Methods for epigenetic reprogramming in crops

Abstract

The disclosure relates to methods for obtaining plants that exhibit useful traits by suppressing HDA6, CUE1, and / or SAL1 gene expression in plants. Methods for identifying genetic loci that provide for useful traits in plants and plants produced with those loci are also provided. In addition, plants that exhibit, contain, or harbor the useful traits, parts of the plants including seeds, and products of the plants are provided as well as methods of using the plants. Recombinant DNA vectors and transgenic plants comprising those vectors that provide for HDA6, CUE1, and SAL1 gene suppression are also provided.

Description

Agent Ref: P14842WO00 TITLE: METHODS FOR EPIGENETIC REPROGRAMMING IN CROPS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional application U.S. Serial No.63 / 656,798, filed June 6, 2024, which is incorporated herein by reference in its entirety. SEQUENCE LISTING XML

[0002] The instant application contains a sequence listing, which has been submitted in XML file format by electronic submission and is hereby incorporated by reference in its entirety. The XML file, created on June 3, 2025, is named P14842WO00.xml and is 220,534 bytes in size. TECHNICAL FIELD

[0003] The present disclosure relates generally to plant epigenetic reprogramming for improving agronomic traits. BACKGROUND

[0004] The ability of plants to adjust to environment plays a vital role in ecosystem resilience and food security. Plants are able to sense changes in their environments ranging from seasonal adjustments in daylength and light quality, temperature fluctuations, availability of moisture, and disease and pathogen pressures and to adjust their growth accordingly. While there is extensive evidence in support of cellular signaling and phytohormone contributions to this systemic process in plants, less is known about intracellular mechanisms of environmental sensing and nuclear, heritable responses in specific plant tissues and stages of development. SUMMARY

[0005] Methods for producing a plant having a useful trait comprising (a) crossing a first plant to a second plant or selfing the first plant, wherein the first plant comprises suppressed expression of an endogenous HDA6, CUE1, and / or SAL1 gene; (b) screening a population of progeny plants obtained from the cross or self of step (a) for the useful trait; and (c) selecting one or more progeny plants having the useful trait are provided. In certain embodiments, theAgent Ref: P14842WO00 first plant comprises a loss-of-function mutation in the endogenous HDA6, CUE1, and / or SAL1 gene. In certain embodiments, the first plant comprises a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA having complementarity to the endogenous HDA6, CUE1, and / or SAL1 gene. In certain embodiments, the first plant comprises a catalytically inactive Cas protein, optionally wherein the Cas protein is fused to a transcriptional repressor domain, and a guide RNA molecule comprising a spacer RNA molecule that targets the endogenous HDA6, CUE1, and / or SAL1 gene.

[0006] Plants and progeny thereof that exhibit a useful trait made by the aforementioned methods are provided. Plant parts obtained from the plants and progeny thereof and processed plant products obtained from the plant part are also provided.

[0007] Plants and plant cells having suppressed expression of an endogenous HDA6, CUE1, and / or SAL1 gene, wherein the plant or plant cell comprises: (i) a loss-of-function mutation in the endogenous HDA6, CUE1, and / or SAL1 gene; (ii) a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA having complementarity to the endogenous HDA6, CUE1, and / or SAL1 gene; and / or (iii) a catalytically inactive Cas protein, optionally wherein the Cas protein is fused to a transcriptional repressor domain, and a guide RNA molecule comprising a spacer RNA molecule that targets the endogenous HDA6, CUE1, and / or SAL1 gene are provided. Methods for obtaining the aforementioned plants and plant cells are also provided.

[0008] Guide RNA molecules comprising a spacer RNA molecule that targets (i) the endogenous HDA6 gene of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; (ii) the endogenous CUE1 gene of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; and (iii) the endogenous SAL1 gene of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 are provided. Genome engineering systems comprising a Cas protein in association with the aforementioned guide RNA molecules are provided. DNA molecules encoding the aforementioned guide RNA molecule are also provided.

[0009] DNA molecules encoding a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, or an anti-sense RNA having complementarity to (i) the endogenous HDA6 gene of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63;Agent Ref: P14842WO00 (ii) the endogenous CUE1 gene of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; and (iii) the endogenous SAL1 gene of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 are provided. BRIEF DESCRIPTION OF THE FIGURES

[0010] The following drawings form part of the specification and are included to further demonstrate certain embodiments. In some instances, embodiments can be best understood by referring to the accompanying figures in combination with the detailed description presented herein. The description and accompanying figures may highlight a certain specific example, or a certain embodiment. However, one skilled in the art will understand that portions of the example or embodiment may be used in combination with other examples or embodiments.

[0011] FIG.1A-D shows sensory plastid proteins MSH1, PPD3, CUE1, and SAL1 target specific gene networks. FIG.1A is a diagram of a hypothetical model of sensory plastid proteins signaling to the nucleus through a gatekeeper to adjust plant phenotype. FIG.1B is a Venn diagram of targeted networks of differentially methylated and / or differentially expressed hub genes identified by k-means clustering in msh1, PPD3-OX (dwarf phenotype), cue1, and sal1. FIG.1C is a dot plot of representative networks unique in each genotype. FIG.1D is a dot plot of representative networks common in four genotypes.

[0012] FIG.2A-E shows the DNA methylome of hda6 compared to sensory plastid-induced DNA methylome changes. FIG.2A shows the proportion of differentially methylated positions (DMPs) to total cytosine sites in gene body and promoter, TEs, RNAs, and others in hda6 and wildtype at 12-hr daylength. Sum of wildtype (WT) methylation level was used as reference to obtain DMPs. FIG.2B is a genome browser image of methylation levels with a gene (TOR) associated with CHG DMR and neighboring genes without CHG hypermethylation in hda6. Methylation level is computed at DMPs as ctreatment / (ctreatment+ttreatment) − ccontrol / (ccontrol+tcontrol), where c=methylated readcounts, t=unmethylated readcounts. One biological replicate was used as representative for each condition. FIG.2C shows hub gene networks of CHG DMR-associated genes in hda6 at 12-hr daylength. FIG. 2D is a Venn diagram of numbers of hda6 CHG DMR-associated genes, DMGs, DEGs overlapping with msh167 hub genes, DMGs, and DEGs. FIG.2E shows core hubs of msh1, PPD3OX dwarf, msh1, and cue1 DMGs that are also hda6 CHG DMR-associated genes.Agent Ref: P14842WO00

[0013] FIG.3A-H shows the phenotype of double mutants msh1 / hda6, ppd3 / hda6, cue1 / hda6, sal1 / hda6 with single mutants in 12-hr and 16-hr daylength. All double mutants were generated at 12-hr daylength except for msh1 / hda6, which is lethal at 12-hr. FIG.3A shows plants at 23 DAP in 12-hr and 16-hr daylength. FIG.3B shows the growth curve from 21 to 25 DAP at 12-hr and 16-hr daylengths. n=18-23. Individual plants were followed throughout time and shown in individual lines. Student’s t-test was performed at each time point and daylength between wildtype and mutants. Asterisks refer to p<0.05(*), p<0.01(**), p<0.001(***). FIG.3C is a diagram of how msh1 / hda6 was obtained at 16-hr daylength with segregation data at F4. F0 and F1 were grown at 12-hr daylength. FIG.3D shows plants at reproductive stage in 12-hr and 16-hr daylengths. FIG.3E shows abnormal development of ppd3 / hda6 at 12-hr daylength. FIG.3F shows cue1 / hda6 at reproductive stage. Phenotype is more severe in 16-hr with flowering at dwarf rosette stage with shorter stature. FIG.3G shows silique length of cue1 / hda6 compared to wildtype and single mutants in 12-hr and 16- hr daylengths. Closed triangle represents mean and horizontal bold line represents median. Tukey’s range test was performed at p<0.05 for all pair-wise comparisons and displayed in letters. n=80-100. FIG.3H shows lethality of sal1 / hda6 at 12-hr with segregation data at F3. The asterisk (*) indicates that the hda6 used as parent did not have full 12-hr methylome, as described in FIG.6 (Gen2 hda6).

[0014] FIG.4A-F shows the hda6 and sensory plastid-perturbed DNA methylome show similar responses to daylength. FIG.4A shows the proportion of differentially methylated positions (DMPs) to total cytosine sites in gene body and promoter, TEs, RNAs, and others of hda6 and wildtype at 12-hr and 16-hr daylength. DMPs of wild type control corresponding to each genotype were shown on the left. Sum of wildtype (WT) methylation level in each daylength was used as reference to obtain DMPs. FIG.4B is a genome browser image of methylation levels with a gene (TOR) associated with CHG DMR at 12-hr but not at 16-hr in hda6. Methylation level is computed at DMPs as ctreatment / (ctreatment+ttreatment) − ccontrol / (ccontrol+tcontrol), where c=methylated readcounts, t=unmethylated readcounts. One biological replicate was used as representative for each condition. FIG.4C is a Venn diagram of numbers of hda6 vs WT DMGs at 12-hr and 16-hr daylength. FIG.4D shows hub gene networks of hda6 vs WT at 12-hr and 16-hr daylength derived from DEGs and DMGs. Representative networks in each section of Venn diagram are shown in the dot plots. FIG.4E shows hub gene networks of DMGs at least three of msh1, PPD3OX (dwarf), cue1, and sal1Agent Ref: P14842WO00 at 12-hr daylength. Hub genes were identified by k-means clustering and filtered by using genes in GO:0010467 gene expression and GO:0010468 regulation of gene expression networks. FIG.4F shows hub gene networks of DMGs common in cue1 and msh1 DMGs at 16-hr daylength. Hub genes were identified by k-means clustering and filtered by using genes in GO:0010467 gene expression network.

[0015] FIG.5A-G shows hda6 epigenetic memory of daylength. FIG.5A shows a pedigree to obtain 12-hr hda6 and 16-hr hda6 with 12-hr methylome memory. Conditions in bold font were used for sequencing. FIG.5B shows a pedigree to remove 12-hr daylength memory in hda6 through heterozygous state. Conditions in bold font were used for sequencing. FIG.5C shows a pedigree to remove 12-hr daylength memory in msh1 / hda6 by growing at 16-hr daylength for 2 generations. Conditions in bold font were used for sequencing. FIG.5D is a genome browser image of methylation levels with a gene (TOR) associated with 12-hr CHG DMR in hda6. Methylation level is computed at DMPs as ctreatment / (ctreatment+ttreatment) − ccontrol / (ccontrol+tcontrol), where c=methylated readcounts, t=unmethylated readcounts. One wildtype reference is shown to represent control. One biological replicate was used as representative for each condition. FIG.5E shows the proportion of differentially methylated positions (DMPs) to total cytosine sites in gene body and promoter, TEs, RNAs, and others in WT, hda612-hr, hda6 Gen216-hr, Gen212-hr, and msh1 / hda6 Gen1. Three biological replicates were used and each replicate is shown in individual bars. DMPs of wild type control corresponding to each genotype were shown on the left. Sum of wildtype methylation level was used as reference to obtain DMPs. FIG.5F shows the Gen1 and Gen2 msh1 / hda6 plants at reproductive stage. FIG.5G shows the relative rosette size of Gen1 and Gen2 msh1 / hda6 plants relative to wildtype. The size of each plant was divided by average size of wildtype at each time point. Individual plants were followed throughout time and shown in individual lines. Black dashed line indicates where rosette size was same as Col-0 wild type.

[0016] FIG.6A-E shows abiotic and biotic stress response in the msh1 and hda6 interaction. FIG.6A is a summary of the heat stress experiment. FIG.6B shows WT, hda6, hda6 Gen2, msh1, msh1 / hda6 plants in control and heat stress conditions at 12-hr daylength. The experiment was conducted separately for hda6. FIG.6C shows flowering time in control and heat stress conditions at 12-hr daylength. The experiment was conducted separately for hda6. Data were collected every 2 d. Closed triangle represents mean and horizontal bold line represents median. Tukey’s range test was performed at p<0.05 for all pair-wise comparisonsAgent Ref: P14842WO00 and displayed in letters. FIG.6D shows representative plants of the indicated Arabidopsis genotypes infected by Gc UCSC1 and quantification of disease susceptibility. Pictures were taken at 11 days post-inoculation (dpi). Six weeks-old plants were inoculated with Gc UCSC1. FIG.6E is a diagram of the model of HDA6 as the gatekeeper for sensory plastid-to- nucleus signaling to adjust plant phenotype. MSH1 and PPD3 perturbations adjust energy allocation between growth versus defense, with PPD3 predominantly affecting the meristem. CUE1 is involved in light sensing and circadian clock, and SAL1 in RNA metabolism. HDA6 interacts with circadian clock components CCA1 and LHY. DETAILED DESCRIPTION

[0017] So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the disclosure pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present disclosure without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present disclosure, the following terminology will be used in accordance with the definitions set out below.

[0018] The phrase “allelic variant” as used herein refers to a polynucleotide or polypeptide sequence variant that occurs in a particular gene at particular locus in a different strain, variety, or isolate of a given organism.

[0019] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0020] As used herein, the phrase “chromosomal modification” refers to any of: a) an “altered chromosomal loci” and an “altered chromosomal locus”; b) “mutated chromosomal loci”, a “mutated chromosomal locus”, “chromosomal mutations” and a “chromosomal mutation”; or c) a transgene.Agent Ref: P14842WO00

[0021] As used herein, the phrases “altered chromosomal loci” (plural) or “altered chromosomal locus (singular) refer to portions of a chromosome that have undergone a heritable and reversible epigenetic change relative to the corresponding parental chromosomal loci. Heritable and reversible genetic changes in altered chromosomal loci include, but are not limited to, methylation of chromosomal DNA, and in particular, methylation of cytosine residues to 5-methylcytosine residues, and / or post-translational modification of histone proteins, and in particular, histone modifications that include, but are not limited to, acetylation, methylation, ubiquitination, phosphorylation, and sumoylation (covalent attachment of small ubiquitin-like modifier proteins). As used herein, “chromosomal loci” refer to loci in chromosomes located in the nucleus of a cell.

[0022] As used herein, the phrase “clonal propagate” refers to a plant or progeny thereof obtained from a plant cell. Clonal propagates can be obtained by methods including but not limited to regenerating whole plants from plant cells, plant embryos, cuttings, and the like. Various techniques used for such clonal propagation include, but are not limited to, meristem culture, somatic embryogenesis, thin cell layer cultures, adventitious shoot culture, and callus culture.

[0023] As used herein, the phrase “crop plant” includes, but is not limited to, cereal, seed, grain, fruit, and vegetable crop plants.

[0024] As used herein, the phrases “mutated chromosomal loci” (plural), “mutated chromosomal locus” (singular), “chromosomal mutations” and “chromosomal mutation” refer to portions of a chromosome that have undergone a heritable genetic change in a nucleotide sequence relative to the nucleotide sequence in the corresponding parental chromosomal loci. Mutated chromosomal loci comprise mutations that include, but are not limited to, nucleotide sequence inversions, insertions, deletions, substitutions, or combinations thereof. In certain embodiments, the mutated chromosomal loci can comprise mutations that are reversible. In this context, reversible mutations in the chromosome can include, but are not limited to, insertions of transposable elements, defective transposable elements, and certain inversions. In certain embodiments, the chromosomal loci comprise mutations that are irreversible. In this context, irreversible mutations in the chromosome can include, but are not limited to, deletions.

[0025] As used herein, the phrase “heterologous sequence”, when used in the context of an operably linked promoter, refers to any sequence or any arrangement of a sequence that isAgent Ref: P14842WO00 distinct from the sequence or arrangement of the sequence with the promoter as it is found in nature.

[0026] As used herein, the term “heterotic group” refers to genetically related germplasm that produce superior hybrids when crossed to genetically distinct germplasm of another heterotic group.

[0027] As used herein, a “loss-of-function mutation” can include an amorphic allele or a hypomorphic allele of a gene.

[0028] As used herein, the phrase “amorphic allele” refers to an allele of a gene having no gene activity in comparison to the wild-type allele of the gene. Amorphic alleles are also known as null alleles.

[0029] As used herein, the phrase “hypomorphic allele” refers to an allele of a gene with less gene activity than a wild-type allele but more gene activity than an amorphic allele.

[0030] As used herein, the term “isomorphic allele” refers to an allele of a gene having wild- type gene activity.

[0031] As used herein, the term “progeny” refers to any one of a first, second, third, or subsequent generation obtained from a parent plant or plant cell.

[0032] As used herein the terms “microRNA” or “miRNA” refers to both a miRNA that is substantially similar to a native miRNA that occurs in a plant as well as to an artificial miRNA. In certain embodiments, a transgene can be used to produce either a miRNA that is substantially similar to a native miRNA that occurs in a plant or an artificial miRNA.

[0033] As used herein, the phrase “obtaining a nucleic acid associated with the altered chromosomal locus” refers to any method that provides for the physical separation or enrichment of the nucleic acid associated with the altered chromosomal locus from covalently linked nucleic that has not been altered. In this context, the nucleic acid does not necessarily comprise the alteration (e.g., methylation) but at least comprises one or more of the nucleotide base or bases that are altered. Nucleic acids associated with an altered chromosomal locus can thus be obtained by methods including, but not limited to, molecular cloning, PCR, or direct synthesis based on sequence data.

[0034] The phrase “operably linked” as used herein refers to the joining of nucleic acid sequences such that one sequence can provide a required function to a linked sequence. In the context of a promoter, “operably linked” means that the promoter is connected to a sequence of interest such that the transcription of that sequence of interest is controlled and regulatedAgent Ref: P14842WO00 by that promoter. When the sequence of interest encodes a protein and when expression of that protein is desired, “operably linked” means that the promoter is linked to the sequence in such a way that the resulting transcript will be efficiently translated. If the linkage of the promoter to the coding sequence is a transcriptional fusion and expression of the encoded protein is desired, the linkage is made so that the first translational initiation codon in the resulting transcript is the initiation codon of the coding sequence. Alternatively, if the linkage of the promoter to the coding sequence is a translational fusion and expression of the encoded protein is desired, the linkage is made so that the first translational initiation codon contained in the 5′ untranslated sequence associated with the promoter is linked such that the resulting translation product is in frame with the translational open reading frame that encodes the protein desired. Nucleic acid sequences that can be operably linked include, but are not limited to, sequences that provide gene expression functions (i.e., gene expression elements such as promoters, 5′ untranslated regions, introns, protein coding regions, 3′ untranslated regions, polyadenylation sites, and / or transcriptional terminators), sequences that provide DNA transfer and / or integration functions (i.e., site specific recombinase recognition sites, integrase recognition sites), sequences that provide for selective functions (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scoreable marker functions (i.e., reporter genes), sequences that facilitate in vitro or in vivo manipulations of the sequences (i.e., polylinker sequences, site specific recombination sequences, homologous recombination sequences), and sequences that provide replication functions (i.e., bacterial origins of replication, autonomous replication sequences, centromeric sequences).

[0035] As used herein, the phrases “suppressing expression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene(s)”, “HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression is suppressed,” and similar refer to any genetic manipulation that provides for decreased levels of functional HDA6, CUE1, SAL1, MSH1, and / or PPD3 activity in a plant or plant cell relative to the levels of functional HDA6, CUE1, SAL1, MSH1, and / or PPD3 activity that occur in an otherwise isogenic plant or plant cell that had not been subjected to this genetic manipulation.

[0036] As used herein, the term “transgene,” in the context of a chromosomal modification, refers to any DNA from a heterologous source that has been integrated into a chromosome that is stably maintained in a host cell. In this context, heterologous sources for the DNA include, but are not limited to, DNAs from an organism distinct from the host cell organism,Agent Ref: P14842WO00 species distinct from the host cell species, varieties of the same species that are either distinct varieties or identical varieties, DNA that has been subjected to any in vitro modification, recombinant DNA, and any combination thereof.

[0037] As used herein, the term “non-regenerable” refers to a plant part or plant cell that cannot give rise to a whole plant.

[0038] Methods for introducing heritable and epigenetic and / or genetic variation that result in plants that exhibit useful traits are provided herewith along with plants, plant seeds, plant parts, plant cells, and processed plant products obtainable by these methods. In certain embodiments, methods provided herewith can be used to introduce epigenetic and / or genetic variation into varietal or non-hybrid plants that result in useful traits as well as useful plants, plant parts including, but not limited to, seeds, plant cells, and processed plant products that exhibit, carry, or otherwise reflect benefits conferred by the useful traits. In certain embodiments, methods provided herewith can be used to introduce epigenetic and / or genetic variation into plants that are also amenable to hybridization.

[0039] In certain embodiments, methods provided herewith involve suppressing expression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 target genes, restoring expression of a functional HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene, and selecting progeny plants that exhibit one or more useful traits. The nuclear gene CAB UNDEREXPRESSED (CUE1) encodes a component of the sensory plastid proteome and acts as a second-site suppressor of DNA demethylase repressor of silencing 1 (ros1). SAL1, a nuclear-encoded sensory plastid protein, is a 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1. The protein is dual-targeted to mitochondria and plastids and accumulates in root meristem and epidermal sensory plastids. SAL1 participates by degrading adenosine 3,5-bisphosphate (PAP), a plastid-to-nucleus retrograde signaling molecule that inhibits 5’-to-3’ exoribonucleases. The plant nuclear gene MutS HOMOLOG 1 (MSH1) encodes a mitochondrial- and plastid- targeted DNA binding protein that functions in organellar DNA surveillance and recombination. Through spatial regulation, MSH1 protein accumulates in sensory plastids, with little or no detectable localization to mesophyll chloroplasts, and mutation or suppression of MSH1 triggers epigenetic reprogramming of the plant for heightened stress response. The putative MSH1-interacting protein PsbP DOMAIN-CONTAINING PROTEIN 3 (PPD3) also shows sensory plastid association and influence on nuclear epigenetic effects following PPD3 mutation, RNAi suppression or overexpression. In certain embodiments,Agent Ref: P14842WO00 these useful traits are associated with either one or more altered chromosomal loci that have undergone heritable and reversible epigenetic changes.

[0040] In certain embodiments, methods for selectively suppressing expression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 target genes in sub-populations of cells found in plants that contain plastids referred to herein as “sensory plastids” are provided. Sensory plastids are plastids that occur in cells that exhibit preferential expression of at least the MSH1 promoter. In certain embodiments, MSH1 and other promoters active in sensory plastids can thus be operably linked to a heterologous sequence that perturbs plastid function to effect selective suppression of genes in cells containing the sensory plastids. In addition to the distinguishing characteristic of expressing MSH1, such cells containing sensory plastids can also be readily identified as their plastids are only about 30-40% of the size of the chloroplasts contained within mesophyll cells. Other promoters believed to be active in sensory plastids include, but are not limited to, CUE1, SAL1, and PPD3 gene promoters. Selective suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes in cells containing sensory plastids can trigger epigenetic changes that provide useful plant traits.

[0041] Examples of HDA6, CUE1, and SAL1 genes from Arabidopsis with the accession number for the corresponding sequences in the Arabidopsis genome database (on the world wide web at the address “arabidopsis.org”) and orthologous HDA6, CUE1, and SAL1 genes that can be targeted for suppression in other crop plants are provided in Table 1. Examples of MSH1 and PPD3 target genes are disclosed in US Patent No.10058044, incorporated herein by reference in its entirety. Orthologous genes from many crop species can be obtained through the BLAST comparison of the protein sequences of the Arabidopsis genes to the genomic databases (NCBI and publicly available genomic databases for specific crop species), as well as from the specific names of the genes. Specifically, the genome sequences are available for apple, arugula, asparagus, banana, barley, bean, Brassica, cacao, Camelina, carrot, cashew, cassava, chickpea, clementine, coffee, cotton, cucumber, flax, grape, lettuce, lentil, maize, millet, olive, papaya, peach, peanut, pecan, pineapple, potato, rice, sorghum, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet orange, tomato, wheat, yam, and other species at the following internet or world wide web address: phytozome- next.jgi.doe.gov.

[0042] Table 1Agent Ref: P14842WO00

[0043] In general, methods provided herewith for introducing epigenetic and / or genetic variation in plants simply require that HDA6, CUE1, SAL1, MSH1, and / or PPD3 target geneAgent Ref: P14842WO00 expression be suppressed for a time sufficient to introduce the variation and / or in appropriate subsets of cells (e.g., cells containing sensory plastids). As such, a wide variety of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression methods can be employed to practice the methods provided herewith and the methods are not limited to a particular suppression technique.

[0044] Sequences of HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes or fragments thereof from Arabidopsis and various crop plants are provided herewith (e.g., in Table 1). In certain embodiments, such genes may be used directly in either the homologous or a heterologous plant species to provide for suppression of the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 target gene in either the homologous or heterologous plant species. A non- limiting, exemplary demonstration where an exemplary MSH1 gene from one species was shown to be effective in suppressing the endogenous MSH1 gene in both a homologous and a heterologous species is provided by Sandhu et al.2007, where a transgene that provides for an MSH1 inhibitory RNA (RNAi) with tomato MSH1 sequences was shown to inhibit the endogenous MSH1 genes of both tomato and tobacco. A transgene that provides for a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene inhibitory RNA (RNAi) with Arabidopsis, soybean, canola, or maize HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene sequences can be used in certain embodiments to inhibit the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes from other plants including, but not limited to, cotton, canola, wheat, barley, flax, oat, rye, turf grass, sugarcane, alfalfa, banana, broccoli, cabbage, carrot, cassava, cauliflower, celery, citrus, a cucurbit, eucalyptus, garlic, grape, millet, onion, lettuce, pea, peanut, pepper, potato, poplar, pine, sunflower, safflower, sorghum, blackberry, blueberry, sugar beet, sweet potato, tobacco, strawberry, sugar beet, sweet potato, Jatropha, Camelina, and Agave can be obtained by a variety of techniques and used to suppress expression of either the corresponding HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene in those plants or the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene in a distinct plant.

[0045] Methods for obtaining HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes for various plants include, but are not limited to, techniques such as: i) searching amino acid and / or nucleotide sequence databases comprising sequences from the plant species to identify the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene by sequence identity comparisons; ii) cloning the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene by either PCR from genomic sequences or RT-PCR from expressed RNA; iii) cloning the HDA6, CUE1, SAL1, MSH1,Agent Ref: P14842WO00 and / or PPD3 gene from a genomic or cDNA library using PCR and / or hybridization based techniques; iv) cloning the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene from an expression library where an antibody directed to the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene protein is used to identify the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene containing clone; v) cloning the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene by complementation of an HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene mutant or HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene deficient plant; or vi) any combination of (i), (ii), (iii), (iv), and / or (v). The DNA sequences of the target genes can be obtained from the promoter regions or transcribed regions of the target genes by PCR isolation from genomic DNA, or PCR of the cDNA for the transcribed regions, or by commercial synthesis of the DNA sequence. RNA sequences can be chemically synthesized or, more preferably, by transcription of suitable DNA templates. Recovery of the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene from the plant can be readily determined or confirmed by constructing a plant transformation vector that provides for suppression of the gene, transforming the plants with the vector, and determining if plants transformed with the vector exhibit the characteristic responses that are typically observed when HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression is suppressed.

[0046] In certain embodiments, HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes or fragments thereof used in the methods provided herein will have nucleotide sequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to one or more of the HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes or fragments thereof provided herein that include, but are not limited to, genes provided in Table 1 and orthologs thereof found in various crop plants. In certain embodiments, the HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes or fragments thereof used in the methods provided herein encode HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene proteins or portions thereof will have amino acid sequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to one or more of the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene proteins provided herein that include, but are not limited to, the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene proteins encoded by genes provided in Table 1.

[0047] It is anticipated that HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene nucleic acid fragments of 18 to 20 nucleotides, but more preferably 21 nucleotides or more, can be used toAgent Ref: P14842WO00 effect suppression of the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene. In certain embodiments, HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene nucleic acid fragments of at least 18, 19, 20, or 21 nucleotides to about 50, 100, 200, 500, or more nucleotides can be used to effect suppression of the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene. Regions of 20, 50, 100, 500, or more bp are suitable for this purpose, with lengths of 100 to 300 bases of the target gene sequences preferable, and lengths of 300 to 500 bp or more being most preferable. For use in a hairpin or inverted repeat knockdown design, a spacer region with a sequence not related to the sequence of the genome of the target plant can be used. A hairpin construct containing 300 to 500 bp or more of a target gene sequence in the antisense orientation, followed by a spacer region whose sequence is not critical but can be an intron or non-intron. If the spacer is an intron, the castor bean catalase intron, which is effectively spliced in both monocots and dicots (Tanaka, Mita et al. Nucleic Acids Res 18(23): 6767-6770, 1990), is known to those skilled in the art and is useful for certain embodiments. After the spacer the same target gene sequence in the sense orientation is present, such that the antisense and sense strands can form a double stranded RNA after transcription of the transcribed region. The target gene sequences are followed by a polyadenylation region.3′ polyadenylation regions known to those skilled in the art to function in monocots and dicot plants include but are not limited to the Nopaline Synthase (NOS) 3′ region, the Octopine Synthase (OCS) 3′ region, the Cauliflower Mosaic Virus 35S 3′ region, the Mannopine Synthase (MAS) 3′ region. Additional 3′ polyadenylation regions from monocotyledonous genes such as those from rice, sorghum, wheat, and maize are available to those skilled in the art to provide similar polyadenylation region and function in DNA constructs. In certain embodiments, a transgene designed to suppress a target gene in dicots is designed to have the following order: promoter / antisense to target gene / catalase intron / sense gene A / polyadenylation region. In certain embodiments, a gene is designed to suppress a target gene in monocots can have the following order: promoter / intron for monocots / antisense to target gene / catalase intron / sense gene A / polyadenylation region.

[0048] Sequences that provide for suppression of a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene can include sequences that exhibit complementarity to either strand of the promoter, 5′ or 3′ untranslated region, intron, coding regions, and / or any combination thereof. A target gene promoter region for gene suppression can include the transcription start site, the TATA box, and upstream regions. The promoter region for gene silencing can be about 20,Agent Ref: P14842WO00 50, 80, or 100 nucleotides in length, and more preferably is about 100 to 500 nucleotides in length. The promoter region used for such suppression can be from different regions in the upstream promoter, preferably containing at least about 500 nucleotides upstream from the start of transcription, and most preferably containing at least about 500 nucleotides upstream from the start of translation of the native coding region of the native gene. This would include the UTR which may or may not be part of the promoter. A description of various recombinant DNA constructs that target promoter and / or adjoining regions of target genes are described in U.S. Pat. No.8,293,975, which is incorporated herein by reference in its entirety.

[0049] In certain embodiments, suppression of a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene in a plant is effected with a transgene. Transgenes that can be used to suppress expression of a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene include, but are not limited to, transgenes that produce dominant-negative mutants of a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene, a small inhibitory RNA (siRNA), a microRNA (miRNA), a co- suppressing sense RNA, and / or an anti-sense RNA that provide for inhibition of the endogenous MSH1 and DRM2 gene. U.S. patents incorporated herein by reference in their entireties that describe suppression of endogenous plant genes by transgenes include U.S. Pat. Nos.7,109,393, 5,231,020 and 5,283,184 (co-suppression methods); and U.S. Pat. Nos. 5,107,065 and 5,759,829 (antisense methods). In certain embodiments, transgenes specifically designed to produce double-stranded RNA (dsRNA) molecules with homology to the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene can be used to decrease expression of the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene. In such embodiments, the sense strand sequences of the dsRNA can be separated from the antisense sequences by a spacer sequence, preferably one that promotes the formation of a dsRNA (double-stranded RNA) molecule. Examples of such spacer sequences include, but are not limited to, those set forth in Wesley et al., Plant J., 27(6):581-90 (2001), and Hamilton et al., Plant J., 15:737-746 (1998). One non-limiting example of a vector that has been shown to provide for suppression of an MSH1 target gene in tobacco and tomato has been described by Sandhu et al., 2007 where an intron sequence separates the sense and antisense strands of the MSH1 target gene sequence. The design of recombinant DNA constructs for suppression of gene expression are also described in Helliwell, C. and P. Waterhouse (2003). “Constructs and methods for high- throughput gene silencing in plants.” Methods 30(4): 289-295.Agent Ref: P14842WO00

[0050] In certain embodiments, transgenes that provide for HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression can comprise regulated promoters that provide for either induction or downregulation of operably linked HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene inhibitory sequences. In this context, HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene inhibitory sequences can include, but are not limited to, dominant-negative mutants of a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene, a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA that provide for inhibition of the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene of a plant. Such promoters can provide for suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene during controlled time periods by either providing or withholding the inducer or down regulator. Inducible promoters include, but are not limited to, a PR-1a promoter (U.S. Patent Application Publication Number 20020062502) or a GST II promoter (WO 1990 / 008826 A1). In certain embodiments, both a transcription factor that can be induced or repressed as well as a promoter recognized by that transcription factor and operably linked to the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene inhibitory sequences are provided. Such transcription factor / promoter systems include, but are not limited to: i) RF2a acidic domain- ecdysone receptor transcription factors / cognate promoters that can be induced by methoxyfenozide, tebufenozide, and other compounds (U.S. Patent Application Publication Number 20070298499); ii) chimeric tetracycline repressor transcription factors / cognate chimeric promoters that can be repressed or de-repressed with tetracycline (Gatz, C., et al. (1992). Plant J.2, 397-404), and the like.

[0051] In certain embodiments, a promoter that provides for selective expression of a heterologous sequence that suppresses expression of the target gene in cells containing sensory plastids is used. In certain embodiments, this promoter is a CUE1, SAL1, MSH1, or PPD3 promoter. In certain embodiments, this promoter is an MSH1 or a PPD3 promoter and the operably linked heterologous sequence suppresses expression of a target gene provided in US Patent No.10767188, incorporated herein by reference in its entirety. MSH1 promoters that can be used to express heterologous sequences in cells containing sensory plastids include, but are not limited to, the Arabidopsis, sorghum, tomato, and maize promoters provided in US Patent No.10767188, incorporated herein by reference in its entirety, as well as functional derivatives thereof that likewise provide for expression in cells that contain sensory plastids. PPD3 promoters that can be used to express heterologous sequences in cellsAgent Ref: P14842WO00 containing sensory plastids include, but are not limited to, the Arabidopsis, rice, and tomato promoters provided in US Patent No.10767188, incorporated herein by reference in its entirety as well as functional derivatives thereof that provide for expression in cells that contain sensory plastids. In certain embodiments, deletion derivatives of the CUE1, SAL1, MSH1, or PPD3 promoters comprising about 1500 bp, 1000 bp, or about 750 bp can also be used to express heterologous sequences. In certain embodiments, deletion derivatives of the CUE1, SAL1, MSH1, or PPD3 promoters comprising about 800 bp, 600 bp, or about 500 bp can also be used to express heterologous sequences. In certain embodiments, the aforementioned promoters and an additional 200, 500, or 1000 base pairs of the endogenous 5′ promoter sequences can be used to express heterologous sequences. Additional 200, 500, or 1000 base pairs of the endogenous 5′ promoter sequences can be obtained by methods including, but not limited to, retrieval of sequences from databases provided herein and recovery of the adjoining promoter DNA by PCR amplification of genomic template sequences or by direct synthesis. In certain embodiments, recombinant DNA constructs for suppression of dicot target genes can comprise a CUE1, SAL1, MSH1, or PPD3 promoter from a dicotyledonous species such as Arabidopsis, soybean, or canola, is attached to a hairpin construct containing 300 to 500 bp or more of a target gene sequence in the antisense orientation, followed by a spacer region whose sequence is not critical but can be an intron or non-intron. The castor bean catalase intron (Tanaka, Mita et al. Nucleic Acids Res 18(23): 6767-6770, 1990), can be used as a spacer in certain embodiments. After the spacer the same target gene sequence in the sense orientation is present, such that the antisense and sense strands can form a double stranded RNA after transcription of the transcribed region. The target gene sequences are followed by a polyadenylation region. Various 3′ polyadenylation regions known to function in monocots and dicot plants include but are not limited to the Nopaline Synthase (NOS) 3′ region, the Octopine Synthase (OCS) 3′ region, the Cauliflower Mosaic Virus 35S 3′ region, the Mannopine Synthase (MAS) 3′ region. In certain embodiments recombinant DNA constructs for suppression of monocot target genes can comprise CUE1, SAL1, MSH1, or PPD3 promoter from a monocot species such as rice, maize, sorghum or wheat can either be attached directly to the hairpin region or to a monocot intron before the hairpin region. Monocot introns that are beneficial to gene expression when located between the promoter and coding region are the first intron of the maize ubiquitin (described in U.S. Pat. No.6,054,574, which is incorporated herein by reference in itsAgent Ref: P14842WO00 entirety) and the first intron of rice actin 1 (McElroy, Zhang et al. Plant Cell 2(2): 163-171, 1990). Additional introns that are beneficial to gene expression when located between the promoter and coding region are the maize hsp70 intron (described in U.S. Pat. No.5,859,347, which is incorporated herein by reference in its entirety), and the maize alcohol dehydrogenase 1 gene introns 2 and 6 (described in U.S. Pat. No.6,342,660, which is incorporated herein by reference in its entirety).

[0052] In certain embodiments, transgenic plants are provided where the transgene that provides for HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression is flanked by sequences that provide for removal for the transgene. Such sequences include, but are not limited to, transposable element sequences that are acted on by a cognate transposase. Non- limiting examples of such systems that have been used in transgenic plants include the cre- lox and FLP-FRT systems.

[0053] HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression can be readily identified or monitored by molecular techniques. In certain embodiments where the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene is intact but its expression is inhibited, production or accumulation of the RNA encoding HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene can be monitored. Molecular methods for monitoring HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene RNA expression levels include, but are not limited to, use of semi-quantitative or quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) techniques. Various quantitative RT-PCR procedures including, but not limited to, Taqman™ reactions (Applied Biosystems), use of Scorpion™ or Molecular Beacon™ probes, or any of the methods disclosed in Bustin, S. A. (Journal of Molecular Endocrinology (2002) 29, 23-39) can be used. It is also possible to use other RNA quantitation techniques such as Quantitative Nucleic Acid Sequence Based Amplification (Q-NASBA™) or the Invader™ technology (Third Wave Technologies).

[0054] In certain embodiments where HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression is achieved by use of a loss-of-function mutation in the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene of a plant, the presence or absence of that loss-of- function mutation in the genomic DNA can be readily determined by a variety of techniques. Certain techniques can also be used that provide for identification of the mutation in a hemizygous state (i.e., where one chromosome carries the mutated HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene and the other chromosome carries the wild type HDA6, CUE1,Agent Ref: P14842WO00 SAL1, MSH1, and / or PPD3 gene). Mutations in HDA6, CUE1, SAL1, MSH1, and / or PPD3 DNA sequences that include insertions, deletions, nucleotide substitutions, and combinations thereof can be detected by a variety of effective methods including, but not limited to, those disclosed in U.S. Pat. Nos.5,468,613, 5,217,863; 5,210,015; 5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876; 5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039; 7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of which are incorporated herein by reference in their entireties. For example, mutations can be detected by hybridization to allele-specific oligonucleotide (ASO) probes as disclosed in U.S. Pat. Nos.5,468,613 and 5,217,863. U.S. Pat. No.5,210,015 discloses detection of annealed oligonucleotides where a 5′ labelled nucleotide that is not annealed is released by the 5′-3′ exonuclease activity. U.S. Pat. No.6,004,744 discloses detection of the presence or absence of mutations in DNA through a DNA primer extension reaction. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations where single or multiple nucleotide variations in nucleic acid sequence can be detected by a process in which the sequence containing the nucleotide variation is amplified, affixed to a support and exposed to a labeled sequence-specific oligonucleotide probe. Mutations can also be detected by probe ligation methods as disclosed in U.S. Pat. No.5,800,944 where sequence of interest is amplified and hybridized to probes followed by ligation to detect a labeled part of the probe. U.S. Pat. Nos. 6,613,509 and 6,503,710, and references found therein provide methods for identifying mutations with mass spectroscopy. These various methods of identifying mutations are intended to be exemplary rather than limiting as the methods of the present invention can be used in conjunction with any polymorphism typing method to identify the presence of absence of mutations in a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene in genomic DNA samples. Furthermore, genomic DNA samples used can include, but are not limited to, genomic DNA isolated directly from a plant, cloned genomic DNA, or amplified genomic DNA.

[0055] Loss-of-function mutations in endogenous plant HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes, including HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes provided in Table 1, can be obtained from a variety of sources and by a variety of techniques. A homologous replacement sequence containing one or more loss of function mutations in the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene and homologous sequences at both ends of the double stranded break can provide for homologous recombination and substitution of theAgent Ref: P14842WO00 resident wild-type HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene sequence in the chromosome with a HDA6, CUE1, SAL1, MSH1, and / or PPD3 replacement sequence with the loss of function mutation(s). Such loss of function mutations include, but are not limited to, insertions, deletions, and substitutions of sequences within a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene that result in either a complete loss of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene function or a loss of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene function sufficient to elicit alterations (i.e., heritable and reversible epigenetic changes) in other chromosomal loci or mutations in other chromosomal loci. Loss-of-function mutations in a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene include, but are not limited to, frameshift mutations, pre-mature translational stop codon insertions, deletions of one or more functional domains, and the like. Methods for substituting endogenous chromosomal sequences by homologous double stranded break repair have been reported in tobacco and maize (Wright et al., Plant J.44, 693, 2005; D'Halluin, et al., Plant Biotech. J.6:93, 2008). A homologous replacement HDA6, CUE1, SAL1, MSH1, and / or PPD3 sequence comprising a loss-of-function mutation (i.e., which provides a loss of function mutation in a HDA6, CUE1, SAL1, MSH1, and / or PPD3 target gene sequence) can also be introduced into a targeted nuclease cleavage site by non-homologous end joining or a combination of non-homologous end joining and homologous recombination (reviewed in Puchta, J. Exp. Bot.56, 1, 2005; Wright et al., Plant J.44, 693, 2005). In certain embodiments, at least one site specific double stranded break can be introduced into the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 target gene by a meganuclease. Genetic modification of meganucleases can provide for meganucleases that cut within a recognition sequence that exactly matches or is closely related to specific endogenous target gene sequence (WO / 06097853A1, WO / 06097784A1, WO / 04067736A2, U.S.20070117128A1). Methods for introduction of the loss-of-function mutations in HDA6, CUE1, SAL1, MSH1, and / or PPD3 thus includes use of site-specific nucleases including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENS), clustered regularly interspaced short palindromic repeat (CRISPR)- associated Cas nuclease (e.g., Cas9, Cas12a, Cms1, S. aureus Cas9 variants, a Cas9, a nCas9 nickase, a type V Cas nuclease, a Cas12a nuclease, a nCas12a nickase, a Cas12d (CasY), a Cas12e (CasX), a Cas12b (C2c1), a Cas12c (C2c3), a Cas12i, a Cas12f, a Cas12j, a Cas14, or eSpCas9 nuclease) in combination with guide RNAs, and the like. Methods where these site- specific nucleases, and in particular, CRISPR / Cas systems comprising a Cas nuclease and aAgent Ref: P14842WO00 guide RNA directed to HDA6, CUE1, SAL1, MSH1, and / or PPD3 are contemplated. Cpf1 or Csm1 nucleases are disclosed in US Patent Application Publication 20180148735, which is incorporated herein by reference in its entirety, and can be used to obtain HDA6, CUE1, SAL1, MSH1, and / or PPD3 loss-of-function mutations. CRISPR-Cas systems disclosed in US Patent Application Publications 20150344912, 20160138008, 20180179547, 20200172886, and 20220282244, which are incorporated herein by reference in its entirety, can also be used to obtain HDA6, CUE1, SAL1, MSH1, and / or PPD3 loss-of-function mutations. It is thus anticipated that one can select or design a nuclease that will cut within a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene sequence which can result in a loss-of- function mutation through non-homologous end joining (NHEJ)-mediated repair. In addition to its use within genome editing, a CRISPR-Cas system can also be used for control of gene expression. This application, referred to as CRISPR inhibition or CRISPRi, allows sequence- specific repression of a gene. CRISPR interference utilizes a catalytically inactive (dead) Cas variant (termed “dCas”) lacking nuclease activity. For example, dCas9 contains mutations of catalytically active residues (D10 and H840) and does not have nuclease activity. The dCas- gRNA complex retains the ability to bind to the target DNA sequence but cannot introduce any breaks in the DNA strand. CRISPRi can sterically repress transcription, e.g., by blocking transcriptional initiation or elongation. CRISPRi can also repress transcription via an effector domain. Fusing a repressor domain to a catalytically inactive Cas protein can further repress transcription. For example, the Krüppel associated box (KRAB) domain can be fused to a catalytically inactive Cas protein to repress transcription of the target gene. By varying the gRNA sequence, one can control the target DNA sequence for the dCas-gRNA complex and thereby regulate the expression of virtually any gene. In certain embodiments, at least one site specific double stranded break can be introduced in the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 target gene sequence with a zinc finger nuclease. The use of engineered zinc finger nuclease to provide homologous recombination in plants has also been disclosed (WO 03 / 080809, WO 05 / 014791, WO 07014275, WO 08 / 021207). In certain embodiments, mutations in endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 target gene genes can be identified through use of the TILLING technology (Targeting Induced Local Lesions in Genomes) as described by Henikoff et al. where traditional chemical mutagenesis would be followed by high-throughput screening to identify plants comprising point mutations or other mutations in the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 target geneAgent Ref: P14842WO00 (Henikoff et al., Plant Physiol.2004, 135:630-636). The recovery of mutations in endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes is specifically provided herein. In certain embodiments where crop plants comprise two or more HDA6, CUE1, SAL1, MSH1, and / or PPD3 (e.g., certain crop plants provided in Table 1), loss-of-function mutations can be introduced in one, two, three, four, five, six, seven, or more of the HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes present in the crop plant. In certain embodiments where a crop plant (e.g., certain crop plants provided in Table 1) has a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene which gives rise to multiple transcripts (e.g., undergoes alternative splicing), loss-of function mutations comprising deletions and / or frameshift mutations located near the 5’ end of the HDA6, CUE1, SAL1, MSH1, and / or PPD3 coding region can be introduced in the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene.

[0056] Any of the recombinant DNA constructs provided herein can be introduced into the chromosomes of a host plant via methods such as Agrobacterium-mediated transformation, Rhizobium-mediated transformation, Sinorhizobium-mediated transformation, particle- mediated transformation, DNA transfection, DNA electroporation, or “whiskers”-mediated transformation. Aforementioned methods of introducing transgenes are well known to those skilled in the art and are described in U.S. Patent Application No.20050289673 (Agrobacterium-mediated transformation of corn), U.S. Pat. No.7,002,058 (Agrobacterium- mediated transformation of soybean), U.S. Pat. No.6,365,807 (particle mediated transformation of rice), and U.S. Pat. No.5,004,863 (Agrobacterium-mediated transformation of cotton), each of which are incorporated herein by reference in their entirety. Methods of using bacteria such as Rhizobium or Sinorhizobium to transform plants are described in Broothaerts, et al., Nature.2005, 10; 433(7026):629-33. It is further understood that the recombinant DNA constructs can comprise cis-acting site-specific recombination sites recognized by site-specific recombinases, including Cre, Flp, Gin, Pin, Sre, pinD, Int-B13, and R. Methods of integrating DNA molecules at specific locations in the genomes of transgenic plants through use of site-specific recombinases can then be used (U.S. Pat. No. 7,102,055). Those skilled in the art will further appreciate that any of these gene transfer techniques can be used to introduce the recombinant DNA constructs into the chromosome of a plant cell, a plant tissue or a plant.

[0057] Methods of introducing plant minichromosomes comprising plant centromeres that provide for the maintenance of the recombinant minichromosome in a transgenic plant canAgent Ref: P14842WO00 also be used in practicing this invention (U.S. Pat. No.6,972,197 and U.S. Patent Application Publication 20120047609). In certain embodiments, the transgenic plants harbor the minichromosomes as extrachromosomal elements that are not integrated into the chromosomes of the host plant. It is anticipated that such mini-chromosomes may be useful in providing for variable transmission of a resident recombinant DNA construct that suppresses expression of a HDA6, CUE1, SAL1, MSH1, and / or PPD3 target gene.

[0058] Methods where HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression is effected in cultured plant cells are also provided herein. In certain embodiments, HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression is effected in cultured plant cells by introducing a nucleic acid that provides for such suppression into the plant cells. Nucleic acids that can be used to provide for suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene in cultured plant cells include, but are not limited to, transgenes that produce a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA directed to the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene. Nucleic acids that can be used to provide for suppression of a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene in cultured plant cells include, but are not limited to, a small inhibitory RNA (siRNA) or a microRNA (miRNA) directed against the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene. RNA molecules that provide for inhibition of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene can be introduced by electroporation. Introduction of inhibitory RNAs to cultured plant cells to inhibit target genes can in certain embodiments be accomplished as disclosed in Vanitharani et al. (Proc Natl Acad Sci USA., 2003, 100(16):9632-6), Qi et al. (Nucleic Acids Res.2004 Dec.15; 32(22):e179), or J. Cheon et al. (Microbiol. Biotechnol. (2009), 19(8), 781-786).

[0059] Methods where HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression is effected in vegetatively or clonally propagated plant materials are also provided herein. Such vegetatively or clonally propagated plant materials can include, but are not limited to, cuttings, cultured plant materials, and the like. In certain embodiments, recovery of such plant or clonally propagated plant materials that have been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression can be accomplished by methods that allow for transient suppression of the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene. In certain non- limiting examples, plant or clonally propagated plant materials that have been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression are recovered by placingAgent Ref: P14842WO00 recombinant DNA constructs that suppress a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene in vectors that provide for their excision or segregation. In certain embodiments, such excision can be facilitated by use of transposase-based systems or such segregation can be facilitated by use of mini-chromosomes. In certain embodiments, such excision or segregation can be facilitated by linking a transgene that provides for a “conditional-lethal” counter selection to the transgene that suppresses a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene in the recombinant DNA construct. Vegetatively or clonally propagated plant materials that have been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression and lacking recombinant DNA constructs that suppress a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene can then be screened and / or selected for useful traits. Also provided are methods where vegetatively or clonally propagated plant materials are obtained from a plant resulting from a self or outcross or from a cultured plant cell, where either the plant or plant cell had been subjected to suppression of a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene. Such vegetatively or clonally propagated plant materials obtained from such plants resulting from a self or outcross or from a plant cell that have been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression can also be screened and / or selected for useful traits. Also provided herein are methods where a sexually reproducing plant or plant population comprising useful traits is vegetatively or clonally propagated, and a plant or a plant population derived therefrom is then used to produce seed or a seed lot.

[0060] Plants subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression, as well as the progeny thereof, can exhibit a variety of nuclear chromosomal DNA methylation patterns that are absent from control plants that were not subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression. Such methylation patterns can include, but are not limited to, CG hypermethylation, pericentromeric CHG hypermethylation, and / or additional characteristic methylation patterns observed in plants or progeny thereof that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression. Such methylation patterns can also include, but are not limited to, changes in 5- hydroxymethylation and in particular, the occurrence of 5-hydroxymethylcytosine (5-hmC). Changes in 5-hmC can be monitored by immunoassays (Quest 5-hmC™ DNA ELISA Kit, Zymo Research Corp.; or EpiSeeker™ hydroxymethylated DNA Quantification Kit, Abeam, Inc.). It is anticipated that plants, plant parts, and processed plant products provided herein or produced by the methods provided herein can be identified by comparing methylationAgent Ref: P14842WO00 patterns in the genomic DNA of such materials to the methylation patterns of control plants, plant parts, and processed plant products.

[0061] HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression can also be readily identified or monitored by traditional methods where plant phenotypes are observed. It is further contemplated that in certain embodiments, a combination of both molecular, biochemical, and traditional methods can be used to identify or monitor HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression in plants.

[0062] In certain embodiments, sub-populations of plants comprising the useful traits and epigenetic changes induced by suppression of the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene can be selected and bred as a population. Such populations can then be subjected to one or more additional rounds of selection for the useful traits and / or epigenetic changes to obtain subsequent sub-populations of plants exhibiting the useful trait. Any of these sub-populations can also be used to generate a seed lot. In certain embodiments, plants can be selfed or outcrossed to obtain an F1 generation. A bulk selection at the F1, F2, and / or F3 generation can thus provide a population of plants exhibiting the useful trait and / or epigenetic changes or a seed lot. In certain embodiments, it is also anticipated that populations of progeny plants or progeny seed lots comprising a mixture of inbred and hybrid germplasms can be derived from populations comprising hybrid germplasm (i.e., plants arising from cross of one inbred line to a distinct inbred line). In certain embodiments, such sub-populations can comprise grafted plants comprising a scion grafted to rootstock that had been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression. Sub-populations of grafted plants where the rootstock source plant is the progeny of a parental plant that had been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression and that was selected for one or more useful traits can also be selected and bred as a population. Any of the aforementioned subpopulations can comprise 2 or more, 10 or more, 50 or more, 100 or more, 1000 or more, or 10,000 or more plants. Seed lots thus obtained from these methods or other methods provided herein can comprise seed wherein at least 25%, 50%, 60%, 70%, 80%, 90%, or 95% of progeny plants grown from the seed exhibit a useful trait. The selection would provide the most robust and vigorous of the population for seed lot production. Seed lots produced in this manner could be used for either breeding or sale. In certain embodiments, a seed lot comprising seed wherein at least 25%, 50%, 60%, 70%, 80%, 90%, or 95% of progeny plants grown from the seed exhibit a useful trait associated with one or more epigenetic changes,Agent Ref: P14842WO00 wherein the epigenetic changes are associated with CG hyper-methylation and / or CHG hyper-methylation at one or more nuclear chromosomal loci in comparison to a control plant that does not exhibit the useful trait, and wherein the seed or progeny plants grown from said seed that is epigenetically heterogeneous are obtained. A seed lot obtainable by these methods can include at least 100, 500, 1000, 5000, or 10,000 seeds.

[0063] In certain embodiments, the methods for introducing heritable epigenetic or genetic variation in a plant or progeny thereof can comprise the step of grafting rootstock obtained from a plant or a parent plant thereof wherein HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression is suppressed to a scion. In certain embodiments, the plant, progeny of the plant, or scion contain(s) one or more epigenetic changes in one or more nuclear chromosomes, wherein the epigenetic changes are absent from nuclear chromosomes of the control plant or are absent from nuclear chromosomes of a plant from which the scion was obtained. In certain embodiments, the epigenetic change(s) are also present in the rootstock that had been subjected to perturbation of plastid function. In certain embodiments, the epigenetic changes in the plant, progeny of the plant, scion, or rootstock are associated with the improvement in the useful trait. In certain embodiments, the epigenetic changes in the plant, progeny of the plant, scion, or rootstock induced by suppression of the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene are associated with the improvement in the useful trait. In certain embodiments, the plant, progeny of the plant, scion, or rootstock contain(s) one or more epigenetic changes in one or more nuclear chromosomes that are absent from nuclear chromosomes of rootstock obtained from a plant or are absent from nuclear chromosomes of a parent plant thereof had not been subjected to perturbation of plastid function. In certain embodiments, the plant, progeny of the plant, scion and / or the rootstock exhibit CG hypermethylation of a region encompassing a HDA6, CUE1, SAL1, MSH1, and / or PPD3 locus in comparison to a control plant that had not been subjected to the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression. In certain embodiments, the plant, progeny of the plant, scion and / or the rootstock exhibit pericentromeric CHG hyper-methylation in comparison to a control plant that had not been subjected to the HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression. In certain embodiments, the plant, progeny of the plant, scion and / or the rootstock exhibit CG hypermethylation and / or CHG hypermethylation at one or more nuclear chromosomal loci in comparison to corresponding nuclear chromosomal loci of a control plant that had not been subjected to the HDA6, CUE1, SAL1, MSH1, and / or PPD3Agent Ref: P14842WO00 gene suppression. Also provided are plants or progeny thereof obtained by any of the aforementioned methods. Also provided are plant parts obtained from the plant or progeny thereof that were made by any of the aforementioned methods.

[0064] Also provided herein are grafted plants comprising a scion to which a rootstock had been grafted, wherein the rootstock is obtained from a plant or a parent plant thereof wherein HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression is suppressed, as well as progeny plants and clonal propagates obtained from the grafted plant. Such rootstocks can be also used to introduce epigenetic and / or genetic variation into varietal or non-hybrid plants that result in useful traits as well as useful plants, plant parts including, but not limited to, seeds, plant cells, and processed plant products that exhibit, carry, or otherwise reflect benefits conferred by the useful traits. In certain embodiments, such rootstocks can also be used to introduce epigenetic and / or genetic variation into plants that are also amenable to hybridization.

[0065] Rootstocks useful for introducing epigenetic and / or genetic variation into plants can be obtained from a variety of rootstock source plants wherein HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression is suppressed. In certain embodiments, the rootstock source plant is a plant that had itself been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression. In certain embodiments, the rootstock source plant is the progeny of a parental plant that had itself been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression. Various methods of making rootstock source plants by suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression are provided herein. Plants that can serve as rootstock source plants and methods of making such plants are also disclosed in US Patent Application Publication No.20120284814, which is incorporated herein by reference in its entirety, and elsewhere in this disclosure. Rootstock source plants in which MSH1 and DRM2 gene expression are simultaneously suppressed are disclosed in International Patent Publication WO 2024 / 226935 A1, which is incorporated herein by reference in its entirety.

[0066] In certain embodiments where the rootstock source plant, or a parental plant thereof, had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression, a population of progeny plants obtained from the grafted plant are screened and individual progeny plants are selected for one or more useful traits. Such populations of progeny plants can be obtained by methods including, but not limited to, selfing orAgent Ref: P14842WO00 outcrossing the grafted plant comprising the rootstock to obtain seed that give rise to the population. Such populations of progeny plants can also be obtained by methods including, but not limited to, growing a population of plants that are derived from independent clonal propagates obtained from the grafted plant comprising the rootstock. Such selected individual progeny plants that exhibit the useful trait can then be sexually or asexually propagated to yield populations of plants that exhibit the useful trait or seed lots that exhibit or harbor the useful trait. Such sexual propagation can be accomplished by selfing or outcrossing the selected individual progeny plants that exhibit the useful trait.

[0067] In certain embodiments where the rootstock source plant is the progeny of a parental plant that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression, the rootstock source plant itself can be a plant that was selected for one or more useful traits. Grafting rootstock from a plant that had been selected for a useful trait to a scion that does not exhibit the trait can impart the trait to the resultant grafted plant or to progeny thereof. Resultant grafted plants or progeny thereof that exhibit the useful trait can then be sexually or asexually propagated to yield populations of plants that exhibit the useful trait or seed lots that exhibit or harbor the useful trait.

[0068] In grafted plants or progeny thereof, suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression in the rootstock can be continuous and ongoing or can be transient. Non-limiting and exemplary methods for effecting continuous and ongoing suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression in the rootstock include suppressing expression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes with loss-of-function mutations in the endogenous gene and / or with a transgene that yields a product that suppresses expression of the endogenous gene. Alternatively, the suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression in the rootstock can be transient or have occurred in a parental plant from which the rootstock was obtained but not in the rootstock that was used in the graft. Non-limiting and exemplary methods for effecting transiently suppressing HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene function in the rootstock include suppressing expression of an endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene with a transgene that provides for inducible or repressible expression of a product that suppresses expression of the endogenous HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene, with a transgene that can be excised, or with a heterozygous transgene insert that is removed from the rootstock by segregation. Any of the methods described herein forAgent Ref: P14842WO00 restoring plastid function after perturbation can be used to generate rootstock used in certain embodiments.

[0069] Grafting can be effected by any method that provides for establishment of a vascular connection between the rootstock and the scion. Methods of grafting that can be used to effect the connection between the scion and the rootstock include, but are not limited to, apical graftage, side graftage, bark graftage, and root graftage. Such methods for effecting grafts of scions to rootstock are disclosed in “Plant Propagation: Principles and Practices; Chapter 12: Techniques of Grafting” Ed. Hartman, Kester, Davies, and Geneve, 7thEdition. Methods for effecting grafts of monocot plant scions to rootstocks that can be used with the scions and rootstocks provided herein are disclosed in Muzik and La Rue, The Grafting of Large Monocotyledonous Plants, Science 116, No.3022: 589-591, 1952.

[0070] Rootstocks subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression or obtained from a parental plant that had been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression can exhibit modifications of one or more nuclear chromosomes. In certain embodiments, such rootstocks can exhibit characteristic DNA methylation and / or gene transcription patterns that occur in plants subjected to suppression of an MSH1 target gene. Such characteristic DNA methylation and / or gene transcription patterns that occur in plants or seeds subjected to suppression of an MSH1 target gene can include, but are not limited to, those patterns disclosed in US Patent No.10767188, which is incorporated herein by example in its entirety. In certain embodiments, a rootstock, a scion grafted thereto, and / or a plant cell, a seed, a progeny plant, plant populations, seed populations, and / or processed products obtained therefrom that has been subject to suppression of a HDA6, CUE1, SAL1, and / or PPD3 gene will exhibit methylation repatterning similar to methylation repatterning observed when MSH1 is suppressed (Kundariya et al.2020 and Kundariya et al.2022). Such methylation repatterning can be assessed by comparing the methylation status of a sample from rootstocks, scions of plants grafted to root stocks, plants or seed that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes, or a sample from progeny plants or seed derived therefrom, to a sample from control plants or seed that had not been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 genes. In this and certain other contexts, such control plants include, but are not limited to, plants, grafted plants, scions thereof and rootstocks thereof that had not been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3Agent Ref: P14842WO00 gene suppression. In certain embodiments, such aforementioned changes in the methylation patterns exhibited by scions that are grafted to the rootstocks, or exhibited by a plant cell, a seed, a progeny plant, plant populations, seed populations, and / or processed products obtained from the grafted plant, be used to monitor the effectiveness of the graft in transmitting desirable epigenetic changes or to identify a plant cell, a seed, a progeny plant, plant populations, seed populations, and / or processed products obtained from the grafted plant.

[0071] Also provided herein are various methods for producing a plant exhibiting a useful trait that comprise crossing grafted plants comprising a scion grafted to rootstock that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression with another plant, or crossing progeny plants obtained from the grafted plant with another plant, and selecting one or more progeny plants obtained from the cross for an improvement in the useful trait in comparison to a control plant. In certain embodiments, the second plant can also be a grafted plant comprising a scion grafted to rootstock that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression, a progeny plants obtained from a grafted plant comprising a scion grafted to rootstock that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression, any other ungrafted plant that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression, or any other ungrafted plant obtained from one or more parental plants that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression. Such second plants can be plants that were selected for a useful trait and that were progeny of any plant or grafted plant that had subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression. Control plants used as comparators to identify progeny of the cross that exhibit an improvement in the useful trait include, but are not limited to: progeny of a cross between a plant which lacks a graft to the rootstock and a plant that is isogenic to the second plant, progeny of a self of a plant that lacks a graft to the rootstock, progeny of a self of the second plant; progeny of a cross between a plant that is isogenic to the plant source of the scion of the grafted plant and a plant that is isogenic to the second plant; and, progeny of a cross between a plant that is isogenic to the plant source of the scion of the grafted plant and that is isogenic to the plant source of a scion of the second plant when the second plant is a grafted plant. Also provided are methods where at least the scion of the first plant is from a different heterotic group than the secondAgent Ref: P14842WO00 plant or where at least the scion of the first plant is from the same heterotic group as the second plant.

[0072] Also provided herein are various methods for producing a plant exhibiting a useful trait that comprise selfing grafted plants comprising a scion grafted to rootstock that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression with another plant, or selfing progeny plants obtained from the grafted plant, and selecting one or more progeny plants obtained from the self for an improvement in the useful trait in comparison to a control plant to produce a plant exhibiting a useful trait. In certain embodiments, the selfed plant is a grafted plant where the rootstock source plant is the progeny of a parental plant that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression and the rootstock source plant itself was selected for and exhibits one or more useful traits. Control plants used as comparators to identify progeny of the self that exhibit an improvement in the useful trait include, but are not limited to: progeny of a self of a plant which lacks a graft to the rootstock, progeny of a self of a plant that has a graft to rootstock that had not been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression, and progeny of a self of a plant that is isogenic to the plant source of the scion of the grafted plant.

[0073] In certain embodiments, useful traits provided herein can be exhibited to a greater extent in subsequent generations of plants that are obtained from any of the grafted plants, parental plants, or parental plant cells that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression that are provided herein by either crossing or selfing. As such, a given initial plant obtained from a parent plant that was subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression can be selfed to obtain first, second, third, or later generations of progeny that exhibit a given useful trait to a greater extent in comparison to either the initial plant or in comparison to a control plant. An initial grafted plant comprising a scion grafted to rootstock subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression or to rootstock obtained from a parent plant that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression can be selfed to obtain first, second, third, or later generations of progeny that exhibit a given useful trait to a greater extent in comparison to either the grafted initial plant or in comparison to a control plant. In certain embodiments, a given initial plant obtained from a parent plant that was subjected to suppression of HDA6,Agent Ref: P14842WO00 CUE1, SAL1, MSH1, and / or PPD3 gene expression can be outcrossed to obtain F1, F2, F3, or later generations of progeny that exhibit a given useful trait to a greater extent in comparison to either the initial plant or in comparison to a control plant. In certain embodiments, a useful trait harbored by an initial plant or an initial grafted plant is not exhibited, or is exhibited to a lesser degree or extent, in the initial plant or an initial grafted plant. However, the useful trait harbored by such an initial plant or an initial grafted plant is exhibited or is exhibited to a greater extent in progeny obtained by outcrossing the initial plant or the initial grafted plant to another plant. A useful trait harbored by such an initial plant or an initial grafted plant can also be exhibited or is exhibited to a greater extent in progeny obtained by selfing the initial plant or the initial grafted plant. In certain embodiments, plants or grafted plants that are selfed or outcrossed can be inbred lines. In certain embodiments, a useful trait harbored by an inbred line is not exhibited, or is exhibited to a lesser degree or extent, in the inbred line. However, the useful trait harbored by such inbred lines is exhibited or is exhibited to a greater extent in progeny obtained by outcrossing the inbred line to another plant. An initial grafted plant comprising a scion grafted to rootstock subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression or to rootstock obtained from a parent plant that had been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression can be outcrossed to obtain F1, F2, F3, or later generations of progeny that exhibit a given useful trait to a greater extent in comparison to either the initial grafted plant or in comparison to a control plant. Outcrosses of such initial plants or grafted plants can be to isogenic plants or to genetically distinct plants. In the methods provided herein, initial or subsequent generations of progeny obtained from such selfs or crosses can thus be selected for useful traits. The methods provided herein also permit the identification of plants that harbor, but do not necessarily exhibit to a full extent, various useful traits.

[0074] Clonal propagates can be obtained by methods including, but not limited to, regenerating whole plants from plant cells, plant embryos, cuttings, and the like that are obtained from scions of the grafted plants provided herein or progeny thereof. Various techniques used for such clonal propagation include, but are not limited to, meristem culture, somatic embryogenesis, thin cell layer cultures, adventitious shoot culture, and callus culture. In certain embodiments, clonal propagation is effected by placing sterile plant cells, plant embryos, cuttings, and the like in sterile plant culture media containing suitable salts, sugars,Agent Ref: P14842WO00 and plant growth regulators to support regeneration of a plant or plant part. Such techniques suitable for clonal propagation are often referred to as “micropropagation.” Typically, cytokinins are used to stimulate shoot formation while auxins are used to stimulate root formation in the cultured material. Techniques that can be used for clonal propagation of potato plants provided herein include, but are not limited to, methods where sterile cuttings from tubers are multiplied in a modified Murashige-Skoog media to produce micropropagated plants that can be explanted to soil to produce micro-tubers that can then serve as seed potato tubers (Ahloowalia, Euphytica 75:163, 1994). Other methods that can be used for clonal propagation of potato plants provided herein include, but are not limited to, methods where nodal, meristem, or shoot tip tissues are cultured and multiplied (Rosell, G. et al. Potato Research 30:111, 1987, and references cited therein). Still other methods that can be used for clonal propagation of potato plants provided herein include, but are not limited to, methods where nodal segments are cultured in a bioreactor to mass produce microtubers that can then serve as seed potato tubers (Piao et al., Current Science 84 (8): 1129, 2003). Techniques that can be used for clonal propagation of sugar beet plants provided herein include, but are not limited, to petiole explant propagation (Grieve, et al. Plant Growth Regulation 21:15, 1997), or propagation of leaf blades, apical meristems, stalk, embryo, or hypocotyls (Mezei, S. et al. Biotechnology & Biotechnological Equipment, 20:1, 9-14, 2006).

[0075] Altered chromosomal loci that can confer useful traits can be identified and selected by performing appropriate comparative analyses of reference plants that do not exhibit the useful traits and test plants obtained from a parental plant or plant cell that had been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression and obtaining either the altered loci or plants comprising the altered loci. It is anticipated that a variety of reference plants and test plants can be used in such comparisons and selections. In certain embodiments, the reference plants that do not exhibit the useful trait include, but are not limited to, any of: a) a wild-type plant; b) a distinct subpopulation of plants within a given F2 population of plants of a given plant line (where the F2 population is any applicable plant type or variety); c) an F1 population exhibiting a wild type phenotype (where the F1 population is any applicable plant type or variety); and / or, d) a plant that is isogenic to the parent plants or parental cells of the test plants prior to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene in those parental plants or plant cells (i.e., the reference plant is isogenic to the plants or plant cells that were later subjected to HDA6, CUE1, SAL1, MSH1,Agent Ref: P14842WO00 and / or PPD3 gene suppression to obtain the test plants). In certain embodiments, the test plants that exhibit the useful trait include, but are not limited to, any of: a) any non-transgenic segregants that exhibit the useful trait and that were derived from parental plants or plant cells that had been subjected to transgene mediated HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression, b) a distinct subpopulation of plants within a given F2 population of plants of a given plant line that exhibit the useful trait (where the F2 population is any applicable plant type or variety); (c) any progeny plants obtained from the plants of (a) or (b) that exhibit the useful trait; or d) a plant or plant cell that had been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression that exhibit the useful trait.

[0076] In general, an objective of these comparisons is to identify differences in the small RNA profiles and / or methylation of certain chromosomal DNA loci between test plants that exhibit the useful traits and reference plants that do not exhibit the useful traits. Altered loci thus identified can then be isolated or selected in plants to obtain plants exhibiting the useful traits.

[0077] In certain embodiments, altered chromosomal loci can be identified by identifying small RNAs that are up or down regulated in the test plants (in comparison to reference plants). This method is based in part on identification of altered chromosomal loci where small interfering RNAs direct the methylation of specific gene targets by RNA-directed DNA methylation (RdDM). The RNA-directed DNA methylation (RdDM) process has been described (Chinnusamy V et al. Sci China Ser C-Life Sci. (2009) 52(4): 331-343). Any applicable technology platform can be used to compare small RNAs in the test and reference plants, including, but not limited to, microarray-based methods (Franco-Zorilla et al. Plant J. 200959(5):840-50), deep sequencing based methods (Wang et al. The Plant Cell 21:1053- 1069 (2009)), and the like.

[0078] In certain embodiments, altered chromosomal loci can be identified by identifying histone proteins associated with a locus and that are methylated or acylated in the test plants (in comparison to reference plants). The analysis of chromosomal loci associated with methylated or acylated histones can be accomplished by enriching and sequencing those loci using antibodies that recognize methylated or acylated histones. Identification of chromosomal regions associated with methylation or acetylation of specific lysine residues of histone H3 by using antibodies specific for H3K4me3, H3K9ac, H3K27me3, and H3K36me3Agent Ref: P14842WO00 has been described (Li et al., Plant Cell 20:259-276, 2008; Wang et al. The Plant Cell 21:1053-1069 (2009).

[0079] In certain embodiments, altered chromosomal loci can be identified by identifying chromosomal regions (genomic DNA) that has an altered methylation status in the test plants (in comparison to reference plants). An altered methylation status can comprise either the presence or absence of methylation in one or more chromosomal loci of a test plant comparison to a reference plant. Any applicable technology platform can be used to compare the methylation status of chromosomal loci in the test and reference plants. Applicable technologies for identifying chromosomal loci with changes in their methylation status include, but not limited to, methods based on immunoprecipitation of DNA with antibodies that recognize 5-methylcytidine, methods based on use of methylation dependent restriction endonucleases and PCR such as McrBC-PCR methods (Rabinowicz, et al. Genome Res.13: 2658-26642003; Li et al., Plant Cell 20:259-276, 2008), sequencing of bisulfite-converted DNA (Frommer et al. Proc. Natl. Acad. Sci. U.S.A.89 (5): 1827-31; Tost et al. BioTechniques 35 (1): 152-156, 2003), methylation-specific PCR analysis of bisulfite treated DNA (Herman et al. Proc. Natl. Acad. Sci. U.S.A.93 (18): 9821-6, 1996), deep sequencing based methods (Wang et al. The Plant Cell 21:1053-1069 (2009)), methylation sensitive single nucleotide primer extension (MsSnuPE; Gonzalgo and Jones Nucleic Acids Res.25 (12): 2529-2531, 1997), fluorescence correlation spectroscopy (Umezu et al. Anal Biochem. 415(2):145-50, 2011), single molecule real time sequencing methods (Flusberg et al. Nature Methods 7, 461-465), high resolution melting analysis (Wojdacz and Dobrovic (2007) Nucleic Acids Res.35 (6): e41), and the like.

[0080] Methods for introducing various chromosomal modifications that can confer a useful trait into a plant, as well as the plants, plant parts, and products of those plant parts are also provided herein. Chromosomal alterations and / or chromosomal mutations induced by suppression of a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene can be identified as described herein. Once identified, chromosomal modifications including, but not limited to, chromosomal alterations, chromosomal mutations, or transgenes that provide for the same genetic effect as the chromosomal alterations and / or chromosomal mutations induced by suppression of a HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene can be introduced into host plants to obtain plants that exhibit the desired trait. In this context, the “same genetic effect” means that the introduced chromosomal modification provides for an increase and / or aAgent Ref: P14842WO00 reduction in expression of one or more endogenous plant genes that is similar to that observed in a plant that has been subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression and exhibits the useful trait. In certain embodiments where an endogenous gene is methylated in a plant subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression and exhibits both reduced expression of that gene and a useful trait, chromosomal modifications in other plants that also result in reduced expression of that gene and the useful trait are provided. In certain embodiments where an endogenous gene is demethylated in a plant subjected to HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene suppression and exhibits both increased expression of that gene and a useful trait, chromosomal modifications in other plants that also result in increased expression of that gene and that useful trait are provided.

[0081] In certain embodiments, the chromosomal modification that is introduced is a chromosomal alteration. Chromosomal alterations including, but not limited to, a difference in a methylation state can be introduced by crossing a plant comprising the chromosomal alteration to a plant that lacks the chromosomal alteration and selecting for the presence of the alteration in F1, F2, or any subsequent generation progeny plants of the cross. In certain embodiments, the chromosomal alterations in specific target genes can be introduced by expression of a siRNA or hairpin RNA targeted to that gene by RNA directed DNA methylation (Chinnusamy V et al. Sci China Ser C-Life Sci. (2009) 52(4): 331-343; Cigan et al. Plant J 43929-940, 2005; Heilersig et al. (2006) Mol Genet Genomics 275437-449; Miki and Shimamoto, Plant Journal 56(4):539-49; Okano et al. Plant Journal 53(1):65-77, 2008).

[0082] In certain embodiments, the chromosomal modification is a chromosomal mutation. Chromosomal mutations that provide for reductions or increases in expression of an endogenous gene of a chromosomal locus can include, but are not limited to, insertions, deletions, and / or substitutions of nucleotide sequences in a gene. Chromosomal mutations can result in decreased expression of a gene by a variety of mechanisms that include, but are not limited to, introduction of missense codons, frame-shift mutations, premature translational stop codons, promoter deletions, mutations that disrupt mRNA processing, and the like. Chromosomal mutations that result in increased expression of a gene include, but are not limited to, promoter substitutions, removal of negative regulatory elements from the gene, and the like. Chromosomal mutations can be introduced into specific loci of a plant by any applicable method. Applicable methods for introducing chromosomal mutations inAgent Ref: P14842WO00 endogenous plant chromosomal loci include, but are not limited to, homologous double stranded break repair (Wright et al., Plant J.44, 693, 2005; D'Halluin, et al., Plant Biotech. J. 6:93, 2008), non-homologous end joining or a combination of non-homologous end joining and homologous recombination (reviewed in Puchta, J. Exp. Bot.56, 1, 2005; Wright et al., Plant J.44, 693, 2005), meganuclease-induced, site specific double stranded break repair (WO / 06097853A1, WO / 06097784A1, WO / 04067736A2, U.S.20070117128A1), and zinc finger nuclease mediated homologous recombination (WO 03 / 080809, WO 05 / 014791, WO 07014275, WO 08 / 021207). In certain embodiments, desired mutations in endogenous plant chromosomal loci can be identified through use of the TILLING technology (Targeting Induced Local Lesions in Genomes) as described (Henikoff et al., Plant Physiol.2004, 135:630-636).

[0083] In certain embodiments, chromosomal modifications that provide for the desired genetic effect can comprise a transgene. Transgenes that can result in decreased expression of a gene by a variety of mechanisms that include, but are not limited to, dominant-negative mutants, a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA and the like. U.S. patents incorporated herein by reference in their entireties that describe suppression of endogenous plant genes by transgenes include U.S. Pat. Nos.7,109,393, 5,231,020 and 5,283,184 (co-suppression methods); and U.S. Pat. Nos.5,107,065 and 5,759,829 (antisense methods). In certain embodiments, transgenes specifically designed to produce double-stranded RNA (dsRNA) molecules with homology to the endogenous gene of a chromosomal locus can be used to decrease expression of that endogenous gene. In such embodiments, the sense strand sequences of the dsRNA can be separated from the antisense sequences by a spacer sequence, preferably one that promotes the formation of a dsRNA (double-stranded RNA) molecule. Examples of such spacer sequences include, but are not limited to, those set forth in Wesley et al., Plant J., 27(6):581- 90 (2001), and Hamilton et al., Plant J., 15:737-746 (1998). Vectors for inhibiting endogenous plant genes with transgene-mediated expression of hairpin RNAs are disclosed in U.S. Patent Application Nos.20050164394, 20050160490, and 20040231016, each of which is incorporated herein by reference in their entirety.

[0084] Transgenes that result in increased expression of a gene of a chromosomal locus include, but are not limited to, a recombinant gene fused to heterologous promoters that are stronger than the native promoter, a recombinant gene comprising elements such asAgent Ref: P14842WO00 heterologous introns, 5′ untranslated regions, 3′ untranslated regions that provide for increased expression, and combinations thereof. Such promoter, intron, 5′ untranslated, 3′ untranslated regions, and any necessary polyadenylation regions can be operably linked to the DNA of interest in recombinant DNA molecules that comprise parts of transgenes useful for making chromosomal modifications as provided herein.

[0085] Examples of promoters useful for expression of transgenes include, but are not limited to, enhanced or duplicate versions of the viral CaMV35S and FMV35S promoters (U.S. Pat. No.5,378,619, incorporated herein by reference in its entirety), the cauliflower mosaic virus (CaMV) 19S promoters, the rice Act1 promoter and the Figwort Mosaic Virus (FMV) 35S promoter (U.S. Pat. No.5,463,175; incorporated herein by reference in its entirety). Exemplary introns useful for transgene expression include, but are not limited to, the maize hsp70 intron (U.S. Pat. No.5,424,412; incorporated by reference herein in its entirety), the rice Act1 intron (McElroy et al., 1990, The Plant Cell, Vol.2, 163-171), the CAT-1 intron (Cazzonnelli and Velten, Plant Molecular Biology Reporter 21: 271-280, September 2003), the pKANNIBAL intron (Wesley et al., Plant J.200127(6):581-90; Collier et al., 2005, Plant J 43: 449-457), the PIV2 intron (Mankin et al. (1997) Plant Mol. Biol. Rep.15(2): 186-196) and the “Super Ubiquitin” intron (U.S. Pat. No.6,596,925, incorporated herein by reference in its entirety; Collier et al., 2005, Plant J 43: 449-457). Exemplary polyadenylation sequences include, but are not limited to, and Agrobacterium tumor-inducing (Ti) plasmid nopaline synthase (NOS) gene and the pea ssRUBISCO E9 gene polyadenylation sequences.

[0086] Plant lines and plant populations obtained by the methods provided herein can be screened and selected for a variety of useful traits by using a wide variety of techniques. In certain embodiments, individual progeny plant lines or populations of plants obtained from the selfs or outcrosses of plants where HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression was suppressed to other plants are screened and selected for the desired useful traits.

[0087] In certain embodiments, the plant is selected from the group consisting of a crop plant, a tree, a bush, and a vine. In certain embodiments, the crop plant is selected from the group consisting of corn, soybean, cotton, canola, wheat, rice, tomato, tobacco, millet, potato, sugarbeet, cassava, alfalfa, barley, oat, sugarcane, sunflower, strawberry, and sorghum. In certain embodiments, the tree is selected from the group consisting of an apple, apricot, grapefruit, orange, peach, pear, plum, lemon, coconut, poplar, eucalyptus, date palm, palmAgent Ref: P14842WO00 oil, pine, and olive tree. In certain embodiments, the bush is selected from the group consisting of a blueberry, raspberry, and blackberry bush. In certain embodiments, the vine is a grape vine.

[0088] In certain embodiments, the screened and selected trait is improved plant yield. In certain embodiments, such yield improvements are improvements in the yield of a plant line relative to one or more parental line(s) (e.g., a control plant or check which has not been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression) under non-stress conditions. Non-stress conditions comprise conditions where water, temperature, nutrients, minerals, and light fall within typical ranges for cultivation of the plant species. Such typical ranges for cultivation comprise amounts or values of water, temperature, nutrients, minerals, and / or light that are neither insufficient nor excessive. In certain embodiments, such yield improvements are improvements in the yield of a plant line relative to parental line(s) (e.g., a control plant or check which has not been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression) under abiotic stress conditions. Such abiotic stress conditions include, but are not limited to, conditions where water, temperature, nutrients, minerals, and / or light that are either insufficient or excessive. Abiotic stress conditions would thus include, but are not limited to, drought stress, osmotic stress, nitrogen stress, phosphorous stress, mineral stress, heat stress, cold stress, and / or light stress. In this context, mineral stress includes, but is not limited to, stress due to insufficient or excessive potassium, calcium, magnesium, iron, manganese, copper, zinc, boron, aluminum, or silicon. In this context, mineral stress includes, but is not limited to, stress due to excessive amounts of heavy metals including, but not limited to, cadmium, copper, nickel, zinc, lead, and chromium.

[0089] Improvements in yield in plant lines obtained by the methods provided herein can be identified by direct measurements of wet or dry biomass including, but not limited to, grain, lint, leaves, stems, or seed. Improvements in yield can also be assessed by measuring yield related traits that include, but are not limited to, 1000 or 100 seed weight, a harvest index, and seed weight. In certain embodiments, such yield improvements are improvements in the yield of a plant line relative to one or more parental line(s) and can be readily determined by growing plant lines obtained by the methods provided herein in parallel with the parental plants. In certain embodiments, field trials to determine differences in yield whereby plots of test and control plants are replicated, randomized, and controlled for variation can beAgent Ref: P14842WO00 employed (Giesbrecht F G and Gumpertz M L.2004. Planning, Construction, and Statistical Analysis of Comparative Experiments. Wiley. New York; Mead, R.1997. Design of plant breeding trials. In Statistical Methods for Plant Variety Evaluation. eds. Kempton and Fox. Chapman and Hall. London). Methods for spacing of the test plants (i.e., plants obtained with the methods of this invention) with check plants (e.g., parental or other controls which have not been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression) to obtain yield data suitable for comparisons are provided in references that include, but are not limited to, any of Cullis, B. et al. J. Agric. Biol. Env. Stat.11:381-393; and Besag, J. and Kempton, R A.1986. Biometrics 42: 231-251).

[0090] In certain embodiments, the screened and selected trait is improved resistance to biotic plant stress relative to the parental lines. Biotic plant stress includes, but is not limited to, stress imposed by plant fungal pathogens, plant bacterial pathogens, plant viral pathogens, insects, nematodes, and herbivores. In certain embodiments, screening and selection of plant lines that exhibit resistance to fungal pathogens including, but not limited to, an Alternariaa Diplodia sp., an Erysiphe sp., a Fusarium sp., Gaeumanomyces sp., Helminthosporium sp., Macrophomina sp., a Nectria sp., a Peronospora sp., a Phakopsora sp., Phialophora sp., a Phoma sp., a Phymatotrichum sp., a Phytophthora sp., a Plasmopara sp., a Puccinia sp., a Podosphaera sp., a Pyrenophora sp., a Pyricularia sp, a Pythium sp., a Rhizoctonia sp., a Sclerotium sp., a Sclerotinia sp., a Septoria sp., a Thielaviopsis sp., an Uncinula sp, a Venturia sp., and a Verticillium sp. are provided. In certain embodiments, screening and selection of plant lines that exhibit resistance to bacterial pathogens including, but not limited to, an Erwinia sp., a Pseudomonas sp., and a Xanthamonas sp. are provided. In certain embodiments, screening and selection of plant lines that exhibit resistance to insects including, but not limited to, aphids and other piercing / sucking insects such as Lygus sp., lepidopteran insects such as Armigera sp., Helicoverpa sp., Heliothis sp., and Pseudoplusia sp., and coleopteran insects such as Diabroticus sp. are provided. In certain embodiments, screening and selection of plant lines that exhibit resistance to nematodes including, but not limited to, Meloidogyne sp., Heterodera sp., Belonolaimus sp., Ditylenchus sp., Globodera sp., Naccobbus sp., and Xiphinema sp. are provided.

[0091] Other useful traits that can be obtained by the methods provided herein include various seed quality traits including, but not limited to, improvements in either theAgent Ref: P14842WO00 compositions or amounts of oil, protein, or starch in the seed. Still other useful traits that can be obtained by methods provided herein include, but are not limited to, increased biomass, non-flowering, male sterility, digestibility, seed filling period, maturity (either earlier or later as desired), reduced lodging, and plant height (either increased or decreased as desired). Still other useful traits that can be obtained by methods provided herein include, but are not limited to, delayed leaf senescence, increased flower number, improved architecture for high density planting, improved photosynthesis, increased root mass, increased cell number, improved seedling vigor, improved seedling size, increased rate of cell division, improved metabolic efficiency, and increased meristem size. Such aforementioned improvements are in comparison to check plants (e.g., parental or other controls which have not been subjected to suppression of HDA6, CUE1, SAL1, MSH1, and / or PPD3 gene expression). Embodiments

[0092] The following numbered embodiments also form part of the present disclosure:

[0093] 1. A method for producing a plant having a useful trait, the method comprising: (a) crossing a first plant to a second plant or selfing the first plant, wherein the first plant comprises suppressed expression of an endogenous HDA6, CUE1, and / or SAL1 gene; (b) screening a population of progeny plants obtained from the cross or self of step (a) for the useful trait; and (c) selecting one or more progeny plants having the useful trait.

[0094] 2. The method of embodiment 1, wherein the first plant comprises a loss-of-function mutation in the endogenous HDA6, CUE1, and / or SAL1 gene.

[0095] 3. The method of embodiment 1 or embodiment 2, wherein the first plant comprises a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA having complementarity to the endogenous HDA6, CUE1, and / or SAL1 gene.

[0096] 4. The method of any one of embodiments 1-3, wherein the first plant comprises a catalytically inactive Cas protein, optionally wherein the Cas protein is fused to a transcriptional repressor domain, and a guide RNA molecule comprising a spacer RNA molecule that targets the endogenous HDA6, CUE1, and / or SAL1 gene.

[0097] 5. The method of any one of embodiments 1-4, wherein the endogenous HDA6 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 51, 52, 53,Agent Ref: P14842WO00 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; or wherein the endogenous HDA6 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.

[0098] 6. The method of any one of embodiments 1-5, wherein the endogenous CUE1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; or wherein the endogenous CUE1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

[0099] 7. The method of any one of embodiments 1-6, wherein the endogenous SAL1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36; or wherein the endogenous SAL1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0100] 8. The method of any one of embodiments 1-7, wherein the first plant comprises suppressed expression of: (i) an endogenous HDA6 and CUE1 gene; (ii) an endogenous HDA6 and SAL1 gene; (iii) an endogenous HDA6 and MSH1 gene; or (iv) an endogenous HDA6 and PPD3 gene.

[0101] 9. The method of any one of embodiments 1-8, wherein the plant is selected from the group consisting of maize, soybean, canola, cotton, wheat, rice, tomato, tobacco, millet, potato, sugarbeet, cassava, alfalfa, barley, oat, sugarcane, sunflower, strawberry, and sorghum.

[0102] 10. The method of any one of embodiments 1-9, wherein the plant is a soybean plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 52, 53, or 54 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 65, 66, or 67 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 2, 3, or 4 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 13, 14, or 15 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 24,Agent Ref: P14842WO00 25, or 26 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 38, 39, or 40 or an allelic variant thereof.

[0103] 11. The method of any one of embodiments 1-9, wherein the plant is a canola plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 55, 56, 57, 58, 59, 60, or 61 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 68, 69, 70, 71, 72, 73, or 74 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 5 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 16 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 27, 28, 29, 30, 31, or 32 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 41, 42, 43, 44, 45, or 46 or an allelic variant thereof.

[0104] 12. The method of any one of embodiments 1-9, wherein the plant is a maize plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 62 or 63 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 75 or 76 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 6, 7, 8, 9, 10, or 11 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 17, 18, 19, 20, 21, or 22 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 33, 34, 35, or 36 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 47, 48, 49, or 50 or an allelic variant thereof.

[0105] 13. The method of any one of embodiments 1-12, wherein the useful trait is selected from the group consisting of improved yield, delayed flowering, non-flowering, increased biotic stress resistance, increased abiotic stress resistance, enhanced lodging resistance, enhanced growth rate, enhanced biomass, enhanced tillering, enhanced branching, delayed flowering time, delayed senescence, increased flower number, improved architecture for high density planting, improved photosynthesis, increased root mass, increased cell number, improved seedling vigor, improved seedling size, increased rate of cell division, improved metabolic efficiency, and increased meristem size in comparison to a control plant.

[0106] 14. The method of any one of embodiments 1-13, wherein the useful trait exhibits nuclear inheritance.Agent Ref: P14842WO00

[0107] 15. The method of any one of embodiments 1-14, further comprising producing seed or a progeny plant from the one or more progeny plants selected in step (c), wherein the produced seed or produced progeny plant have the useful trait.

[0108] 16. The method of any one of embodiments 1-15, further comprising producing a seed lot from the one or more progeny plants selected in step (c) or from one or more progeny plants obtained therefrom.

[0109] 17. A plant or progeny thereof that exhibits a useful trait that is made by the method of any one of embodiments 1-16.

[0110] 18. A plant part obtained from the plant or progeny thereof of embodiment 17, optionally wherein the part is a seed or grain.

[0111] 19. A processed plant product obtained from the plant part of embodiment 18.

[0112] 20. A plant or plant cell having suppressed expression of an endogenous HDA6, CUE1, and / or SAL1 gene, wherein the plant or plant cell comprises: (i) a loss-of-function mutation in the endogenous HDA6, CUE1, and / or SAL1 gene; (ii) a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA having complementarity to the endogenous HDA6, CUE1, and / or SAL1 gene; and / or (iii) a catalytically inactive Cas protein, optionally wherein the Cas protein is fused to a transcriptional repressor domain, and a guide RNA molecule comprising a spacer RNA molecule that targets the endogenous HDA6, CUE1, and / or SAL1 gene.

[0113] 21. The plant or plant cell of embodiment 20, wherein the endogenous HDA6 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; or wherein the endogenous HDA6 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.

[0114] 22. The plant or plant cell of embodiment 20 or embodiment 21, wherein the endogenous CUE1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; or wherein the endogenous CUE1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at leastAgent Ref: P14842WO00 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

[0115] 23. The plant or plant cell of any one of embodiments 20-22, wherein the endogenous SAL1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36; or wherein the endogenous SAL1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0116] 24. The plant or plant cell of any one of embodiments 20-23, wherein the plant or plant cell comprises suppressed expression of: (i) an endogenous HDA6 and CUE1 gene; (ii) an endogenous HDA6 and SAL1 gene; (iii) an endogenous HDA6 and MSH1 gene; or (iv) an endogenous HDA6 and PPD3 gene.

[0117] 25. The plant or plant cell of any one of embodiments 20-24, wherein the plant is selected from the group consisting of maize, soybean, canola, cotton, wheat, rice, tomato, tobacco, millet, potato, sugarbeet, cassava, alfalfa, barley, oat, sugarcane, sunflower, strawberry, and sorghum.

[0118] 26. The plant or plant cell of any one of embodiments 20-25, wherein the plant or plant cell is a soybean plant or plant cell, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 52, 53, or 54 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 65, 66, or 67 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 2, 3, or 4 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 13, 14, or 15 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 24, 25, or 26 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 38, 39, or 40 or an allelic variant thereof.

[0119] 27. The plant or plant cell of any one of embodiments 20-25, wherein the plant or plant cell is a canola plant or plant cell, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 55, 56, 57, 58, 59, 60, or 61 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 68, 69, 70, 71, 72, 73, or 74 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQAgent Ref: P14842WO00 ID NO: 5 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 16 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 27, 28, 29, 30, 31, or 32 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 41, 42, 43, 44, 45, or 46 or an allelic variant thereof.

[0120] 28. The plant or plant cell of any one of embodiments 20-25, wherein the plant or plant cell is a maize plant or plant cell, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 62 or 63 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 75 or 76 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 6, 7, 8, 9, 10, or 11 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 17, 18, 19, 20, 21, or 22 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 33, 34, 35, or 36 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 47, 48, 49, or 50 or an allelic variant thereof.

[0121] 29. A method for obtaining a plant or plant cell of any one of embodiments 20-28, the method comprising introducing into a plant or plant cell: (i) a loss-of-function mutation in the endogenous HDA6, CUE1, and / or SAL1 gene; (ii) a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA having complementarity to the endogenous HDA6, CUE1, and / or SAL1 gene; and / or (iii) a catalytically inactive Cas protein, optionally wherein the Cas protein is fused to a transcriptional repressor domain, and a guide RNA molecule comprising a spacer RNA molecule that targets the endogenous HDA6, CUE1, and / or SAL1 gene.

[0122] 30. The method of embodiment 29, wherein the loss-of-function mutation is introduced with one or more gene editing molecules.

[0123] 31. The method of embodiment 30, wherein the gene editing molecules comprise: (i) a Cas protein and a guide RNA directed to the gene; (ii) a transcription activator-like effector nuclease (TALEN) directed to the gene; (iii) a zinc-finger nuclease (ZFN) directed to the gene; or (iv) any one of (i), (ii), or (iii) and DNA donor template.

[0124] 32. The method of any one of embodiments 29-31, wherein the loss-of-function mutation is introduced by random mutagenesis, and wherein the method further comprisingAgent Ref: P14842WO00 screening progeny subjected to the mutagenesis by a DNA analysis technique to identify a plant comprising the loss-of-function mutation.

[0125] 33. The method of any one of embodiments 29-32, wherein the endogenous HDA6 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; or wherein the endogenous HDA6 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.

[0126] 34. The method of any one of embodiments 29-33, wherein the endogenous CUE1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; or wherein the endogenous CUE1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

[0127] 35. The method of any one of embodiments 29-34, wherein the endogenous SAL1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36; or wherein the endogenous SAL1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0128] 36. The method of any one of embodiments 29-35, wherein the plant is selected from the group consisting of maize, soybean, canola, cotton, wheat, rice, tomato, tobacco, millet, potato, sugarbeet, cassava, alfalfa, barley, oat, sugarcane, sunflower, strawberry, and sorghum.

[0129] 37. The method of any one of embodiments 29-36, wherein the plant is a soybean plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 52, 53, or 54 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 65, 66, or 67 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 2, 3, or 4 or an allelic variant thereof, orAgent Ref: P14842WO00 wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 13, 14, or 15 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 24, 25, or 26 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 38, 39, or 40 or an allelic variant thereof.

[0130] 38. The method of any one of embodiments 29-36, wherein the plant is a canola plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 55, 56, 57, 58, 59, 60, or 61 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 68, 69, 70, 71, 72, 73, or 74 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 5 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 16 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 27, 28, 29, 30, 31, or 32 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 41, 42, 43, 44, 45, or 46 or an allelic variant thereof.

[0131] 39. The method of any one of embodiments 29-36, wherein the plant is a maize plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 62 or 63 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 75 or 76 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 6, 7, 8, 9, 10, or 11 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 17, 18, 19, 20, 21, or 22 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 33, 34, 35, or 36 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 47, 48, 49, or 50 or an allelic variant thereof.

[0132] 40. The method of any one of embodiments 29-39, further comprising selecting a plant comprising: (i) the loss-of-function mutation in the endogenous HDA6, CUE1, and / or SAL1 gene; (ii) the siRNA, miRNA, co-suppressing sense RNA, and / or an anti-sense RNA having complementarity to the endogenous HDA6, CUE1, and / or SAL1 gene; and / or (iii) the catalytically inactive Cas protein.

[0133] 41. The method of any one of embodiments 29-40, further comprising crossing the plant or a progeny thereof to a second plant or selfing the plant.

[0134] 42. The method of embodiment 41, further comprising screening a population of progeny plants obtained from the cross or self for the presence of one or more differentially methylated and / or differentially expressed genes which are differentially methylated and / orAgent Ref: P14842WO00 differentially expressed genes in plants wherein expression of a MSH1 and / or PPD3 gene is suppressed.

[0135] 43. The method of embodiment 41 or embodiment 42, further comprising screening a population of progeny plants obtained from the cross or self for a useful trait.

[0136] 44. The method of embodiment 43, wherein the useful trait is selected from the group consisting of improved yield, delayed flowering, non-flowering, increased biotic stress resistance, increased abiotic stress resistance, enhanced lodging resistance, enhanced growth rate, enhanced biomass, enhanced tillering, enhanced branching, delayed flowering time, delayed senescence, increased flower number, improved architecture for high density planting, improved photosynthesis, increased root mass, increased cell number, improved seedling vigor, improved seedling size, increased rate of cell division, improved metabolic efficiency, and increased meristem size in comparison to a control plant.

[0137] 45. A guide RNA molecule comprising a spacer RNA molecule that targets (i) the endogenous HDA6 gene of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; (ii) the endogenous CUE1 gene of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; or (iii) the endogenous SAL1 gene of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36.

[0138] 46. A genome engineering system comprising a Cas protein in association with a guide RNA molecule of embodiment 45.

[0139] 47. The genome engineering system of embodiment 46, wherein the Cas protein is catalytically inactive, optionally wherein the Cas protein is fused to a transcriptional repressor domain.

[0140] 48. A DNA molecule encoding the guide RNA molecule of embodiment 45.

[0141] 49. A DNA molecule encoding a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, or an anti-sense RNA having complementarity to: (i) the endogenous HDA6 gene of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; (ii) the endogenous CUE1 gene of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; or (iii) the endogenous SAL1 gene of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36.Agent Ref: P14842WO00 EXAMPLES

[0142] Plastids serve vital roles in energy generation and cellular signaling in plant cells. An important but less well defined subgroup of plastids, termed ‘sensory plastids’, differs from photosynthetic mesophyll chloroplasts in size, proteome and spatial distribution. Sensory plastids, found in the epidermis, vascular parenchyma, reproductive and meristem tissues, are thought to participate directly in environmental sensing and signaling. Their proteome is enriched in stress response proteins, and perturbation of sensory plastids can result in programmed adjustments in gene expression.

[0143] These examples investigated the differential effects of sensory plastid perturbation by parallel disruption of these four nuclear genes known or suspected to encode sensory plastid proteins, MSH1, PPD3, CUE1 and SAL1, to compare their influences on nuclear epigenomic behavior. The four sensory plastid perturbations were found to elicit distinct nuclear epigenomic responses, with each mutant’s effect epistatically influenced by HISTONE DEACETYLASE 6 (HDA6), an RdDM factor that participates in localized chromatin condensation during plant defense. The association of HDA6 with sensory plastid signaling could be directly linked to daylength, implicating circadian clock components in the sensory plastid-to-HDA6 epistatic interaction, and treatment-associated DNA methylation data identified candidate gene networks that underpinned the distinct phenotype transitions observed. These data allowed the modeling of environmentally-acquired adjustments in heritable plant phenotype as plastid-triggered events. Example 1: CUE1 is a confirmed sensory plastid protein

[0144] Whereas MSH1 and PPD3 are known to accumulate and function within sensory plastids, there was interest to include additional sensory plastid candidate proteins to test broader potential nuclear effects. SAL1 was confirmed as a sensory plastid component based on extensive previous studies. However, CUE1 has not been previously linked to plant environmental stress response.

[0145] CUE1 was identified exclusively in proteomic data of sensory plastids but not mesophyll chloroplasts, and transcripts are detected in vascular tissue, flowers, siliques, and root tip. To confirm CUE1 as a sensory plastid protein, CUE1::CUE1::Venus transformants in cue1 background were obtained by crossing cue1 with CUE1:Venus / Col-0 by recombineering. The CUE1 signal was present in sensory plastids as diffused and aggregatedAgent Ref: P14842WO00 peripheral signals, agreeing with its annotation as a plastid inner envelope protein. CUE1 signal was detected in the root tip, epidermis, vasculature, reproductive tissue, and shoot apical meristems in seedlings, similar to MSH1 and PPD3. No signal was detected in leaf mesophyll chloroplasts. Example 2: MSH1, PPD3, CUE1, or SAL1 perturbation triggers DNA methylation changes in distinct gene networks

[0146] DNA methylation changes can be measured to assess response to stress or developmental transitions. This example focused on changes in density-independent, context- agnostic differentially methylated positions (DMPs) with highest probability of treatment association. This methylation variation was referred to as treatment-associated gene body methylation (gbMT). This approach enabled the identification of genes and their associated networks that undergo de novo changes in DNA methylation (DMGs) and expression in response to various mutant or stress conditions.

[0147] Similar to the methylation repatterning previously described in msh1 and PPD3OX, DNA gbMTmethylation repatterning occurred in cue1 and sal1 when compared to Col-0 wild type Arabidopsis. To compare targeted gene networks responsive to each mutation, k-means clustering and STRINGdb network enrichment analysis were applied to a combined dataset of DMGs and RNAseq-derived differentially expressed genes (DEGs) for each mutant to derive candidate network hub genes related to gene expression regulation. By overlapping networks enriched in hub genes, networks in common or distinct to msh1, PPD3OX (dwarf type), cue1, and sal1 were identified (FIG.1B-D).

[0148] Several gene networks were exclusive to each genotype, supporting the distinct functions of MSH1, PPD3, SAL1 and CUE1 proteins (FIG.1C). Most of the msh1-exclusive networks were related to abiotic and biotic stress response, in agreement with previous studies of msh1 and msh1 heritable memory phenotypes that display enhanced stress tolerance with delayed growth, maturity and flowering (FIG.1C). Stress response and phytohormone pathways are prominent in previously reported msh1-derived methylome and gene expression datasets, agreeing with these data.

[0149] The most prominent networks exclusive to PPD3OX related to development, cell cycle, and meristem identity (FIG.1C). PPD3OX presents as a mixed population of dwarf and enlarged plants, and it was previously shown that defense and growth-related networksAgent Ref: P14842WO00 are divergently regulated in the dwarfed and vigorous plants, corresponding to phenotype. Several of the genes that distinguish dwarf and large plants by methylation repatterning were associated with meristem development; PPD3 accumulates in root tips, and the ppd3 mutant displays shortened root meristems as its most prominent phenotype.

[0150] Among the cue1-exclusive hub gene networks, circadian rhythm was outstanding (FIG.1C). While previous reports show that the msh1 mutant phenotype can be strongly influenced by daylength, circadian clock and light response pathways were especially enriched in the cue1 data. CUE1 (aka PHOSPHOENOLPYRUVATE / PHOSPHATE TRANSLOCATOR 1; PPT1) is a phosphoenolpyruvate (PEP) transporter that appears specific to sensory plastids, while its paralog PPT2 localizes to the mesophyll chloroplast. These data imply that CUE1 may have neofunctionalized beyond its primary metabolic function in PEP transport.

[0151] The identified sal1-exclusive gene networks were predominantly related to RNA processing and stability (FIG.1C), consistent with its known function in RNA surveillance through the SAL1-PAP-XRN pathway. These data provided assurance that the methylome analysis methods that were applied gave sufficient resolution to relate derived gene network data to mutant phenotype effects. Overlap of datasets from the four mutants revealed shared features in sensory plastid disruption. These overlapping pathways included Metabolism, Translation, RNA splicing, Transcription, and Development, with evident connections to RdDM-related pathways (FIG.1D). Example 3: The hda6 methylome reveals targeted chromatin regions corresponding to sensory plastid-induced DMGs

[0152] The msh1 mutant is known to be strongly epistatic with the RdDM component HDA6, such that the msh1 / hda6 double mutant is not recoverable at 12-hour daylength. Therefore, hda6 methylation analysis was included in this study. Loss of HDA6 function was associated with marked gene body hypermethylation in non-CG context, predominantly CHG (FIG.2A). This non-CG gene body hypermethylation in hda6 was not universal but occurred in a subset of genes (FIG.2B). It was assumed that the CHG differentially methylated regions identified designate sites where HDA6 stabilizes chromatin in concert with histone methyltransferase SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG 4 (SUVH4) and CHROMOMETHYLASE 3 (CMT3). CMT3, which directs CHG methylation, forms a positive feedback loop withAgent Ref: P14842WO00 histone H3K9 methylation by SUVH4 to suppress transcription. HDA6 interacts with SUVH4 / 5 / 6.

[0153] For this analysis, the de novo CHG hypermethylated sites provided a dataset of putative HDA6 gene targets within the Arabidopsis genome. hda6 also showed hypomethylation of TE- and rRNA-encoding regions, with most notable effects in CHG context for TEs, and CG and CHG contexts for rRNAs (FIG.2A). These patterns of change have been described previously.

[0154] The hda6 CHG hypermethylation was found at 2949 sites located within 1 kb upstream and downstream of genes, with 97% (2862) in gene bodies. These genes were referred to as CHG DMR-associated genes and, within this dataset, 2396 (87%) were present in at least one of the four sensory plastid perturbation datasets. K-means clustering and network analysis of the hda6 CHG DMR-associated gene dataset identified hub genes as central regulators like TARGET OF RAPAMYCIN (TOR), BRAHMA (BRM), SPLAYED (SYD), PICKLE (PKL), histone modifiers, and RdDM components (FIG.2C). The networks enriched in hub genes included Translation (GO:0006412), RNA splicing (GO:0008380), Developmental process (GO:0032502), and Response to stress (GO:0006950), all enriched in sensory plastid-induced conditions (FIG.1 and FIG.2C). Of the 262 core hub genes derived from the hda6 CHG DMR-associated dataset, 162 (72%) were present as core hub genes in at least one of the msh1, PPD3OX dwarf, cue1, and sal1 sensory plastid conditions (FIG.2C).

[0155] In parallel, gbMTvariation was assessed in the hda6 mutant grown at 12-hr daylength to identify 6201 DMGs. In previous studies, it was found that DMG data are generally enriched for genes functioning upstream to DEGs in signaling pathways, and it was speculated the same trend in the present datasets. Overlaps between hda6 datasets (CHG DMR-associated genes, DMGs, and DEGs) and sensory plastid mutant datasets (core hub DMGs, DMGs, and DEGs) in every possible combination allowed us to evaluate the relationship of hda6 and sensory plastid disruptions (FIG.2D). In the case of msh1, 67 msh1 core hub genes were identified in association with msh1-induced methylome changes. For PPD3OX dwarf, cue1, and sal1, applying DMG datasets to k-means clustering analysis and selecting genes involved in gene expression regulation (GO:0010467 Gene expression and GO:0010468 Regulation of gene expression) allowed us to derive core hub gene datasets.

[0156] The msh1-hda6 comparisons showed 91% of the msh1 core hub genes overlapping with hda6 CHG DMR-associated genes, while only 67% overlapped with hda6 DMGs andAgent Ref: P14842WO00 10% with hda6 DEGs. For msh1 DMGs, the overlap was largest with hda6 DMGs (52%) compared to CHG DMR-associated genes (37%) or DEGs (19%). Likewise, overlap for msh1 DEGs was larger with hda6 DEGs (31%) than with CHG DMR-associated genes (11%) or DMGs (19%) (FIG.2D). These data gave us an estimate of overlapping methylome responses induced by msh1 and hda6 mutations, with pronounced (91%) intersection between msh1 core network hubs and the hda6 CHG hypermethylation sites.

[0157] Similar overlap was observed for PPD3OX dwarf DEG, DMG, and core hub gene sets in hda6 comparisons. However, for sal1 and cue1, derived core hub genes showed greater overlap with hda6 DMGs than with the CHG hub genes. Overlapping the four mutant core hub DMG datasets with hda6 CHG DMR-associated hub genes identified several genes with overlap in more than one mutant dataset (FIG.2E), raising the possibility of HDA6 participation in the epigenomic effects induced by all four distinct sensory plastid perturbations. Example 4: Epistatic interactions of hda6 and sensory plastid mutants are daylength- dependent

[0158] Growth of the four sensory plastid-associated mutants, msh1, ppd3, cue1 and sal1, showed more pronounced phenotype effects when grown at 12-hr daylength than at 16-hr (FIG.3). It was also noted that HDA6 expression overlapped spatially with sensory plastid localization, including within ovules. HDA6 interacts directly with components of the circadian clock complex, implying that the daylength-sensitive responses observed might reflect HDA6 interaction with sensory plastid signaling.

[0159] An msh1 / hda6 double mutant is not obtainable at 12-hr daylength, with segregation of progeny significantly skewed toward homozygous wild type segregants, implying that incompatibility is gametophytic. To investigate this interaction in more detail, the msh1 / hda6 double mutant genetic experiment was repeated in plants growing at 16-hr daylength, with successful segregation for msh1 / hda6 double mutants consistent with expected ratios (FIG. 3C), indicating daylength-dependence of the msh1 / hda6 epistasis. To confirm that this short- day incompatibility was sensory plastid-derived, the hda6 mutant was crossed to mitochondrial and plastid hemicomplemented msh1 lines. Segregation was significantly skewed toward homozygous wild type under conditions for plastid MSH1 depletion, but not for mitochondrial, confirming sensory plastid-dependence of the effect.Agent Ref: P14842WO00

[0160] hda6 epistatic effects were also evident with other sensory plastid-associated mutants. Derived ppd3 / hda6 double mutants showed abnormal floral and silique development effects influenced by daylength (FIG.3E). At 12-hr but not 16-hr daylength, approximately 10% of ppd3 / hda6 plants showed aerial rosettes and abnormal floral branching (FIG.3E). Aerial rosettes are characteristic of msh1 and ppd3 at short daylength.

[0161] Surprisingly, the cue1 / hda6 double mutant showed more severe phenotypic effects at 16-hr than at 12-hr. Epistatic interaction was evident as extreme delays in growth in the double mutant plants compared to parental single mutants, with severe dwarfing that persisted to the reproductive stage and shortened siliques compared to wild type and single mutants at 16-hr daylength (FIG.3F). Silique length in cue1 / hda6 at 12-hr was comparable to hda6 at 12-hr. These growth data, when combined with gene network and gene expression analysis of the cue1 mutant, led to the conclusion that CUE1, and its interaction with HDA6, primarily impacts daylength response of the plant.

[0162] In parallel to msh1, the sal1 / hda6 could not be obtained from genetic crossing experiments conducted at 12-hr daylength (FIG.3G). Because SAL1 and HDA6 are linked at ca.1cM genetic distance, sal1+ / -hda6- / -F2plants were obtained and their progeny were genotyped. All derived F3plants in these experiments genotyped as sal1+ / +hda6- / -, again supporting gametophytic epistasis. The sal1 phenotype was more severely delayed in growth and flowering at 12-hr than 16-hr daylength. Example 5: hda6 and sensory plastid mutant DNA methylomes respond to daylength

[0163] The observed effects of daylength on hda6 epistatic interactions prompted us to investigate circadian influence on methylome behavior. hda6 and wild type methylome data were obtained from plants grown at 16-hr daylength for comparison with the 12-hr data described above. Seeds used for the 16-hr experiment were derived from parental plants grown in 12-hr daylength conditions.

[0164] At 16-hr daylength, the CHG DMR ‘islands’ previously seen in 12-hr samples were absent (FIG.4B). TEs in hda6 also showed greater levels of hypomethylation in 16-hr than 12-hr growth conditions, particularly in CHG context. Therefore, the hda6 CHG islands served as an epigenetic marker for 12-hr hda6 state, although DMG numbers were comparable in 12-hr and 16-hr conditions (FIG.4C).Agent Ref: P14842WO00

[0165] The hda612-hr DMG dataset showed more diverse hub networks, including networks identified to be specific to msh1 (stress response), sal1 (RNA metabolism), PPD3OX dwarf (meristem and cell division), and cue1 (light response, FIG.4D). Many hub networks found in 16-hr daylength were also at 12-hr daylength and shared in common with all sensory plastid conditions, including Translation (GO:0006412), Ribosome biogenesis (GO:0042254), RNA splicing (GO:0008380), and Transcription (GO:0006351).

[0166] Of the 6201 DMGs detected in hda6 at 12-hr daylength, 366 were found in all four sensory plastid conditions, and 1523 were in at least three. Hub genes and networks derived from DMGs in at least three of the four sensory plastid conditions showed marked overlap with those derived from hda6, including important regulators like TOR, INCREASED SIZE EXCLUSION LIMIT2 (ISE2), GIGANTEA (GI), and components of the RdDM pathway (FIG. 4E). These data support a hypothesis of sensory plastid intersection with HDA6 in triggering nuclear epigenetic changes.

[0167] To assess the change in DNA methylome features in sensory plastid mutants at 16-hr daylength, DNA methylome datasets were developed for msh1 and cue1 plants grown at 16- hr daylength and 2917 and 2833 genes, respectively, were identified that were differentially methylated, with 1770 genes in common.1191 (67%) of these common DMGs were also differentially methylated in hda6 at 16-hr daylength. Hub genes derived from the overlap of msh1 and cue1 datasets formed a weak network with Translation (GO:0006412), rRNA metabolic process (GO:0016072), and mRNA splicing via spliceosome (GO:0000397), but lacked the strong stress- and light-related pathways characteristic of 12-hr datasets despite the comparable number of genes used (FIG.4F).

[0168] The daylength responsive methylome changes in the sensory plastid mutants resembled hda6 effects, with sensory plastid-triggered networks for stress response, growth, and chromatin remodeling prominent in 12-hr but not 16-hr datasets. The difference between 12-hr and 16-hr methylome datasets in all mutants tested was not in number of DMGs, but in the genes and gene networks targeted, with those involved in environmental stress response pronounced at 12-hr conditions (FIG.4).

[0169] Gene expression also showed marked changes in response to daylength in hda6 and sensory plastid-perturbed plants. In hda6, msh1, and sal1, the majority of DEG networks enriched in stress response were upregulated at 12-hr daylength, while networks related to light stimulus were downregulated. This trend was less pronounced or reversed at 16-hrAgent Ref: P14842WO00 daylength. In contrast, cue1 showed an opposite trend, with stress response upregulated at 16- hr and downregulated at 12-hr. This observation supports a distinct relationship of CUE1 with HDA6 in daylength sensing and light response compared to other sensory plastid proteins. PPD3OX 12-hr data showed stress response up (dwarfed) or downregulated (large plants) depending on phenotype, which agreed with previous indications of PPD3 as a modulator of growth and defense. Example 6: The hda6 mutation confers epigenetic memory of daylength

[0170] The striking differences in DNA methylome features seen in hda6 at 12-hr and 16-hr daylengths suggested that hda6 may participate in heritable memory. To investigate how hda612-hr methylome features establish, and whether they could be maintained through transgenerational memory, hda6 methylome features in plants grown at 12-hr daylength for multiple generations versus conditions where 12-hr memory has been erased were compared (FIG.5A-C). To erase 12-hr memory, 12-hr hda6 were taken through a heterozygous state by crossing and then reestablished the homozygous mutant. This was achieved using available genotyped F2progeny from an F1cue1+ / -hda6+ / -self-cross, grown at 12-hr daylength, and selected for cue1+ / +hda6- / -individuals (FIG.5B). The derived F3generation plants were then grown at 12- and 16-hr daylengths. When grown at 16-hr, the hda612-hr signature methylome features were no longer detected (FIG.5D-E). In fact, these derived F3plants were more strongly devoid of hda616-hr signature methylome features of TE CHG hypomethylation than was seen in 16-hr hda6 plants coming from a 12-hr daylength-grown parent. This observation confirmed that removal of 12-hr memory could be achieved by transit through the heterozygous state. In contrast, growth of the F3hda6- / -plants at 12-hr daylength partially reestablished the 12-hr signature features within one plant growth cycle (FIG.5). These data were interpreted as an indication that two plant generations are required to fully reestablish daylength memory in hda6.

[0171] To confirm the two-generation requirement for memory reset, the msh1 / hda6 double mutant was grown at 16-hr daylength for two generations to remove 12-hr memory and then grown the next generation at 12-hr daylength. These plants were designated Gen112-hr msh1 / hda6 (FIG.5C). As expected, the 12-hr Gen1 msh1 / hda6 showed only partial reestablishment of the CHG gene body hypermethylation characteristic of 12-hr hda6 (FIG. 5D-E). Interestingly, growth of Gen2 msh1 / hda6 plants showed delayed development,Agent Ref: P14842WO00 particularly at reproductive stage, indicating that reestablishment of 12-hr hda6 methylome signatures was accompanied by the incompatibility phenotype of lethality in msh1 / hda612-hr daylength crossing experiments (FIG.5F-G). To confirm that the msh1 / hda6 double mutant 12-hr incompatibility is associated with reproductive development, reciprocal daylength shift experiments were also carried out. Example 7: Abiotic and biotic stress response phenotypes in the msh1 and hda6 interaction

[0172] The pronounced and overlapping enrichment of abiotic and biotic stress response pathways in methylome and gene expression datasets from msh1 and hda6 predicts that these mutants, singularly and in combination, have the capacity to elicit phenotypic stress response. Abiotic and biotic stress were applied to wild type, msh1, hda6, and the msh1 / hda6 double mutant to test this prediction. Because msh1 and hda6 both triggered changes in overlapping stress response pathways, these effects were expected to be confirmed through acquired plant phenotypes, particularly in the double mutant.

[0173] The heat stress experiments involved growth of plants at 28°C and 30°C for sustained periods as shown in FIG.6. Under 12-hr daylength, all genotypes showed early flowering under heat relative to control conditions. However, hda6 and the msh1 / hda6 double mutant flowered significantly later than wild type and msh1, reflecting enhanced heat tolerance in these lines (FIG.6). The tolerant msh1 / hda6 plants showed evidence of curly leaves, suggesting a mitochondrial influence. Similarly, hda6 and msh1 / hda6 flowered significantly later than wildtype in 16-hr daylength. This outcome was unexpected because the DNA methylation and gene expression data for msh1 and hda6 suggested greater stress response in 12-hr than 16-hr daylength. It was suspected that the observed tolerance in 16-hr plants was a consequence of memory. To test this hypothesis, the derived F3 hda6 devoid of 12-hr methylome signatures were used for the heat treatment experiments. Under both 12-hr and 16-hr daylength conditions, the F3 hda6 plants grown in heat stress showed no significant differences in flowering time compared to wild type, while hda6 possessing 12-hr methylome features flowered later (FIG.6). These data support the hypothesis of 12-hr memory in the stress experiment, but also underline the importance of hda612-hr effects on heat tolerance.

[0174] 4508 genes were identified that were either differentially expressed or methylated in msh1 / hda6 in heat relative to control conditions, with 1850 overlapping the wild type heatAgent Ref: P14842WO00 dataset. As expected, many of the overlapping genes were related to stress. Of the 4508 identified genes, 3194 were differentially expressed and 1413 differentially methylated.

[0175] These experiments were then extended to assess potential for biotic stress tolerance (FIG.1C). To evaluate biotic stress response, Arabidopsis leaves were infected with powdery mildew (Golovinomyces cichoracearum (syn. Erysiphe cichoracearum)), which invades epidermal cells, where sensory plastids reside. Powdery mildew resistance in the powdery mildew resistant 4 (pmr4) mutant depends on the salicylic acid pathway, a pronounced signal in msh1 datasets (FIG.1C). At 16-hr daylength, the msh1 / hda6 mutant showed the most effective resistance, followed by msh1 and hda6, suggesting that epistatic interaction of msh1 and hda6 influences biotic stress response (FIG.6D). This trend was more marked at 12-hr daylength as expected. These data provided evidence in support of a link between methylome changes effected by sensory plastid perturbation and / or HDA6 disruption and predictable changes in plant phenotype. More importantly, obviation of 12-hr memory in the hda6 mutant enabled association of these phenotypic changes with hda6 methylome behavior Example 8: Discussion

[0176] Sensory plastids are distinct from mesophyll chloroplasts in size, behavior, and protein enrichment. Here, distinct functions of four sensory plastid proteins were uncovered based on treatment-associated DNA methylation and gene expression analysis, suggesting that sensory plastids can influence a range of distinct signaling properties of the cell (FIG.1). Based on the results, MSH1 as a DNA-binding protein has neofunctionalized to modulate energy allocation in stress response and growth. PPD3, as a PsbP domain-containing protein and subunit of Photosystem II, also influences meristem growth, cell cycle regulation, and growth-vs-defense modulation in association with MSH1. The CUE1 gene, encoding a PEP translocator, also functions to influence circadian rhythm and light sensing. SAL1 as a bifunctional protein with 3’,5’-bisphosphate nucleotidase and inositol polyphosphate 1- phosphatase activities, participates in RNA metabolism to regulate various stress responses through a SAL1-PAP-XRN pathway. Importantly, PAP signaling depends on CUE1 to import the precursor of tyrosine and tocopherol biosynthesis required for PAP metabolism in plastids. Environment-responsive functions of sensory plastid proteins, distinct from their paralog functions in primary metabolism or housekeeping, support a model of proteinAgent Ref: P14842WO00 neofunctionalization during plastid spatial differentiation. Data from this study indicate that this process enables plants to effectively respond to their environment.

[0177] Evidence for epistatic interaction of hda6 with sensory plastid mutants was shown (FIG.3). HDA6 directly interacts with key regulators of phytohormone signaling components CORONATINE INSENSITIVE1 (COI1) and JASMONATE-ZIM-PROTEIN1 (JAZ1), DNA METHYLTRANSFERASE1 (MET1) involved in the RdDM pathway, and the circadian clock components CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). HDA6 is also involved in response to biotic stress, salt stress, ethylene and ABA signaling, leaf development, and flowering control. The model places HDA6 as the nuclear integrator of sensory plastid signals, a nuclear ‘gatekeeper’ of sorts, which sheds new light on HDA6 properties. Studies have reported changes in rRNA methylation in association with HDA6 activity. hda6 is hyperacetylated in rDNA repeats, where DNA methylation changes are context-dependent. DNA methylation in rRNA-encoding regions was shown to be daylength-dependent, adjusting as the plant shifts from growth (and heightened translation) to stress response (FIG.4).

[0178] hda6 plants grown in 12-hr and 16-hr daylengths showed most prominent differences occurring in CHG context for hypermethylation of gene bodies or hypomethylation of TEs (FIG.4). While CHG hypomethylation in pericentromeric regions, where TEs are enriched, has been reported previously for hda6, this study made clear that these features are daylength-regulated. CHG hypermethylation at 12-hr daylength was highly targeted to a subset of genes, compared to the more generally distributed, low density CG gene body methylation with little daylength sensitivity (FIG.4B). Only 434 of the 2962 CHG DMR- associated genes were differentially expressed, implying that CHG hypermethylation to stabilize chromatin under hda6 mutant conditions may serve to suppress changes in expression of these genes.

[0179] The phenotypes of hda6, msh1, ppd3, cue1, and sal1 were more severe and stress- responsive in shorter daylength when stress response is more critical (FIG.3). In fact, both the msh1 / hda6 and sal1 / hda6 double mutants were unobtainable under 12-hr daylength, emphasizing the essential role of hda6 in chromatin stabilization under short days. The observed epistatic interactions of hda6 with all four sensory plastid mutants, and the overlap in methylome and gene expression data derived from the mutants, support the model that hda6 participates in integrating sensory plastid stress signals.Agent Ref: P14842WO00

[0180] The hda6 short-day DNA methylome changes and heat stress tolerant phenotype show heritable memory, requiring at least two generations to transition between short-day and long-day states. The msh1 mutant also shows a weak phenotype in the first generation following the heterozygote, with DNA methylation variation accumulating with advancement of generations at 12-hr daylength. These data are consistent with a model of methylome adjustment during reproduction, requiring two cycles of gametogenesis, and phenotype adjustment as a function of methylome transition.

[0181] Daylength changes occur through season and latitude, and plants adjust growth and flowering accordingly. HDA6 regulates flowering time by directly interacting with FLOWERING LOCUS D (FLD). Plant growth, regulated by the photosynthetic period, is regulated by the level of metabolic compounds and MYO-INOSITOL-1-PHOSPHATE SYNTHASE 1 (MIPS1) expression. MIPS1 is differentially expressed in hda6 at 12-hr daylength and in msh1 at 12-hr and 16-hr daylengths. FAR-RED-IMPAIRED RESPONSE1 (FAR1) and FAR-RED ELONGATED HYPOCOTYL3 (FHY3) are RdDM targets that regulate MIPS1 expression and plant growth, and the far1 / fhy3 double mutant phenotype is more severe in short day. FAR1 and FHY3 are hda6 CHG DMR-associated genes and represent DMGs in hda6, msh1, and the PPD3OX dwarf. FAR1 is also a DMG in cue1. The myo- inositol pathway is linked to growth versus defense adjustment by SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE1 (SnRK1) and jasmonate signaling. SnRK1 is a regulator of growth versus defense by its antagonistic relationship with TOR, a differentially methylated hub gene in hda6, msh1, PPD3OX, and cue1. SAL1 is an inositol phosphatase, and the sal1 mutation decreases levels of various sugars. These related metabolic pathways were common hub networks in sensory plastid-perturbed conditions and in hda6, further supporting a model of sensory plastid- and HDA6-mediated regulation of growth vs defense adjustments in response to daylength.

[0182] It is not yet clear how sensory plastids communicate their signals to HDA6. Changes in redox status and related gene networks are observed in msh1 and ppd3, and the msh1 sensory plastid translatome is enriched in calcium signaling pathways. Nitric oxide is another signaling molecule produced in plastids, and cue1 (aka nitrous oxide overexpressor 1, nox1) shows high nitric oxide levels in roots and leaves. S-nitrosoglutathione (GSNO) is a nitric oxide signaling compound, and mutation of GSNO REDUCTASE (GSNOR) induces similar gene expression and histone modification patterns as hda6 to influence the growth andAgent Ref: P14842WO00 defense transition in response to light. SAL1 and CUE1 are both involved in the PAP retrograde pathway, implying yet another means of inter-organellar signaling. Example 9: Methods Plant materials and growth conditions

[0183] The Arabidopsis thaliana msh1 (SAIL-877-F01), ppd3 (GK-121C07), cue1 (SALK_116454), sal1 (fry 1-6, SALK_020882.55.50), and hda6-7 (CS66154) mutants in Col-0 background were obtained from Arabidopsis Biological Resource Center (ABRC). Seeds were stratified in water at 4 °C for 2 - 3 d and sown on soil and grown in growth chambers with 22 / 20 °C with 12-hr or 16-hr daylength and 120 − 150 μmol m−2 s−1light. The cue1 / hda6 and ppd3 / hda6 double mutants were generated by crossing single mutant parents in 12-hr, using cue1 or ppd3 as paternal and hda6 as maternal parent. The msh1 / hda6 double mutant was obtained by genotyping the progeny of msh1+ / -hda6- / -at 16-hr daylength. For the attempt to obtain sal1 / hda6 double mutant, sal1 was crossed with F3 hda6 from a cue1xhda6 cross as described in FIG.5B. The progeny of sal1+ / -hda6+ / -genotyped to isolate sal1+ / -hda6- / -, of which the seeds were used to genotype for sal1. F0, F1 and F2 plants were grown in 12-hr to obtain all double mutants.

[0184] For the heat stress experiment, plants were grown at normal condition (22 / 20 °C) for 10 days for germination. Then, treatment plants were moved to 28 / 26 °C.

[0185] The pPPD3:PPD3:GFP and pMSH1:MSH1:GFP transgenic lines were generated in previous studies. pCUE1:CUE1:Venus / WT was ordered from ABRC (CS67180) and crossed to cue1-TDNA to generate pCUE1:CUE1:Venus / cue1. Plants with the cue1 phenotype complemented were confirmed as homozygous cue1 mutants by using an LP primer at the downstream of Venus insertion. For pHDA6:HDA6:eYFP / hda6, a full genomic copy of HDA6 without its native stop codon was entry cloned into pCR8™ / GW / TOPO™ TA Cloning Kit (ThermoFisher Scientific, Catalog number K250020). After sequence verification, the entry clone was transferred to the destination plasmid pGWB540 using gateway recombination. The construct was transformed into the hda6-7 mutant using Agrobacterium strain GV3101 and selected using hygromycin. Tissue collection for whole-genome bisulfite and RNA sequencing

[0186] Plants were grown until the bolting stage and harvested at 8 h after dawn. Harvested tissue was flash-frozen in liquid nitrogen, ground with mortar and pestle, and aliquoted inAgent Ref: P14842WO00 separate tubes for DNA extraction (NucleoSpin Plant II Kit; Macherey-Nagel) and mRNA extraction (NucleoSpin RNA Plant Kit; Macherey-Nagel). The samples were sequenced in different batches. Library was constructed and sequenced as paired-end 100-bp for the Batch 1 methylome and paired-end 150-bp for other batches and RNA-seq, resulting in at least 10 Gb for WGBS and 9 Gb for RNA-seq. DNBSeq (BGI-tech) was used for Batch 1 and Illumina NovaSeq X Plus or NovaSeq6000 (Illumina) was used for other batches. The bisulfite conversion rate was calculated with the readcounts in the plastid genome. Methylome and transcriptome data analysis

[0187] Raw whole-genome bisulfite sequencing data was trimmed using Trim Galore! (v0.6.6) with Cutadapt (v2.8) and aligned to the TAIR10 reference genome using Bismark (v0.23.1) with Bowtie 2 (v3.5.1) and Samtools (v1.10). The read counts from Bismark were split into CG, CHG, and CHH contexts for downstream analysis with MethylIT (v0.3.2.7) on R (v4.3.2). For msh1 and PPD3OX 12-hr methylome and gene expression, DMGs and DEGs were generated as described in previous studies. Reference genome was generated as the sum of three replicates of control samples and used to estimate divergence with percentile=0.9999 as threshold to remove outliers. Hellinger divergence was used to determine the best fitted probability distribution model, which was used to get potential DMPs with tv.cut=0.2. Then, the final cutpoint to obtain DMPs was determined with estimateCutPoint function by using the following parameters: column=c (hdiv= TRUE, wprob= TRUE, pos= TRUE), div.col=9, clas.perf=TRUE, classifier1=”pca.logistic”, n.pc=3, center=TRUE, scale= TRUE. Finally, selectDIMP was used to obtain DMPs using Hellinger divergence (div.col=9).

[0188] To calculate the percentage of cytosines differentially methylated in genomic features (gene body, TE, RNA, and other), the number of DMPs was divided by the number of cytosines on the whole genome annotated as each genomic feature in TAIR 10. Promoters were defined as 2kb upstream of genes.

[0189] Differentially methylated genes (DMGs) were identified as genes with at least seven DMPs per individual sample, five DMPs per 1kb, log2FC>1, and maximum coefficient of variance for each group as 0.5, using DMPs within 1kb upstream and downstream of genes. For msh1 and PPD3OX DMGs, same cutoffs were used as published in previous studies16,18.

[0190] Differentially methylated regions (DMRs) were identified by using dmpClusters and dmpClustering functions in MethylIT. First, DMP clusters were identified independently by using “fixed.int” and “relaxed” for dmpClusters and dmpClustering. Parameters were set asAgent Ref: P14842WO00 maxDist=50, minNumDMPs=10, maxClustDist=100 for dmpClusters and win.size=50, step.size=50, minNumDMPs=10, maxClustDist=100 for dmpClustering. Then, whether the DMP clusters were differentially methylated between control and treatment samples was tested with countTest2 with at least eight DMPs per individual, ten DMPs per 1kb, log2FC>1, and maximum coefficient of variance for each group as 1. DMR-associated genes were identified as genes containing DMRs.

[0191] Raw RNA sequencing data was trimmed using Trim Galore! (v0.6.6, --length 30) with Cutadapt (v2.8) and aligned to the TAIR10 reference genome using STAR (v2.7.10a,-- twopassMode Basic -- outFilterMultimapNmax 1). Read count data was generated with QoRTs (v1.3.6, --minMAPQ 25). DEGs were identified with edgeR (v3.42.4) using │logFC │≥ 0.5 and adjusted p-value < 0.05 with Benjamini & Hochberg adjustment. For heat stress datasets,│logFC│≥ 1 was used instead of 0.5. Read counts were obtained from Shao et al. 2017 and Jeh et al.2023 for msh112-hr and PPD3OX 12-hr (dwarf and large), respectively. The list of DEGs in sal1 at 16-hr daylength was obtained from publicly available data. Laser scanning confocal microscopy and transmission electron microscopy

[0192] For laser scanning confocal microscopy Zeiss LSM510 was used with 488nm laser for eGFP and Venus and 561nm for mVenus. Chlorophyll autofluorescence was imaged simultaneously with a separate channel. HFT 488 or HFT 405 / 488 / 561 / 633 were used as primary dichroics and NFT 545 or NFT 565 were used as secondary dichroics. Gene network enrichment analysis

[0193] Cytoscape (v3.9.0 in Java 11.0.6) was used for gene network analysis as described in previous studies. In summary, gene lists were imported using STRING protein public database as full STRING networks using confidence (score) cutoff=0.40 and maximum additional interactors=0. ‘Analyze network’ in ‘tools’ section was used and nodes with degree=0 were removed. Then, the K-means cluster function in clusterMaker2 app (v2.3.4) was used with the following parameters: maximum number of clusters as 4 to estimate k using silhouette, 300 iterations, Euclidean distance, 7 node attributes (AverageShortestPathLength, BetweennessCentrality, ClosenessCentrality, ClusteringCoefficient, Degree, Eccentricity, and TopologicalCoefficient). The cluster with the node having the largest degree was selected for STRING functional enrichment with stringApp (v2.0.2), and resulting GO Biological Process networks were used for downstreamAgent Ref: P14842WO00 analysis. Genes in GO:0010467 (Gene expression) and GO:0010468 (Regulation of gene expression) were selected as hub network genes to yield <300 genes. If there were more than 300 genes, k-means clustering was performed one more time before STRING functional enrichment. The genes associated with gene expression were used for another STRING functional enrichment. Representative GO terms were selected for the hub network genes, and nodes were rearranged for visualization of those GO terms. References

[0194] Abdelnoor, R. V. et al. Substoichiometric shifting in the plant mitochondrial genome is influenced by a gene homologous to MutS. Proceedings of the National Academy of Sciences 100, 5968–5973 (2003).

[0195] Ageeva-Kieferle, A. et al. Nitric oxide coordinates growth, development, and stress response via histone modification and gene expression. Plant Physiol 187, 336–360 (2021).

[0196] An, C. et al. Regulation of jasmonate signaling by reversible acetylation of TOPLESS in Arabidopsis. Mol Plant 15, 1329–1346 (2022).

[0197] Beltrán, J. et al. Specialized Plastids Trigger Tissue-Specific Signaling for Systemic Stress Response in Plants. Plant Physiol 178, 672–683 (2018).

[0198] Chen, H., Zhang, B., Hicks, L. M. & Xiong, L. A nucleotide metabolite controls stress-responsive gene expression and plant development. PLoS One 6, (2011).

[0199] Chen, L. T., Luo, M., Wang, Y. Y. & Wu, K. Involvement of Arabidopsis histone deacetylase HDA6 in ABA and salt stress response. J Exp Bot 61, 3345–3353 (2010).

[0200] Crisp, P. A. et al. RNA polymerase II read-through promotes expression of neighboring genes in SAL1-PAP-XRN retrograde signaling. Plant Physiol 178, 1614–1630 (2018).

[0201] Davila, J. I. et al. Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis. BMC Biol 9, 64 (2011).

[0202] De Moraes, C. M., Schultz, J. C., Mescher, M. C. & Tumlinson, J. H. Induced plant signaling and its implications for environmental sensing. in Journal of Toxicology and Environmental Health - Part A vol.67819–834 (2004).

[0203] Devoto, A. et al. COI1 links jasmonate signalling and fertility to the SCF ubiquitin- ligase complex in Arabidopsis. Plant Journal 32, 457–466 (2002).Agent Ref: P14842WO00

[0204] Earley, K. W. et al. Mechanisms of HDA6-mediated rRNA gene silencing: Suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation. Genes Dev 24, 1119–1132 (2010).

[0205] Erdmann, R. M. & Picard, C. L. RNA-Directed DNA Methylation. PLoS Genetics vol. 16 (2020).

[0206] Estavillo, G. M. et al. Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis. Plant Cell 23, 3992–4012 (2011).

[0207] Fang, X. et al. Chloroplast-to-Nucleus Signaling Regulates MicroRNA Biogenesis in Arabidopsis. Dev Cell 48, 371-382.e4 (2019).

[0208] Gy, I. et al. Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors. Plant Cell 19, 3451–3461 (2007).

[0209] He, Y. et al. Nitric Oxide Represses the Arabidopsis Floral Transition. Science (1979) 305, 1968–1971 (2004).

[0210] Hirsch, J. et al. A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5′->3′ exoribonuclease (XRN) activities in Arabidopsis thaliana roots. PLoS One 6, (2011).

[0211] Hung, F. Y. et al. The Arabidopsis LDL1 / 2-HDA6 histone modification complex is functionally associated with CCA1 / LHY in regulation of circadian clock genes. Nucleic Acids Res 46, 10669–10681 (2018).

[0212] Ishiga, Y. et al. The SAL-PAP chloroplast retrograde pathway contributes to plant immunity by regulating glucosinolate pathway and phytohormone signaling. Molecular Plant-Microbe Interactions 30, 829–841 (2017).

[0213] Jeh, H. E. et al. Sensory plastid‐associated PsbP DOMAIN‐CONTAINING PROTEIN 3 triggers plant growth‐ and defense‐related epigenetic responses. The Plant Journal (2023) doi:10.1111 / tpj.16233.

[0214] Knappe, S. et al. Characterization of two functional phosphoenolpyruvate / phosphate translocator (PPT) genes in Arabidopsis - AtPPT1 may be involved in the provision of signals for correct mesophyll development. Plant Journal 36, 411–420 (2003).

[0215] Kundariya, H. et al. MSH1-induced heritable enhanced growth vigor through grafting is associated with the RdDM pathway in plants. Nat Commun 11, 1–14 (2020).Agent Ref: P14842WO00

[0216] Kundariya, H., Sanchez, R., Yang, X., Hafner, A. & Mackenzie, S. A. Methylome decoding of RdDM-mediated reprogramming effects in the Arabidopsis MSH1 system. Genome Biol 23, (2022).

[0217] Kurihara, Y. et al. Surveillance of 39 noncoding transcripts requires FIERY1 and XRN3 in arabidopsis. G3: Genes, Genomes, Genetics 2, 487–498 (2012).

[0218] Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nature Reviews Genetics vol.11204–220 (2010).

[0219] Liu, X., Luo, M. & Wu, K. Epigenetic interplay of histone modifications and DNA methylation mediated by HDA6. Plant Signal Behav 7, 633–635 (2012).

[0220] Liu, Y. et al. Arabidopsis FHY3 and FAR1 regulate the balance between growth and defense responses under shade conditions. Plant Cell 31, 2089–2106 (2019).

[0221] Lundquist, P. K., Rosar, C., Bräutigam, A. & Weber, A. P. M. Plastid signals and the bundle sheath: Mesophyll development in reticulate mutants. Molecular Plant vol.714–29 (2014).

[0222] Lunn, J. E. Compartmentation in plant metabolism. in Journal of Experimental Botany vol.5835–47 (2007).

[0223] Luo, M. et al. Histone Deacetylase HDA6 Is Functionally Associated with AS1 in Repression of KNOX Genes in Arabidopsis. PLoS Genet 8, (2012).

[0224] Ma, L. et al. Arabidopsis FHY3 and FAR1 Regulate Light-Induced myo-Inositol Biosynthesis and Oxidative Stress Responses by Transcriptional Activation of MIPS1. Mol Plant 9, 541–557 (2016).

[0225] Mackenzie, S. A. & Mullineaux, P. M. Plant environmental sensing relies on specialized plastids. Journal of Experimental Botany vol.737155–7164 (2022).

[0226] Margalha, L., Confraria, A. & Baena-González, E. SnRK1 and TOR: Modulating growth–defense trade-offs in plant stress responses. J Exp Bot 70, 2261–2274 (2019).

[0227] Mathieu, O., Reinders, J., Čaikovski, M., Smathajitt, C. & Paszkowski, J. Transgenerational Stability of the Arabidopsis Epigenome Is Coordinated by CG Methylation. Cell 130, 851–862 (2007).

[0228] Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: An epigenetic pathway of increasing complexity. Nature Reviews Genetics vol.15394–408 (2014).Agent Ref: P14842WO00

[0229] Micali, C., Göllner, K., Humphry, M., Consonni, C. & Panstruga, R. The Powdery Mildew Disease of Arabidopsis: A Paradigm for the Interaction between Plants and Biotrophic Fungi. Arabidopsis Book 6, e0115 (2008).

[0230] Nabi, R. B. S. et al. Nitric oxide regulates plant responses to drought, salinity, and heavy metal stress. Environ Exp Bot 161, 120–133 (2019).

[0231] Nakagawa, T. et al. Improved gateway binary vectors: High-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem 71, 2095–2100 (2007).

[0232] Nishimura, M. T. et al. Loss of a Callose Synthase Results in Salicylic Acid- Dependent Disease Resistance. Science (1979) 301, 969–972 (2003).

[0233] Ojolo, S. P. et al. Regulation of plant growth and development: a review from a chromatin remodeling perspective. Frontiers in Plant Science vol.9 (2018).

[0234] Phua, S. Y. et al. The arabidopsis SAL1-PAP pathway: A case study for integrating chloroplast retrograde, light and hormonal signaling in modulating plant growth and development? Front Plant Sci 9, (2018).

[0235] Probst, A. V. et al. Arabidopsis histone deacetylase HDA6 is required for maintenance of transcriptional gene silencing and determines nuclear organization of rDNA repeats. Plant Cell 16, 1021–1034 (2004).

[0236] Quintero, F. J., Garciadeblás, B. & Rodríguez-Navarro, A. The SAL1 Gene of Arabidopsis, Encoding an Enzyme with 3′(2′),5′-Bisphosphate Nucleotidase and Inositol Polyphosphate 1-Phosphatase Activities, Increases Salt Tolerance. The Plant Cell vol.8 (1996).

[0237] Sanchez, R., Yang, X., Maher, T. & Mackenzie, S. A. Discrimination of DNA methylation signal from background variation for clinical diagnostics. Int J Mol Sci 20, (2019).

[0238] Shao, M. R., Kumar Kenchanmane Raju, S., Laurie, J. D., Sanchez, R. & Mackenzie, S. A. Stress-responsive pathways and small RNA changes distinguish variable developmental phenotypes caused by MSH1 loss. BMC Plant Biol 17, (2017).

[0239] Shen, J., Ren, X., Cao, R., Liu, J. & Gong, Z. Transcriptional gene silencing mediated by a plastid inner envelope phosphoenolpyruvate / phosphate translocator CUE1 in arabidopsis. Plant Physiol 150, 1990–1996 (2009).Agent Ref: P14842WO00

[0240] To, T. K. et al. Arabidopsis hda6 regulates locus-directed heterochromatin silencing in cooperation with met1. PLoS Genet 7, (2011).

[0241] Valluru, R. & Van den Ende, W. Myo-inositol and beyond - Emerging networks under stress. Plant Science vol.181387–400 (2011).

[0242] Verma, V., Ravindran, P. & Kumar, P. P. Plant hormone-mediated regulation of stress responses. BMC Plant Biol 16, (2016).

[0243] Vílchez, J. I. et al. DNA demethylases are required for myo-inositol-mediated mutualism between plants and beneficial rhizobacteria. Nat Plants 6, 983–995 (2020).

[0244] Virdi, K. S. et al. Arabidopsis MSH1 mutation alters the epigenome and produces heritable changes in plant growth. Nat Commun (2015) doi:10.1038 / ncomms7386.

[0245] Virdi, K. S. S. et al. MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development. Mol Plant 9, 245–260 (2016).

[0246] Wang, Q. et al. AtHDA6 functions as an H3K18ac eraser to maintain pericentromeric CHG methylation in Arabidopsis thaliana. Nucleic Acids Res 49, 9755–9767 (2021).

[0247] Wang, Q., Liu, W., Leung, C. C., Tarté, D. A. & Gendron, J. M. Plants distinguish different photoperiods to independently control seasonal flowering and growth. Science (1979) 383, 605–605 (2024).

[0248] Wang, Y. et al. HISTONE DEACETYLASE 6 represses pathogen defence responses in Arabidopsis thaliana. Plant Cell Environ 40, 2972–2986 (2017).

[0249] Wang, Y. et al. Linking mitochondrial and chloroplast retrograde signalling in plants. Philosophical Transactions of the Royal Society B: Biological Sciences 375, (2020).

[0250] Wilson, P. B. et al. The nucleotidase / phosphatase SAL1 is a negative regulator of drought tolerance in Arabidopsis. Plant Journal 58, 299–317 (2009).

[0251] Wu, K., Zhang, L., Zhou, C., Yu, C. W. & Chaikam, V. HDA6 is required for jasmonate response, senescence and flowering in Arabidopsis. J Exp Bot 59, 225–234 (2008).

[0252] Xiong, L. et al. FIERY1 encoding an inositol polyphosphate 1-phosphatase is negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes Dev 15, 1971– 1984 (2001).

[0253] Xu, Y.-Z. et al. MutS HOMOLOG1 Is a Nucleoid Protein That Alters Mitochondrial and Plastid Properties and Plant Response to High Light. Plant Cell 23, 3428–3441 (2011).

[0254] Xu, Y.-Z. et al. The Chloroplast Triggers Developmental Reprogramming When MUTS HOMOLOG1 Is Suppressed in Plants. Plant Physiol 159, 710–720 (2012).Agent Ref: P14842WO00

[0255] Yang, X. et al. Segregation of an MSH1 RNAi transgene produces heritable non- genetic memory in association with methylome reprogramming. Nat Commun 11, (2020).

[0256] You, C. et al. FIERY1 promotes microRNA accumulation by suppressing rRNA- derived small interfering RNAs in Arabidopsis. Nat Commun 10, 4424 (2019).

[0257] Yu, C. W. et al. HISTONE DEACETYLASE6 acts in concert with histone methyltransferases SUVH4, SUVH5, and SUVH6 to regulate transposon silencing. Plant Cell 29, 1970–1983 (2017).

[0258] Yu, C. W. et al. HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis. Plant Physiol 156, 173–184 (2011).

[0259] Zhang, H., Lang, Z. & Zhu, J. K. Dynamics and function of DNA methylation in plants. Nature Reviews Molecular Cell Biology vol.19489–506 (2018).

[0260] Zhou, R., Benavente, L. M., Stepanova, A. N. & Alonso, J. M. A recombineering- based gene tagging system for Arabidopsis. Plant Journal 66, 712–723 (2011).

[0261] Zhu, Z. et al. Derepression of ethylene-stabilized transcription factors (EIN3 / EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis. Proc Natl Acad Sci U S A 108, 12539–12544 (2011).

Claims

Agent Ref: P14842WO00 What is claimed is:

1. A method for producing a plant having a useful trait, the method comprising: (a) crossing a first plant to a second plant or selfing the first plant, wherein the first plant comprises suppressed expression of an endogenous HDA6, CUE1, and / or SAL1 gene; (b) screening a population of progeny plants obtained from the cross or self of step (a) for the useful trait; and (c) selecting one or more progeny plants having the useful trait.

2. The method of claim 1, wherein the first plant comprises a loss-of-function mutation in the endogenous HDA6, CUE1, and / or SAL1 gene.

3. The method of claim 1, wherein the first plant comprises a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA having complementarity to the endogenous HDA6, CUE1, and / or SAL1 gene.

4. The method of claim 1, wherein the first plant comprises a catalytically inactive Cas protein, optionally wherein the Cas protein is fused to a transcriptional repressor domain, and a guide RNA molecule comprising a spacer RNA molecule that targets the endogenous HDA6, CUE1, and / or SAL1 gene.

5. The method of claim 1, wherein the endogenous HDA6 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; or wherein the endogenous HDA6 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.

6. The method of claim 1, wherein the endogenous CUE1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; or wherein the endogenous CUE1 gene encodes a polypeptide having at least 80%, atAgent Ref: P14842WO00 least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

7. The method of claim 1, wherein the endogenous SAL1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36; or wherein the endogenous SAL1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

8. The method of claim 1, wherein the first plant comprises suppressed expression of: (i) an endogenous HDA6 and CUE1 gene; (ii) an endogenous HDA6 and SAL1 gene; (iii) an endogenous HDA6 and MSH1 gene; or (iv) an endogenous HDA6 and PPD3 gene.

9. The method of claim 1, wherein the plant is selected from the group consisting of maize, soybean, canola, cotton, wheat, rice, tomato, tobacco, millet, potato, sugarbeet, cassava, alfalfa, barley, oat, sugarcane, sunflower, strawberry, and sorghum.

10. The method of claim 1, wherein the plant is a soybean plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 52, 53, or 54 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 65, 66, or 67 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 2, 3, or 4 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 13, 14, or 15 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 24, 25, or 26 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 38, 39, or 40 or an allelic variant thereof.Agent Ref: P14842WO00 11. The method of claim 1, wherein the plant is a canola plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 55, 56, 57, 58, 59, 60, or 61 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 68, 69, 70, 71, 72, 73, or 74 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 5 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 16 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 27, 28, 29, 30, 31, or 32 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 41, 42, 43, 44, 45, or 46 or an allelic variant thereof.

12. The method of claim 1, wherein the plant is a maize plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 62 or 63 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 75 or 76 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 6, 7, 8, 9, 10, or 11 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 17, 18, 19, 20, 21, or 22 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 33, 34, 35, or 36 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 47, 48, 49, or 50 or an allelic variant thereof.

13. The method of claim 1, wherein the useful trait is selected from the group consisting of improved yield, delayed flowering, non-flowering, increased biotic stress resistance, increased abiotic stress resistance, enhanced lodging resistance, enhanced growth rate, enhanced biomass, enhanced tillering, enhanced branching, delayed flowering time, delayed senescence, increased flower number, improved architecture for high density planting, improved photosynthesis, increased root mass, increased cell number, improved seedling vigor, improved seedling size, increased rate of cell division, improved metabolic efficiency, and increased meristem size in comparison to a control plant.

14. The method of claim 1, wherein the useful trait exhibits nuclear inheritance.Agent Ref: P14842WO00 15. The method of claim 1, further comprising producing seed or a progeny plant from the one or more progeny plants selected in step (c), wherein the produced seed or produced progeny plant have the useful trait.

16. The method of claim 1, further comprising producing a seed lot from the one or more progeny plants selected in step (c) or from one or more progeny plants obtained therefrom.

17. A plant or progeny thereof that exhibits a useful trait that is made by the method of claim 1.

18. A plant part obtained from the plant or progeny thereof of claim 17, optionally wherein the part is a seed or grain.

19. A processed plant product obtained from the plant part of claim 18.

20. A plant or plant cell having suppressed expression of an endogenous HDA6, CUE1, and / or SAL1 gene, wherein the plant or plant cell comprises: (i) a loss-of-function mutation in the endogenous HDA6, CUE1, and / or SAL1 gene; (ii) a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA having complementarity to the endogenous HDA6, CUE1, and / or SAL1 gene; and / or (iii) a catalytically inactive Cas protein, optionally wherein the Cas protein is fused to a transcriptional repressor domain, and a guide RNA molecule comprising a spacer RNA molecule that targets the endogenous HDA6, CUE1, and / or SAL1 gene.

21. The plant or plant cell of claim 20, wherein the endogenous HDA6 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; or wherein the endogenous HDA6 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.Agent Ref: P14842WO00 22. The plant or plant cell of claim 20, wherein the endogenous CUE1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; or wherein the endogenous CUE1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

23. The plant or plant cell of claim 20, wherein the endogenous SAL1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36; or wherein the endogenous SAL1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

24. The plant or plant cell of claim 20, wherein the plant or plant cell comprises suppressed expression of: (i) an endogenous HDA6 and CUE1 gene; (ii) an endogenous HDA6 and SAL1 gene; (iii) an endogenous HDA6 and MSH1 gene; or (iv) an endogenous HDA6 and PPD3 gene.

25. The plant or plant cell of claim 20, wherein the plant is selected from the group consisting of maize, soybean, canola, cotton, wheat, rice, tomato, tobacco, millet, potato, sugarbeet, cassava, alfalfa, barley, oat, sugarcane, sunflower, strawberry, and sorghum.

26. The plant or plant cell of claim 20, wherein the plant or plant cell is a soybean plant or plant cell, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 52, 53, or 54 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 65, 66, or 67 or an allelic variant thereof;Agent Ref: P14842WO00 (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 2, 3, or 4 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 13, 14, or 15 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 24, 25, or 26 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 38, 39, or 40 or an allelic variant thereof.

27. The plant or plant cell of claim 20, wherein the plant or plant cell is a canola plant or plant cell, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 55, 56, 57, 58, 59, 60, or 61 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 68, 69, 70, 71, 72, 73, or 74 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 5 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 16 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 27, 28, 29, 30, 31, or 32 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 41, 42, 43, 44, 45, or 46 or an allelic variant thereof.

28. The plant or plant cell of claim 20, wherein the plant or plant cell is a maize plant or plant cell, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 62 or 63 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 75 or 76 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 6, 7, 8, 9, 10, or 11 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 17, 18, 19, 20, 21, or 22 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 33, 34, 35, or 36 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 47, 48, 49, or 50 or an allelic variant thereof.Agent Ref: P14842WO00 29. A method for obtaining a plant or plant cell of claim 20, the method comprising introducing into a plant or plant cell: (i) a loss-of-function mutation in the endogenous HDA6, CUE1, and / or SAL1 gene; (ii) a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and / or an anti-sense RNA having complementarity to the endogenous HDA6, CUE1, and / or SAL1 gene; and / or (iii) a catalytically inactive Cas protein, optionally wherein the Cas protein is fused to a transcriptional repressor domain, and a guide RNA molecule comprising a spacer RNA molecule that targets the endogenous HDA6, CUE1, and / or SAL1 gene.

30. The method of claim 29, wherein the loss-of-function mutation is introduced with one or more gene editing molecules.

31. The method of claim 30, wherein the gene editing molecules comprise: (i) a Cas protein and a guide RNA directed to the gene; (ii) a transcription activator-like effector nuclease (TALEN) directed to the gene; (iii) a zinc-finger nuclease (ZFN) directed to the gene; or (iv) any one of (i), (ii), or (iii) and DNA donor template.

32. The method of claim 29, wherein the loss-of-function mutation is introduced by random mutagenesis, and wherein the method further comprising screening progeny subjected to the mutagenesis by a DNA analysis technique to identify a plant comprising the loss-of-function mutation.

33. The method of claim 29, wherein the endogenous HDA6 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; or wherein the endogenous HDA6 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.

34. The method of claim 29, wherein the endogenous CUE1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, orAgent Ref: P14842WO00 11; or wherein the endogenous CUE1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

35. The method of claim 29, wherein the endogenous SAL1 gene comprises a DNA molecule having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36; or wherein the endogenous SAL1 gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

36. The method of claim 29, wherein the plant is selected from the group consisting of maize, soybean, canola, cotton, wheat, rice, tomato, tobacco, millet, potato, sugarbeet, cassava, alfalfa, barley, oat, sugarcane, sunflower, strawberry, and sorghum.

37. The method of claim 29, wherein the plant is a soybean plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 52, 53, or 54 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 65, 66, or 67 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 2, 3, or 4 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 13, 14, or 15 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 24, 25, or 26 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 38, 39, or 40 or an allelic variant thereof.

38. The method of claim 29, wherein the plant is a canola plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 55, 56, 57, 58, 59, 60, or 61 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 68, 69, 70, 71, 72, 73, or 74 or an allelic variant thereof;Agent Ref: P14842WO00 (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 5 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 16 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 27, 28, 29, 30, 31, or 32 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 41, 42, 43, 44, 45, or 46 or an allelic variant thereof.

39. The method of claim 29, wherein the plant is a maize plant, and wherein: (i) the HDA6 gene comprises the DNA molecule of SEQ ID NO: 62 or 63 or an allelic variant thereof, or wherein the endogenous HDA6 gene encodes the polypeptide of SEQ ID NO: 75 or 76 or an allelic variant thereof; (ii) the CUE1 gene comprises the DNA molecule of SEQ ID NO: 6, 7, 8, 9, 10, or 11 or an allelic variant thereof, or wherein the endogenous CUE1 gene encodes the polypeptide of SEQ ID NO: 17, 18, 19, 20, 21, or 22 or an allelic variant thereof; and / or (iii) the SAL1 gene comprises the DNA molecule of SEQ ID NO: 33, 34, 35, or 36 or an allelic variant thereof, or wherein the endogenous SAL1 gene encodes the polypeptide of SEQ ID NO: 47, 48, 49, or 50 or an allelic variant thereof.

40. The method of claim 29, further comprising selecting a plant comprising: (i) the loss- of-function mutation in the endogenous HDA6, CUE1, and / or SAL1 gene; (ii) the siRNA, miRNA, co-suppressing sense RNA, and / or an anti-sense RNA having complementarity to the endogenous HDA6, CUE1, and / or SAL1 gene; and / or (iii) the catalytically inactive Cas protein.

41. The method of claim 29, further comprising crossing the plant or a progeny thereof to a second plant or selfing the plant.

42. The method of claim 41, further comprising screening a population of progeny plants obtained from the cross or self for the presence of one or more differentially methylated and / or differentially expressed genes which are differentially methylated and / or differentially expressed genes in plants wherein expression of a MSH1 and / or PPD3 gene is suppressed.Agent Ref: P14842WO00 43. The method of claim 41, further comprising screening a population of progeny plants obtained from the cross or self for a useful trait.

44. The method of claim 43, wherein the useful trait is selected from the group consisting of improved yield, delayed flowering, non-flowering, increased biotic stress resistance, increased abiotic stress resistance, enhanced lodging resistance, enhanced growth rate, enhanced biomass, enhanced tillering, enhanced branching, delayed flowering time, delayed senescence, increased flower number, improved architecture for high density planting, improved photosynthesis, increased root mass, increased cell number, improved seedling vigor, improved seedling size, increased rate of cell division, improved metabolic efficiency, and increased meristem size in comparison to a control plant.

45. A guide RNA molecule comprising a spacer RNA molecule that targets (i) the endogenous HDA6 gene of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; (ii) the endogenous CUE1 gene of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; or (iii) the endogenous SAL1 gene of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36.

46. A genome engineering system comprising: a Cas protein in association with a guide RNA molecule of claim 45.

47. The genome engineering system of claim 46, wherein the Cas protein is catalytically inactive, optionally wherein the Cas protein is fused to a transcriptional repressor domain.

48. A DNA molecule encoding the guide RNA molecule of claim 45.

49. A DNA molecule encoding a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, or an anti-sense RNA having complementarity to: (i) the endogenous HDA6 gene of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63; (ii) the endogenous CUE1 gene of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; orAg geentnt Re ef:f: P14842W0O000 (ii iii)) the e endog geennoouuss SAL LI1 gen nee of f SEQ ID NO O:: 233,, 244,, 255,, 266,, 277,, 288,, 299,, 300,, 311,, 322,, 333,, 344,, 35, , or r 366..

Images