Compositions and methods for conferring resistance to pestalotia leaf spot and fruit rot of strawberries

Abstract

Described are genetically modified strawberry plants that have increased resistance to Pestalotia Leaf Spot and Fruit Rot caused by Neopestalotiopsis. Also described are RNAi and CRISPR systems that can be used to generate the genetically modified strawberry plants.

Description

Compositions and Methods for Conferring Resistance to Pestalotia Leaf Spot and Fruit Rot of StrawberriesCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U. S. Provisional Application No. 63 / 642,244, filed May 3, 2024, which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. 2022-51181- 38328 awarded by The United States Department of Agriculture, National Institutes of Food & Agriculture. The government has certain rights in the invention.

[0003] This invention was made in whole or in part from funding received under a contract from the Florida Strawberry Research & Education Foundation.SEQUENCE LISTING

[0004] The Sequence Listing written in file T19295W0001_SeqListing.xml is 56.5 kilobytes in size, was created April 21, 2025, and is hereby incorporated by reference.BACKGROUND

[0005] Pestalotia Leaf Spot and Fruit Rot is disease caused by infection with Neopestalotiopsis. a fungal pathogen. Various species of Neopestcilotiopsis have been known to affect strawberries but had previously been considered a weak pathogen mostly infecting stressed plants. Since 2018, more severe outbreaks of Pestalotia Leaf Spot and Fruit Rot have been observed in strawberries in Florida and have rapidly spread throughout the southeast. The fungus spreads rapidly under rainy conditions causing lesions and necrosis on leaves and fruit of strawberry7plants. Once introduced in a field, Neopestcilotiopsis can survive in the strawberry residue materials until the following season. Severe crop losses have been reported in many commercial fields. The popular ‘Florida Brilliance’ cultivar is highly susceptible to the new pathogen, which has been identified as a more virulent and aggressive strain of Neopestcilotiopsis .

[0006] Little is known about strawberry' resistance mechanisms to Neopestalotiopsis infections. Pestalotia Leaf Spot and Fruit Rot poses significant challenges to the commercial strawberry industry'. There remains an unmet need for the identification of potential gene targets for resistance against Neopestalotiopsis infections and new strawberry' varieties that are less susceptible to Pestalotia Leaf Spot and Fruit Rot.SUMMARY

[0007] Pestalotia Leaf Spot and Fruit Rot, caused by Neopestalotiopsis spp.. is an emerging pathogen affecting strawberry (Fragaria) production. Described are compositions and methods for generating genetically modified Fragaria plants that are resistant to Neopestalotiopsis .Described are specific genes responsible Neopestalotiopsis susceptibility' in Fragaria plants. In diploid Fragaria vesca the genes include FvH4_2g29450 (also termed FvOCP3) and FvH4_2g29460 (also termed FvEDRN2). In octaploid Fragaria x ananassa the genes include Fxa2Agl 724640, Fxa2Bg958120, Fxa2Cg808210, Fxa2Dg2512710 (homeologs of FvH4_2g29450), Fxa2Ag 1724630, Fxa2Cg808220, and Fxa2Dg2512700 (homeologs of FvH4_2g29460) (cultivar Florida Brilliance (Fb): Genome Database of Rosaceae, Fragaria x ananassa 'Florida Brilliance' Genome vl.O Assembly & Annotation (Han et al. 2025 GigaScience, Volume 14, 2025, giaf005)) or Fxa2AglO2916, Fxa2Bg202737, Fxa2Cg201149, Fxa2Dg202538 (homeologs of FvH4_2g29450), Fxa2AglO2917, Fxa2Cg201148, and Fxa2Dg202539 (homeologs of FvH4_2g29460) (cultivar Royal Royce (Rr): Royal Royce vl.O, phytozome genome ID: 701. NCBI taxonomy ID: 3747 reference genome).

[0008] Described are genetically modified plants in which the plants have decreased expression of (a) a FyH4_2g29450 gene and / or one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or (b) a FvH4_2g29460 gene and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs. In some embodiments, the Fragaria plant is a diploid Fragaria vesca plant having decreased expression of the FvH4 2g29450 gene and / or the FvH4_2g29460 gene. In some embodiments, the Fragaria plant is a polyploid Fragaria plant having decreased expression of one or more Fragaria FvH4_2g29450 orthologs or homeologs and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs. In some embodiments, the Fragaria plant is an octoploid Fragaria x ananassa plant having decreased expression of (a) one or more Fragaria FvH4_2g29450 homeologs selected from the group consisting of: a Fxa2Agl 724640 gene (Florida Brilliance (Fb)) or Fxa2AglO2916 gene (Royal Royce (Rd)), a Fxa2Bg958120 gene (Fb) or Fxa2Bg202737 gene (Rr), a Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr), and a Fxa2Dg2512710 gene (Fb) or Fxa2Dg202538 gene (Rr); and / or (b) one or more Fragaria FvH4_2g29460 homeologs selected from the group consisting of: a Fxa2Agl 724630 gene (Fb) or Fxa2Agl02917 gene (Rr), a Fxa2Cg808220 gene (Fb) or Fxa2Cg201148 gene (Rr), and a Fxa2Dg2512700 gene (Fb) or Fxa2Dg202539 gene (Rr). In some embodiments, the Fragaria plant is an octoploid Fragaria x ananassa plant having decreased expression of (a) the Fxa2Agl724640 gene (Fb) or Fxa2Agl02916 gene (Rr), the Fxa2Bg958120 gene (Fb) or Fxa2Bg202737 gene (Rr), the Fxa2Cg808210 gene (Fb) orFxa2Cg201149 gene (Rr), and the Fxa2Dg2512710 gene (Fb) or Fxa2Dg202538 gene (Rr); and / or (b) the Fxa2Agl724630 gene (Fb) or Fxa2AglO2917 gene (Rr), the Fxa2Cg808220 gene (Fb) or Fxa2Cg201148 gene (Rr), and the Fxa2Dg2512700 gene (Fb) or Fxa2Dg202539 gene (Rr).

[0009] In some embodiments, the genetically modified Fragaria plants have increased resistance to Pestalotia Leaf Spot and Fruit Rot caused by infection w Neopestalotiopsis spp. compared to control Fragaria plants that do not have decreased expression of the FvH4_2g29450 gene and / orthe one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or the FvH4_2g29460 gene and / or the one or more Fragaria FvH4_2g29460 orthologs or homeologs.

[0010] In some embodiments, the Fragaria plant expresses: (a) one or more RNAi constructs targeting the FvH4_2g29450 gene and / or the one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or (b) one or more RNAi constructs targeting the FvH4_2g29460 gene and / or the one or more Fragaria FvH4_2g29460 orthologs or homeologs. In some embodiments, the Fragaria plant expresses: (a) an RNAi construct targeting the Fxa2Agl 724640 gene (Fb) or Fxa2AglO2916 gene (Rr). the Fxa2Bg958120 gene (Fb) or Fxa2Bg202737 gene (Rr), the Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr), and the Fxa2Dg2512710 gene (Fb) or Fxa2Dg202538 gene (Rr); and / or (b) an RNAi construct targeting the Fxa2Agl 724630 gene (Fb) or Fxa2Agl 02917 gene (Rr), the Fxa2Cg808220 gene (Fb) or Fxa2Cg201148 gene (Rr), and the Fxa2Dg2512700 gene (Fb) or Fxa2Dg202539 gene (Rr). In some embodiments, the Fragaria plant expresses one more RNAi constructs comprising SEQ ID NO: 25 and / or SEQ ID NO:26. In some embodiments, a single RNAi construct targets (inhibits expression of) the Fxa2Agl724640 gene (Fb) or Fxa2Agl02916 gene (Rr) (SEQ ID NO: 11). the Fxa2Bg958120 gene (Fb) or Fxa2Bg202737 gene (Rr) (SEQ ID NO: 13), the Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr) (SEQ ID NO: 15), and the Fxa2Dg2512710 gene (Fb) or Fxa2Dg202538 gene (Rr) (SEQ ID NO: 17). In some embodiments, a single RNAi construct targets (inhibits expression of) the Fxa2Agl 724630 gene (Fb) or Fxa2AglO2917 gene (Rr) (SEQ ID NO: 19), the Fxa2Cg808220 gene (Fb) or Fxa2Cg201148 gene (Rr) (SEQ ID NO: 21). and the Fxa2Dg2512700 gene (Fb) or Fxa2Dg202539 gene (Rr) (SEQ ID NO: 23).

[0011] In some embodiments the genetically modified Fragaria plant has a loss of function mutation in (a) the FvH4_2g29450 gene and / or the one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or (b) the FvH4 2g29460 gene and / or the one or more Fragaria FvH4_2g29460 orthologs or homeologs. In some embodiments, the loss of function mutationis a CRISPR-induced loss of function mutation. In some embodiments, the Fragaria plant is genetically modified through the use of a CRISPR / Cas system. In some embodiments, the Fragaria plant is genetically modified through the use of a CRISPR / Cas system at (a) the Fxa2Agl 724640 gene (Fb) or Fxa2AglO2916 gene (Rr), the Fxa2Bg958120 gene (Fb) or Fxa2Bg202737 gene (Rr), the Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr), and the Fxa2Dg2512710 gene (Fb) or Fxa2Dg202538 gene (Rr); and / or (b) the Fxa2Agl724630 gene (Fb) or Fxa2AglO2917 gene (Rr). the Fxa2Cg808220 gene (Fb) or Fxa2Cg201148 gene (Rr). and the Fxa2Dg2512700 gene (Fb) or Fxa2Dg202539 gene (Rr). In some embodiments, the Fragaria plant contains a deletion at: (a) the FvH4_2g29450 gene and / or the one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or (b) the FvH4_2g29460 gene and / or the one or more Fragaria FvH4 2g29460 orthologs or homeologs. In some embodiments, the Fragaria plant is an octoploid Fragaria x ananassa plant and contains a deletion at: (a) one or more of: a Fxa2Agl724640 gene (Fb) or Fxa2AglO2916 gene (Rr), a Fxa2Bg958120 gene (Fb) or Fxa2Bg202737 gene (Rr), a Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr), and a Fxa2Dg2512710 gene (Fb) or Fxa2Dg202538 gene (Rr); and / or (b) one or more of: a Fxa2Agl724630 gene (Fb) or Fxa2AglO2917 gene (Rr), a Fxa2Cg808220 gene (Fb) or Fxa2Cg201148 gene (Rr), and a Fxa2Dg2512700 gene (Fb) or Fxa2Dg202539 gene (Rr).

[0012] Described are methods for increasing resistance to Pestalotia Leaf Spot and Fruit Rot in Fragaria plants comprising decreasing expression of (a) a FvH4 2g29450 gene and / or one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or (b) a FvH4_2g29460 gene and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs in the plant or a progenitor of the plant. In some embodiments, decreasing expression comprises introducing into the plant or a progenitor of the plant one or more expression vectors encoding one or more RNAi constructs. In some embodiments, the one or more RNAi constructs comprises SEQ ID NO: 25 and / or SEQ ID NO: 26.

[0013] In some embodiments, decreasing expression comprises introducing a CRISPR system into a Fragaria plant cell, wherein the CRISPR system targets: (a) the FvH4_2g29450 gene and / or one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or (b) the FvH4_2g29460 gene and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs. In some embodiments, the CRISPR system targets one or more of: a FvH4_2g29450 gene (SEQ ID NO: 7), a FvH4_2g29460 gene (SEQ ID NO: 9), a Fxa2Ag 1724640 (Fb) or Fxa2AglO2916 gene (Rr) gene (SEQ ID NO: 11), a Fxa2Bg958120 gene (Fb) or Fxa2Bg202737 gene (Rr) (SEQ ID NO: 13), a Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr) (SEQ ID NO: 15),aFxa2Dg2512710 gene (Fb) orFxa2Dg202538 gene (Rr) (SEQ ID NO: 17), a Fxa2Agl 724630 gene (Fb) or Fxa2AglO2917 gene (Rr) (SEQ ID NO: 19), a Fxa2Cg808220 gene (Fb) or Fxa2Cg201148 gene (Rr) (SEQ ID NO: 21), and a Fxa2Dg2512700 gene (Fb) or Fxa2Dg202539 gene (Rr) (SEQ ID NO: 23). In some embodiments, a single CRISPR system targets the Fxa2Agl724640 gene (Fb) or Fxa2AglO2916 gene (Rr) (SEQ ID NO: 11), the Fxa2Bg958120 gene (Fb) or Fxa2Bg202737 gene (Rr) (SEQ ID NO: 13), the Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr) (SEQ ID NO: 15), and the Fxa2Dg2512710 gene (Fb) or Fxa2Dg202538 gene (Rr) (SEQ ID NO: 17). In some embodiments, a single CRISPR system targets the Fxa2Agl724630 gene (Fb) or Fxa2AglO2917 gene (Rr) (SEQ ID NO: 19), the Fxa2Cg808220 gene (Fb) or Fxa2Cg201148 gene (Rr) (SEQ ID NO: 21), and the Fxa2Dg2512700 gene (Fb) or Fxa2Dg202539 gene (Rr) (SEQ ID NO: 23).

[0014] Provided are nucleic acids for increasing resistance to Pestalotia Leaf Spot and Fruit Rot in a Fragaria plant comprising an RNAi construct targeting: (a) a FvH4_2g29450 gene and / or one or more Fragaria FvH4_2g29450 orthologs or homeologs; or (b) a FvH4_2g29460 gene and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs. In some embodiments, the nucleic acid comprises SEQ ID NO: 25 or SEQ ID NO: 26.BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1 depicts screening candidate resistance genes against emerging Neopestalotiopsis spp with T-DNA knockout mutants in Arabidopsis. (A) Phenotypes observed 7 days post-inoculation (dpi) with Neopestalotiopsis in plug trays. (B) Analysis of the necrosis rate post-fungal inoculation using ImageJ software. Statistical analysis was performed using one-way ANOVA.

[0016] FIG. 2 depicts the multiple alignment of amino acid sequences for Neopestalotiopsis resistance-related candidate gene AT5G11270 (AtOCP3) with the orthologs in F. vesca and F. x ananassa (cv. "Royal Royce').

[0017] FIG. 3 depicts the multiple alignment of amino acid sequences for Neopestalotiopsis resistance-related candidate gene AT5G11280 (uncharacterized gene) with the orthologs in F. vesca and F. x ananassa (cv. 'Royal Royce').

[0018] FIG. 4 depicts disease symptoms on octoploid strawberry leaves with empty vector or FaOCP3 transient expression following a Neopestalotiopsis inoculation assay.

[0019] FIG. 5. FIG. 5. RNAi -mediated knock-down of OCP3 or EDRN2 reduced disease severity in ‘Florida Brilliance'. Representative images of F. ^ananassa cv. Florida Brilliance plants with RNAi-mediated silencing of OCP3 and EDRN2. Empty vector controls showed severe disease symptoms (susceptible), while OCP3 RNAi-treated plants exhibited reduced symptoms (resistant). EDRN2 RNAi-treated plants displayed higher levels of resistance.

[0020] FIG. 6A. Representative images control F. ^ananassa cv. Florida Brilliance plants illustrating and susceptibility to Neopestalotiopsis.

[0021] FIG. 6B. Representative images of F ^ananassa cv. Florida Brilliance plants with RNAi-mediated silencing of V .OCP3 illustrating increased resistance to Neopestalotiopsis .

[0022] FIG. 6C. Representative images of F ananassa cv. Florida Brilliance plants with RNAi-mediated silencing of FaEDRN2 illustrating increased resistance to Neopestalotiopsis.

[0023] FIG. 7 depicts a graph of the fungal entry rate of octoploid strawberry leaves with empty vector or FaOCP3 transient expression following ^Neopestalotiopsis inoculation assay.

[0024] FIG. 8. RNAi-mediated knock-down of OCP3 or EDRN2 reduced disease severity in ‘Florida Brilliance’. Disease severity quantification using ImageJ analysis. Empty vector controls had the highest disease score, while OCP3 and EDRN2 RNAi-treated plants showed lower scores. The double knockdown of OCP3 and EDRN2 resulted in the highest reduction of disease symptoms.DETAI ED DESCRIPTION1. Definitions

[0025] Unless otherwise defined, all terms of art, notations, and other scientific terminology used herein are intended to have the meanings commonly understood by those ofskill 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 cloning 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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 analogues 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 (<?.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.

[0030] 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.

[0031] The term “expression vector” or “expression construct” or “expression cassette” refers to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell or organism. Nucleic acid sequences necessary for expression in prokary otes usually include a promoter, an operator (optional), and a ribosome binding site, as well as other sequences. Eukaryotic cells are generally known to utilize promoters, enhancers, and termination and polyadenylation signals, although some elements may be deleted, and other elements added without sacrificing the necessary expression.

[0032] The terms "identical" or percent "identify," in the context of tw o 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% identify, preferably 75%, 80%, 85%, 90%, or 95% identify over a specifiedregion, 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.

[0033] 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 number 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 (z.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.

[0034] The term “isolated” with respect to proteins, nucleic acids, and cells includes proteins, nucleic acids, and cells that are relatively purified with respect to other cellular or organism components that may normally be present in situ, up to and including a substantially pure preparation of the protein, nucleic acid, or cell. The term “isolated” may include proteins and nucleic acids that have no naturally occurring counterpart or proteins or nucleic acids that have been chemically synthesized and are thus substantially uncontaminated by other proteins or nucleic acids. The term “isolated” may include proteins, nucleic acids, or cells that have been separated or purified from most other cellular components or organism components with which they are naturally accompanied (e.g., but not limited to, other cellular proteins, nucleic acids, or cellular or extracellular components).

[0035] The term “in vitro” includes artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube or an isolated cell or cell line). The term “in vivo” includes natural environments (e.g.. a cell or organism or body) and to processes or reactions that occur within a natural environment. The term “ex vivo” includes cells that have been removed from the body of an individual and processes or reactions that occur within such cells.

[0036] 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 isgenerally 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).

[0037] The terms “elite breeding population” or “elite genotype” or “elite line” refer to specific genotypes of plants that have been bred by plant breeding programs to have similar essential alleles for desirable end use characteristics, agronomics, disease resistance, and adaptation in the target region in which they are to be grown.

[0038] 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.

[0039] 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.

[0040] "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.

[0041] "Closely linked" refers to one or more genes or markers that are located within about 2.0 Mb of one another on the same chromosome. Thus, two closely linked genes or markers may be separated, for example, by about 2.00 Mb, about 1.95 Mb, about 1.90 Mb, about 1.85 Mb, about 1.80 Mb, about 1.75 Mb, about 1.70 Mb, about 1.65 Mb, about 1.60 Mb, about 1.55Mb, about 1.50 Mb, about 1.45 Mb, about 1.40 Mb, about 1.35 Mb, about 1.30 Mb, about 1.25Mb, about 1.20 Mb, about 1.15 Mb, about 1.10 Mb, about 1.05 Mb. about 1.00 Mb. about 0.95Mb, about 0.90 Mb, about 0.85 Mb, about 0.80 Mb, about 0.75 Mb, about 0.70 Mb, about 0.65Mb, about 0.60 Mb, about 0.55 Mb, about 0.50 Mb, about 0.45 Mb, about 0.40 Mb, about 0.35 Mb, about 0.30 Mb, about 0.25 Mb, about 0.20 Mb, about 0. 15 Mb, about 0. 10 Mb. about 0.05 Mb, about 0.025 Mb, or about 0.01 Mb

[0042] "Tightly linked" refers to one or more genes or markers that are located within about 1.0 Mb of one another on the same chromosome. Thus, two tightly linked genes or markers may be separated, for example, by about 1.00 Mb, about 0.95 Mb, about 0.90 Mb, about 0.85 Mb, about 0.80 Mb, about 0.75 Mb, about 0.70 Mb, about 0.65 Mb. about 0.60 Mb. about 0.55 Mb about 0.5 Mb, about 0.45 Mb, about 0.4 Mb, about 0.35 Mb, about 0.3 Mb, about 0.25 Mb, about 0.2 Mb, about 0.15 Mb, about 0.1 Mb, or about 0.05 Mb.

[0043] "Extremely tightly linked" refers to one or more genes or markers that are located within about 100 kb of one another on the same chromosome. Thus, two extremely tightly linked genes or markers may be separated, for example, by about 100 kb, about 95 kb, about 90 kb, about 85 kb, about 80 kb, about 75 kb, about 70 kb, about 65 kb, about 60 kb, about 55 kb, about 50 kb, about 45 kb, about 40 kb, about 35 kb, about 30 kb, about 25 kb, about 20 kb, about 15 kb, about 10 kb, about 5 kb, or about 1 kb.

[0044] “Introgression” or “introgressing” of a locus means introduction of a locus from a donor plant comprising the locus of interest into a recipient plant by standard breeding techniques, wherein selection can be done phenotypically by means of observation of a plant characteristic including, but not limited to, characteristics such as the intemodal length or plant height, or selection can be done with the use of markers through marker-assisted breeding, or combinations of these. The process of introgressing is often referred to as "backcrossing" when the process is repeated tw o 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 Fl or any further generation from a cross between the recipient plant and the donor plant, suitably by using markers as identified herein. The skilled person is, how ever, familiar with creating and using new molecular markers that can identify or are linked to a locus of interest.

[0045] 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).

[0046] An “ortholog” or “orthologous” genes refers to genes in different species that evolved from a common ancestral gene by speciation. Orthologs ty pically retain the same function in the course of evolution.

[0047] A “paralog” or “paralogous” genes refers to genes related by duplication within a genome. Paralogs can evolve new functions in the course of evolution.

[0048] A “homeolog” or “homeologous” genes refer to genes found in the same species that originated by a speciation event. Homeologs can evolve in polyploidy species from allopolyploidization. a whole-genome duplication via hybridization followed by genome doubling. For example, an octaploid strawberry may contain four homeologous genes that evolved from allopolyploidization, which are homologous to a single gene in the diploid strawberry.

[0049] The term “polyploidy” refers to an organism with more than one pair of chromosomes. Polyploid plants include tetrapioid, pentapioid, hexapioid, octoploid and decaploid plants.

[0050] 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.”

[0051] “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.

[0052] 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.

[0053] 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.

[0054] The term "Neopestalotiopsis spp” or "Neopestalotiopsis ” refers to a genus of fungal pathogens in the Sporocadaceae family. A more aggressive strain of Neopestalotiopsis has recently been identified as a pathogen causing severe outbreaks of Pestalotia Leaf Spot and Fruit Rot in strawberries.

[0055] “Pestalotia Leaf Spot and Fruit Rot” refers the disease caused in strawberry by infection with a Neopestalotiopsis species which has recently become commercially significant. The disease affects the roots, crown, and leaves of strawberry plants, resulting in lesions on fruit and blight-like necrosis of leaves. The fungus thrives in warm and humid conditions and can cause complete plant collapse and death.

[0056] A “AtOCP3” or “AT5G11270” gene refers to the gene encoding OVEREXPRESSOR OF CATIONIC PEROXIDASE 3, ahomeodomain transcription factor in Arabidopsis thaliana located at 3595274-3597237 bp on chromosome 5.

[0057] A “AT5G11280” gene refers to the gene encoding an uncharacterized protein in Arabidopsis thaliana located at 3597216-3598687 bp on chromosome 5.

[0058] A “FvH4_2g29450” or “FaOCP3” gene refers to the ortholog of AtOCP3 gene in Fragaria vesca.

[0059] A “FvH4 2g29460” or “FaAT5Gl 1280” (also termed FaEDRN2) gene refers to the ortholog of AT5G1 1280 in Fragaria vesca.

[0060] “Fxa2Ag 1724640” (Fb) or “Fxa2AglO2916” (Rr), “Fxa2Bg958120” (Fb) or “Fxa2Bg202737” (Rr), “Fxa2Cg808210” (Fb) or “Fxa2Cg201149” gene (Rr), and “Fxa2Dg2512710” (Fb) or “Fxa2Dg202538” (Rr) genes in Fragaria x ananassa are homeologs and are each orthologs of the “FvH4_2g29450” gene and the “AtOCP3” gene. They are located on chromosomes 2 A, 2B, 2C, and 2D respectively.

[0061] “Fxa2Ag 1724630” (Fb) or “Fxa2AglO2917” (Rr), “Fxa2Cg808220” (Fb) or “Fxa2Cg201148” (Rr), and “Fxa2Dg2512700” (Fb) or “Fxa2Dg202539” (Rr) genes in Fragaria x ananassa are homeologs and are each orthologs of the “FvH4_2g29460” gene and the “AT5G11280” gene. They are located on chromosomes 2A, 2C, and 2D respectively.

[0062] 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, aCas 12a nuclease, a Casl2b nuclease, a Casl2c nuclease, a CasY nuclease, a CasX nuclease, a Casl2i nuclease, or an engineered RNA-guided DNA endonuclease.

[0063] 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).

[0064] The term "CRISPR RNA (crRNA)" has been described in the art (e.g.. in Makarova et al. (2011) Nat Rev Microbiol 9 A67-47T, Makarova et al. (2011) Biol Direct 6:38; Bhaya et al. (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).

[0065] 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 (z.e., target sequence). Non-limiting examples of PAMs include NGG, NNGRRT, NN[A / C / T]RRT. NGAN, NGCG, NGAG. NGNG, NGC, and NGA.

[0066] 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.

[0067] 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.

[0068] A "regenerant" is a plant produced from a plant tissue cell, such as a genetically modified plant tissue cell.II. Overview

[0069] Described are compositions for genetically modifying a strawberry plant to increase resistance of the plant to Pestalotia Leaf Spot and Fruit Rot caused by Neopestalotiopsis . Described are compositions for reducing expression of a FvH4_2g29450 gene or a FvH4_2g29460 gene or one or more orthologs thereof. Also described are methods of using the compositions for producing plants having ^Neopestalotiopsis -resistant phenotype. In some embodiments, the plant is a Fragaria plant. A Fragaria plant includes any member of the Fragaria genus suitable for commercial strawberry production. A Fragaria plant can be a diploid species, a tetrapioid species, a hexapioid species, an octoploid species, or any other ploidy species or hybrid thereof. A Fragaria plant can be, but is not limited to, Fragaria veseu. Fragaria x bifera. Fragaria x bringhurstii. Fragaria virgintana. Fragaria chiloensis. or Fragaria x ananassa. In some embodiments, the Fragaria plant is a Fragaria x ananassa plant.

[0070] Decreasing the expression of a FvH4_2g29450 gene or a FvH4_2g29460 gene or one or more orthologs thereof in a Fragaria plant increases resistance to Neopestalotiopsis and decreases severity of Pestalotia Leaf Spot and Fruit Rot.

[0071] A “FvH4_2g29450 gene” or “FaOCP3 gene” is an ortholog of the Arabidopsis thaliana OVEREXPRESSOR OF CATIONIC PEROXIDASE 3 (AtOCP3) in the diploid Fragaria vesca. The FvH4_2g29450 gene encodes a protein having the amino acid sequence of SEQ ID NO: 8. The FvH4_2g29450 gene comprises the nucleic acid sequence of SEQ ID NO: 7.

[0072] Orthologs of the FvH4_2g29450 gene include, but are not limited to, Fxa2Agl724640 (Fb) or Fxa2AglO2916 (Rr), Fxa2Bg958120 (Fb) or Fxa2Bg202737, Fxa2Cg808210 (Fb) or Fxa2Cg201149 (Rr), and Fxa2Dg2512710 (Fb) or “Fxa2Dg202538” (Rr) genes which are homeologs in the octoploid Fragaria x ananassa. The Fxa2AglO2916 gene encodes a protein having the amino acid sequence of SEQ ID NO: 12. The Fxa2AglO2916 gene comprises the nucleic acid sequence of SEQ ID NO: 11. The Fxa2Bg202737 gene encodes a protein having the amino acid sequence of SEQ ID NO: 14. The Fxa2Bg202737 gene comprises the nucleic acid sequence of SEQ ID NO: 13. The Fxa2Cg201149 gene encodes a protein having the amino acid sequence of SEQ ID NO: 16. The Fxa2Cg201149 gene comprises the nucleic acid sequence of SEQ ID NO: 15. The Fxa2Dg202538gene encodes a proteinhaving the amino acid sequence of SEQ ID NO: 18. The Fxa2Dg202538 gene comprises the nucleic acid sequence of SEQ ID NO: 17.

[0073] A '‘FvH4_2g29460 gene’’ or “FaAT5G11280 gene” is an ortholog of the Arabidopsis thaliana AT5G11280 in the diploid Fragaria vesca. The “FvH4_2g29460 gene” encodes a protein having the amino acid sequence of SEQ ID NO: 10. The “FvH4_2g29450 gene” comprises the nucleic acid sequence of SEQ ID NO: 9.

[0074] Orthologs of the FvH4_2g29460 gene include, but are not limited to. Fxa2Agl 724630 (Fb) or Fxa2Agl 02917 (Rr), Fxa2Cg808220 (Fb) or Fxa2Cg201148 (Rr), and Fxa2Dg2512700 (Fb) or Fxa2Dg202539 (Rr) genes which are homeologs in the octoploid Fragaria * ananassa. The Fxa2Agl02917 gene encodes a protein having the amino acid sequence of SEQ ID NO: 20. The Fxa2AglO2917 gene comprises the nucleic acid sequence of SEQ ID NO: 19. The Fxa2Cg201148 gene (Rr)encodes a protein having the amino acid sequence of SEQ ID NO: 22. The Fxa2Cg201148 gene comprises the nucleic acid sequence of SEQ ID NO: 21. The Fxa2Dg202539 gene encodes a protein having the amino acid sequence of SEQ ID NO: 24. The Fxa2Dg202539 gene comprises the nucleic acid sequence of SEQ ID NO: 23.

[0075] In some embodiments, decreasing expression of an ortholog of AtOCPB in a polyploid Fragaria plant (e.g, FvH4_2g29450) comprises decreasing expression of all homeologs of the AlOCPS ortholog in the polyploid Fragaria plant. In some embodiments, decreasing expression of an ortholog of At5G 11280 in a polyploid Fragaria plant (e.g, FvH4_2g29460) comprises decreasing expression of all homeologs of i e At5Gl 1280 ortholog in the polyploid Fragaria plant.

[0076] The approach used herein involves methods for decreasing expression of one or more of a FvH4_2g29450 gene or one or more orthologs or homeologs thereof, and / or a FvH4_2g29460 gene or one or more orthologs or homeologs thereof by RNAi inhibition or CRISPR / Cas targeted mutagenesis. In some embodiments, decreased expression of the FvH4_2g29450 gene or the one or more orthologs or homeologs thereof, and / or the FvH4_2g29460 gene or the one or more orthologs or homeologs thereof results in increased resistance to Neopestalotiopsis and / or Pestalotia Leaf Spot and Fruit Rot.

[0077] In some embodiments, constructs and systems for RNAi of one or more of a FvH4_2g29450 gene, a FvH4_2g29460 gene, or orthologs or homeologs thereof are described.

[0078] In polyploid Fragaria plants, homeologous genes may be highly conserved and nearly identical in sequence. Thus, multiple homeologous genes may be targeted with a single RNAi construct or a single guide RNA sequence. For example, a single RNAi constructcomprising SEQ ID NO: 25 targets and decreases expression of the four homeologs Fxa2AglO2916 (SEQ ID NO: 11), Fxa2Bg202737 (SEQ ID NO: 13), Fxa2Cg201149 (SEQ ID NO: 15), and Fxa2Dg202538 (SEQ ID NO: 17) in octaploid Fragaria x ananassa (cultivar Rr). The RNAi construct comprising SEQ ID NO: 26 targets and decreases expression of the three homeologs Fxa2AglO2917 (SEQ ID NO: 19), Fxa2Cg201148 (SEQ ID NO: 21), and Fxa2Dg202539 (SEQ ID NO: 23) in octaploid Fragaria * ananassa.

[0079] In some embodiments, methods of producing Neopestalotiopsis resistant Fragaria plants and methods of genetically modifying a Fragaria plant to produce a Neopestalotiopsis resistant plant using a CRISPR system are described.III. RNAi / CRISPR systemsA. RNA Interference

[0080] Described herein are nucleic acids for producing Neopestalotiopsis resistant Fragaria plants using a RNAi system. The described nucleic acids can be used to inhibit or knockdown the expression of (a) the FvH4_2g29450 gene and / or the one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or (b) the FvH4_2g29460 gene and / or the one or more Fragaria FvH4_2g29460 orthologs or homeologs in a Fragaria plant.

[0081] Sequence-selective, post-transcriptional inactivation of expression of a target gene can be achieved in a wide variety of eukaryotes by introducing double-stranded RNA (dsRNA) corresponding to the target gene, a phenomenon termed RNA interference (RNAi). RNAi occurs when an organism recognizes dsRNA molecules and processes the dsRNA into small RNA fragments of 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length, called small interfering RNAs (siRNAs). The siRNAs then diffuse or are carried throughout the organism, including across cellular membranes, where associated with RISC and hybridize to mRNAs (or other RNAs), resulting in cleavage of the RNA. Most plant interfering RNAs (siRNAs and miRNAs) show extensive base pairing to. and guide cleavage of their target mRNAs (Jones- Rhoades et al. (2006) Annu. Rev. Plant Biol. 57, 19-53; Llave et al. (2002) Proc. Natl. Acad. Sci. USA 97, 13401 -10406). In other instances, interfering RNAs may bind to target RNA molecules having imperfect complementarity, causing translational repression without mRNA degradation.

[0082] The term “RNAi” or “RNA interference’" refers to the process of sequence-specific post-transcriptional gene silencing (e.g., in nematodes), mediated by double-stranded RNA (dsRNA). “DsRNA” refers to RNA that is partially or completely double stranded. Double stranded RNA is also referred to as small interfering RNA (siRNA), small interfering nucleicacid (siNA), microRNA (miRNA), and the like. In the RNAi process, dsRNA comprising a first (antisense) strand that is complementary to a portion of a target gene and a second (sense) strand that is fully or partially complementary to the first antisense strand is introduced into an organism (e.g., plants and / or crops), by, e.g., transformation, injection, spray, brush, mechanical abrasion, laser etching or immersion, etc. After introduction into the organism, the target gene-specific dsRNA is processed into relatively small fragments (siRNAs) and can subsequently become distributed throughout the organism, leading to a loss-of-function mutation having a phenotype that, over the period of a generation, may come to closely resemble the phenotype arising from a complete or partial deletion of the target gene.

[0083] This approach takes advantage of the discovery that siRNA can trigger the degradation of mRNA corresponding to the siRNA sequence. RNAi is a remarkably efficient process whereby dsRNA induces the sequence-specific degradation of homologous mRNA in animals and plant cells (Hutvagner and Zamore (2002), Curr. Opin. Genet. Dev., 12, 225-232; Sharp (2001), Genes Dev., 15, 485-490).

[0084] In some embodiments, RNAi constructs are described that inhibit expression of the FvH4_2g29450 gene, the FvH4_2g29460 gene, one or more Fragaria FvH4_2g29450 orthologs or homeologs; or one or more Fragaria FvH4_2g29460 orthologs or homeologs. In some embodiments, the RNAi construct is designed to target regions that contain high sequence identity (at least 95%, at least 98% or at least 99%) between homeologous genes in order to suppress multiple homeologs in a polyploid plant.

[0085] In some embodiments, the RNAi construct targets the Fxa2Agl 724640 gene (Fb) or Fxa2AglO2916 gene (Rr), the Fxa2Bg958120 gene (Fb) or Fxa2Bg202737 gene (Rr), the Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr), and the Fxa2Dg2512710 gene (Fb) or Fxa2Dg202538 gene (Rr). In some embodiments, the RNAi construct comprises SEQ ID NO: 25.

[0086] In some embodiments, RNAi construct targets the Fxa2Agl 724630 gene (Fb) or Fxa2AglO2917 gene (Rr), the Fxa2Cg808220 gene (Fb) or Fxa2Cg201148 gene (Rr), and the Fxa2Dg2512700 gene (Fb) or Fxa2Dg202539 gene (Rr). In some embodiments, the RNAi construct comprises SEQ ID NO: 26.

[0087] Additional RNAi constructs that target the FvH4_2g29450 gene, the FvH4_2g29460 gene, one or more Fragaria FvH4_2g29450 orthologs or homeologs; or one or more Fragaria FvH4_2g29460 orthologs or homeologs, are readily made using methods available in the art. An RNAi construct, such as a siRNA, hairpin RNA, short hairpin RNA, or long hairpin RNA, contains a dsRNA in which one strand (an antisense strand) contains aregion that is complementary to a sequence in the target gene mRNA. The region can be a short as about 15-24 nucleotides as in a siRNA or up to several hundred nucleotides in length as in some long hairpin RNAs. An RNAi can contain a region that is complementary to a sequence in a single target gene. An RNAi construct can also contain a region or regions that are complementary' to the same or different sequences present in multiple target genes. For RNAi constructs having a region or regions complementary to multiple target genes, a single region in the RNAi construct can be complementary to a common sequence present in each of one or more target genes, the RNAi construct can be chimeric in containing multiple regions that are complementary' to different sequences in one or more target genes, or a combination thereof. Algorithms have been developed to identify sequences in target genes suitable for use in RNAi constructs (e.g., GenScript siRNA Target Finder and / or BLOCK-iT™ RNAi Designer) and are readily adapted for identifying additional RNAi constructs for inhibiting the FvH4_2g29450 gene, the FvH4_2g29460 gene, one or more Fragaria FvH4_2g29450 orthologs or homeologs; or one or more Fragaria FvH4_2g29460 orthologs or homeologs.

[0088] Without wishing to be bound by theory, the double stranded hairpin RNA is believed to be recognized by the endoribonuclease Dicer which cleaves the double-stranded hairpin RNA into short double-stranded RNA fragments called small interfering RNAs (siRNAs), which are then used by the RNA induced silencing complex (RISC) in a homology7search to target the specific endogenous genes for silencing.B. CRISPR / Cas Systems

[0089] Described herein are CRISPR systems for producing genetically modified Fragaria plant with increased resistance to Pestalotia Leaf Spot and Fruit Rot caused by infection with Neopestalotiopsis spp. The described CRISPR systems can be used to disrupt or introduce loss of function mutations in (a) a FvH4_2g29450 gene and / or one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or (b) a FvH4_2g29460 gene and / or one or more Fragaria FvH4 2g29460 orthologs or homeologs.

[0090] 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. The RNA- guided DNA endonuclease enzyme can be, but is not limited to, Cas9. 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 RNAcontains 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 can be delivered to a plant or plant cell via a bacterium. The bacterium can be, but is not limited to. Agrobacterium tumefaciens.

[0091] The CRISPR system is designed to target one or more of the described genes. 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.

[0092] Guide sequences suitable for forming gRNAs or crRNAs for CRISPR system mediated genetic modification of genes to achieve Pestalotia Leaf Spot and Fruit Rot resistant Fragaria plants are described. Suitable guide sequences include 17-25 nucleotide sequences in any of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, and 23, or a complement thereof 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 17-25 nucleotide sequence immediately 5' of a 5'-NGG-3' in the coding sequence of a FvH4_2g29450 gene (SEQ ID NO: 7), a FvH4_2g29460 gene (SEQ ID NO: 9), one or more Fragaria FvH4 2g29450 orthologs or homeologs, or one or more Fragaria FvH4_2g29460 orthologs or homeologs can be used in forming a gRNA. In some embodiments, the 17-25 nucleotide sequence is designed as a gRNA to simultaneously target one or more of Fxa2Agl724640 (Fb) or Fxa2AglO2916 gene (Rr) (SEQ ID NO: 11), Fxa2Bg958120 (Fb) or Fxa2Bg202737 (Rr) (SEQ ID NO: 13), Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr) (SEQ ID NO: 15), and Fxa2Dg2512710 (Fb) or Fxa2Dg202538 (Rr) (SEQ ID NO: 17). In some embodiments, the 17-25 nucleotide sequence is designed as a gRNA to simultaneously target two or more of Fxa2Agl724640 (Fb) or Fxa2AglO2916 gene (Rr) (SEQ ID NO: 11), Fxa2Bg958120 (Fb) or Fxa2Bg202737 (Rr) (SEQ ID NO: 13), Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr) (SEQ ID NO: 15), and Fxa2Dg2512710 (Fb) or Fxa2Dg202538 (Rr) (SEQ ID NO: 17). In some embodiments, the 17-25 nucleotide sequence is designed as a gRNA to simultaneously target three or more of Fxa2Ag 1724640 (Fb) or Fxa2AglO2916 gene (Rr) (SEQ ID NO: 11), Fxa2Bg958120 (Fb) or Fxa2Bg202737 (Rr) (SEQ ID NO: 13), Fxa2Cg808210 gene (Fb) or Fxa2Cg201149 gene (Rr) (SEQ ID NO: 15), and Fxa2Dg2512710 (Fb) or Fxa2Dg202538 (Rr) (SEQ ID NO: 17). In someembodiments, the 17-25 nucleotide sequence is designed as a gRNA to simultaneously target Fxa2Agl724640 (Fb) or Fxa2AglO2916 (Rr) (SEQ ID NO: 11), Fxa2Bg958120 (Fb) or Fxa2Bg202737 (Rr) (SEQ ID NO: 13), Fxa2Cg808210 (Fb) or Fxa2Cg201149 (Rr) (SEQ ID NO: 15), and Fxa2Dg2512710 (Fb) or Fxa2Dg202538 (Rr) (SEQ ID NO: 17).

[0093] In some embodiments, the 17-25 nucleotide sequence is designed as a gRNA to simultaneously target one or more of Fxa2Agl 724630 (Fb) or Fxa2AglO2917 (Rr) (SEQ ID NO: 19). Fxa2Cg808220 (Fb) or Fxa2Cg201148 (Rr) (SEQ ID NO: 21), and Fxa2Dg2512700 (Fb) or Fxa2Dg202539 (Rr) (SEQ ID NO: 23). In some embodiments, the 17-25 nucleotide sequence is designed as a gRNA to simultaneously target two or more of Fxa2Ag 1724630 (Fb) or Fxa2AglO2917 (Rr) (SEQ ID NO: 19), Fxa2Cg808220 (Fb) or Fxa2Cg201148 (Rr) (SEQ ID NO: 21), and Fxa2Dg2512700 (Fb) or Fxa2Dg202539 (Rr) (SEQ ID NO: 23). In some embodiments, the 17-25 nucleotide sequence is designed as a gRNA to simultaneously target Fxa2Agl724630 (Fb) or Fxa2Agl02917 (Rr) (SEQ ID NO: 19), Fxa2Cg808220 (Fb) or Fxa2Cg201148 (Rr) (SEQ ID NO: 21). and Fxa2Dg2512700 (Fb) or Fxa2Dg202539 (Rr) (SEQ ID NO: 23).

[0094] In some embodiments, a gRNA suitable for targeting Fxa2AglO2916, Fxa2Bg202737, Fxa2Cg201149, and Fxa2Dg202538 comprises SEQ ID NO: T1 and / or SEQ ID NO:28.

[0095] Described are guide RNA for generating a Pestalotia Leaf Spot and Fruit Rot resistant Fragaria plant for use with a CRISPR system, wherein the guide RNA targets: a FvH4_2g29450 gene (SEQ ID NO: 7); a FvH4_2g29460 gene (SEQ ID NO: 9); one or more of a Fxa2Agl02916 gene (SEQ ID NO: 11), a Fxa2Bg202737 gene (SEQ ID NO: 13), a Fxa2Cg201149 gene (SEQ ID NO: 15), and a Fxa2Dg202538 gene (SEQ ID NO: 17); or one or more of a Fxa2AglO2917 gene (SEQ ID NO: 19), a Fxa2Cg201148 gene (SEQ ID NO: 21), and a Fxa2Dg202539gene (SEQ ID NO: 23). In some embodiments, the guide RNA comprises SEQ ID NO: 27 or SEQ ID NO: 28.

[0096] Deletions or insertions in the flanking regions may alter expression of a FvH4_2g29450 gene, a FvH4_2g29460 gene, one or more Fragaria FvH4_2g29450 orthologs or homeologs, and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs leading to plants displaying increased resistance to Neopestalotiopsis. 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.

[0097] CRISPR modification of a Fragaria plant 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 derived 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.

[0098] Two or more guide RNAs can be used with the same RNA-guided DNA endonuclease (e.g., Cas nuclease) or different RNA-guided DNA endonucleases.

[0099] In some embodiments, two or more gRNAs targeting two or more different genes are used. The two or more gRNAs can be used with the same RNA-guided DNA endonuclease or different RNA-guided DNA endonucleases.

[0100] In some embodiments, three or more gRNAs targeting three or more different genes are used. The three or more gRNAs can be used with the same RNA-guided DNA endonuclease or different RNA-guided DNA endonucleases.

[0101] In some embodiments, tw o or more gRNAs targeting a single gene can be used. The two or more gRNAs can be used with the same RNA-guided DNA endonuclease (Cas nuclease) or different RNA-guided DNA endonucleases.

[0102] Any of the above-described guide RNAs can be provided as an RNA or a DNA encoding the RNA. 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.

[0103] In some embodiments, a CRISPR system comprises one or more guide RNAs and a nucleic acid encoding an RNA-guided DNA endonuclease.

[0104] 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.

[0105] 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.

[0106] 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. Once a plant cell has been successfully transformed, it may be cultivated to regenerate a transgenic plant (regenerant).

[0107] 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.

[0108] In some embodiments, introducing the CRISPR system into the Fragaria plant cell comprises electroporation, microprojectile bombardment, biolistic transformation, microinjection, protoplast transformation, an Agrobacterium tumefaciens vector transformation or an Agrobacterium rhizogenes vector transformation. 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 DNA into Agrobacteria 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.

[0109] To transgenic plants may be used to generate subsequent generations e.g. Ti, T2, etc.) by selfing of primary or secondary transformants, or by sexual crossing of primary or secondary transformants with other plants (transformed or untransformed).IV. Genetically Modified Plants

[0110] A genetically modified Fragaria plant can be any member of the Fragaria genus suitable for commercial strawberry production. A Fragaria plant can be a diploid species, a tetrapioid species, a hexapioid species, an octoploid species, a decaploid species or any other ploidy species or hybrid thereof. A Fragaria plant can be, but is not limited to. Fragaria vesca. Fragaria x bifera, Fragaria x bringhurstii, Fragaria virginiana, Fragaria chiloensis, orFragaria x ananassa. In some embodiments, the Fragaria plant is a Fragaria x ananassa plant.

[0111] Described are methods of producing Neopestalotiopsis resistant plants comprising introducing into a plant, a plantlet, a plant tissue, a callus, or a plant cell, one or more RNAi constructs targeting a FvH4_2g29450 gene, a FvH4_2g29460 gene, one or more Fragaria FvH4_2g29450 orthologs or homeologs, and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs. The RNAi constructs can comprise any of the described RNAi constructs. In some embodiments, genetically modified plants created using a RNAi construct are described. In some embodiments, the RNAi construct comprises or consists of an intronhairpin RNA construct.

[0112] In some embodiments, methods are described for producing a Fragaria plant that has increase resistance to Neopestalotiopsis comprising introducing into the plant or plant cell one or more RNAi constructs targeting one or more of the FvH4_2g29450 gene, the FvH4_2g29460 gene, one or more Fragaria FvH4_2g29450 orthologs or homeologs, and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs. The RNAi constructs can comprise any of the described RNAi constructs. In some embodiments, these RNAi constructs inhibit or knockdown the expression of a FvH4_2g29450 gene, a FvH4_2g29460 gene, one or more Fragaria FvH4_2g29450 orthologs or homeologs, and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs.

[0113] Plants produced using the described RNAi constructs display increased resistance to Pestalotia Leaf Spot and Fruit Rot caused by infection with Neopestalotiopsis spp. compared to a control Fragaria plant that does not have decreased expression of the targeted gene (e.g., an unmodified WT cultivar).

[0114] In some embodiments, the plants are modified using a CRISPR system. Other methods known in the art may also be used to produce modified Fragaria plants with a loss of function mutation in a FvH4_2g29450 gene, a FvH4_2g29460 gene, one or more Fragaria FvH4_2g29450 orthologs or homeologs, and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs.

[0115] In some embodiments, one or more CRISPR systems targeting a FvH4_2g29450 gene, a FvH4_2g29460 gene, one or more Fragaria FvH4_2g29450 orthologs or homeologs, and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs are introduced into a plant or a progenitor of the plant. The CRISPR system can comprise any of the described CRISPR systems. In some embodiments, these CRISPR systems disrupt or introduce a loss of function mutation in the FvH4_2g29450 gene, the FvH4_2g29460 gene, one or more FragariaFvH4_2g29450 orthologs or homeologs, and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs.

[0116] In some embodiments, plants produced using the described CRISPR systems display increased resistance to Pestalotia Leaf Spot and Fruit Rot caused by infection with Neopestalotiopsis spp. compared to a control Fragaria plant that does not have decreased expression of the targeted gene (e.g., an unmodified WT cultivar).

[0117] Genetically modified Fragaria plants 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 grow th control, enhanced animal and / or human nutrition, improved processing traits, or improved flavor, among others.V. Detection of a Modified Gene

[0118] Modification of a FvH4_2g29450 gene, a FvH4_2g29460 gene, one or more Fragaria FvH4_2g29450 orthologs or homeologs, and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs can be detected or confirmed by any means known in the art for detecting genetic modifications in plants.

[0119] 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.

[0120] 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.VI. Sequences

[0121] The nucleotide and amino acid sequences 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 acidsequence 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.Table 1. Sequences

[0122] 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 w ere specifically and individually indicated to be so incorporated byreference. 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.EXAMPLES

[0123] 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. Screening Neopestalotiopsis Resistance Genes via T-DNA Tagging in Mutant Lines.

[0124] To investigate resistance mechanisms against the newly emerged Neopestalotiopsis species, candidate genes were identified through Arabidopsis mutant screening following a reverse genetics approach. Publicly available databases were used to identify several candidate genes for fungal resistance and plant pathogen resistance. The genes AT1G08720 (AtEDRl), AT5G11270 (AtOCP3), and AT5G11280 (an uncharacterized gene) were selected for screening for Neopestalotiopsis resistance in Arabidopsis thaliana. Transfer DNA (T-DNA) insertion mutants were generated using T-DNA insertion lines CS67959 for AT1G08720, SALK 201649C for AT5G11270, and SALK 003729C for AT5G11280. For sowing, the seeds were subjected to a 24-hour cold treatment and then transferred to MS agar medium. Each mutant line was sown with 20 replicates per plug tray. The mutant lines were initially sterilized with 70% ethanol for 30 seconds, followed by three rinses in distilled water. The Arabidopsis plants were subjected to a 24-hour cold treatment and then transferred to MS agar medium. The transplanting stage occurred once cotyledons emerged, at which point the seedlings were transferred to plug trays. When 5-6 true leaves had developed, they were inoculated with Neopestalotiopsis spp. After transplanting and the emergence of 15 true leaves, Agrobacterium infiltration was performed on all leaves. Four days post-infiltration, the plants were inoculated with Neopestalotiopsis spp.

[0125] Isolates, including Neopestalotiopsis spp. strains including 21-59, 21-60, 21-61, and 21 -62, were sourced from the Strawberry Pathology Laboratory at the Gulf Coast Research and Education Center, University of Florida (GCREC-UF). The isolates w ere cultivated following the methodology’ described by Baggio JS, et al.. Plant Disease 2023, 107(7):2177-2184 involving the growth of mycelial discs on 50% strength potato-dextrose-agar (PDA, Oxoid Ltd., Basingstoke, U.K.) medium. The cultures were incubated at 23°C fluorescent lighting mimicking daylight conditions for a duration of 7 days. The inoculum prepared for the spray method was suspended in sterilized deionized water containing 0.1% Tween 20, achieving a 5 x 105spores / mL concentration. After inoculation, all the trays were covered with dome lids to maintain humidity conditions.

[0126] Symptoms of the disease could be observed four days post-inoculation. To validate the disease entry' rate, observations were taken five days later on Arabidopsis leaves. The diseased area was quantified using ImageJ software.

[0127] Four days post-inoculation with Neopestalotiopsis spp., the observed necrosis rates in Arabidopsis lines revealed varying degrees of fungal resistance. The wild type Col-0 exhibited an 18% necrosis rate following fungal inoculation, while the AtEDRl knockout mutant showed a significantly higher rate of 65%. In contrast, the AtOCP3 knockout mutant presented a much lower necrosis rate of 5.1%, and the AT5G11280 knockout mutant demonstrated a remarkably minimal necrosis rate of 0.004% (Figure 1).Example 2. Identification of Orthologous Genes in Strawberry

[0128] Orthologs of the genes AT5G11270 (AtOCP3) and AT5G11280 were identified in strawberries. Initially, the search was conducted on the diploid strawberry Fragaria vesca genome to identify orthologous genes, using the Genome Database for Rosaceae (GDR, https: / / www.rosaceae.org) and Phytozome (https: / / phytozome-next.jgi.doe.gov / ). AT5G11270 (AtOCP3) showed high homology7with FvH4_2g29450 from the diploid strawberry (FIG. 2), while AT5G11280 was highly homologous to FvH4_2g29460 (FIG. 3).

[0129] Subsequently, based on this data, homologous and homeologous copies were identified in the octoploid strawberry Fragaria * ananassa by aligning the sequences of FvH4_2g29450 (FaOCP3) and FvH4_2g29460 (also termed FaAT5G11280 or FaEDRN2) to the reference genomes of the 'Florida Brilliance' and 'Royal Royce' varieties. The analysis identified four homeologous copies of FvH4_2g29450 on chromosomes 2A, 2B. 2C, and 2D: Fxa2Agl724640 (Fb) or Fxa2AglO2916 (Rr), Fxa2Bg958120 (Fb) or Fxa2Bg202737 (Rr), Fxa2Cg808210 (Fb) or Fxa2Cg201149 (Rr), and Fxa2Dg2512710 (Fb) or Fxa2Dg202538 (Rr) (FIG. 2). In contrast, FvH4_2g29460 was found to have three homeologous copies on chromosomes 2A, 2C, and 2D: Fxa2Agl724630 (Fb) or Fxa2AglO2917 (Rr), Fxa2Cg808220 (Fb) or Fxa2Cg201148 (Rr). and Fxa2Dg2512700 (Fb) or Fxa2Dg202539 (Rr) (FIG. 3). One notable point was that all copies of FaOCP3 and FaAT5G11280, except for those on Chr. 2B, were found to be adjacent to each other on the same chromosome. Considering the possibility that this might be specific to the reference sequence, we validated our findings against the existing octoploid strawberry reference genome ‘Royal Royce’. The results confirmed that, similar to ‘Brilliance’, the genes were indeed positioned next to each other. Subsequently, we assessed the sequence similarity of FvH4_2g29450 (FaOCP3) and FaAT5G11280 located on the Chr.2A, C, and D genomes. The results showed low sequence similarities, with 25.25% for Chr.2A, 17.943% for Chr.2C, and 35.277% for Chr.2D (Figure 3B). These findings demonstrate that the two genes are indeed distinct entities.Example 3. Assessment of the Candidate Gene FaOCP3 and FaEDRN2 in Strawberry Leaves Using RNAi Transient Assay.

[0130] To elucidate the resistance mechanism against Neopestalotiopsis spp., a transient knockdown assay using RNAi was performed, specifically targeting the genes annotated as FaOCP3 and FaEDRN2. The pssRNAit program was utilized to design specific RNAi fragments targeting the strawberry ortholog of the AT5G11270 gene (FaOCP3) and FaEDRN2 using the acquired gene sequences in the octoploid F x ananassa. The Gateway cloning system (Invitrogen, MA, USA) was used to clone 300 and 350 bp target gene fragments of FaOCP3 and FaEDRN2, respectively, into pK7GWIWG2 RNAi vectors for use in transient assays. The varieties ‘Florida Brilliance’ and ‘Florida Medallion’ were tested in the transient assay.

[0131] Propagation was achieved through the runner propagation method. After transplanting and the emergence of 15 true leaves, Agrobacterium infiltration was performed on all leaves with pK7G::FaOCP3 or pK7G::FaEDRN2, or an empty vector control.

[0132] The final RNAi clones were introduced into the Agrobacterium strain EHA105 via electroporation. For conducting transient assays, clones transformed with the target genes were manipulated by culturing at 28°C in LB media supplemented with 50 mg / L spectinomycin and 10 mg / L rifampicin until reaching an OD600 of 0.6-0.8. Subsequently, the bacteria were centrifuged, and the resulting pellets were resuspended in activation buffer (10 mM MES, 10 mM MgCh, and 200 pM acetosyringone, pH 5.6) reached OD600 of 0.8. The pK7G::FaOCP3 or pK7G::FaEDRN2 inoculum was incubated for approximately 3 hours on a rotary shaker at room temperature (70 rpm) prior to leaf infiltration. For the inoculation with activated Agrobacterium, 0.05% Silwet-77 was added, and the entire plant w as submerged upside down in the inoculum, followed by a 5-minute treatment using the vacuum infiltration method. Subsequently, plastic lids were used to maintain humidity. After infiltration, plants were maintained in high humidity conditions under dark conditions for two days, followed by a 16- hour light / 8-hour dark photoperiod.

[0133] Three to four days post-Agrobacterium inoculation, the plants were sprayed with the Neopestalotiopsis pathogen. Five days later, symptoms developed, and a reduction in disease necrosis symptoms was observed in plants inoculated with FaOCP3 or FaEDRN2 compared to those treated with the empty vector (FIGs. 4-6). The area of disease symptoms was quantified using ImageJ, and statistical analysis was conducted using one-way ANOVA.

[0134] RNA was extracted from leaf samples collected on 4-day post-infiltration using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich). RNA purity (260 / 280 ratio of 1.8-2.2) was verified, followed by DNase I treatment and reverse transcription into cDNA. qRT-PCRwas performed using FaGAPDH as a housekeeping gene, and relative gene expression levels were quantified using the AACq method.

[0135] As shown in FIGs. 7-8, a significant reduction in fungal entry rate was observed for the leaves with FaOCP3 and / or FaEDRN2 knockdown as compared to the control. Disease symptoms were reduced in both FaOCP3 and / or FaEDRN2 knock-down plants compared to empty vector control plants.

Claims

Claims:

1. A genetically modified Fragaria plant, wherein the Fragaria plant has decreased expression of:(a) a FvH4_2g29450 gene and / or one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or;(b) a FvH4_2g29460 gene and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs.

2. The genetically modified Fragaria plant of claim 1, wherein:(a) the Fragaria plant is a diploid Fragaria vesca plant having decreased expression of the FvH4 2g29450 gene and / or the FvH4 2g29460 gene; or(b) the Fragaria plant is a polyploid Fragaria plant having decreased expression of one or more Fragaria FvH4_2g29450 orthologs or homeologs and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs.

3. The genetically modified Fragaria plant of claim 2, wherein the Fragaria plant is an octoploid Fragaria x ananassa plant having decreased expression of:(a) one or more Fragaria FvH4_2g29450 homeologs selected from the group consisting of: a Fxa2Agl724640 gene, a Fxa2Bg958120 gene or Fxa2Bg202737 gene, a Fxa2Cg808210 gene or Fxa2Cg201149 gene, and a Fxa2Dg2512710 gene or Fxa2Dg202538 gene; and / or(b) one or more Fragaria FvH4_2g29460 homeologs selected from the group consisting of: a Fxa2Agl724630 gene or Fxa2AglO2917 gene, a Fxa2Cg808220 gene or Fxa2Cg201148 gene, and a Fxa2Dg2512700 gene Fxa2Dg202539 gene.

4. The genetically modified Fragaria plant of claim 3, wherein the Fragaria x ananassa plant has decreased expression of:(a) two or more Fragaria FvH4_2g29450 homeologs selected from the group consisting of: the Fxa2Agl 724640 gene, the Fxa2Bg958120 gene or Fxa2Bg202737 gene, the Fxa2Cg808210 gene or Fxa2Cg201149 gene, and the Fxa2Dg2512710 gene or Fxa2Dg202538 gene; and / or(b) two or more Fragaria FvH4_2g29460 homeologs selected from the group consisting of: the Fxa2Agl724630 gene or Fxa2AglO2917 gene, the Fxa2Cg808220 gene or Fxa2Cg201148 gene, and the Fxa2Dg2512700 gene or Fxa2Dg202539 gene.

5. The genetically modified Fragaria plant of claim 4, wherein the Fragaria x ananassa plant has decreased expression of:(a) three or more Fragaria FvH4_2g29450 homeologs selected from the group consisting of: the Fxa2Agl724640 gene, the Fxa2Bg958120 gene or Fxa2Bg202737 gene, the Fxa2Cg808210 gene or Fxa2Cg201149 gene, and the Fxa2Dg2512710 gene or Fxa2Dg202538 gene; and / or(b) the Fxa2Agl724630 gene or Fxa2AglO2917 gene, the Fxa2Cg808220 gene or Fxa2Cg201148 gene, and the Fxa2Dg2512700 gene or Fxa2Dg202539 gene.

6. The genetically modified Fragaria plant of claim 5. wherein the Fragaria ananassa plant has decreased expression of:(a) the Fxa2Agl724640 gene, the Fxa2Bg958120 gene or Fxa2Bg202737 gene, the Fxa2Cg808210 gene or Fxa2Cg201149 gene, and the Fxa2Dg2512710 gene or Fxa2Dg202538 gene; and / or(b) the Fxa2Agl724630 gene or Fxa2AglO2917 gene, the Fxa2Cg808220 gene or Fxa2Cg201148 gene, and the Fxa2Dg2512700 gene or Fxa2Dg202539 gene.

7. The genetically modified Fragaria plant of any one of claims 1-6, wherein the genetically modified Fragaria plant has increased resistance to Pestalotia Leaf Spot and Fruit Rot caused by infection with Neopestalotiopsis spp. compared to a control Fragaria plant that does not have decreased expression of the FvH4_2g29450 gene and / or the one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or the FvH4_2g29460 gene and / or the one or more Fragaria FvH4_2g29460 orthologs or homeologs.

8. The genetically modified Fragaria plant of any one of claims 1 -7, wherein the Fragaria plant expresses:(a) one or more RNAi constructs targeting the FvH4_2g29450 gene and / or the one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or(b) one or more RNAi constructs targeting the FvH4_2g29460 gene and / or the one or more Fragaria FvH4_2g29460 orthologs or homeologs.

9. The genetically modified Fragaria plant of claim 8, wherein the Fragaria plant expresses:(a) an RNAi construct targeting the Fxa2Ag 1724640 gene, the Fxa2Bg958120 gene or Fxa2Bg202737 gene, the Fxa2Cg808210 gene or Fxa2Cg201149 gene, and the Fxa2Dg2512710 gene or Fxa2Dg202538 gene: and / or(b) an RNAi construct targeting the Fxa2Agl724630 gene or Fxa2AglO2917 gene, the Fxa2Cg808220 gene or Fxa2Cg201148 gene, and the Fxa2Dg2512700 gene or Fxa2Dg202539 gene.

10. The genetically modified Fragaria plant of claim 9, wherein the one or more RNAi constructs comprise SEQ ID NO: 25 and / or SEQ ID NO:26.

11. The genetically modified Fragaria plant of any one of claims 1-7, wherein the genetically modified Fragaria plant has loss of function mutation in(a) the FvH4_2g29450 gene and / or the one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or(b) the FvH4 2g29460 gene and / or the one or more Fragaria FvH4 2g29460 orthologs or homeologs.

12. The genetically modified Fragaria plant of claim 11, wherein the loss of function mutation is a CRISPR-induced loss of function mutation.

13. The genetically modified Fragaria plant of claim 12, wherein the Fragaria plant has been genetically modified through the use of a CRISPR / Cas system at:(a) the FvH4_2g29450 gene and / or the one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or(b) the FvH4_2g29460 gene and / or the one or more Fragaria FvH4_2g29460 orthologs or homeologs.

14. The genetically modified Fragaria plant of claim 13, wherein the Fragaria plant is an octoploid Fragaria x ananassa plant and has been genetically modified through the use of a CRISPR / Cas system at(a) one or more of: a Fxa2Agl 724640 gene, a Fxa2Bg958120 gene or Fxa2Bg202737 gene, a Fxa2Cg808210 gene or Fxa2Cg201149 gene, and a Fxa2Dg2512710 gene or Fxa2Dg202538 gene; and / or(b) one or more of: a Fxa2Agl724630 gene or Fxa2AglO2917 gene, a Fxa2Cg808220 gene or Fxa2Cg201148 gene, and a Fxa2Dg2512700 gene or Fxa2Dg202539 gene.

15. The genetically modified Fragaria plant of claim 14, wherein the Fragaria plant has been genetically modified through the use of a CRISPR / Cas system at(a) the Fxa2Agl724640 gene, the Fxa2Bg958120 gene or Fxa2Bg202737 gene, the Fxa2Cg808210 gene or Fxa2Cg201149 gene, and the Fxa2Dg2 12710 gene or Fxa2Dg202538 gene; and / or(b) the Fxa2Agl724630 gene or Fxa2AglO2917 gene, the Fxa2Cg808220 gene or Fxa2Cg201148 gene, and the Fxa2Dg2512700 gene or Fxa2Dg202539 gene.

16. The genetically modified Fragaria plant of claim 14 or 15, wherein the Fragaria plant contains a deletion at:(a) the FvH4 2g29450 gene and / or the one or more Fragaria FvH4 2g29450 orthologs or homeologs; and / or(b) the FvH4_2g29460 gene and / or the one or more Fragaria FvH4_2g29460 orthologs or homeologs.

17. The genetically modified Fragaria plant of claim 16, wherein the Fragaria plant is an octoploid Fragaria x ananassa plant and contains a deletion at:(a) one or more of: a Fxa2Agl 724640 gene, aFxa2Bg958120 gene or Fxa2Bg202737 gene, a Fxa2Cg808210 gene or Fxa2Cg201149 gene, and a Fxa2Dg2512710 gene or Fxa2Dg202538 gene; and / or(b) one or more of: a Fxa2Agl724630 gene or Fxa2AglO2917 gene, a Fxa2Cg808220 gene or Fxa2Cg201148 gene, and a Fxa2Dg2512700 gene or Fxa2Dg202539 gene.

18. A method for increasing resistance to Pestalotia Leaf Spot and Fruit Rot in a Fragaria plant, the method comprising decreasing expression of(a) a FvH4_2g29450 gene and / or one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or(b) a FvH4_2g29460 gene and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs in the plant or a progenitor of the plant.

19. The method of claim 18, wherein decreasing expression comprises introducing into the plant or the progenitor of the plant one or more expression vectors encoding one or more RNAi constructs targeting(a) the FvH4_2g29450 gene and / or the one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or(b) the FvH4_2g29460 gene and / or the one or more Fragaria FvH4_2g29460 orthologs or homeologs.

20. The method of claim 19, wherein the one or more RNAi constructs comprises SEQ ID NO: 25 and / or SEQ ID NO: 26.

21. The method of claim 18, wherein decreasing expression comprises introducing a CRISPR system into & Fragaria plant cell, wherein the CRISPR system targets:(a) the FvH4_2g29450 gene and / or the one or more Fragaria FvH4_2g29450 orthologs or homeologs; and / or(b) the FvH4_2g29460 gene and / or the one or more Fragaria FvH4_2g29460 orthologs or homeologs.

22. The method of claim 21, wherein the CRISPR system targets one or more of: aFvH4_2g29450 gene (SEQ ID NO: 7); a FvH4_2g29460 gene (SEQ ID NO: 9); aFxa2Agl724640 gene (SEQ ID NO: 11), a Fxa2Bg202737 gene (SEQ ID NO: 13), aFxa2Cg201149 gene (SEQ ID NO: 15), a Fxa2Dg202538 gene (SEQ ID NO: 17); aFxa2AglO2917 gene (SEQ ID NO: 19), a Fxa2Cg201148 gene (SEQ ID NO: 21), and aFxa2Dg202539 gene (SEQ ID NO: 23).

23. The method of claim 22 wherein the CRISPR system comprises one or more guide RNAs targeting one or more of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, and 23.

24. The method of claim 23 wherein the CRISPR system comprises a guide RNA comprising SEQ ID NO: 27 and / or a guide RNA comprising SEQ ID NO: 28.

25. A nucleic acid for increasing resistance to Pestalotia Leaf Spot and Fruit Rot in a Fragaria plant comprising an RNAi construct targeting:(a) a FvH4_2g29450 gene and / or one or more Fragaria FvH4_2g29450 orthologs or homeologs; or(b) a FvH4_2g29460 gene and / or one or more Fragaria FvH4_2g29460 orthologs or homeologs.

26. The nucleic acid of claim 25. wherein the RNAi construct comprises SEQ ID NO: 25 or SEQ ID NO: 26.

27. A CRISPR system for genetically modifying a Fragaria plant comprising one or more guide RNAs targeting one or more of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21. and 23.

28. The CRISPR system of claim 27, wherein CRISPR system comprises a guide RNA comprising SEQ ID NO: 27 and / or a guide RNA comprising SEQ ID NO: 28.

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