAM sequence located on the immediate 3′ end of the guide sequence. Other PAM sequences include, but are not limited to, NNNNGATT (Neisseria meningitidis), NNAGAA (Streptococcus thermophilus), and NAAAAC (Treponema denticola). Guide sequences for CRISPR systems having nucleases with different PAM sequence requirements are identified as described above for zCas9, substituting the different PAM sequences.

[0109] Two or more guide RNAs can used with the same RNA-guided DNA endonuclease (e.g., Cas nuclease) or different RNA-guided DNA endonucleases.

[0110] Any of the above-described guide RNAs can be provided as an RNA or a DNA encoding the RNA.

[0111] In some embodiments, a CRISPR system comprises one or more guide RNAs and a nucleic acid encoding an RNA-guided DNA endonuclease. In some embodiments, a CRISPR system comprises one or more guide RNAs, a nucleic acid encoding an RNA-guided DNA endonuclease, and a DNA donor template.

[0112] In some embodiments, a CRISPR system comprises one or more guide RNAs and a one or more nucleic acids encoding two or more different RNA-guided DNA endonucleases. In some embodiments, a CRISPR system comprises one or more guide RNAs, one or more nucleic acids encoding two or more different RNA-guided DNA endonucleases, and at least one DNA donor template.

[0113] In some embodiments, a CRISPR system comprises a guide RNA and an RNA- guided DNA endonuclease in a complex. In some embodiments, a CRISPR system comprises a guide two or more RNAs each in a complex with an RNA-guided DNA endonuclease. In some embodiments, a CRISPR system comprises a guide RNA and an RNA-guided DNA endonuclease in a complex and a DNA donor template. IV. Modified Plants

[0114] Described are genetically modified plants comprising a modified PVR1 gene or genomic locus. Also described are methods of making the genetically modified plants and plants made using the described methods. In some embodiments, the plants have reduced male fertility (i.e., male sterility). In some embodiments, the methods comprise using a CRISPR system to genetically modify an endogenous PVR1 gene or genomic locus. Thus, methods of reducing male fertility, or introducing male sterility, in a plant are described. Also described are methods for restoring male fertility in a plant in which the endogenous PVR1 gene or genomic locus was previously modified. In some embodiments, CRISPR is used to genetically 25 LEGAL02 / 44913848v1modify the plant. Other methods known in the art may also be used to modify an endogenous PVR1 genomic locus..

[0115] Described are methods of generating genetically modified male sterile or fertile male plants comprising introducing into a plant, a plant tissue, or a plant cell, one or more of the described CRISPR systems. In some embodiments, genetically modified male sterile plants created using a CRISPR system are described. In some embodiments, the CRISPR system is a CRISPR / Cas system.

[0116] In some embodiments, methods are described for producing a male sterile or fertile male plant, the methods comprising the step of introducing into the plant one or more of the described CRISPR systems.

[0117] Nucleic acids may be introduced into a plant cell or cells using a number of methods known in the art, including but not limited to electroporation, DNA bombardment or biolistic approaches, microinjection, via the use of various DNA-based vectors such as Agrobacterium tumefaciens and Agrobacterium rhizogenes vectors, and CRISPR or CRISPR / Cas9. Various methods for introducing the transgene expression vector constructs of the invention into a plant or plant cell are well known to those skilled in the art, and any method capable of transforming the target plant or plant cell may be utilized. Once a plant cell has been successfully transformed, it may be cultivated to regenerate a transgenic plant (regenerant).

[0118] In some embodiments, Agrobacterium tumefaciens is used to deliver CRISPR system nucleic acids to a plant. Agrobacterium-mediated transformation of a large number of plants are extensively described in the literature (see, for example, Agrobacterium Protocols, Wan, ed., Humana Press, 2ndedition, 2006). Various methods for introducing DNAAgrobacteria are known, including electroporation, freeze / thaw methods, and triparental mating. In some embodiments, a pMON316-based vector is used in the leaf disc transformation system of Horsch et al. Other commonly used transformation methods include, but are not limited to, microprojectile bombardment, biolistic transformation, and protoplast transformation of naked DNA by calcium, polyethylene glycol (PEG) or electroporation (Paszkowski et al., 1984, EMBO J.3: 2727-2722; Potrykus et al., 1985, Mol. Gen. Genet.199: 169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82: 5824-5828; Shimamoto et al., 1989, Nature, 338: 274- 276.

[0119] T0transgenic plants may be used to generate subsequent generations (e.g., T1, T2, etc.) by selfing of primary or secondary transformants, or by sexual crossing of primary or secondary transformants with other plants (transformed or untransformed). 26 LEGAL02 / 44913848v1

[0120] The described CRISPR systems can be used to genetically modify an endogenous PCR1 genomic locus or to introduce one heterologous PVR1 gene into a plant. The plant can be a plant having another trait of interest. Delivery of the CRISPR system leads to insertions, deletions, or substitutions near a target sequence.

[0121] The transgenic plant line can also contain one or more genes for herbicide tolerance, increased yield, insect control, other fungal disease resistance, virus resistance, bacterial disease resistance, germination and / or seedling growth control, enhanced animal and / or human nutrition, improved processing traits, or improved flavor, among others. V. Detection of a Modified Gene

[0122] Modification of a plant to modify a PVR1 genomic locus using any of the described CRISPR constructs can be detected or confirmed by any means known in the art for detecting genetic modifications.

[0123] In some embodiments, a modification can be detected in a genomic DNA sample. Genomic DNA samples include, but are not limited to, genomic DNA isolated directly from a plant, cloned genomic DNA, or amplified genomic DNA.

[0124] Genetic analysis methods include, but are not limited to, polymerase chain reaction (PCR)-based detection methods (for example, TaqMan assays), microarray methods, mass spectrometry-based methods and / or nucleic acid sequencing methods, including whole genome sequencing. In some embodiments, the detection of genetic modification in a sample of DNA, RNA, or cDNA may be facilitated through the use of nucleic acid amplification methods. Such methods specifically increase the concentration of polynucleotides that span a target site, or include that site and sequences located either distal or proximal to it. Such amplified molecules can be readily detected by gel electrophoresis, fluorescence detection methods, or other means.

[0125] In some embodiments, modification of a PVR1 genomic locus is detected by hybridization to allele-specific oligonucleotide (ASO) probes. ASO probes are described in US5,468,613 and US5,217,863, each of which is incorporated herein by reference. Single or multiple nucleotide variations in nucleic acid sequence can be detected in nucleic acids by a process in which the sequence containing the nucleotide variation is amplified, spotted on a membrane and treated with a labeled allele-specific oligonucleotide probe.

[0126] In some embodiments, a modified PVR1 genomic locus or gene is detected by a probe ligation method. Probe ligation methods are described in US5,800,944 (incorporated herein by reference) where a sequence of interest is amplified and hybridized to probes followed by ligation to detect a labeled part of the probe. 27 LEGAL02 / 44913848v1

[0127] In some embodiments, microarrays can be used for detection of a modified PVR1 genomic locus or gene. Typing of target sequences by microarray-based methods is described in US6,799,122, US6,913,879, and US6,996,476, each of which is incorporated herein by reference.

[0128] In some embodiments, a modified PVR1 genomic locus or gene can be directly identified or sequenced using nucleic acid sequencing technologies. Methods for nucleic acid sequencing are known in the art and include parallel bead arrays, sequencing by ligation, capillary electrophoresis, electronic microchips, "biochips," microarrays, parallel microchips, and single-molecule arrays.

[0129] In some embodiments, the modified PVR1 genomic locus or gene in a plant may be detected through the use of a nucleotide probe.

[0130] A polynucleotide probe may be labeled or unlabeled. A wide variety of techniques are readily available in the art for labeling a nucleotide probe. Nucleotide labels include, but are not limited to, radiolabeling, fluorophores, haptens, antibodies, antigens, enzymes, enzyme substrates, enzyme cofactors, and enzyme inhibitors. A label may provide a detectable signal by itself (e.g., a radiolabel or fluorophore) or in conjunction with other agents.

[0131] A probe may be an exact copy of a marker to be detected. A probe may also be a nucleic acid molecule comprising, or consisting of, a nucleotide sequence which is substantially identical to an inserted nucleic acid sequence. The term "substantially identical" may refer to nucleotide sequences that are more than 85% identical. For example, a substantially identical nucleotide sequence may be 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence.

[0132] A probe may also be a nucleic acid molecule that is "specifically hybridizable" or "specifically complementary" to an exact copy of the marker to be detected ("DNA target"). "Specifically hybridizable" and "specifically complementary" are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the nucleic acid molecule and the DNA target. A nucleic acid molecule need not be 100% complementary to its target sequence to be specifically hybridizable. A nucleic acid molecule is specifically hybridizable when there is a sufficient degree of complementarity to avoid non- specific binding of the nucleic acid to non-target sequences under conditions where specific binding is desired. Thus, an oligonucleotide probe is "specifically hybridizable" to a marker allele if stable and specific binding occurs between the oligonucleotide probe and the marker allele (e.g., a SNP marker) under stringent hybridization conditions, but stable and specific 28 LEGAL02 / 44913848v1binding does not occur between the oligonucleotide probe and the wild-type allele at the marker position.

[0133] In some embodiments, a probe comprises a pair of primers designed to produce an amplification product, wherein the amplification product is directly or indirectly determinative for the presence or absence of a AFR and / or ARN resistance marker. VI. Marker-Assisted Selection

[0134] Described are markers and methods for marker-assisted selection (MAS) of a PVR1 genomic locus or gene. Use of MAS in breeding programs can be used to facilitate modification of rhomboid-like protease loci into diverse genetic backgrounds.

[0135] Described is a PVR1 genomic locus encoding a rhomboid protein that cleave XA21. Loss of PVR1 activity results in reduce male fertility. The Oryza sativa PVR1 genomic locus maps to a 2.2-kb interval between 20,207,332 bp and 20,209,555 bp on chromosome 10.

[0136] PVR1 gene markers useful for genotyping (mapping, tracking, identifying, analyzing) a PVR1 genomic locus in a Oryza sativa plants are described. In some embodiments, a PVR1 gene marker comprises a detectable genetic marker linked, closely linked, tightly linked, or extremely tightly to a PVR1 genomic locus. In some embodiments, a Oryza sativa PVR1 gene marker comprises a detectable genetic marker linked, closely linked, tightly linked, or extremely tightly linked to a genomic sequence encompassed by 20,207,332 bp and 20,209,555 bp on chromosome 10 of a Oryza sativa plant. In some embodiments, a Oryza sativa PVR1 gene marker comprises a detectable genetic marker extremely tightly linked to a genomic sequence encompassed by 20,207,332 bp and 20,209,555 bp on chromosome 10 of a Oryza sativa plant. In some embodiments, a Oryza sativa PVR1 gene marker is a detectable genetic marker linked, closely linked, tightly linked, or extremely tightly linked SEQ ID NO: 37 in a Oryza sativa plant. In some embodiments, a Oryza sativa PVR1 gene marker is a detectable genetic marker extremely tightly linked to SEQ ID NO: 37.

[0137] In some embodiments, a PVR1 rhomboid protease marker comprises a PCR amplification product, a single nucleotide polymorphism (SNP), a restriction fragment length polymorphism (RFLP), an amplified fragment length polymorphism (AFLP), a simple sequence repeat (SSR), a simple sequence length polymorphism (SSLP), an insertion / deletion polymorphism (indel), a variable number tandem repeat (VNTRs), or a random amplified polymorphic DNA (RAPD) linked, closely linked, tightly linked, or extremely tightly linked to a PVR1 genomic locus. 29 LEGAL02 / 44913848v1

[0138] With the described PVR1 gene markers, plants with modified male fertility can be rapidly and efficiently identified. VII. Brief Description of the Sequences

[0139] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5′ end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3′ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. When a nucleotide sequence encoding an amino acid sequence is provided, it is understood that codon degenerate variants thereof that encode the same amino acid sequence are also provided. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.

[0140] Amino acid sequence of Oryza sativa ssp. japonica PVR1 (NCBI Reference Sequence: XP_015614650.1): MAAAAARYDVEKGGRKREGEEERCGSPAAVAQYPQREGEREWVPWLVPAILVANVVVFAVAMYVNNCPSHASRGG ACVAGFLRRFSFQPLSENPLLGPSSATLQKMGALVWDKVVHEHQGWRLVTCIWLHAGVVHLLANMLSLVLIGLRL EQQFGYMRIGIIYLVSGIGGSVLSSLFIRNSISVGASGALFGLLGAMLSELFTNWTIYTNKAAALVTLLIVIAIN LAIGILPHVDNFAHIGGFLTGFLLGFIFLMRPHYGWMQRYVLPSSVKYTSKKYLAYQWILLAVASVLAVIGFAVG LSMLFRGVNANERCHWCHYLSCIPTSRWTCGN (SEQ ID NO: 35)

[0141] DNA Coding Sequence of Oryza sativa ssp. japonica PVR1: atggcagcggcggcggcgaggtacgacgtggagaagggagggaggaagagggagggggaggaggagaggtgcggt tcgccggcggcggtggcgcagtacccgcagcgggagggggagagggagtgggtgccgtggctcgtcccggccatc ctcgtcgccaacgtcgtcgtcttcgccgttgccatgtacgtcaacaactgcccctcccacgcctctcgcggcggc gcgtgcgtcgccggattcctccgccgcttctccttccagcctctctccgagaacccgctcctcggcccctcctcc gccacattgcagaagatgggagcactggtatgggacaaggttgttcatgagcatcagggatggaggctagtgacc tgcatctggctgcacgccggtgttgttcatctgcttgccaacatgctaagcctcgtgctcatcggactcaggctc gagcagcagtttggatacatgagaattggcatcatctaccttgtttctggcattggaggcagcgttctttcatcg ctgttcatcagaaacagcatctccgtcggtgcttctggcgctctctttggtcttcttggagccatgctgtcggag cttttcaccaactggaccatatacacgaacaaggctgcagctctagtgacgctcttgatcgtcatcgcgatcaac ctcgccatcggcatcctccctcacgtcgacaacttcgcccacatcggagggttcttgactgggttcctcctcgga ttcatcttcctgatgcgcccacactacggatggatgcagcgatatgtcctgccttcctctgtcaagtacacctcc aagaagtacctggcttatcaatggatcttgctggccgtggcctcggtcctagctgtcatcggattcgccgttggg ctgtcgatgctcttcagaggggtgaatgctaatgaacgctgccattggtgccactacctgagctgcattcctacc tcgagatggacctgcgggaactga (SEQ ID NO: 36) 30 LEGAL02 / 44913848v1

[0142] Genomic DNA Sequence of Oryza sativa ssp. japonica PVR1 (NCBI Reference Sequence: NC_029265.1): agtccctgtatccaaacaccatctaaatcattctagcatttaccacattcgtattgatgttaatgaaactagaca catatatatatatatatatatatatatatatatgtatgtatgtatgtatgtatgtatataaagattcattaacat caatatgaatacgaaaaatgctagaataattcattaacattaatatgaatatgaaaaatgctagaattacactgt gaaacggaggaagtactgttaaacttgctcttgcattgcgcaaaaacaacgaggcccccctaattttgggcgtcc tggcatcttccgccgctacgtccgtcctcgtgccagctcgcgcggcggaagctgggaaaccgaacggaaccacca gtccgcgccaaaccgcgtcgcgcccctcccaccccacgcgcgcgccggaaacaacgccccaactacgcccccatg tcgccctcccctcccaccacccgcccgcttatttacccgcgtcgccaccaccaccaccaccaccactcgcagccg tttccacttcaccattcaccaacgcaaacgcaaacgcaacgcagcggcggtagacgaagcagcagcaggtagcgg ttcttggtcggttcgccatggcagcggcggcggcgaggtacgacgtggagaagggagggaggaagagggaggggg aggaggagaggtgcggttcgccggcggcggtggcgcagtacccgcagcgggagggggagagggagtgggtgccgt ggctcgtcccggccatcctcgtcgccaacgtcgtcgtcttcgccgttgccatgtacgtcaacaactgcccctccc acgcctctcgcggcggcgcgtgcgtcgccggattcctccgccgcttctccttccagcctctctccgagaacccgc tcctcggcccctcctccgccacgtaagtgctttattccttcttctcggagacttgggatgttgttcgtgatgaaa tatggggatggttcttgaatcagtagagcgttttcttgtgcacagattgcagaagatgggagcactggtatggga caaggttgttcatgagcatcagggatggaggctagtgacctgcatctggctgcacgccggtgttgttcatctgct tgccaacatgctaagcctcgtgctcatcggactcaggctcgagcagcagtttggatacagtaagcacagctttct ttgagtacaaaattactctccaaaattcactgacacgattagtggagtactgtatctgaatctgaattgacactt acgtttttcttgcagtgagaattggcatcatctaccttgtttctggcattggaggcagcgttctttcatcgctgt tcatcagaaacagcatctccgtcggtgcttctggcgctctctttggtcttcttggagccatgctgtcggagcttt tcaccaactggaccatatacacgaacaaggtgacattttcgcgcacattttctgcaccagagaagaaaacatgca tcctcacattagtcatatttcaatctgttcatgctgctacatccattcgactggtagctcaatctcatatgtatc gaaattgcaatgcaggctgcagctctagtgacgctcttgatcgtcatcgcgatcaacctcgccatcggcatcctc cctcacgtcgacaacttcgcccacatcggagggttcttgactgggttcctcctcggattcatcttcctgatgcgc ccacactacggatggatgcagcgatatgtcctgccttcctctgtcaagtacacctccaagaagtacctggcttat caatggatcttgctggccgtggcctcggtcctagctgtcatcgggtaagcaatttattaagcattacgtcttcac aaagcaaaaccttttttgtttgttatgatcttccactgattatgccgtacaaaatggagcccaatacggcaatac caatcaccatcatcctgcttgtaactgaagcagttaaactcaattgcaaattgcaagaatcgagactgttttttt tattataacattggatcatgtagatttcaatgtgtttggtatctaatttagtcaggtgggcgggcagcagagccg ttggtgttgcctcctgcctgtcccatgttcagagattcatgtgaaatccatagagcatttcaatgcagcactaga taatcattttccccatgccattgccggccttttgtttcaaggagtaacatgctcaatccagcctaacatgcaacc tgctaatatcttattgtgctccagttaatgcaagtcttttgccggccttttgtttcaaggagtatataatactgg ctatagcatttgtgtcagtatggttggtttgattcaatttttcaagtttgaactgacgattgtgctgctgacctg tgcagattcgccgttgggctgtcgatgctcttcagaggggtgaatgctaatgaacgctgccattggtgccactac ctgagctgcattcctacctcgagatggacctgcgggaactgaagatttagtagaaacgcaacaaactgaagacat taaggagatttattgaaagtgtttgatgcacagacatacaattttactgtatataacttatacagacataccctc ccaggggtttagtcagttcacttgattgattcattctgttcgtttgtctgaattatttggaatagaaacatcttc tccttgctcgcataggagtgattagcgatcttgagattcgatgcatgcagaaccgagttgccgttaccgttctgg 31 LEGAL02 / 44913848v1catagccaagtgtgggttgcggacagaatggcagtctcgtgtagattttacaccgtgtttagttccaattttttt cttcaaatttccaactttttcattacataaaaactttcttacacacacaaactttcaatttttcggtcacatcgt tctaatttcaaccaaactttcaatttttgtgtgaactaaacacagcctaaagtataaattggtttgaatggaatg caagctgtccaaaaataattatgccattagaccagcaatgctttacaccgtagtgttgcatgctttagatgccta aaaagcctgtgacaacacaatccagccacatcatcaataaaacatataagctctatggagttgcatcactatgat ttcatcatgttcaaatgaatggaattgatttcctgacctataaaacaaagcacctcaaaagcgtatctacggta (SEQ ID NO: 37) Table 1. Orthologs of SEQ ID NOs:36 and 37.

[0143] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. 32 LEGAL02 / 44913848v1EXAMPLES

[0144] The following examples are provided to illustrate certain particular features and / or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described. Example 1. Materials and Methods

[0145] (A) Plant materials and growth conditions. Rice (Oryza sativa L.) ssp. japonica cv. Taipei309 (TP309) was used to generate all transgenic lines in this study. Germination of rice seeds and growth of plants were as described previously. For phenotypic characterization of pvr1-1 Myc-Xa21L and pvr1-2 Myc-Xa21 lines, plants were first grown to the early booting stage and then moved to a temperature-controlled growth chamber (under LED light (200 μmol m–2s–1) 507 with a 13-h-light (28°C) / 11-h-dark (24°C) photoperiod and 70% relative humidity). pvr1-b 508 Myc-Xa21H and the control plants (Myc-Xa21H) were maintained by continuous ratooning in a greenhouse (29°39'55"N, 82°20'10"W) for characterization. Nicotiana benthamiana plants were grown as described previously (Shamsunnaher et al. “Rice immune sensor XA21 differentially enhances plant growth and survival under distinct levels of drought.” Sci Rep 10, 16938 (2020)). The crosses between pvr1-b Myc-Xa21H (pollen recipient) and wild-type TP309 (pollen donor) were performed by removing anthers from pvr1-b Myc-Xa21H florets followed by randomly casting pollen from TP309 onto the florets.

[0146] (B) Identification of the PVR1 gene. PVR1 was identified as an upregulated differentially expressed gene from a comparison of the transcriptomes of previously characterized plants expressing Myc-XA21-FLAG (B7-12) and the vector control (A36) under water-deficit stress conditions by RNA-seq analysis. For stress treatments, plants were grown in a shared soil-holding tray prepared with evenly distributed holes on the bottom for water uptake. The tray was maintained in a large tank filled with water in the greenhouse under natural light conditions in the winter season for 1 month. The plant tray was then transferred to a bench and watering was withheld for 20 days.

[0147] To identify DEGs between the sterile pvr1 Myc-Xa21Hand fertile Myc-Xa21Hlines, spikelets at anthesis from five plants were harvested from each line and pooled to minimize individual variation. Total RNA was extracted using TRIzol reagent (Ambion, Austin, TX, USA) according to the manufacturer’s instructions.

[0148] Leaves from five plants were harvested for each line and pooled to minimize individual variation. Total RNA was extracted using TRIzol reagent (Ambion) according to the manufacturer’s instructions. The isolated total RNA was treated with RNase-free DNase I 33 LEGAL02 / 44913848v1(Qiagen) to eliminate genomic DNA contamination, followed by purification using a RNeasy MinElute Cleanup Kit (Qiagen). The purified RNA was submitted to the Center for Cancer Computational Biology at Dana Farber Cancer Institute for RNA-seq library construction and sing-end (50 base read length) sequencing using the HiSeq 2000 platform (Illumina).

[0149] For RNA-seq analysis, around 40 million reads were generated per sample. After trimming for adapters using the cutadapt program, the reads were aligned to the Oryza sativa ‘Nipponbare’ reference genome (Y. Kawahara et al., Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice (N Y) 6, 4 (2013)) using TopHat version 2.013 (C. Trapnell, et al., TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105-1111 (2009)). Ambiguous reads that mapped to more than one region in the genome or those with a MAPQ score below 10 were removed by samtools software. Transcript quantification was carried out with the Partek Genomics Suite (version 6.4, Partek Inc) to obtain raw read counts and normalized read counts (Reads per kilobase per million mapped reads [RPKM]) (A. Mortazavi, et al., Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5, 621-628 (2008)). Differential gene expression was analyzed using generalized linear model approaches implemented in the BioConductor package edgeR (D.J. McCarthy, et al., Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res 40, 4288-4297 (2012)). Significantly differentially expressed genes were selected based on the following criteria: absolute fold-change of over 2, P value below 0.05, and at least one sample with RPKM value greater than 1. Genes with a fold-change greater than or equal to 2 were considered upregulated, while those with a fold-change less than or equal to −2 were considered down-regulated.

[0150] (C) RT-qPCR analysis. Total RNA was extracted using TRIzol reagent (Ambion) followed by purification using an RNAeasy MiniElute Cleanup Kit (Qiagen) according to the manufacturer’s instructions. Single-stranded cDNA was synthesized with 2 µg total RNA using a QuantiTect Reverse Transcription Kit (Qiagen). qPCR was performed using a LightCycler 480 System (Roche) according to the manufacturer’s instructions under the following conditions: 95 °C, 2 min; (95 °C, 5 sec; 60 °C, 20 sec) × 45 cycles, 72 °C, 5 min. The gene expression levels were calculated by the ΔΔCt method using the geometric mean of rice UBIQUITIN5 and ACTIN expression levels to normalize the data.

[0151] (D) Plasmid constructs. 34 LEGAL02 / 44913848v1

[0152] (i) PVR1pro:GUS. A 2,497-bp promoter fragment of the PVR1 gene was PCR- amplified with the primers OsRhmbd17P-6New / OsRhmbd17P-2New and cloned into the binary vector pCmH-GUS using HindIII and BglII restriction sites.

[0153] (ii) Xa21pro:GUS. A 2,204-bp promoter fragment of the Xa21 gene was PCR- amplified with Xa21Pro-1 / Xa21Pro-2 and cloned into the binary vector pCmH-GUS using HindIII-BamHI. The promoter fragment used here was shown to be sufficient for directing XA21-mediated resistance to Xoo (W. Y. Song et al., A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 270, 1804-1806 (1995)) and reduced seed setting in pvr1 Myc-Xa21 mutants.

[0154] (iii) Myc-XA21-FLAG (Agrobacterium tumefaciens-mediated infiltration). The construct was made by using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs). The XB3Ank sequence of the construct pC1300SH-XB3Ank-3xFLAG (X. Huang, et al., Members of the XB3 family from diverse plant species induce programmed cell death in Nicotiana benthamiana. PLoS One 8, e63868 (2013)) was replaced with the 5' and 3' halves of the coding region of Xa21 amplified with primer pairs XA21N-1 / XA21N-2 and XA21N- 3 / XA21N-4, respectively.

[0155] (iv) HA-PVR1 (Agrobacterium-mediated infiltration). RT-PCR was used to amplify the PVR1 coding sequence from total RNA isolated from rice cultivar TP309 using primers PVR1N-1 / PVR1N-2. The resulting cDNA was cloned into the binary vector pC1300S- 3xHA using BamHI and SpeI restriction sites.

[0156] (v) HA-PVR1Cat(Agrobacterium-mediated infiltration). Site-directed mutagenesis was used to replace the Ser-187 residue of PVR1 with alanine using primers PVR1S187A- 1 / PVR1S187A-2 and the plasmid HA-PVR1 above as template.

[0157] (vi) HA-OsRBL3a (Agrobacterium-mediated infiltration). RT-PCR was used to amplify the OsRBL3a coding sequence from total RNA isolated from rice cultivar TP309 using primers OsRBL3a-1 / OsRBL3a-2. The resulting cDNA was cloned into the binary vector pC1300S-3xHA using BamHI and SpeI restriction sites.

[0158] (vii) XA21P652L,P655L(Agrobacterium-mediated infiltration). A Xa21 fragment of 1,084 bp encoding a protein possessing the Pro-652-to-Leu652 and Pro-655-to-Leu-655 mutations in the transmembrane domain was synthesized. The resulting fragment was used to replace the corresponding region of Myc-XA21-FLAG using BamHI and DraII restriction sites.

[0159] (viii) XA21P652Land XA21P655L(Agrobacterium-mediated infiltration). NEBuilder® HiFi DNA Assembly Master Mix was used to generate the mutant constructs. Xa21 fragments 35 LEGAL02 / 44913848v1containing each mutation were individually PCR-amplified and used to replace the corresponding region of Myc-XA21-FLAG digested with BamHI and HindIII. The primers used for the amplification were XA21P652L, XA21N-5 / XA21N-6 and XA21N-7 / XA21N-8; and XA21P655L, XA21N-5 / XA21N-9 and XA21N-10 / XA21N-8.

[0160] (ix) GFP-PVR1 (subcellular localization). eGFP was PCR-amplified with primers eGFP-3 / eGFP-4 and cloned into HA-PVR1 using SacI and BamHI restriction sites.

[0161] All the constructs used in this study have been verified by DNA sequencing. All the primers used in this study are listed in Table 2. Table 2. Primer sequences for PCR, qPCR, and cloning.36 LEGAL02 / 44913848v137 LEGAL02 / 44913848v1

[0162] (E) Histochemical GUS staining. GUS staining was carried out as described (P. Anbu, L. Arul, Beta glucuronidase activity in early stages of rice seedlings and callus: A comparison with Escherichia coli beta glucuronidase expressed in the transgenic rice. International Journal for Biotechnology and Molecular Biology Research 4, 52-59 (2013)). Freshly collected spikelets were immersed in GUS staining solution (100 mM sodium phosphate pH 7.0, 10 mM EDTA, 0.1% [vol / vol] Triton X-100, 1 mM potassium ferrocyanide, 1 mM potassium ferricyanide, 20% [vol / vol] methanol, 2 mM X-Gluc), vacuum-infiltrated for 10 min, and incubated overnight at 37°C. The stained samples were incubated in 100% (v / v) ethanol for 10 hours to remove the chlorophylls.

[0163] (F) Rhomboid cleavage of XA21 in N. benthamiana. Infiltration of N. benthamiana with Agrobacterium strain EHA105 harboring each construct of interest for transient gene expression was performed as previously described (Huang X et al. “Members of the XB3 family from diverse plant species induce programmed cell death in Nicotiana benthamiana.” PLoS One 8, e63868 (2013)). Overnight bacterial cultures were harvested by centrifugation ina microfuge at 4,000 g for 10 min at room temperature. Harvested cells wereresuspended ininfiltration buffer (10 mM MES pH 5.6, 10 mM MgCl2, and 150 mM acetosyringone), adjusted to an OD600of 0.5, and incubated at room temperature for 3 hours. Infiltration was carried out using the leaves of 4-week-old N. benthamiana plants with 1-ml needleless syringes. Infiltrated leaf tissues were collected at 40 hours post infiltration for protein extraction using extraction buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol [v / v], 0.5% [v / v] Triton X-100, 2 mM EDTA, 2% [w / v] polyvinylpolypyrrolidone [PVPP], 2 mM DTT, and 1 mM 38 LEGAL02 / 44913848v1phenylmethylsulfonyl fluoride). Protein concentration was determined using a Pierce™ BCAProtein Assay Kit.

[0164] To generate an antibodyrecognizingthe C-terminal portion of XA21, the intracellulardomain of XA21 was expressed as a glutathione S-transferase fusion in Escherichia coli and the purified fusion protein was used as immunogen in mice. Approximately 20 μg plant protein per lane was resolved by SDS-PAGE and analyzed by immunoblotting using 1:5,000 anti-Myc (9E10, UF-Hybridoma), 1:1,000 α-XA21ICD (this study), and 1:000 anti-HA (3F10, Roche). The immune complexes were detected using horseradish peroxidase–linked whole antibody (NXA931, Sigma) and Amersham ECL Prime Western Blotting Detection Reagent according to the manufacturer’s instructions.

[0165] (G) Immunoblot analysis of rice samples.Immunoblot analysis was performed asdescribed previously (Xu WH et al. “The autophosphorylated Ser686, Thr688, and Ser689 residues in the intracellular juxtamembrane domain of XA21 are implicated in stability control of rice receptor-like kinase.” Plant J 45, 740-751 (2006)). Total protein was extracted by grinding approximately 100 mg rice samples in liquid nitrogen and thawing them in 100 μL extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% [v / v] Triton X- 100, 5% [v / v] β-mercaptoethanol, 0.1 mM DTT, 100 mM PMSF, and complete protease inhibitors [Roche]). Cell debris was removed by centrifugation at 12,000 g for 10 min at 4 °C. Protein concentration was determined with a Pierce™ BCA Protein Assay Kit. Approximately 20 μg protein per lane was subjected to immunoblot assays using 1:2,000 anti-Myc (9E10, UF- Hybridoma) and 1:10,000 anti-ATPase (Agrisera) antibodies. Protein samples subjected to immunoblot assays using anti-ATPase antibodies were heated at 58 °C before loading.

[0166] (H) Generation of rice pvr1 mutants by the CRISPR / Cas9 system.Homozygous pvr1mutant plant lines (pvr1-1 and -2) were generated in TP309 using CRISPR / Cas9.

[0167] A single guide RNA (sgRNA)targetinga site upstream of the sequence encoding thecatalytic Ser-187 residue of PVR1 was selected, frame-shift mutations generated by the CRISPR construct cause premature termination of translation, leading to a truncated PVR1 lacking protease activity (FIG. 1A-B). A synthetic double-stranded fragment for the spacer sequence of the sg RNA was cloned at the BsaI site in the binary vector pRGEB32Xh-Bar that was constructed by replacing the selective marker hygromycin resistance gene (hptII) of the CRISPR vector pRGEB32 (addgene, plasmid #63142) with a Basta resistance gene (bar). The resulting construct was transformed into rice cultivar TP309 or the line 4021-3 (containing the 39 LEGAL02 / 44913848v1Myc-Xa21 and hptII genes) using Agrobacterium-mediated transformation according to the procedure described previously, except that transgenic lines were selected using bialaphos.

[0168] To identifymutations, genomic DNA was isolated from the transgenic lines generatedabove using the cetyl trimethylammonium bromide (CTAB) method. PCR was carried out to amplify the targeted PVR1 region using primers PVR1CR-1 / PVR1CR-2 followed by DNA sequencing.

[0169] (I) Subcellular localization of PVR1 using rice protoplasts. Protoplasts were isolated from 8-day-old, dark-grown seedlings of rice cultivar TP309 as described (Y. Zhang et al., A highly efficient rice green tissue protoplast system for transient gene expression and studying light / chloroplast-related processes. Plant Methods 7, 30 (2011)). Sixteen hours after transfection with DNA constructs of interest, the protoplasts were visualized using a Zeiss LSM800 confocal laser scanning microscope. FM4-64 staining was performed by incubating the dye (final concentration 10 μM) with transfected protoplasts for 10 min at room temperature. eGFP and FM4-64 were excited with the 488-nm laser line. Fluorescence emissions were captured at 410–535 nm for eGFP and 650–700 nm for FM4-64. Images were analyzed using ZEN 2.0 software.

[0170] (J) Xoo inoculation. The incompatible Xoo strain PXO99Awas used to inoculate 2- week-old rice seedlings by the leaf-clipping method. Bacterial culture was suspended into water, and the concentration was adjusted to an OD600= 0.5. Inoculated seedlings were incubated in water under fluorescent light at 27 °C. Lesion length was scored at 12 days post inoculation (dpi) and seedling survival was determined at 5 weeks post inoculation. Diseased leaves were curved and displayed grayish color at 12 dpi.

[0171] (K) Spikelet fertility, pollen viability, and anther dehiscence assays. Spikelet fertility was determined based on the number of filled and empty seeds of representative panicles from at least three plants per line.

[0172] Spikelets were dissected and assayed as described (S. Thangasamy et al., Rice SIZ1, a SUMO E3 ligase, controls spikelet fertility through regulation of anther dehiscence. New Phytol 189, 869-882 (2011)). For pollen viability analysis, approximately 10 anthers from five plants per line were randomly collected and squashed in a centrifuge tube. The released pollen grains were stained with 1% (w / v) iodine-potassium iodide (I2-KI) and visualized using an Olympus BX43 LED Fluorescence Microscope (Olympus). For anther dehiscence / indehiscence observations, spikelets were collected 2 h or 10 days after anthesis (DAA). Following dissection, the spikelets were submerged in I2-KI solution and visualized 40 LEGAL02 / 44913848v1using an Olympus microscope as above to count the number of pollen grains on the stigmas. Anther indehiscence of a spikelet was scored when I2-KI-stained pollen grains were seen inside the anthers, but not on the stigmas. Anther dehiscence was defined as when more than five pollen grains were observed on the stigmas, and weak anther dehiscence was defined as when fewer than five pollen grains were seen on the stigmas.

[0173] To confirm the presence of pollen grains in the anthers, cross sections of anthers were obtained by paraffin embedding followed by sectioning. Collected anthers were fixed in Dietrich’s Formalin Acetic Acid for 16 h at room temperature. Fixed samples were processed with a Leica tissue processor. After embedding in paraffin, samples were sectioned to 10 μm with a Rotary Microtome (HM 355 S) and visualized using the Olympus microscope as above.

[0174] Statistical analysis was performed in R Studio v.4.1.2. One-way analysis of variance (ANOVA) was performed using the agricolae R package. Post_hoc comparisons were done using Tukey’s honestly significant difference test (HSD) for significance.

[0175] (L)Alkaline phosphatase (AP)-TMD shedding assay. Stable PVR1 and PVR1S187Acelllines were generated by transfecting the pcDNA3.1-PVR1 and pcDNA3.1-PVR1S187Aconstructs (see above), respectfully, into HEK293 cells (ATCC, Manassas, VA, USA), followed by selecting with G418 (500 ng / ml) for 3 weeks. Stable clones were expanded as individual cell lines and maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic-antimycotic (ThermoFisher Scientific). AP-TMD shedding assays were performed in 96-well plates as described (Grieve AG et al. "Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation." Mol Cell 81, 4784-4798 (2021)). Briefly, The HEK293 cells that stably expressed PVR1 or PVR1S187Awere transfected with the pcDNA3.1_TMDscreen-TMDs constructs (30 ng each) using lipofectamine 2000 reagent (Invitrogen) according to manufacturer’s instructions. Cells were left for 24 h to express protein, and then exchanged into 200 ml optiMEM overnight. Supernatants were collected. Cell lysates were extracted using Triton X-100 buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris (pH 7.5), 10 μg / ml aprotinin and 10 μg / ml leupeptin). AP activity was detected in the supernatants or cell lysates by adding equal volumes of p-nitrophenyl phosphate (PNPP buffer) followed by measurement of absorbance at 405 nm using a BioTek Microplate Reader (Agilent Technologies, Inc. Santa Clara, CA). The percentage of the total material shed from each well (i.e., signal from supernatant divided by total signal from lysate and supernatant) was then used to calculate AP release. 41 LEGAL02 / 44913848v1

[0176] (M) Quantification and statistical analysis. Quantifications of Western blots were performed using ImageJ. Volcano blots were generated using SRPLOT. One-way ANOVA tests were performed to compare multiple groups of data. Student’s t-tests were carried out to compare two independent groups of data. Example 2. XA-21-dependent expression of OsRBL3b

[0177] XA21 has been previously shown to confer tolerance to water-deficit stress. Transcriptome deep sequencing (RNA-seq) analysis identified OsRBL3b (PVR1) as being up- regulated by water-deficit stress in transgenic plants expressing XA21 (XA21 plants) relative to control plants (Table 3). The OsRBL3b has seven putative TMDs and belongs to the RhoA1 subfamily of rice rhomboid-like proteases (OsRBLs) that are orthologous to members of the mammalian RhoA2 subfamily (e.g., human rhomboid-like proteases RHBDL1-3) based on phylogenetic analysis (FIGs.2A-2C and 3) (Li Q et al. “Differential evolution of members of the rhomboid gene family with conservative and divergent patterns.” New Phytol 206, 368-380 (2015)). Topological analysis suggested that OsRBL3b has catalytic sites located close to the extracellular side of its TMD4 and TMD6, which would allow the cleavage of substrates atsites near the N termini of their TMDs (FIGs. 2A-C).To identify the subcellular localizationof OsRBL3b, we performed fluorescence imaging of the rice protoplasts transfected with aGFP (green fluorescent protein)-OsRBL3b construct.Similar to the mammalian RHBDL2,OsRBL3b was determined to reside in the plasma membrane of rice protoplasts transfectedasconfirmed by its colocalization with FM4-64 plasma-membrane marker (FIGs.4A-B). Because XA21 resides in the plasma membrane, we predicted that XA21 may be a substrate of OsRBL3b (PVR1). Table 3: Top 29 upregulated differentially expressed genes in XA21 plants (B7-12) relative to the control (A36) in response to water-deficit stress.42 LEGAL02 / 44913848v1*The predicted Os10g37760.2 sequence from the MSU Rice Genome Annotation Project Release 7 is incorrect, missing the N-terminal part of PVR1. Os10g0521900 as predicted in the Rice Annotation Project Database (RAP-DB) is confirmed by our RT-PCR analysis. Example 3. Preferential expression of OsRBL3b / PVR1 in spikelets

[0178] Microarray datafromRiceXpro showed that OsRBL3b transcripts accumulatedhighly in spikelets, but declined rapidly after anthesis (FIG.5). By contrast, OsRBL3b mRNAs were markedly less abundant in the leaves at both the vegetative and reproductive stages. Notably, five other rice RhoA1 genes (OsRBL3a, OsRBL3c, OsRBL3e, OsRBL1, and OsRBL4) also showed spatiotemporal expression, including in the leaf sheath, root, stem, inflorescence, anther, pistil, lemma / palea, ovary, embryo, and / or endosperm, but low transcript levels werefound in the leaf blades, which is the major site for plant–microbe interactions.These findingstogether with our previous hypothesis on XA21 cleavage led to the prediction that OsRBL3b functions in the rice spikelet to minimize accumulation of the immune receptor and maintain 43 LEGAL02 / 44913848v1physiological homeostasis in vulnerable sites. We therefore renamed OsRBL3b as Protecting Physiologically Vulnerable Site from Cell Surface Immune Receptor 1 (PVR1).

[0179] To test ourhypothesis, we used RT-qPCR to measure the expression of PVR1 in apreviously characterized transgenic line (U0-137) expressing a construct encoding an N-terminal c-Myc-tagged XA21 (Myc-XA21) from the native promoter at a relatively low level (Myc-Xa21L). We detected high PVR1 transcript levels specifically during flower development, but not in rice leaf tissues at distinct developmental stages (FIG.6A). We then confirmed that the XA21 protein is readily detectable in the spikelet of this line as well as another previously characterized line (4021-3) expressing the same construct at a higher level (Myc-Xa21H) (FIG. 6B). We generated reporter lines harboring the PVR1 or Xa21 promoter driving the β-glucuronidase (GUS) reporter gene. In these lines, expression of PVR1 and Xa21 in the rice spikelet largely overlapped, although PVR1 was specifically expressed in the central core of the anther and the style (FIG.7). Table 4. Expression profiling of rice RhoA1 members with tissue-specific expression in rice44 LEGAL02 / 44913848v1AntherExample 4. PVR1 mediates cleavage of XA21.

[0180] To determine whether XA21 can be cleaved by PVR1, we co-expressed constructs encoding epitope-tagged XA21 (Myc-XA21-FLAG) (FIG.8A) and PVR1 (HA-PVR1) fusion proteins in Nicotiana benthamiana. We observed cleavage of XA21 in the infiltrated leaf tissues in a PVR1-dependent manner (FIG.8B, D). Mutation of the catalytic serine residue of PVR1 (Ser-187) to alanine abolished cleavage (FIG. 8B). By contrast, OsRBL3a, which is closely related to PVR1 (FIG.3), was unable to cleave XA21. These results demonstrate that XA21 is a substrate of PVR1. Example 5. Proline residues in the TMD of XA21 are required for PVR1 cleavage. 45 LEGAL02 / 44913848v1

[0181] Helical instability caused by the presence of helix-breaking residues (e.g., proline and glycine) at the N-terminal half of TMDs is a hallmark of rhomboid substrates and has been used as a criterion to predict new substrates. XA21 possesses two prolines, Pro-652 and Pro- 655, in this region (FIG.8C). Replacing Pro-652 with the hydrophobic residue leucine slightly decreased the cleavage of XA21 by PVR1, while changing Pro-655 to leucine markedly compromised cleavage (FIGs. 8C and 9). Mutation of both Pro-652 and Pro-655 to leucine fully abolished cleavage. Example 6. PVR1 is required for seed set in rice carrying XA21 immune receptor.

[0182] Xa21 was originally identified from the wild rice species Oryza longistaminata, and remains absent from most of the domestic wild-type cultivars, including the susceptible line O. sativa ssp. Japonica var.Taipei309 (TP309). To investigate the biological function of PVR1- mediated cleavage of XA21, we generated homozygous pvr1 mutants (pvr1-1 and -2) in TP309 using clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9-mediated gene editing. The mutants we obtained carried frameshift mutations resulting in premature termination of translation; if they accumulated, these truncated PVR1 products lacked the catalytic Ser-187 residue (FIG. 1A-B). Thus, they likely harbored loss-of-function alleles of PVR1. As a control, we obtained wild-type siblings (WTsib) segregated from the heterozygous pvr1-1 PVR1 line for characterization. To generate XA21 plants with PVR1 mutations, we then crossed pvr1-1 and pvr1-2 to Myc-Xa21L(hygromycin resistant). F1 plants were selfed to produced progeny. We identified homozygous pvr1-1 Myc-Xa21Land pvr1-2 Myc-Xa21Llines (pvr1 Myc-Xa21L) in the F4 generation by hygromycin selection for Myc-Xa21Land by PCR genotyping followed by sequencing pvr1 mutations. In addition, we transformed the same CRISPR / Cas9 construct into Myc-Xa21Hand recovered one biallelic mutant line, pvr1-b Myc- Xa21H.

[0183] In the absence of Xoo, the pvr1 Myc-Xa21Llines showed markedly reduced grain set when grown under low temperature seasons (i.e., spring or fall) in a greenhouse or in a temperature-controlled growth chamber (24–28 °C) (Average rates of seed-set for the pvr1 Myc-Xa21Lwas ~31%) compared to >83% for the controls (WTsib, pvr1-1, pvr1-2, and Myc- Xa21L) (FIGs. 10A, C). In summer, the seed-setting rates of pvr1 Myc-Xa21Lplants were similar to those of the controls (FIGs.11A-D). The temperature sensitivity of pvr1Myc-Xa21Lfertility can be explained by our previous findings showing that low-temperature treatment promotes XA21 functions without significantly affecting its abundance. The pvr1-2 and Myc- Xa21Lplants showed slightly lower seed-setting rates (~83% and ~87%, respectively) than did 46 LEGAL02 / 44913848v1WTsibor pvr1-1 (~96% and ~97%, respectively) when grown in a growth chamber at the same low temperature (FIGs.10A, C). Apart from the fertility defect, pvr1 Myc-Xa21Lplants grew normally (FIG.10B). These findings indicate that PVR1 is required for normal seed setting in plants carrying XA21.

[0184] The pvr1-b Myc-Xa21Hmutant was completely sterile in all seasons over seven growth years (FIGs.12A-B). Of note, transgenic rice lines ectopically expressing Xa21-GFP from the maize ubiquitin promoter displayed normal seed set. It has been shown that non- specific promoters may not provide strong tissue-specific expression of target genes. Our observations suggest that the impact of Xa21 on seed set requires the expression of this immune receptor gene in specific tissues.

[0185] The pvr1 Myc-Xa21Llines showed markedly reduced grain set when grown under low temperature seasons (i.e., some springs or falls) in a greenhouse or in a temperature-controlled growth chamber at low temperatures [24 / 28 °C (dark / light)]. The average rates of grain-set for the pvr1 Myc-Xa21Lwas ~31% compared to > 83-97% for the controls (WTsib, pvr1-1, pvr1-2, and Myc-Xa21L) (FIGs.10A-C). In summer, the grain-setting rates of pvr1 Myc-Xa21Lplants were similar to those of the controls). These observations are in line with the temperature- sensitive nature of XA21 signaling (Chen QH et al. "Reversible activation of XA21-mediated resistance by temperature." European Journal of Plant Pathology 153, 1177-1184 (2019)). The pvr1-2 line and Myc-Xa21Lline showed slightly lower grain-setting rates (~83% and ~87%, respectively) than did WTsibor pvr1-1 (~96% and ~97%, respectively) when grown in a growth chamber at the same low temperatures (FIG.10C). Apart from the fertility defect, pvr1 Myc- Xa21Lplants grew normally (FIG. 10B). These findings indicate that PVR1 is required for normal grain setting in plants carrying XA21 and that the influence of XA21 on rice grain set is dose dependent.

[0186] We inoculated two-week-old seedlings of pvr1 Myc-Xa21Lwith the incompatible Xoo strain PXO99A. As expected, PVR1 mutations did not significantly affect XA21-mediated resistance as compared to Myc-Xa21L(FIGs. 10H-I). In contrast, the susceptible control line WTsib(lacking Xa21) was later killed by bacterial infection at the seedling stage (FIG.13). Example 7. PVR1 is negatively regulates XA21 accumulation in the rice spikelet.

[0187] Consistent with the cleavage presented above (FIG. 8), XA21 protein levels were higher in the spikelets in all the pvr1 mutants than in wild-type (FIGs. 10D-E, 12C-D). We failed to detect XA21 cleavage intermediates using antibodies that recognize either the N- or C-terminal regions of Myc-XA21, very likely due to further degradation of the products. RT- 47 LEGAL02 / 44913848v1qPCR analysis showed that Xa21 transcripts accumulated to comparable levels in the spikelets of pvr1 Myc-Xa21 and Myc-Xa21 (FIGs. 10F, 12E). These findings indicate that PVR1 negatively regulates steady-state accumulation of XA21 in spikelets and that rice reproduction is sensitive to excessive accumulation of XA21. In leaves, regardless of the presence of a functional PVR1, XA21 abundance and resistance against Xoo were not significantly affected (FIGs. 10F-H, 13). By contrast, the susceptible control line WTsib(lacking Xa21) was later killed by bacterial infection at the seedling stage (FIG.13). Example 8. PVR1 is required for XA21-dependent pollen viability and dehiscence in rice.

[0188] To understand mechanisms underlying the partial sterility of pvr1 Myc-Xa21 lines, we first examined the starch contents of their pollen grains, a commonly used marker for pollen viability. Under low-temperature conditions, the partially sterile pvr1 Myc-Xa21Lmutants produced fewer viable pollen grains (~38% for pvr1-1 Myc-Xa21Land ~34% for pvr1-2 Myc- Xa21L) relative to the more fertile controls (> 82% for WTsib, pvr1-1, pvr1-2 and Myc-Xa21L) (FIGs.14A, 15B). The sterile line pvr1-b Myc-Xa21Hproduced only ~18% viable pollen grains (FIG.15A). These findings indicate that higher levels of XA21 impair starch accumulation in pollen grain in a dose-dependent manner.

[0189] To locate the defect(s) that causes pvr1 Myc-Xa21 sterility, we tested whether the female organ of pvr1-b Myc-Xa21Hremains functional by crossing the sterile pvr1-b Myc- Xa21H(pollen recipient) to the wild-type TP309 that can supply functional pollen. The mutant plants were able to set seeds, indicating that sterility of the pvr1-b Myc-Xa21His mainly due to defects in its male organ. Since the sterile pvr1-b Myc-Xa21Hproduced some viable pollen grains, we examined the ability of anthers from this mutant to release pollen at anthesis (anther dehiscence), which is essential for pollination and fertilization. None of the spikelets dissected from the pvr1-b Myc-Xa21Hmutant dehisced 2 hours after anthesis (HAA) (n > 200) or even at 10 days after flowering (n > 30) (FIG.12B). Pollen was still detectable in the anthers of this line. In agreement with the indehiscent anthers, we observed no pollen grains on the stigmas of the dissected spikelets from pvr1-b Myc-Xa21Hmutant plants. Thus, anther indehiscence alone is sufficient to explain the sterility of pvr1-b Myc-Xa21Hplants.

[0190] We next examined the partially sterile lines pvr1 Myc-Xa21Land their controls (WTsib, pvr1-1, pvr1-2 and Myc-Xa21L) grown in the growth chamber at 24–28 °C. Among more than 100 randomly chosen spikelets, indehiscent ones were rare in the controls (≤ 5%) (FIG.14A, C). By contrast, ~25–28% of spikelets from the pvr1 Myc-Xa21Llines were indehiscent at 2 HAA. Higher percentages of weakly dehiscent spikelets (defined as ≤ 5 pollen grains / stigma) 48 LEGAL02 / 44913848v1were also observed from the pvr1 Myc-Xa21L lines than from the controls. Collectively, our findings demonstrate that impaired anther dehiscence was a major reason for reduced seed setting in these two mutants. Additional fertility defects (e.g., reduced pollen viability) likely also contribute to the phenotype. Example 10. Upregulation of NLR genes and downregulation of JA signaling and responsive genes in the sterile pvr1-b Myc-Xa21Hspikelets.

[0191] To gain the molecular insights into elevated XA21-triggered male sterility, we performed transcriptome profiling on the sterile pvr1 Myc-Xa21Hand fertile Myc-Xa21Hlines using spikelets at anthesis collected from plants grown in the greenhouse during summer (optimal for rice growth). A total of 6331 differentially expressed genes (DEGs) were identified between these two lines using an FDR (False Discovery Rate) value of < 0.05 and log2FC cut- off criteria of >1 and <−1. Among the 473 annotated as NLR receptors in the rice reference genome, 399 were detected in our RNA-seq analysis. Interestingly, 66 of these genes were found to be significantly upregulated in the sterile pvr1-b Myc-Xa21Hspikelets (FIG.17A). In contrast, only 12 NLRs were found as downregulated. These findings demonstrate that a large number of NLR genes are transcriptionally auto-activated in the pvr1 mutant spikelets with elevated XA21 protein levels.

[0192] We also observed downregulation of nine JA-responsive and signaling genes from distinct families (FIG. 17B), namely the genes encoding five JASMONATE ZIM DOMAIN (JAZ) transcriptional repressors, the CORONATINE INSENSITIVE1a (COI1a) JA receptor and the transcription factors OsbHLH148, RERJ1, and OsWRKY71, in pvr1-b Myc-Xa21Hrelative to Myc-Xa21H. The downregulation of seven of these genes with RPKM (reads per kilo base per million mapped reads) values > 1 was verified by qRT-PCR analysis (FIG.17C). JA signaling has been implicated in positively regulating pollen development and anther dehiscence in plants. Of note, none of the genes encoding the rest of the JA receptors (i.e., OsCOI1b and OsCOI2) or the other JAZ repressors identified in rice were upregulated in the anther indehiscent line (FIG.17B). These results indicate that JA signaling is compromised in the spikelets of the sterile pvr1-b Myc-Xa21Hmutant during anthesis. Example 11. PVR1 can cleave canonical TMDs of a subset of plant RLKs.

[0193] Canonical TMDs contain predominantly hydrophobic amino acids to maintain an α- helical structure. Non-canonical TMDs with helix-breaking residues (e.g., proline and glycine) at their N-terminal half are a hallmark of rhomboid substrates, which has been used as a 49 LEGAL02 / 44913848v1criterion to predict rhomboid cleavage targets. XA21 possesses two prolines, Pro-652 and Pro- 655, in this region. Replacing Pro-652 with the hydrophobic residue leucine slightly decreased the cleavage of XA21 by PVR1, while changing Pro-655 to leucine markedly compromised the cleavage (FIG. 8C). Mutation of both Pro-652 and Pro-655 to leucine residues fully abolished cleavage (FIG.8C).

[0194] We tested whether PVR1 also cleaves other non-canonical TMDs using an alkaline phosphatase (AP)-based colorimetric assay (Grieve AG 2021). The TMDs of seven well- studied Arabidopsis RLKs were individually fused in frame with the C-terminus of AP containing an N-terminal signal peptide, allowing the expressed fusion protein to be localized to the plasma membrane. Rhomboid-mediated cleavage of the TMDs will result in a release and accumulation of AP in the extracellular medium, which can be detected by a colorimetric assay. To validate the cleavage assay, we co-expressed PVR1 with the XA21TMD or the uncleavable mutant XA21TMDP652L, P655Lin mammalian HEK 293 cells. Consistent with the cleavage data from the N. benthamiana-based system, PVR1 hydrolyzed XA21TMD, but not XA21TMDP652L, P655L(FIG. 18). Furthermore, the catalytically deficient mutant PVR1S187Afailed to cleave the XA21TMD.

[0195] As shown in FIG.18, PVR1 was also found to cleave the TMDs of the receptor kinase AtBRI1 and its co-receptor AtBAK1 / AtSERK3, both involved in perceiving the steroid hormone brassinolide (BR) during cell elongation. The other identified PVR1 substrates included the TMDs of Arabidopsis AtEFR, AtCERK1 and AtSERK2. RLK-SERK complexes are a common theme in plant signaling that is of central importance to hormone and immune responses. Over-accumulation of AtBAK1 / AtSERK3 in Arabidopsis causes autoimmune cell- death and developmental defects, such as growth arrest and reduced grain production. Under our conditions, PVR1 failed to cleave the TMDs of AtFLS2 and AtLYK5, which both act as immune receptors. Therefore, PVR1 and its plant orthologs are likely to cleave multiple RLKs. Discussion

[0196] Based on our data above, we have envisioned a model for organ-specific control of the immune receptor XA21 in rice. We propose that XA21 acts as a quantitative mediator that can drastically reduce fertility largely via impairing floret functions, including anther dehiscence and pollen viability (FIG.16). XA21 is also a substrate of the rhomboid-like PVR1 protease and the resulting cleavage triggers degradation of the immune receptor. In spikelets, it is the abundant PVR1 that prevents the excessive accumulation of XA21 over a threshold that would otherwise interfere with fertility. In leaves, PVR1 has little influence on XA21 50 LEGAL02 / 44913848v1accumulation, as PVR1 transcript levels are low throughout development, which ensures host immunity at the main entry site of Xoo. Thus, differential expression of PVR1 to fine-tune localized levels of XA21 solves the survival-or-reproduction dilemma in the major food crops.

[0197] We identified spikelets as a site vulnerable to abundant levels of XA21 protein yet protected by the rhomboid protease PVR1 in rice. Mutations leading to loss of PVR1 function caused markedly increased accumulation of XA21 protein, which coincided with elevated XA21-triggered male sterility, the upregulation of 66 NLR genes, and the downregulation of nine JA-responsive and -signaling genes in the absence of Xoo. In addition to PVR1, temperature markedly influenced the manifestation of reduced seed setting in the pvr1 Myc- Xa21Llines. We have shown that levels of the XA21 protein in spikelets are crucial for XA21- induced male sterility. Thus, our results provide the first evidence that over-accumulation of a cell-surface immune receptor can trigger autoimmunity in plants.

[0198] Our findings thus indicate that PVR1 plays a crucial role in preventing autoimmune responses in the spikelets of XA21-expressing plants.

[0199] Like PVR1, five other rice RhoA1 genes show specific spatiotemporal expression, including the leaf sheath, root, stem, inflorescence, anther, pistil, lemma / palea, ovary, embryo, and / or endosperm, but low transcript levels in leaf blades, the major battleground for plant– microbe interactions (FIG.5). Together, these data suggest that members of RhoA1 rhomboids might be part of a cellular proteostasis network that spatiotemporally controls the level of potentially many immune-related or even unrelated TMD proteins at multiple sites in rice and other plants. Notably, the rice fungal resistance gene PigmR, which encodes an NLR receptor, also has a negative effect on seed set through unknown mechanisms (Deng Y et al. “Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance.” Science 355, 962-965 (2017)). Moreover, in a naturally occurring resistant line, the PigmR-mediated yield penalty is counteracted by PigmS, a susceptible, pollen / panicle-expressed member of the Pigm gene family whose product (PigmS) is capable of attenuating PigmR homodimerization. It is thus likely that rice has evolved distinct mechanisms to protect its reproduction from the penalties caused by the cell surface and intracellular immune receptors.

[0200] Male-sterile plants form the basis of hybrid rice breeding for high yields (Chen L et al. “Male sterility and fertility restoration in crops.” Annu Rev Plant Biol 65, 579-606 (2014)). The pvr1-b Myc-Xa21Hmutant plants are completely sterile during all growth seasons. The pvr1 Myc-Xa21Lmutants, with lower levels of XA21, are capable of producing seeds. Thus, 51 LEGAL02 / 44913848v1the pvr mutant plants identified herein could be used in plant breeding programs and in the development of both male-sterile and fertility-restoration plants for a two-line breeding system.

[0201] Rice is a staple food for more than half of the human population, represents 50% of the agronomic biomass produced worldwide, and serves as an important model for cereal crops, including rice, maize, wheat, barley, and sorghum. The pvr mutations identified for rice are predicted to have similar effects in other grain crops. 52 LEGAL02 / 44913848v1

Claims

Claims:

1. A genetically modified plant comprising a modified PVR1 gene.

2. The genetically modified plant of claim 1, wherein the genetic modification comprises a deletion, insertion, substitution, or frameshift mutation of the PVR1 gene.

3. The genetically modified plant of claim 2, wherein the genetic modification results in decreased expression of the PVR1 gene.

4. The genetically modified plant of claim 2, wherein the genetic modification results in decreased activity of a protein encoded by the PVR1 gene.

5. The genetically modified plant of claim 4, wherein the genetic modification comprises a deletion, insertion, substitution, or frameshift mutation at a catalytic serine in the encoded PVR1 rhomboid protease.

6. The genetically modified plant of any one of claims 1-5, wherein the genetically modified plant has decrease male fertility.

7. The genetically modified claim on any one of claims 1-6, wherein the plant is a rice plant.

8. A CRISPR system for decreasing male fertility in a plant an RNA-guided DNA endonuclease or a nucleic acid encoding the RNA-guided DNA endonuclease and a guide RNA (gRNA) or a nucleic acid encoding the gRNA into the plant, wherein the RNA-guided DNA endonuclease and the gRNA form a complex that targets the genomic PVR1 locus.

9. The CRISPR system of claim 8, wherein the CRISPR system comprises a CRISPR class 1 system, a CRISPR class 2 system, a CRISPR / Cas system, a CRISPR / Cas9 system, a CRISPR / zCas9 system or a CRISPR / Cas3 system.

10. The CRISPR system of claim 8 or 9, wherein the gRNA comprises a sequence that hybridizes with a sequence present in a genomic PVR1 locus.

11. The CRISPR system of claim 10, wherein the gRNA comprises a sequence that hybridizes with a sequence present in any of SEQ ID NOs: 36, 37, 81-89, 91-95, and 98-99, or a complement thereof, or an ortholog thereof. 53 LEGAL02 / 44913848v112. The CRISPR system of any one of claims 8-11, wherein the gRNA comprises SEQ ID NO:66 or an RNA equivalent thereof.

13. A method of modifying male fertility in a plant, the method comprising modifying activity of a PVR1 rhomboid protease in a plant cell.

14. The method of claim 13, wherein modifying activity of a PVR1 rhomboid protease comprises: (a) reducing expression of an endogenous PVR1 gene in the plant; or (b) reducing activity of a PVR1 rhomboid protease in the plant.

15. The method of claim 14, wherein reducing expression of the PVR1 gene comprises: (a) introducing an RNA interference (RNAi) molecule or an antisense nucleotide targeting the PVR1 mRNA into the plant or expressing an RNAi nucleic acid sequence or an antisense nucleic acid sequence targeting the PVR1 mRNA in the plant; (b) genetically modifying the plant to form a deletion, insertion, substitution, or frameshift mutation in an endogenous PVR1 gene.

16. The method of claim 14, wherein reducing activity of a PVR1 rhomboid protease comprises genetically modifying an endogenous PVR1 gene in the plant, wherein the genetic modification results in loss of enzymatic activity of the PVR1 rhomboid protease encoded by endogenous PVR1 gene.

17. The method of claim 16, wherein the genetic modification comprises a deletion, insertion, substitution, or frameshift mutation at a catalytic serine in the encoded PVR1 rhomboid protease.

18. The method of claim 17, wherein the genetic modification comprises a deletion of four nucleotides or an insertion of one nucleotide of the endogenous PVR1 gene at a site upstream of the catalytic serine, wherein the catalytic serine corresponds to position 187 of SEQ ID NO:

35.

19. The method of any one of claims 15-18, wherein genetically modifying the endogenous PVR1 gene comprises introducing a CRISPR system into a plant cell, wherein the CRISPR system comprises an RNA-guided DNA endonuclease or a nucleic acid encoding the RNA-guided DNA endonuclease and a gRNA or a nucleic acid encoding the 54 LEGAL02 / 44913848v1gRNA into the plant, wherein the RNA-guided DNA endonuclease and the gRNA form a complex that targets the PVR1 rhomboid protease genomic locus.

20. The method of claim 19, wherein the CRISPR system is selected from the group consisting of: a CRISPR class 1 system, a CRISPR class 2 system, a CRISPR / Cas system, a CRISPR / Cas9 system, a CRISPR / zCas9 system and a CRISPR / Cas3 system.

21. The method of claim 19 or 20, wherein the gRNA comprises a sequence that hybridizes with a sequence present in any of SEQ ID NOs: 36, 37, 81-89, 91-95, and 98-99, or a complement thereof, or an ortholog thereof.

22. The CRISPR system of claim 21, wherein the plant is a rice plant and the gRNA comprises SEQ ID NO:66 or an RNA equivalent thereof.

23. The method of any one of claims 13-22, wherein the plant has reduced anther dehiscence and / or pollen viability compared to a similar plant activity of the PVR1 rhomboid protease has not been modified.

24. The method of claim 23, wherein the mutation reduces the viability of pollen grains of the modified plant by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

25. The genetically modified plant of any one of claims 1-6, the CRISPR system of any one of claims 8-12, or the method of any one of claims 13-21, 23 or 24, wherein the plant is a rice plant, maize plant, sorghum plant, rye plant, barley plant, wheat plant, oat plant, sugarcane plant, soybean plant, canola plant, alfalfa plant, sunflower plant, cotton plant, tobacco plant, peanut plant, tomato plant, or a potato plant. 55 LEGAL02 / 44913848v1

Images