Plants with deeper roots for drought and nitrogen efficiency

Abstract

The disclosure relates to endogenous S-type anion channel genes associated with modified root architecture in plants. Also disclosed are plants comprising one or more mutations in an endogenous S-type anion channel gene along with related methods of modifying root architecture of plants.

Description

TITLE: PLANTS WITH DEEPER ROOTS FOR DROUGHT AND NITROGENEFFICIENCYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional application U.S. Serial No. 63 / 554,705, filed February 16, 2024, which is incorporated herein by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under DE-AR0000826 awarded by the Department of Energy. The government has certain rights in the invention.SEQUENCE LISTING XML

[0003] 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 February 10, 2025, is named P14662WOOO.xml and is 28,430 bytes in size.TECHNICAL FIELD

[0004] The present disclosure relates to compositions and methods for identifying, selecting, and producing plants with modified root architecture.BACKGROUND

[0005] For all crop plants, roots play a critical role in growth. Roots anchor the plants and are the primary site of nutrient and water uptake. Roots are also the main source of C to soil in the form of root tissues and exudates, and thus greatly influence SOM stocks. To perform these functions, primary roots extend into soil, producing a network of branching roots of characteristic form, known as its root system architecture. Deeper roots are major targets for the second green revolution because of their potential to improve crop productivity, increase drought tolerance and decrease nitrogen inputs. Deeper rooted genotypes of maize will be particularly important since this crop is planted on over 92 million acres annually in the US. Elite corn hybrids are also becoming increasingly sensitive to drought and would also benefit from deep roots for that purpose.SUMMARY

[0006] Plants having modified root architecture comprising one or more mutations in an endogenous S-type anion channel gene are provided. In certain embodiments, the endogenous S- type anion channel gene encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3 or 6. In certain embodiments, the endogenous S-type anion channel gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5. In certain embodiments, the modified root architecture comprises increased root biomass, steeper root angle, increased lateral root branching, longer roots, or a combination thereof. In certain embodiments, the plants exhibit improved yield traits or increased tolerance / resistance to abiotic stress relative to a control plant without the one or more mutations in the endogenous S- type anion channel gene. In certain embodiments, the plants exhibit increased tolerance to drought stress or limited nitrogen relative to a control plant without the one or more mutations in the endogenous S-type anion channel gene. Progeny, plant parts, plant cells, seeds, and asexual propagates of the plants are also provided.

[0007] Methods for modifying the root architecture of a plant are provided. In certain embodiments, the methods comprise introducing one or more mutations in an endogenous S- type anion channel gene of the plant. In certain embodiments, the methods comprise reducing expression or activity of the endogenous S-type anion channel gene.

[0008] Methods of producing a plant having modified root architecture comprising crossing the plant of the disclosure with itself or another plant to produce seed; and growing a progeny plant from the seed to produce a plant having modified root architecture are also provided.

[0009] Crops comprising a plurality of the plants of the disclosure planted together in an agricultural field are provided. Methods of producing seeds from a crop comprising cultivating a plurality of the plants of the disclosure as a crop and harvesting seeds from the crop are also provided.

[0010] Commodity plant products prepared from the aforementioned plants, plant parts, and plant cells are provided. Methods for producing a commodity plant product comprising processing the aforementioned plants or plant parts thereof to obtain the product are also provided.

[0011] Methods of introgressing a modified root architecture locus into a plant are provided. In certain embodiments, the methods comprise providing a first plant with the modified root architecture locus, wherein the locus comprises a one or more mutations in an endogenous S-type anion channel gene, providing a second plant, crossing the first plant with the second plant to produce a population of progeny plants; and selecting from the population at least one plant having the modified root architecture locus.

[0012] Methods of identifying or selecting a plant comprising a modified root architecture locus are provided. In certain embodiments, the methods comprise genotyping at least one plant for the presence of one or more mutations in an endogenous S-type anion channel gene or a marker in linkage disequilibrium therewith.

[0013] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent based on the detailed description, which shows and describes illustrative embodiments of the disclosure. Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive.BRIEF DESCRIPTION OF THE FIGURES

[0014] 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.

[0015] FIG. 1 is a schematic representation of the exon-intron structure of ZmSLAH2 / 3 (Zm00004b019600) and the conserved TDT-SLACl-like protein domain on chromosome 3. The Mu transposon insertion site (mul014502) is marked by a triangle.

[0016] FIG. 2 shows increased root system size in the ZmSLAH2 / 3 mutant line grown in the field (mean ± SE), measured as root pulling force (RPF).

[0017] FIG. 3A-D shows phenotypic differences between ZmSLAH2 / 3 mutant and wild-type allele lines from the 2024 mid-flowering measurements of leaf nitrogen content (FIG. 3A), root- mass-ratio (FIG. 3B), root biomass (FIG. 3C), and shoot biomass (FIG. 3D). Significance levels: ‘ .’ / ? < 0.10, *p < 0.05, **p < 0.01.

[0018] FIG. 4A-B shows RSA plasticity among ZmSLAH2 / 3 mutant and wild-type allele lines for plants harvested at mid-flowering in the 2024 nitrogen stress field experiment. FIG. 4A shows images of representative maize root crowns across genotypes and nitrogen treatments. FIG. 4B is a bar plot comparing average standardized (Z-score) RSA trait measurements obtained through 2D imaging and DIRT feature extraction between genotypes and nitrogentreatments (mean ± SE). DIRT abbreviations: 2D Area, Area; 2D Depth, Skeleton Depth; 2D Number Tips, Root Tip Count; 2D Width, Maximum Width.BRIEF DESCRIPTION OF THE SEQUENCES

[0019] SEQ ID NO: 1 is the ZmOOOOl eb 159490 (B73) genomic DNA sequence.

[0020] SEQ ID NO: 2 is the ZmOOOOl eb 159490 (B73) coding sequence.

[0021] SEQ ID NO: 3 is the ZmOOOOl eb 159490 (B73) amino acid sequence.

[0022] SEQ ID NO: 4 is the Zm00004b019600 (W22) genomic DNA sequence.

[0023] SEQ ID NO: 5 is the Zm00004b019600 (W22) coding sequence.

[0024] SEQ ID NO: 6 is the Zm00004b019600 (W22) amino acid sequence.

[0025] SEQ ID NO: 7 and SEQ ID NO: 8 are primer sequences.DETAILED DESCRIPTION

[0026] The capacity of plants to absorb water and nutrients can limit yield. Therefore, one strategy for yield improvement is to breed plants to have an enhanced root system architecture. A steep, rapidly developing root system can allow a plant to optimize uptake of water and nutrients below the shallower soil strata, where water and nutrients are transiently available. Furthermore, early development of long roots may facilitate drought tolerance and reduce waterdeficit related yield costs. Finally, a steeper root system may facilitate high-density planting as by limiting inter-plant competition.

[0027] The present disclosure is directed to generation of plants comprising one or more mutations in an S-type anion channel gene (e.g., ZmSLAH2 / 3). In certain embodiments, modifying an endogenous an S-type anion channel gene leads to improved root system architecture in the plant comprising the modification. In certain embodiments, the modification results in the plant exhibiting one or more of the following phenotypes: increased root biomass, steeper root angle, increased lateral root branching, or longer roots, in any combination. In certain embodiments, the plant comprising the modification in the S-type anion channel gene further exhibits improved yield traits or increased tolerance / resistance to abiotic stress. In certain embodiments, the abiotic stress is drought stress or limited nitrogen.

[0028] 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 thosedescribed 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.

[0029] It is to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms "a," "an" and "the" can include plural referents unless the content clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicate otherwise. The word “or” means any one member of a particular list and also includes any combination of members of that list. Further, all units, prefixes, and symbols may be denoted in its SI accepted form.

[0030] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various embodiments of this disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, P , and 43 / 4. This applies regardless of the breadth of the range.

[0031] As used herein, the term “allele” refers to one of two or more different nucleotides or nucleotide sequences that occur at a specific locus.

[0032] A marker is “associated with” a trait when said trait is linked to it and when the presence of the marker is an indicator of whether and / or to what extent the desired trait or trait form will occur in a plant / germplasm comprising the marker. Similarly, a marker is “associated with” an allele or chromosome interval when it is linked to it and when the presence of the marker is an indicator of whether the allele or chromosome interval is present in a plant / germplasm comprising the marker.

[0033] As used herein, the terms “backcross” and “backcrossing” refer to the process whereby a progeny plant is crossed back to one of its parents one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.). In a backcrossing scheme, the “donor” parent refers to the parental plant with the desiredgene or locus to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al. Marker-assisted Backcrossing: A Practical Example, in TECHNIQUES ET UTILISATIONS DES MARQUEURS MOLECULAIRES LES COLLOQUES, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in Backer oss Breeding, in PROCEEDINGS OF THE SYMPOSIUM “ANALYSIS OF MOLECULAR MARKER DATA,” pp. 41-43 (1994). The initial cross gives rise to the Fl generation. The term “BC1” refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on.

[0034] As used herein, the phrase “biological sample” refers to either intact or non-intact (e.g., milled seed or plant tissue, chopped plant tissue, lyophilized tissue) plant tissue. It may also be an extract comprising intact or non-intact seed or plant tissue. The biological sample can comprise flour, meal, flakes, syrup, oil, starch, and cereals manufactured in whole or in part to contain crop plant by-products. In certain embodiments, the biological sample is “non- regenerable” (i.e., incapable of being regenerated into a plant or plant part).

[0035] As used herein, the terms “cross” or “crossed” refer to the fusion of gametes via pollination to produce progeny (e.g., cells, seeds or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant). The term “crossing” refers to the act of fusing gametes via pollination to produce progeny.

[0036] As used herein, the terms “cultivar” and “variety” refer to a group of similar plants that by structural or genetic features and / or performance can be distinguished from other varieties within the same species.

[0037] As used herein, the terms “desired allele”, “target allele”, and “allele of interest” are used interchangeably to refer to an allele associated with a desired trait. In certain embodiments, a desired allele may be associated with either an increase or a decrease (relative to a control) of or in a given trait, depending on the nature of the desired phenotype.

[0038] An “elite line” or “elite strain” is an agronomically superior line that has resulted from many cycles of breeding and selection for superior agronomic performance. Numerous elite lines are available and known to those of skill in the art of plant breeding. An “elite population” is an assortment of elite individuals or lines that can be used to represent the state of the art in terms of agronomically superior genotypes of a given crop species, such as maize. Similarly, an “elite germplasm” or elite strain of germplasm is an agronomically superior germplasm,typically derived from and / or capable of giving rise to a plant with superior agronomic performance, such as an existing or newly developed elite line. An “elite plant” is any plant from an elite line, such that an elite plant is a representative plant from an elite variety.

[0039] As used herein, the terms “exotic”, “exotic line” and “exotic germplasm” refer to any plant, line or germplasm that is not elite. In general, exotic plants / germplasms are not derived from any known elite plant or germplasm, but rather are selected to introduce one or more desired genetic elements into a breeding program (e.g., to introduce novel alleles into a breeding program).

[0040] The term “endogenous” relates to any gene or nucleic acid sequence that is already present in a cell.

[0041] The terms “enhanced root architecture,” “modified root architecture,” or “improved root architecture” may be used interchangeably and refer to root architecture that provides an improvement in the ability of a plant to uptake water and nutrients, in particular, when the plant is growing under environmental conditions that may limit water and nutrient uptake (e.g., drought conditions) in a plant not comprising the enhanced root architecture. Enhanced or modified root architecture may be characterized by a phenotype that includes, but is not limited to, increased root biomass, steeper root angle, increased lateral root branching and / or longer roots.

[0042] As used herein, the terms “express”, “expresses”, “expressed”, “expression”, and the like, with respect to a nucleic acid molecule and / or a nucleotide sequence (e.g., RNA or DNA) indicates that the nucleic acid molecule and / or a nucleotide sequence is transcribed and, optionally, translated. Thus, a nucleic acid molecule and / or a nucleotide sequence may express a polypeptide of interest or, for example, a functional untranslated RNA.

[0043] The term “gene”, as used herein, refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotide (AMO) and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and / or 5' and 3' untranslated regions). A gene may be “isolated” by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and / or various chemicals used in chemically synthesizing the nucleic acid.

[0044] A “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by the recombination frequencies between them. Recombination between loci can be detected using a variety of markers. A genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. The order and genetic distances between loci can differ from one genetic map to another.

[0045] As used herein, the term “genotype” refers to the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable and / or detectable and / or manifested trait (the phenotype). Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual's genetic constitution at a single locus, at multiple loci, or more generally, the term genotype can be used to refer to an individual's genetic make-up for all the genes in its genome. Genotypes can be indirectly characterized, e.g., using markers and / or directly characterized by nucleic acid sequencing.

[0046] As used herein, the term “germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety, or family), or a clone derived from a line, variety, species, or culture. The germplasm can be part of an organism or cell or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific genetic makeup that provides a foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, as well as plant parts that can be cultured into a whole plant (e.g., leaves, stems, buds, roots, pollen, cells, etc.).

[0047] A “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e., a combination of alleles. Typically, the genetic loci that define a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term “haplotype” can refer to polymorphisms at a particular locus, such as a single marker locus, or polymorphisms at multiple loci along a chromosomal segment.

[0048] As used herein, the term “heterologous” refers to a nucleotide / polypeptide that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same / analogous species, oneor both are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

[0049] As used herein, the term “heterozygous” refers to a genetic status wherein different alleles reside at corresponding loci on homologous chromosomes.

[0050] As used herein, the term “homozygous” refers to a genetic status wherein identical alleles reside at corresponding loci on homologous chromosomes.

[0051] A “hypermorphic mutation” is a mutation that results in increased expression of the gene product and / or increased activity of the gene product.

[0052] A “hypomorphic mutation” is a mutation that results in a partial loss of gene function, which may occur through reduced expression (e.g., reduced protein and / or reduced RNA) or reduced functional performance (e.g., reduced activity), hut not a complete loss of function / activity. A “hypomorphic” allele is a semi -functional allele caused by a genetic mutation that results in production of the corresponding protein that functions at anywhere between 1% and 99% of normal efficiency.

[0053] As used herein, the term “hybrid” in the context of plant breeding refers to a plant that is the offspring of genetically dissimilar parents produced by crossing plants of different lines or breeds or species, including but not limited to the cross between two inbred lines.

[0054] As used herein, the term “inbred” refers to a substantially homozygous plant or variety. The term may refer to a plant or plant variety that is substantially homozygous throughout the entire genome or that is substantially homozygous with respect to a portion of the genome that is of particular interest.

[0055] As used herein, the terms “include”, “includes”, and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0056] As used herein, the terms “introducing”, “introduce”, and “introduced” (and grammatical variations thereof) in the context of a polynucleotide of interest means presenting a nucleotide sequence of interest (e.g., polynucleotide, a nucleic acid construct, or a guide nucleic acid) to a plant, plant part thereof, or cell thereof, in such a manner that the nucleotide sequence gains access to the interior of a cell.

[0057] As used herein, the terms “introgression”, “introgressing”, and “introgressed” refer to both the natural and artificial transmission of a desired allele or combination of desired alleles of a genetic locus or genetic loci from one genetic background to another. For example, a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross betweentwo parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele may be a selected allele of a marker, a QTL, a transgene, or the like. Offspring comprising the desired allele can be backcrossed one or more times (e.g., 1, 2, 3, 4, or more times) to a line having a desired genetic background, selecting for the desired allele, with the result being that the desired allele becomes fixed in the desired genetic background. For example, a marker associated with improved root architecture may be introgressed from a donor into a recurrent parent that does not comprise the marker and does not exhibit improved root architecture. The resulting offspring could then be backcrossed one or more times and selected until the progeny possess the genetic marker(s) associated with improved root architecture in the recurrent parent background.

[0058] A “locus” is a position on a chromosome where a gene or marker or allele is located. In certain embodiments, a locus may encompass one or more nucleotides.

[0059] A “native” or “wild type” nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. Thus, for example, a “wild type mRNA” is an mRNA that is naturally occurring in or endogenous to the reference organism.

[0060] As used herein, a “non-natural mutation” refers to a mutation that is generated though human intervention and differs from mutations found in the same gene that have occurred in nature (e.g., occurred naturally). A “non-natural” mutation as used herein does not include a mutation generated in a gene of a plant species through human intervention if the same mutation is also a naturally occurring mutation in that gene and that plant species.

[0061] A “null allele” is a nonfunctional allele caused by a genetic mutation that results in a complete lack of production of the corresponding protein or produces a protein that is nonfunctional.

[0062] As used herein, “modified”, in the context of plants, seeds, plant components, plant cells, and plant genomes, refers to a state containing changes or variations from their natural or native state. For instance, a “native transcript” of a gene refers to an RNA transcript that is generated from an unmodified gene. Typically, a native transcript is a sense transcript. Modified plants or seeds contain molecular changes in their genetic materials, including either genetic or epigenetic modifications. Typically, modified plants or seeds, or a parental or progenitor line thereof, have been subjected to mutagenesis, genome editing (e.g., without being limiting, via methods usingsite-specific nucleases), genetic transformation (e.g., without being limiting, via methods of Agrobacterium transformation or microprojectile bombardment), or a combination thereof. In certain embodiments, a modified plant provided herein comprises no non-plant genetic material or sequences. In yet another embodiment, a modified plant provided herein comprises no interspecies genetic material or sequences.

[0063] The term “mutation” refers to point mutations (e.g., missense, or nonsense, or insertions or deletions of single base pairs that result in in-frame shifts), insertions, deletions, and / or truncations. When the mutation is a substitution of a residue within an amino acid sequence with another residue, or a deletion or insertion of one or more residues within a sequence, the mutations are typically described by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue.

[0064] A “non-naturally occurring variety of maize” is any variety of maize that does not naturally exist in nature. A “non-naturally occurring variety of maize” may be produced by any method known in the art, including, but not limited to, transforming a maize plant or germplasm, transfecting a maize plant or germplasm and crossing a naturally occurring variety of maize with a non-naturally occurring variety of maize. In certain embodiments, a “non-naturally occurring variety of maize” may comprise one of more heterologous nucleotide sequences. In certain embodiments, a “non-naturally occurring variety of maize” may comprise one or more non- naturally occurring copies of a naturally occurring nucleotide sequence (i.e., extraneous copies of a gene that naturally occurs in maize). In certain embodiments, a “non-naturally occurring variety of maize” may comprise a non-natural combination of two or more naturally occurring nucleotide sequences (i.e., two or more naturally occurring genes that do not naturally occur in the same maize).

[0065] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence” and “polynucleotide” can be used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA. The term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand. The nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. The present disclosure further provides a nucleic acid that is thecomplement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide.

[0066] By “operably linked” or “operably associated,” it is meant that the indicated elements are functionally related to each other, and are also generally physically related. Thus, the term “operably linked” or “operably associated” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Therefore, a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For instance, a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of the nucleotide sequence. Those skilled in the art will appreciate that the control sequences (e.g., promoter) need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered “operably linked” to the nucleotide sequence.

[0067] As used herein, “plant” refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and / or progeny of the same. A progeny plant can be from any filial generation, e.g., Fl, F2, F3, F4, F5, F6, F7, etc. A plant cell is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant.

[0068] The term “plant part,” as used herein, includes but is not limited to reproductive tissues (e.g., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower bud, ovules, seeds, embryos, nuts, kernels, ears, cobs and husks); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristem, axillary bud, cotyledon, hypocotyls, and leaves); vascular tissues (e.g., phloem and xylem); specialized cells such as epidermal cells, parenchyma cells, collenchyma cells, sclerenchyma cells, stomates, guard cells, cuticle, mesophyll cells; callus tissue; and cuttings. The term “plant part” also includes plant cells, including plant cells that are intact in plants and / or parts of plants, plant protoplasts, plant tissues, plant organs, plant cell tissue cultures, plant calli, plant clumps, and the like. As used herein, “shoot” refers to the above ground parts including the leaves and stems. As used herein, the term “tissue culture” encompasses cultures of tissue, cells, protoplasts and callus.

[0069] As used herein, “plant cell” refers to a structural and physiological unit of the plant, which typically comprise a cell wall but also includes protoplasts. A plant cell of the present disclosure can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue (including callus) or a plant organ. In certain embodiments, a plant cell can be an algal cell. A “protoplast” is an isolated plant cell without a cell wall or with only parts of the cell wall. Thus, in certain embodiments, a transgenic cell comprising a nucleic acid molecule and / or nucleotide sequence of the disclosure is a cell of any plant or plant part including, but not limited to, a root cell, a leaf cell, a tissue culture cell, a seed cell, a flower cell, a fruit cell, a pollen cell, and the like. In certain embodiments, the plant part can be a plant germplasm. In certain embodiments, a plant cell can be non-propagating plant cell that does not regenerate into a plant.

[0070] “Plant cell culture” means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.

[0071] “Plant tissue” as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and / or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

[0072] As used herein, a “plant organ” is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.

[0073] As used herein, the terms “progeny” and “progeny plant” refer to a plant generated from a vegetative or sexual reproduction from one or more parent plants. A progeny plant may be obtained by cloning or selfing a single parent plant, or by crossing two parental plants.

[0074] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0075] “Regulatory elements” refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory elements may include, but are not limited to, promoters, translation leadersequences, introns, and polyadenylation recognition sequences. Regulatory elements present on a recombinant DNA construct that is introduced into a cell can be endogenous to the cell, or they can be heterologous with respect to the cell. The terms "regulatory element" and "regulatory sequence" are used interchangeably herein.

[0076] As used herein a “trait” is a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In certain embodiments, this characteristic is visible to the human eye and can be measured mechanically, such as seed or plant size, weight, shape, form, length, height, growth rate and development stage, or can be measured by biochemical techniques, such as detecting the protein, starch, certain metabolites, or oil content of seed or leaves, or by observation of a metabolic or physiological process, for example, by measuring tolerance to water deprivation or particular salt or sugar concentrations, or by the measurement of the expression level of a gene or genes, for example, by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as hyperosmotic stress tolerance or yield. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the plant, however.S-Type Anion Channel Genes

[0077] S-type anion channel genes are provided that are associated with modified root architecture. Such S-type anion channel sequences include the amino acid sequences set forth in SEQ ID NOs: 3 and 6. Also provided are polynucleotide sequences encoding such amino acid sequences, including SEQ ID NOs: 1, 2, 4, and 5. In certain embodiments, the S-type anion channel gene is ZmSLAH2 / 3. The endogenous ZmSLAH2 / 3 gene (ZmOOOOl eb 159490) is located at nucleotides 223,183,535 to 223,193,733 on chromosome 3 of the Zm-B73- REFERENCE-NAM-5.0 assembly version available on the world wide web internet site maizegdb.org. The endogenous ZmSLAH2 / 3 gene (Zm00004b019600) is located at nucleotides 226,297,137 to 226,301,978 on chromosome 3 of the Zm-W22-REFERENCE-NRGENE-2.0 assembly version available on the world wide web internet site maizegdb.org.

[0078] In certain embodiments, the S-type anion channel gene encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to the full length or a fragment of the amino acid sequence of SEQ ID NO: 3 or 6. In certain embodiments, the S-type anionchannel gene comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% to the full length or a fragment of the nucleotide sequence of SEQ ID NO: 1, 2, 4, or 5.

[0079] Different nucleic acids or proteins having homology are referred to herein as “homologues.” The term homologue includes homologous sequences from the same and from other species and orthologous sequences from the same and other species. “Homology” refers to the level of similarity between two or more nucleic acid and / or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins. Thus, the compositions and methods of the disclosure further comprise homologues to the nucleotide sequences and polypeptide sequences of this disclosure. “Orthologous”, as used herein, refers to homologous nucleotide sequences and / or amino acid sequences in different species that arose from a common ancestral gene during speciation. A homologue of a nucleotide sequence of this disclosure has a substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to said nucleotide sequence of the disclosure.

[0080] As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W ., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0081] As used herein, the term “percent sequence identity” or “percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimallyaligned. In certain embodiments, “percent sequence identity” can refer to the percentage of identical amino acids in an amino acid sequence as compared to a reference polypeptide.

[0082] As used herein, the phrase “substantially identical” or “substantial identity” in the context of two nucleic acid molecules, nucleotide sequences or polypeptide sequences, refers to two or more sequences or subsequences that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In certain embodiments, the substantial identity exists over a region of consecutive nucleotides of a nucleotide sequence of the disclosure that is about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 15 nucleotides to about 25 nucleotides, about 30 nucleotides to about 40 nucleotides, about 50 nucleotides to about 60 nucleotides, about 70 nucleotides to about 80 nucleotides, about 90 nucleotides to about 100 nucleotides, about 100 nucleotides to about 200 nucleotides, about 100 nucleotides to about 300 nucleotides, about 100 nucleotides to about 400 nucleotides, about 100 nucleotides to about 500 nucleotides, about 100 nucleotides to about 600 nucleotides, about 100 nucleotides to about 800 nucleotides, about 100 nucleotides to about 900 nucleotides, or more nucleotides in length, and any range therein, up to the full length of the sequence. In certain embodiments, nucleotide sequences can be substantially identical over at least about 20 consecutive nucleotides (e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2500, 3000, 3500, 4000 or more nucleotides). In certain embodiments, two or more S-type anion channel genes may be substantially identical to one another over at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 to about 2000, 2050,2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2510, 2520, 2530, 2540, 2550, 2600,2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350,3400, 3450, 3490, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600,4700, 4800, 4900, or 5000 or more consecutive nucleotides of a S-type anion channel gene, e.g., SEQ ID NO: 1, 2, 4, or 5.

[0083] In certain embodiments, the substantial identity exists over a region of consecutive amino acid residues of a polypeptide of the disclosure that is about 3 amino acid residues toabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues, about 5 amino acid residues to about 25, 30, 35, 40, 45, 50 or 60 amino acid residues, about 15 amino acid residues to about 30 amino acid residues, about 20 amino acid residues to about 40 amino acid residues, about 25 amino acid residues to about 40 amino acid residues, about 25 amino acid residues to about 50 amino acid residues, about 30 amino acid residues to about 50 amino acid residues, about 40 amino acid residues to about 50 amino acid residues, about 40 amino acid residues to about 70 amino acid residues, about 50 amino acid residues to about 70 amino acid residues, about 60 amino acid residues to about 80 amino acid residues, about 70 amino acid residues to about 80 amino acid residues, about 90 amino acid residues to about 100 amino acid residues, or more amino acid residues in length, and any range therein, up to the full length of the sequence. In certain embodiments, polypeptide sequences can be substantially identical to one another over at least about 8, 9, 10, 11, 12, 13, 14, or more consecutive amino acid residues (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400, 450, 500, 550, 600 or more amino acids in length or more consecutive amino acid residues). In certain embodiments, two or more polypeptides encoded by a S-type anion channel gene may be substantially identical to one another over at least about 10 to about 600 consecutive amino acid residues of the amino acid sequence of, for example, SEQ ID NO: 3 or 6; e.g., over at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95, 100, 105, 110, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260,265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355,360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450,455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545,550, 555, 560, 565, 570, 575, 580, 585, 590, 595, or 600 or more consecutive amino acid residues of the amino acid sequence of, for example, SEQ ID NO: 3. In certain embodiments, a substantially identical nucleotide or protein sequence may perform substantially the same function as the nucleotide (or encoded protein sequence) to which it is substantially identical.

[0084] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0085] Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect similar or identical sequences including, but not limited to, the Megalign® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). For example, multiple alignment of the sequences may be performed using the Clustal V method of alignment (Higgins and Sharp (1989) CAB / OS. 5:151- 153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WIND0W=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENAL TY=5, WIND0W=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.

[0086] Alternatively, the Clustal W method of alignment may be used. The Clustal W method of alignment (described by Higgins and Sharp, CAB / OS. 5: 151-153 (1989); Higgins, D. G. et al., Comput. AppL Biosci. 8: 189-191 (1992)) can be found in the MegAlign™ v6.1 program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Default parameters for multiple alignment correspond to GAP PENAL TY= 10, GAP LENGTH PENALTY=0.2, Delay Divergent Sequences=30%, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB. For pairwise alignments the default parameters are Alignment=Slow-Accurate, Gap Penalty=10.0, Gap Length=0.10, Protein Weight Matrix=Gonnet 250 and DNA Weight Matrix=IUB. After alignment of the sequences using the Clustal W program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table in the same program. In certain embodiments, the % sequence identity is determined over the entire length of the molecule (nucleotide or amino acid).

[0087] Those skilled in the art may also find further candidate S-type anion channel genes based on genome synteny and sequence similarity. In certain embodiments, additional gene candidates can be obtained by hybridization or PCR using sequences based on the S-type anion channelnucleotide or amino acid sequences disclosed herein. Two nucleotide sequences may also be considered substantially complementary when the two sequences hybridize to each other under stringent conditions. In certain embodiments, two nucleotide sequences considered to be substantially complementary hybridize to each other under highly stringent conditions.

[0088] In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).

[0089] In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as32P, or any other detectable marker. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0090] By “hybridizing to” or “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

[0091] “Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology -Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize to its target subsequence, but to no other sequences.

[0092] The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tmfor a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42 °C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCl at 72 °C for about 15 minutes. An example of stringent wash conditions is a 0.2* SSC wash at 65 °C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1 *SSC at 45 °C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6*SSC at 40 °C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30 °C Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2* (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

[0093] The following are examples of sets of hybridization / wash conditions that may be used to clone nucleotide sequences that are homologues of reference nucleotide sequences: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCh, 1 mM EDTA at 50 °C with washing in 2* SSC, 0.1% SDS at 50 °C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCN, 1 mM EDTA at 50 °C with washing in l x SSC, 0.1% SDS at 50 °C, more desirably still in 7% sodium dodecylsulfate (SDS), 0.5 M NaPC , 1 mM EDTA at 50 °C with washing in 0.5 SSC, 0.1% SDS at 50 °C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCN, 1 mM EDTA at 50 °C with washing in 0.1 *SSC, 0.1% SDS at 50 °C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPC , 1 mM EDTA at 50 °C with washing in 0.1 SSC, 0.1% SDS at 65 °C.Reducing Expression or Activity of an S-type Anion Channel Gene

[0094] Several embodiments of the disclosure relate to reducing expression of an endogenous S- type anion channel gene in a plant. As used herein “reduced,” “reduction,” or the like refers to any detectable decrease in an experimental group (e.g., modified maize plant with a targeted DNA modification described herein) as compared to a control group (e.g., wild-type maize plant that does not comprise the targeted DNA modification). Methods for reducing expression of genes or gene products are well documented in the art.

[0095] In certain embodiments of the present disclosure, the expression or activity of the target gene is decreased or eliminated by disrupting the gene encoding the S-type anion channel. The gene encoding the S-type anion channel may be disrupted by any method known in the art, for example, by genome editing, transposon tagging, or mutagenizing plants using random or targeted mutagenesis and optionally selecting for plants that have decreased expression or activity.

[0096] In certain embodiments, the target S-type anion channel gene is modified using genome editing technology. Targeted modification of plant genomes through the use of genome editing methods can be used to reduce expression of an S-type anion channel gene through modification of plant genomic DNA. Genome editing methods can enable targeted insertion of one or more nucleic acids of interest into a plant genome. Genome editing uses engineered nucleases such as RNA guided DNA endonucleases or nucleases composed of sequence specific DNA binding domains fused to a non-specific DNA cleavage module. These engineered nucleases enable efficient and precise genetic modifications by inducing targeted DNA double stranded breaks that stimulate the cell’s endogenous cellular DNA repair mechanisms to repair the induced break. Such mechanisms include, for example, error prone non-homologous end joining (NHEJ) and homology directed repair (HDR).

[0097] “Targeted DNA modification” can be used synonymously with targeted DNA mutation and refers to the introduction of a site specification modification that alters or changes the nucleotide sequence at a specific genomic locus of the plant (e.g., maize).

[0098] The targeted DNA modification described herein may be any modification known in the art such as, for example, insertion, deletion, single nucleotide polymorphism (SNP), and or a polynucleotide modification. Additionally, the targeted DNA modification in the genomic locus may be located anywhere in the genomic locus, such as, for example, a coding region of the encoded polypeptide (e.g., exon), a non-coding region (e.g., intron), a regulatory element, or untranslated region.

[0099] In certain embodiments, the at least one mutation in an endogenous S-type anion channel gene may be a deletion (e.g., a deletion of one or more consecutive base pairs, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100, or more (e.g., 110, 120, 130, 140, 150, and the like) consecutive base pairs of SEQ ID NO: 1 or 4. In certain embodiments, a mutation that is a base insertion may be an insertion of at least one base pair (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100, or more) into an endogenous S-type anion channel gene.

[0100] The type and location of the targeted DNA modification of the polynucleotide is not particularly limited so long as the targeted DNA modification results in reduced expression or activity of the protein encoded by the polynucleotide. In certain embodiments, the targeted DNA modification is a deletion of one or more nucleotides, preferably contiguous, of the genomic locus.

[0101] In certain embodiments, a reduction in the expression or activity of the protein encoded by the polynucleotide is due to a targeted DNA modification at a genomic locus of a plant that results in one or more of the following: (a) reduced expression of the polynucleotide; (b) reduced transcriptional activity of the protein encoded by the polynucleotide; (c) generation of one or more alternatively spliced transcripts of the polynucleotide; (d) frameshift mutation in one or more exons of the polynucleotide; (e) deletion of a substantial portion of the polynucleotide or deletion of the full open reading frame of the polynucleotide; (f) repression of an enhancer motif present within a regulatory region encoding the polynucleotide; or (g) modification of one or more nucleotides or deletion of a regulatory element operably linked to the expression of the polynucleotide wherein the regulatory element is present within a promoter, intron, 3'UTR, terminator or a combination thereof.

[0102] In certain embodiments, the genomic locus has more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) targeted DNA modification. For example, the translated region and a regulatory element of a genomic locus may each comprise a targeted DNA modification. In certain embodiments, the plant may have targeted DNA modifications at more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) genomic loci comprising an S-type anion channel gene.

[0103] In certain embodiments, a sequence-specific nucleic acid binding domain (DNA binding domains) of an editing system useful with this disclosure can be from, for example, a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., CRISPR-Cas effector protein), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) and / or an Argonaute protein.

[0104] In certain embodiments, a sequence-specific nucleic acid binding domain / protein may be a CRISPR-Cas effector protein, optionally wherein the CRISPR-Cas effector protein may be from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or a Type VI CRISPR- Cas system. In certain embodiments, a CRISPR-Cas effector protein of the disclosure may be from a Type II CRISPR-Cas system or a Type V CRISPR-Cas system. In certain embodiments, a CRISPR-Cas effector protein may be Type II CRISPR-Cas effector protein, for example, a Cas9 effector protein. In certain embodiments, a CRISPR-Cas effector protein may be Type V CRISPR-Cas effector protein, for example, a Cast 2 effector protein.

[0105] As used herein, a “CRISPR-Cas effector protein” is a protein or polypeptide or domain thereof that cleaves or cuts a nucleic acid, binds a nucleic acid (e.g., a target nucleic acid and / or a guide nucleic acid), and / or that identifies, recognizes, or binds a guide nucleic acid as defined herein. In certain embodiments, a CRISPR-Cas effector protein may be an enzyme (e.g., a nuclease, endonuclease, nickase, etc.) or portion thereof and / or may function as an enzyme. In certain embodiments, a CRISPR-Cas effector protein refers to a CRISPR-Cas nuclease polypeptide or domain thereof that comprises nuclease activity or in which the nuclease activity has been reduced or eliminated, and / or comprises nickase activity or in which the nickase has been reduced or eliminated, and / or comprises single stranded DNA cleavage activity (ss DNAse activity) or in which the ss DNAse activity has been reduced or eliminated, and / or comprises self-processing RNAse activity or in which the self-processing RNAse activity has been reduced or eliminated. A CRISPR-Cas effector protein may bind to a target nucleic acid.

[0106] In certain embodiments, a CRISPR-Cas effector protein may include, but is not limited to, a Cas9, C2cl, C2c3, Casl2a (also referred to as Cpfl), Casl2b, Casl2c, Casl2d, Casl2e,Casl3a, Casl3b, Casl3c, Casl3d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and / or Csf5 nuclease, optionally wherein the CRISPR-Cas effector protein may be a Cas9, Casl2a (Cpfl), Casl2b, Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2g, Casl2h, Casl2i, C2c4, C2c5, C2c8, C2c9, C2cl0, Casl4a, Casl4b, and / or Casl4c effector protein.

[0107] In certain embodiments, a CRISPR-Cas effector protein useful with the disclosure may comprise a mutation in its nuclease active site (e.g., RuvC, HNH, e.g., RuvC site of a Casl2a nuclease domain; e.g., RuvC site and / or HNH site of a Cas9 nuclease domain). A CRISPR-Cas effector protein having a mutation in its nuclease active site, and therefore, no longer comprising nuclease activity, is commonly referred to as “dead,” e.g., dCas. In certain embodiments, a CRISPR-Cas effector protein domain or polypeptide having a mutation in its nuclease active site may have impaired activity or reduced activity as compared to the same CRISPR-Cas effector protein without the mutation, e.g., a nickase, e.g, Cas9 nickase, Casl2a nickase.

[0108] A CRISPR Cas9 effector protein or CRISPR Cas9 effector domain useful with this disclosure may be any known or later identified Cas9 nuclease. In certain embodiments, a CRISPR Cas9 polypeptide can be a Cas9 polypeptide from, for example, Streptococcus spp. (e.g., S. pyogenes, S. thermophiles), Lactobacillus spp., Bifidobacterium spp., Kandleria spp., Leuconostoc spp., Oenococcus spp., Pediococcus spp., Weissella spp., and / or Olsenella spp.

[0109] In certain embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide derived from Streptococcus pyogenes and recognizes the PAM sequence motif NGG, NAG, NGA (Mali et al, Science 2013; 339(6121): 823-826). In certain embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide derived from Streptococcus thermophiles and recognizes the PAM sequence motif NGGNG and / or NNAGAAW (W=A or T) (See, e.g., Horvath et al, Science, 2010; 327(5962): 167-170, and Deveau et al, J Bacteriol 2008; 190(4): 1390-1400). In certain embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide derived from Streptococcus mutans and recognizes the PAM sequence motif NGG and / or NAAR (R=A or G) (See, e.g., Deveau et al, J Bacteriol 2008; 190(4): 1390-1400). In certain embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide derived from Streptococcus aureus and recognizes the PAM sequence motif NNGRR (R=A or G). In certain embodiments, the CRISPR-Cas effector protein may be a Cas9 protein derived from S. aureus, which recognizes the PAM sequence motif N GRRT (R=A or G). In certain embodiments, theCRISPR-Cas effector protein may be a Cas9 polypeptide derived from S. aureus, which recognizes the PAM sequence motif N GRRV (R=A or G). In certain embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide that is derived from Neisseria meningitidis and recognizes the PAM sequence motif N GATT or N GCTT (R=A or G, V=A, G or C) (See, e.g., Hou et ah, PNAS 2013, 1-6). In the aforementioned embodiments, N can be any nucleotide residue, e.g., any of A, G, C or T. In certain embodiments, the CRISPR-Cas effector protein may be a Cast 3a protein derived from Leptotrichia shahii, which recognizes a protospacer flanking sequence (PFS) (or RNA PAM (rPAM)) sequence motif of a single 3' A, U, or C, which may be located within the target nucleic acid.

[0110] In certain embodiments, the CRISPR-Cas effector protein may be derived from Casl2a, which is a Type V Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nuclease. Cast 2a differs in several respects from the more well-known Type II CRISPR Cas9 nuclease. For example, Cas9 recognizes a G-rich protospacer-adjacent motif (PAM) that is 3' to its guide RNA (gRNA, sgRNA, crRNA, crDNA, CRISPR array) binding site (protospacer, target nucleic acid, target DNA) (3'-NGG), while Casl2a recognizes a T-rich PAM that is located 5' to the target nucleic acid (5'-TTN, 5'-TTTN. In fact, the orientations in which Cas9 and Cast 2a bind their guide RNAs are very nearly reversed in relation to their N and C termini. Furthermore, Cast 2a enzymes use a single guide RNA (gRNA, CRISPR array, crRNA) rather than the dual guide RNA (sgRNA (e.g., crRNA and tracrRNA)) found in natural Cas9 systems, and Cast 2a processes its own gRNAs. Additionally, Cast 2a nuclease activity produces staggered DNA double stranded breaks instead of blunt ends produced by Cas9 nuclease activity, and Cast 2a relies on a single RuvC domain to cleave both DNA strands, whereas Cas9 utilizes an HNH domain and a RuvC domain for cleavage.[OHl] A CRISPR Casl2a effector protein / domain useful with this disclosure may be any known or later identified Cast 2a polypeptide (previously known as Cpfl) (see, e.g., U.S. Pat. No. 9,790,490, which is incorporated by reference for its disclosures of Cpfl (Casl2a) sequences). The term “Cast 2a”, “Cast 2a polypeptide” or “Cast 2a domain” refers to an RNA- guided nuclease comprising a Casl2a polypeptide, or a fragment thereof, which comprises the guide nucleic acid binding domain of Casl2a and / or an active, inactive, or partially active DNA cleavage domain of Casl2a. In certain embodiments, a Casl2a useful with the disclosure may comprise a mutation in the nuclease active site (e.g., RuvC site of the Casl2a domain). A Cast 2a domain or Cast 2a polypeptide having a mutation in its nuclease active site, and therefore, no longer comprising nuclease activity, is commonly referred to as deadCas!2a (e.g.,dCasl2a). In certain embodiments, a Casl2a domain or Casl2a polypeptide having a mutation in its nuclease active site may have impaired activity, e.g., may have nickase activity.

[0112] Any deaminase domain / polypeptide useful for base editing may be used with this disclosure. In certain embodiments, the deaminase domain may be a cytosine deaminase domain or an adenine deaminase domain. A cytosine deaminase (or cytidine deaminase) useful with this disclosure may be any known or later identified cytosine deaminase from any organism (see, e.g., U.S. Pat. No. 10,167,457 and Thuronyi et al. Nat. Biotechnol. 37: 1070-1079 (2019), each of which is incorporated by reference herein for its disclosure of cytosine deaminases). Cytosine deaminases can catalyze the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. Thus, in certain embodiments, a deaminase or deaminase domain useful with this disclosure may be a cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil. In certain embodiments, a cytosine deaminase may be a variant of a naturally occurring cytosine deaminase, including but not limited to a primate (e.g., a human, monkey, chimpanzee, gorilla), a dog, a cow, a rat or a mouse.

[0113] In certain embodiments, a cytosine deaminase useful with the disclosure may be an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In certain embodiments, the cytosine deaminase may be an APOBEC 1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, an APOBEC4 deaminase, a human activation induced deaminase (hAID), an rAPOBECl, FERNY, and / or a CDA1, optionally a pmCDAl, an atCDAl (e.g., At2gl9570), and evolved versions of the same.

[0114] In certain embodiments, a nucleic acid construct of this disclosure may further encode an uracil glycosylase inhibitor (UGI) (e.g., uracil-DNA glycosylase inhibitor) polypeptide / domain. Thus, in certain embodiments, a nucleic acid construct encoding a CRISPR-Cas effector protein and a cytosine deaminase domain (e.g., encoding a fusion protein comprising a CRISPR-Cas effector protein domain fused to a cytosine deaminase domain, and / or a CRISPR-Cas effector protein domain fused to a peptide tag or to an affinity polypeptide capable of binding a peptide tag and / or a deaminase protein domain fused to a peptide tag or to an affinity polypeptide capable of binding a peptide tag) may further encode a uracil-DNA glycosylase inhibitor (UGI), optionally wherein the UGI may be codon optimized for expression in a plant. In certain embodiments, the disclosure provides fusion proteins comprising a CRISPR-Cas effector polypeptide, a deaminase domain, and a UGI and / or one or more polynucleotides encoding thesame, optionally wherein the one or more polynucleotides may be codon optimized for expression in a plant. In certain embodiments, the disclosure provides fusion proteins, wherein a CRISPR-Cas effector polypeptide, a deaminase domain, and a UGI may be fused to any combination of peptide tags and affinity polypeptides as described herein, thereby recruiting the deaminase domain and UGI to the CRISPR-Cas effector polypeptide and a target nucleic acid. In certain embodiments, a guide nucleic acid may be linked to a recruiting RNA motif and one or more of the deaminase domain and / or UGI may be fused to an affinity polypeptide that is capable of interacting with the recruiting RNA motif, thereby recruiting the deaminase domain and UGI to a target nucleic acid. A “uracil glycosylase inhibitor” useful with the disclosure may be any protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme.

[0115] An adenine deaminase (or adenosine deaminase) useful with this disclosure may be any known or later identified adenine deaminase from any organism (see, e.g., U.S. Pat. No.10,113,163, which is incorporated by reference herein for its disclosure of adenine deaminases). An adenine deaminase can catalyze the hydrolytic deamination of adenine or adenosine. In certain embodiments, the adenine deaminase may catalyze the hydrolytic deamination of adenosine or deoxy adenosine to inosine or deoxyinosine, respectively. In certain embodiments, the adenosine deaminase may catalyze the hydrolytic deamination of adenine or adenosine in DNA. In certain embodiments, an adenine deaminase encoded by a nucleic acid construct of the disclosure may generate an A^G conversion in the sense (e.g.,template) strand of the target nucleic acid or a T^C conversion in the antisense (e.g., complementary) strand of the target nucleic acid. In certain embodiments, an adenosine deaminase may be a variant of a naturally occurring adenine deaminase. In certain embodiments, the deaminase or deaminase does not occur in nature and may be referred to as an engineered, mutated or evolved adenosine deaminase. In certain embodiments, the adenosine deaminase may be from a bacterium, (e.g., Escherichia coli, Staphylococcus aureus, Haemophilus influenzae, Caulobacter crescentus, and the like).

[0116] In certain embodiments, an adenine deaminase domain may be a wild type tRNA- specific adenosine deaminase domain, e.g., a tRNA-specific adenosine deaminase (TadA) and / or a mutated / evolved adenosine deaminase domain, e.g., mutated / evolved tRNA-specific adenosine deaminase domain (TadA*). In certain embodiments, a TadA domain may be from E. coli. In certain embodiments, the TadA may be modified, e.g., truncated, missing one or more N- terminal and / or C-terminal amino acids relative to a full-length TadA (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal and / or C terminal amino acid residues may be missing relative to a full length TadA.

[0117] A cytosine deaminase catalyzes cytosine deamination and results in a thymidine (through a uracil intermediate), causing a C to T conversion, or a G to A conversion in the complementary strand in the genome. Thus, in certain embodiments, the cytosine deaminase encoded by the polynucleotide of the disclosure generates a C^T conversion in the sense (e.g., template) strand of the target nucleic acid or a G^A conversion in anti sense (e.g., complementary) strand of the target nucleic acid. In certain embodiments, the adenine deaminase encoded by the nucleic acid construct of the disclosure generates an A^G conversion in the sense (e.g.,template) strand of the target nucleic acid or a T^C conversion in the antisense (e.g., complementary) strand of the target nucleic acid.

[0118] The nucleic acid constructs of the disclosure encoding a base editor comprising a sequence-specific nucleic acid binding protein and a cytosine deaminase polypeptide, and nucleic acid constructs / expression cassettes / vectors encoding the same, may be used in combination with guide nucleic acids for modifying target nucleic acid including, but not limited to, generation of C^T or G^A mutations in a target nucleic acid including, but not limited to, a plasmid sequence; generation of C^T or G^A mutations in a coding sequence to alter an amino acid identity; generation of C^T or G^A mutations in a coding sequence to generate a stop codon; generation of C^T or G^A mutations in a coding sequence to disrupt a start codon; generation of point mutations in genomic DNA to generate a mutated S-type anion channel gene.

[0119] The nucleic acid constructs of the disclosure encoding a base editor comprising a sequence-specific nucleic acid binding protein and an adenine deaminase polypeptide, and expression cassettes and / or vectors encoding the same may be used in combination with guide nucleic acids for modifying a target nucleic acid including, but not limited to, generation of A^G or T^C mutations in a target nucleic acid including, but not limited to, a plasmid sequence; generation of A^G or T^C mutations in a coding sequence to alter an amino acid identity; generation of A^G or T^C mutations in a coding sequence to generate a stop codon; generation of A^G or T^C mutations in a coding sequence to disrupt a start codon; generation of point mutations in genomic DNA to disrupt function; and / or generation of point mutations in genomic DNA to disrupt splice junctions.

[0120] In certain embodiments, methods such as prime editing may be used to generate a mutation in an endogenous S-type anion channel gene. In prime editing, RNA-dependent DNApolymerase (reverse transcriptase, RT) and reverse transcriptase templates (RT template) are used in combination with sequence specific nucleic acid binding domains that confer the ability to recognize and bind the target in a sequence-specific manner, and which can also cause a nick of the PAM-containing strand within the target. The nucleic acid binding domain may be a CRISPR-Cas effector protein and in this case, the CRISPR array or guide RNA may be an extended guide that comprises an extended portion comprising a primer binding site (PSB) and the edit to be incorporated into the genome (the template). Similar to base editing, prime editing can take advantageous of the various methods of recruiting proteins for use in the editing to the target site, such methods including both non-covalent and covalent interactions between the proteins and nucleic acids used in the selected process of genome editing.

[0121] The nucleic acid constructs of the disclosure comprising a CRISPR-Cas effector protein or a fusion protein thereof may be used in combination with a guide RNA (gRNA, CRISPR array, CRISPR RNA, crRNA), designed to function with the encoded CRISPR-Cas effector protein or domain, to modify a target nucleic acid. A guide nucleic acid useful with this disclosure comprises at least one spacer sequence and at least one repeat sequence. The guide nucleic acid is capable of forming a complex with the CRISPR-Cas nuclease domain encoded and expressed by a nucleic acid construct of the disclosure and the spacer sequence is capable of hybridizing to a target nucleic acid, thereby guiding the complex (e.g., a CRISPR-Cas effector fusion protein (e.g., CRISPR-Cas effector domain fused to a deaminase domain and / or a CRISPR-Cas effector domain fused to a peptide tag or an affinity polypeptide to recruit a deaminase domain and optionally, a UGI) to the target nucleic acid, wherein the target nucleic acid may be modified (e.g., cleaved or edited) or modulated (e.g., modulating transcription) by the deaminase domain.

[0122] As an example, a nucleic acid construct encoding a Cas9 domain linked to a cytosine deaminase domain (e.g., fusion protein) may be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, wherein the cytosine deaminase domain of the fusion protein deaminates a cytosine base in the target nucleic acid, thereby editing the target nucleic acid. In a further example, a nucleic acid construct encoding a Cas9 domain linked to an adenine deaminase domain (e.g., fusion protein) may be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, wherein the adenine deaminase domain of the fusion protein deaminates an adenosine base in the target nucleic acid, thereby editing the target nucleic acid.

[0123] Likewise, a nucleic acid construct encoding a Casl2a domain (or other selected CRISPR-Cas nuclease, e.g., C2cl, C2c3, Cast 2b, Cast 2c, Cast 2d, Casl2e, Cast 3 a, Cast 3b,Casl3c, Casl3d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and / or Csf5) linked to a cytosine deaminase domain or adenine deaminase domain (e.g., fusion protein) may be used in combination with a Casl 2a guide nucleic acid (or the guide nucleic acid for the other selected CRISPR-Cas nuclease) to modify a target nucleic acid, wherein the cytosine deaminase domain or adenine deaminase domain of the fusion protein deaminates a cytosine base in the target nucleic acid, thereby editing the target nucleic acid.

[0124] A “guide nucleic acid,” “guide RNA,” “gRNA,” “CRISPR RNA / DNA” “crRNA” or “crDNA” as used herein means a nucleic acid that comprises at least one spacer sequence, which is complementary to (and hybridizes to) a target DNA (e.g., protospacer), and at least one repeat sequence (e.g., a repeat of a Type V Casl2a CRISPR-Cas system, or a fragment or portion thereof; a repeat of a Type II Cas9 CRISPR-Cas system, or fragment thereof; a repeat of a Type V C2cl CRISPR Cas system, or a fragment thereof; a repeat of a CRISPR-Cas system of, for example, C2c3, Casl2a (also referred to as Cpfl), Casl2b, Casl2c, Casl2d, Casl2e, Casl3a, Casl3b, Casl3c, Casl3d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and / or Csf5, or a fragment thereof), wherein the repeat sequence may be linked to the 5' end and / or the 3' end of the spacer sequence. The design of a gRNA of this disclosure may be based on a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR-Cas system.

[0125] In certain embodiments, a Casl2a gRNA may comprise, from 5' to 3', a repeat sequence (full length or portion thereof (“handle”); e.g., pseudoknot-like structure) and a spacer sequence.

[0126] In certain embodiments, a guide nucleic acid may comprise more than one repeat sequence-spacer sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeat-spacer sequences) (e.g., repeat-spacer-repeat, e.g., repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer, and the like). The guide nucleic acids of this disclosure are synthetic, human-made and not found in nature. A gRNA can be quite long and may be used as an aptamer (like in the MS2 recruitment strategy) or other RNA structures hanging off the spacer.

[0127] A “repeat sequence” as used herein, refers to, for example, any repeat sequence of a wild-type CRISPR Cas locus (e.g., a Cas9 locus, a Casl2a locus, a C2cl locus, etc.) or a repeatsequence of a synthetic crRNA that is functional with the CRISPR-Cas effector protein encoded by the nucleic acid constructs of the disclosure. A repeat sequence useful with this disclosure can be any known or later identified repeat sequence of a CRISPR-Cas locus (e.g., Type I, Type II, Type III, Type IV, Type V or Type VI) or it can be a synthetic repeat designed to function in a Type I, II, III, IV, V or VI CRISPR-Cas system. A repeat sequence may comprise a hairpin structure and / or a stem loop structure. In certain embodiments, a repeat sequence may form a pseudoknot-like structure at its 5' end (i.e., “handle”). Thus, in certain embodiments, a repeat sequence can be identical to or substantially identical to a repeat sequence from wild-type Type I CRISPR-Cas loci, Type II, CRISPR-Cas loci, Type III, CRISPR-Cas loci, Type IV CRISPR-Cas loci, Type V CRISPR-Cas loci and / or Type VI CRISPR-Cas loci. A repeat sequence from a wild-type CRISPR-Cas locus may be determined through established algorithms, such as using the CRISPRfinder offered through CRISPRdb (see, Grissa et al. Nucleic Acids Res. 35 (Web Server issue): W52-7). In certain embodiments, a repeat sequence or portion thereof is linked at its 3' end to the 5' end of a spacer sequence, thereby forming a repeat-spacer sequence (e.g., guide nucleic acid, guide RNA / DNA, crRNA, crDNA).

[0128] In certain embodiments, a repeat sequence comprises, consists essentially of, or consists of at least 10 nucleotides depending on the particular repeat and whether the guide nucleic acid comprising the repeat is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides, or any range or value therein). In certain embodiments, a repeat sequence comprises, consists essentially of, or consists of about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100 or more nucleotides.

[0129] A repeat sequence linked to the 5' end of a spacer sequence can comprise a portion of a repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous nucleotides of a wild type repeat sequence). In certain embodiments, a portion of a repeat sequence linked to the 5' end of a spacer sequence can be about five to about ten consecutive nucleotides in length (e.g., about 5, 6, 7, 8, 9, 10 nucleotides) and have at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the same region (e.g., 5' end) of awild type CRISPR Cas repeat nucleotide sequence. In certain embodiments, a portion of a repeat sequence may comprise a pseudoknot-like structure at its 5' end (e.g., “handle”).

[0130] A “spacer sequence” as used herein is a nucleotide sequence that is complementary to a region or portion of a target nucleic acid (e.g., target DNA) (e.g., protospacer) (e.g., a portion of consecutive nucleotides of a S-type anion channel gene. A spacer sequence can be fully complementary or substantially complementary (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a region or portion of a target nucleic acid. In certain embodiments, the spacer sequence can have one, two, three, four, or five mismatches as compared to the target nucleic acid, which mismatches can be contiguous or noncontiguous. In certain embodiments, the spacer sequence can have 70% complementarity to a target nucleic acid. In other embodiments, the spacer nucleotide sequence can have 80% complementarity to a target nucleic acid. In still other embodiments, the spacer nucleotide sequence can have 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% complementarity, and the like, to the target nucleic acid (protospacer). In certain embodiments, the spacer sequence is 100% complementary to a region or portion of the target nucleic acid. A spacer sequence may have a length from about 15 nucleotides to about 30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any range or value therein). Thus, in certain embodiments, a spacer sequence may have complete complementarity or substantial complementarity over a region of a target nucleic acid (e.g., protospacer) that is at least about 15 nucleotides to about 30 nucleotides in length. In certain embodiments, the spacer is about 20 nucleotides in length. In certain embodiments, the spacer is about 21, 22, or 23 nucleotides in length.

[0131] In certain embodiments, the 5' region of a spacer sequence of a guide nucleic acid may be identical to a target DNA, while the 3' region of the spacer may be substantially complementary to the target DNA (such as for a Type V CRISPR-Cas system), or the 3' region of a spacer sequence of a guide nucleic acid may be identical to a target DNA, while the 5' region of the spacer may be substantially complementary to the target DNA (such as for a Type II CRISPR-Cas system), and therefore, the overall complementarity of the spacer sequence to the target DNA may be less than 100%. Thus, for example, in a guide for a Type V CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 5' region (i.e., seed region) of, for example, a 20 nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer sequence are substantiallycomplementary (e.g., at least about 70% complementary) to the target DNA. In certain embodiments, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, nucleotides, and any range therein) of the 5' end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to the target DNA.

[0132] As a further example, in a guide for a Type II CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 3' region (i.e., seed region) of, for example, a 20 nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA. In certain embodiments, the first 1 to 10 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, and any range therein) of the 3' end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or any range or value therein)) to the target DNA.

[0133] In certain embodiments, a seed region of a spacer may be about 8 to about 10 nucleotides in length, about 5 to about 6 nucleotides in length, or about 6 nucleotides in length.

[0013] In certain embodiments, the present disclosure provides a guide nucleic acid (e.g., gRNA, gDNA, crRNA, crDNA) that binds to a target site in an endogenous gene S-type anion channel gene, the S-type anion channel gene: (a) comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5; or (b) encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3 or 6. Example spacer sequences useful with a guide of this disclosure may be substantially complementary (e.g., at least 70% complementary) to a fragment or portion (e.g., about 15 consecutive nucleotides to about 30 consecutive nucleotides) of a nucleotide sequence (a) having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1, 2, 4, or 5. In certain embodiments, a system is provided that comprises a guide nucleic acid of the present disclosure and a CRISPR-Caseffector protein that associates with the guide nucleic acid. In certain embodiments, the system may further comprise a tracr nucleic acid that associates with the guide nucleic acid and a CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked. In certain embodiments, a guide nucleic acid is provided that binds to a target site in an endogenous S-type anion channel gene having the gene identification number (gene ID) of ZmOOOOl ebl 59490 / Zm00004b019600 (ZmSLAH2 / 3).

[0135] In certain embodiments, a gene editing system is provided, the gene editing system comprising a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to a S-type anion channel gene. As used herein, “a CRISPR-Cas effector protein in association with a guide nucleic acid” refers to the complex that is formed between a CRISPR-Cas effector protein and a guide nucleic acid in order to direct the CRISPR-Cas effector protein to a target site in a gene. The present disclosure further provides a complex comprising CRISPR-Cas effector protein comprising a cleavage domain and a guide nucleic acid. Also provided herein are expression cassettes comprising (a) polynucleotide encoding CRISPR-Cas effector protein comprising a cleavage domain and (b) a guide nucleic acid that binds to a target site in an endogenous S-type anion channel gene.

[0136] As used herein, a “target nucleic acid”, “target DNA,” “target nucleotide sequence,” “target region,” or a “target region in the genome” refers to a region of a plant's genome that is fully complementary (100% complementary) or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a spacer sequence in a guide nucleic acid of this disclosure. A target region useful for a CRISPR-Cas system may be located immediately 3' (e.g., Type V CRISPR- Cas system) or immediately 5' (e.g., Type II CRISPR-Cas system) to a PAM sequence in the genome of the organism (e.g., a plant genome). A target region may be selected from any region of at least 15 consecutive nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, and the like) located immediately adjacent to a PAM sequence.

[0137] A “protospacer sequence” refers to the target double stranded DNA and specifically to the portion of the target DNA (e.g., or target region in the genome) that is fully or substantially complementary (and hybridizes) to the spacer sequence of the CRISPR repeat-spacer sequences (e.g., guide nucleic acids, CRISPR arrays, crRNAs).

[0138] In the case of Type V CRISPR-Cas (e.g., Casl2a) systems and Type II CRISPR-Cas (Cas9) systems, the protospacer sequence is flanked by (e.g., immediately adjacent to) aprotospacer adjacent motif (PAM). For Type IV CRISPR-Cas systems, the PAM is located at the 5' end on the non-target strand and at the 3' end of the target strand.

[0139] In the case of Type II CRISPR-Cas (e.g., Cas9) systems, the PAM is located immediately 3' of the target region. The PAM for Type I CRISPR-Cas systems is located 5' of the target strand. There is no known PAM for Type III CRISPR-Cas systems. Makarova et al. describes the nomenclature for all the classes, types and subtypes of CRISPR systems (Nature Reviews Microbiology 13:722-736 (2015)). Guide structures and PAMs are described in by R. Barrangou (Genome Biol. 16:247 (2015)).

[0140] Canonical Cast 2a PAMs are T rich. In certain embodiments, a canonical Cast 2a PAM sequence may be 5'-TTN, 5'-TTTN, or 5'-TTTV. In certain embodiments, canonical Cas9 (e.g., S. pyogenes) PAMs may be 5'-NGG-3'. In certain embodiments, non-canonical PAMs may be used but may be less efficient.

[0141] Additional PAM sequences may be determined by those skilled in the art through established experimental and computational approaches. Thus, for example, experimental approaches include targeting a sequence flanked by all possible nucleotide sequences and identifying sequence members that do not undergo targeting, such as through the transformation of target plasmid DNA (Esvelt et al. 2013. Nat. Methods 10: 1116-1121; Jiang et al. 2013. Nat. BiotechnoL 31 :233-239). In certain embodiments, a computational approach can include performing BLAST searches of natural spacers to identify the original target DNA sequences in bacteriophages or plasmids and aligning these sequences to determine conserved sequences adjacent to the target sequence (Briner and Barrangou. 2014. AppL Environ. Microbiol. 80:994- 1001; Mojica et al. 2009. Microbiology 155:733-740).

[0142] In certain embodiments, the present disclosure provides expression cassettes and / or vectors comprising the nucleic acid constructs of the disclosure (e.g., one or more components of an editing system of the disclosure). In certain embodiments, expression cassettes and / or vectors comprising the nucleic acid constructs of the disclosure and / or one or more guide nucleic acids may be provided. In certain embodiments, a nucleic acid construct of the disclosure encoding a base editor (e.g., a construct comprising a CRISPR-Cas effector protein and a deaminase domain (e.g., a fusion protein)) or the components for base editing (e.g., a CRISPR-Cas effector protein fused to a peptide tag or an affinity polypeptide, a deaminase domain fused to a peptide tag or an affinity polypeptide, and / or a UGI fused to a peptide tag or an affinity polypeptide), may be comprised on the same or on a separate expression cassette or vector from that comprising the one or more guide nucleic acids. When the nucleic acidconstruct encoding a base editor or the components for base editing is / are comprised on separate expression cassette(s) or vector(s) from that comprising the guide nucleic acid, a target nucleic acid may be contacted with (e.g., provided with) the expression cassette(s) or vector(s) encoding the base editor or components for base editing in any order from one another and the guide nucleic acid, e.g., prior to, concurrently with, or after the expression cassette comprising the guide nucleic acid is provided (e.g., contacted with the target nucleic acid).

[0143] Fusion proteins of the disclosure may comprise sequence-specific nucleic acid binding domains / proteins, CRISPR-Cas polypeptides, and / or deaminase domains fused to peptide tags or affinity polypeptides that interact with the peptide tags, as known in the art, for use in recruiting the deaminase to the target nucleic acid. Methods of recruiting may also comprise guide nucleic acids linked to RNA recruiting motifs and deaminases fused to affinity polypeptides capable of interacting with RNA recruiting motifs, thereby recruiting the deaminase to the target nucleic acid. Alternatively, chemical interactions may be used to recruit polypeptides (e.g., deaminases) to a target nucleic acid.

[0144] A peptide tag (e.g., epitope) useful with this disclosure may include, but is not limited to, a GCN4 peptide tag (e.g., Sun-Tag), a c-Myc affinity tag, an HA affinity tag, a His affinity tag, an S affinity tag, a methionine-His affinity tag, an RGD-His affinity tag, a FLAG octapeptide, a strep tag or strep tag II, a V5 tag, and / or a VSV-G epitope. In certain embodiments, a peptide tag may also include phosphorylated tyrosines in specific sequence contexts recognized by SH2 domains, characteristic consensus sequences containing phosphoserines recognized by 14-3-3 proteins, proline rich peptide motifs recognized by SH3 domains, PDZ protein interaction domains or the PDZ signal sequences, and an AGO hook motif from plants. Peptide tags are disclosed in WO2018 / 136783 and U.S. Patent Application Publication No. 2017 / 0219596, which are incorporated by reference for their disclosures of peptide tags. Any epitope that may be linked to a polypeptide and for which there is a corresponding affinity polypeptide that may be linked to another polypeptide may be used with this disclosure as a peptide tag. A peptide tag may comprise or be present in one copy or in 2 or more copies of the peptide tag (e.g., multimerized peptide tag or multimerized epitope) (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 9, 20, 21, 22, 23, 24, or 25 or more peptide tags). When multimerized, the peptide tags may be fused directly to one another or they may be linked to one another via one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids, optionally about 3 to about 10, about 4 to about 10, about 5 to about 10, about 5 to about 15, or about 5 to about 20 amino acids, and the like, and any value or range therein. Incertain embodiments, an affinity polypeptide that interacts with / binds to a peptide tag may be an antibody. In certain embodiments, the antibody may be a scFv antibody. In certain embodiments, an affinity polypeptide that binds to a peptide tag may be synthetic (e.g., evolved for affinity interaction) including, but not limited to, an affibody, an anticalin, a monobody and / or a DARPin (see, e.g., Sha et al., Protein Sci. 26(5):910-924 (2017)); Gilbreth (Curr Opin Struc Biol 22(4):413-420 (2013)), U.S. Pat. No. 9,982,053, each of which are incorporated by reference in their entireties for the teachings relevant to affibodies, anticalins, monobodies and / or DARPins.

[0145] In certain embodiments, a guide nucleic acid may be linked to an RNA recruiting motif, and a polypeptide to be recruited (e.g., a deaminase) may be fused to an affinity polypeptide that binds to the RNA recruiting motif, wherein the guide binds to the target nucleic acid and the RNA recruiting motif binds to the affinity polypeptide, thereby recruiting the polypeptide to the guide and contacting the target nucleic acid with the polypeptide (e.g., deaminase). In certain embodiments, two or more polypeptides may be recruited to a guide nucleic acid, thereby contacting the target nucleic acid with two or more polypeptides (e.g., deaminases).

[0146] In certain embodiments, a polypeptide fused to an affinity polypeptide may be a reverse transcriptase and the guide nucleic acid may be an extended guide nucleic acid linked to an RNA recruiting motif. In certain embodiments, an RNA recruiting motif may be located on the 3' end of the extended portion of an extended guide nucleic acid (e.g., 5 '-3', repeat-spacer- extended portion (RT template-primer binding site)-RNA recruiting motif). In certain embodiments, an RNA recruiting motif may be embedded in the extended portion.

[0147] In certain embodiments, an extended guide RNA and / or guide RNA may be linked to one or to two or more RNA recruiting motifs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more motifs, e.g., at least 10 to about 25 motifs), optionally wherein the two or more RNA recruiting motifs may be the same RNA recruiting motif or different RNA recruiting motifs. In certain embodiments, an RNA recruiting motif and corresponding affinity polypeptide may include, but is not limited, to a telomerase Ku binding motif (e.g., Ku binding hairpin) and the corresponding affinity polypeptide Ku (e.g., Ku heterodimer), a telomerase Sm7 binding motif and the corresponding affinity polypeptide Sm7, an MS2 phage operator stem-loop and the corresponding affinity polypeptide MS2 Coat Protein (MCP), a PP7 phage operator stem-loop and the corresponding affinity polypeptide PP7 Coat Protein (PCP), an SfMu phage Corn stemloop and the corresponding affinity polypeptide Com RNA binding protein, a PUF binding site (PBS) and the affinity polypeptide Pumilio / fem-3 mRNA binding factor (PUF), and / or asynthetic RNA-aptamer and the aptamer ligand as the corresponding affinity polypeptide. In certain embodiments, the RNA recruiting motif and corresponding affinity polypeptide may be an MS2 phage operator stem-loop and the affinity polypeptide MS2 Coat Protein (MCP). In certain embodiments, the RNA recruiting motif and corresponding affinity polypeptide may be a PUF binding site (PBS) and the affinity polypeptide Pumilio / fem-3 mRNA binding factor (PUF).

[0148] In certain embodiments, the components for recruiting polypeptides and nucleic acids may those that function through chemical interactions that may include, but are not limited to, rapamycin-inducible dimerization of FRB-FKBP; Biotin-streptavidin; SNAP tag; Halo tag; CLIP tag; DmrA-DmrC heterodimer induced by a compound; bifunctional ligand (e.g., fusion of two protein-binding chemicals together, e.g. dihyrofolate reductase (DHFR).

[0149] TAL effector nucleases (TALEN) are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism. (Miller et al. (2011) Nature Biotechnology 29: 143-148).

[0150] A TALEN comprises a TAL effector DNA binding domain and an endonuclease domain. TAL effectors are proteins of plant pathogenic bacteria that are injected by the pathogen into the plant cell, where they travel to the nucleus and function as transcription factors to turn on specific plant genes. The primary amino acid sequence of a TAL effector dictates the nucleotide sequence to which it binds. Thus, target sites can be predicted for TAL effectors, and TAL effectors can be engineered and generated for the purpose of binding to particular nucleotide sequences.

[0151] Fused to the TAL effector-encoding nucleic acid sequences are sequences encoding a nuclease or a portion of a nuclease, typically a nonspecific cleavage domain from a type II restriction endonuclease such as FokI (Kim et al., 1996). Other useful endonucleases may include, for example, Hhal, Hindlll, Nod, BbvCI, EcoRI, Bgll, and Ahvl. The fact that some endonucleases (e.g., FokI) only function as dimers can be capitalized upon to enhance the target specificity of the TAL effector. For example, in some cases each FokI monomer can be fused to a TAL effector sequence that recognizes a different DNA target sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme. By requiring DNA binding to activate the nuclease, a highly site-specific restriction enzyme can be created.

[0152] Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Endonucleases include restriction endonucleases, which cleave DNA at specific siteswithout damaging the bases, and meganucleases, also known as homing endonucleases (HEases), which like restriction endonucleases, bind and cut at a specific recognition site, however the recognition sites for meganucleases are typically longer, about 18 bp or more (patent application PCT / US 12 / 30061, filed on Mar. 22, 2012). Meganucleases have been classified into four families based on conserved sequence motifs, the families are the LAGLID ADG, GIY-YIG, H-N-H, and His-Cys box families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganuclease is similar to the convention for other restriction endonuclease. Meganucleases are also characterized by prefix F-, I-, or Pl- for enzymes encoded by free-standing ORFs, introns, and inteins, respectively. One step in the recombination process involves polynucleotide cleavage at or near the recognition site. The cleaving activity can be used to produce a double-strand break. For reviews of site-specific recombinases and their recognition sites, see, Sauer (1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:760-7. In some examples the recombinase is from the Integrase or Resolvase families.

[0153] Zinc finger nucleases (ZFNs) are engineered double-strand break inducing agents comprised of a zinc finger DNA binding domain and a double-strand-break-inducing agent domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprising two, three, or four zinc fingers, for example having a C2H2 structure, however other zinc finger structures are known and have been engineered. Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence. ZFNs include an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain, for example nuclease domain from a Type Ils endonuclease such as Fokl. Additional functionalities can be fused to the zinc-finger binding domain, including transcriptional activator domains, transcription repressor domains, and methylases. In some examples, dimerization of nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3 finger domain recognized a sequence of 9 contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind an 18 nucleotide recognition sequence.

[0154] In certain embodiments, transposon tagging is used to decrease or eliminate the activity of the target S-type anion channel gene. Transposon tagging comprises inserting a transposon within an endogenous gene in the pathway to decrease or eliminate expression. In certain embodiments, the expression is decreased or eliminated by inserting a transposon within aregulatory region or coding region of the gene encoding the polypeptide. A transposon that is within an exon, intron, 5' or 3' untranslated sequence, a promoter or any other regulatory sequence of a gene may be used to decrease or eliminate the expression and / or activity of the encoded polypeptide.

[0155] Methods for the transposon tagging of specific genes in plants are well known in the art. See, e.g., Maes et al., (1999) Trends Plant Sci. 4:90-96; Dharmapuri & Sonti, (1999) FEMS Microbiol. Lett. 179:53-59; Meissner et al., (2000) Plant J. 22:265-74; Phogat et al., (2000) J. Biosci. 25:57-63; Walbot, (2000) Curr. Opin. Plant Biol. 2: 103-07; Gai et al., (2000) Nucleic Acids Res. 28:94-96; Fitzmaurice et al., (1999) Genetics 153: 1919-28. In addition, the TUSC process for selecting Mu insertions in selected genes has been described in Bensen et al., (1995) Plant Cell 7:75-84; Mena et al., (1996) Science 274: 1537-40; and U.S. Pat. No. 5,962,764, each of which is herein incorporated by reference.

[0156] Additional methods for reducing or eliminating the expression of endogenous genes in plants are also known in the art and may be similarly applied to the present disclosure. These methods include other forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines, in which the endogenous gene has been mutated or deleted. For examples of these methods see Ohshima et al., (1998) Virology 243:472- 81; Okubara et al., (1994) Genetics 137:867-74; and Quesada et al., (2000) Genetics 154:421- 36, each of which is herein incorporated by reference. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the present disclosure. See McCallum et al., (2000) Nat.BiotechnoL 18:455-57, herein incorporated by reference.

[0157] Mutations may impact gene expression or interfere with the activity of an encoded polypeptide. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues are particularly effective in inhibiting the activity of the encoded protein. Conserved residues of polypeptides suitable for mutagenesis with the goal to eliminate activity have been described. Such mutants may be isolated according to well-known procedures and mutations in different target gene loci may be stacked by genetic crossing. See, e.g., Gruis et al., (2002) Plant Cell 14:2863-82.

[0158] In certain embodiments, reducing the target S-type anion channel gene expression or activity comprises introducing into a plant or plant cell a silencing element, thereby reducing oreliminating the level or expression of a polynucleotide or a polypeptide encoded by the target gene.

[0159] As used herein, “silencing element” refers to a polynucleotide that is capable of reducing or eliminating the level or expression of a target polynucleotide or the polypeptide encoded thereby. The silencing element employed can reduce or eliminate the expression level of the target sequence by influencing the level of the target RNA transcript or, alternatively, by influencing translation and thereby affecting the level of the encoded polypeptide. A single polynucleotide employed in the methods can comprise one or more silencing elements to the same or different target polynucleotides. The silencing element can be produced in vivo (i.e., in a host cell such as a plant) or in vitro.

[0160] Non-limiting examples of silencing elements include, a sense suppression element, an antisense suppression element, a double stranded RNA, a siRNA, an amiRNA, a miRNA, or a hairpin suppression element. Non-limiting examples of silencing elements that can be employed to decrease expression of these target sequences or additionally sequences targeting genes involved in recombination comprise fragments and variants of the sense or antisense sequence or consists of the sense or antisense sequence of wild type polynucleotide or polypeptide sequences, variant polynucleotides, variant polypeptides, cognate promoter sequences, ortholog sequences, variants or fragments thereof. The silencing element can further comprise additional sequences that advantageously effect transcription and / or the stability of a resulting transcript. For example, the silencing elements can comprise at least one thymine residue at the 3' end. This can aid in stabilization. Thus, the silencing elements can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more thymine residues at the 3' end. Enhancer suppressor elements can also be employed in conjunction with the silencing elements.

[0161] In certain embodiments, introducing the silencing element reduces the polynucleotide level and / or the polypeptide level of the target sequence to less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the polynucleotide level, or the level of the polypeptide encoded thereby, of the same target sequence in an appropriate control.

[0162] In certain embodiments, decreasing expression of the target S-type anion channel gene (e.g., ZmSLAH2 / 3) is obtained by sense suppression or cosuppression. For cosuppression, an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding a polypeptide in the “sense” orientation. Over expression of the RNA molecule may result in decreased expression of the native gene. Accordingly, multiple plantlines transformed with the cosuppression expression cassette are screened to identify those that show the desired degree of inhibition of polypeptide expression.

[0163] Typically, a sense suppression element has substantial sequence identity to the target polynucleotide, typically greater than about 65% sequence identity, greater than about 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323; herein incorporated by reference. The sense suppression element can be any length so long as it allows for the suppression of the targeted sequence. In other implementations, the sense suppression element is, for example, 15, 16, 17, 18 19, 20, 22, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 900, 1000, 1100, 1200, 1300 nucleotides or longer of the target polynucleotides. In other implementations, the sense suppression element is, for example, about 15-25, 25-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800 nucleotides or longer of the target polynucleotides.

[0164] The polynucleotide used for cosuppression may correspond to all or part of the sequence encoding the polypeptide, all or part of the 5' and / or 3' untranslated region of a polypeptide transcript or all or part of both the coding sequence and the untranslated regions of a transcript encoding a polypeptide. In certain embodiments where the polynucleotide comprises all or part of the coding region for the polypeptide, the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be translated.

[0165] Cosuppression has been used to inhibit the expression of plant genes to produce plants having undetectable protein levels for the proteins encoded by these genes (see, e.g., Broin et al., (2002) Plant Cell 14: 1417-32), and to inhibit the expression of multiple proteins in the same plant (see, e.g., U.S. Pat. No. 5,942,657). Methods for using cosuppression to inhibit the expression of endogenous genes in plants are described in Flavell et al., (1994) Proc. Natl. Acad. Set. USA 91 :3490-96; Jorgensen et al., (1996) Plant Mol. Biol. 31 :957-73; Johansen & Carrington, (2QQV)Plant Physiol. 126:930-38; Broin et al., (2002) Plant Cell 14: 1417-32; Stoutj esdij k et al., (2002) Plant Physiol. 129: 1723-31; Yu et al., (2003) Phytochemistry 63: 753-63; and U.S. Pat. Nos. 5,034,323, 5,283,184, and 5,942,657, each of which is herein incorporated by reference. The efficiency of cosuppression may be increased by including a poly-dT region in the expression cassette at a position 3' to the sense sequence and 5' of the polyadenylation signal. See, US Patent Application Publication No. 2002 / 0048814, herein incorporated byreference. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, optimally greater than about 65% sequence identity, more optimally greater than about 85% sequence identity, most optimally greater than about 95% sequence identity. See U.S. Pat. Nos. 5,283,184 and 5,034,323, herein incorporated by reference.

[0166] In certain embodiments, decreasing expression of the target S-type anion channel gene (e.g., ZmSLAH2 / 3) is obtained by antisense suppression. For antisense suppression, the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the polypeptide. Over expression of the antisense RNA molecule may result in decreased expression of the target gene. Accordingly, multiple plant lines transformed with the antisense suppression expression cassette are screened to identify those that show the desired degree of inhibition of polypeptide expression.

[0167] The polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the polypeptide, all or part of the complement of the 5' and / or 3' untranslated region of the target transcript or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the polypeptide. In addition, the antisense polynucleotide may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence. In addition, the antisense suppression element may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target polynucleotide. In specific embodiments, the antisense suppression element comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence complementarity to the target polynucleotide. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Furthermore, the antisense suppression element can be complementary to a portion of the target polynucleotide. Generally, sequences of at least 15, 20, 22, 25, 50, 100, 200, 300, 400, 450 nucleotides or greater of the target gene sequence may be used. Methods for using antisense suppression to inhibit the expression of endogenous genes in plants are described, e.g., in Liu et al. (2002) Plant Physiol. 129: 1732-43 and U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is herein incorporated by reference. Efficiency of antisense suppression may be increased by including a poly-dT region in the expression cassette at a position 3' to theantisense sequence and 5' of the polyadenylation signal. See, US Patent Application Publication No. 2002 / 0048814, herein incorporated by reference.

[0168] In certain embodiments, decreasing the expression of the target S-type anion channel gene (e.g., ZmSLAH2 / 3) is obtained by double-stranded RNA (dsRNA) interference. A “double stranded RNA silencing element” or “dsRNA” comprises at least one transcript that is capable of forming a dsRNA. Thus, a “dsRNA silencing element” includes a dsRNA, a transcript or polyribonucleotide capable of forming a dsRNA or more than one transcript or polyribonucleotide capable of forming a dsRNA. “Double stranded RNA” or “dsRNA” refers to a polyribonucleotide structure formed either by a single self-complementary RNA molecule or a polyribonucleotide structure formed by the expression of least two distinct RNA strands. The dsRNA molecule(s) employed in the methods and compositions mediate the decrease of expression of a target sequence, for example, by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner. The dsRNA is capable of decreasing or eliminating the level or expression of a target polynucleotide or the polypeptide.

[0169] The dsRNA can decrease or eliminate the expression level of the target sequence by influencing the level of the target RNA transcript, by influencing translation and thereby affecting the level of the encoded polypeptide, or by influencing expression at the pre- transcriptional level (i.e., via the modulation of chromatin structure, methylation pattern, etc., to alter gene expression). See, e.g., Verdel et al., (2004) Science 303:672-76; Pal-Bhadra et al., (2004) Science 303:669-72; All shire (2002) Science 297: 1818-19; Volpe et al., (2002) Science 297: 1833-37; Jenuwein (2002) Science 297:2215-18; and Hall et al., (2002) Science 297:2232- 37. As used herein, the term “dsRNA” is meant to encompass other terms used to describe nucleic acid molecules that are capable of mediating RNA interference or gene silencing, including, e.g., short-interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), hairpin RNA, short hairpin RNA (shRNA), post-transcriptional gene silencing RNA (ptgsRNA), etc.

[0170] For dsRNA interference, a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.

[0171] Expression of the sense and antisense molecules may be accomplished by designing the expression cassette to comprise both a sense sequence and an antisense sequence. Alternatively, separate expression cassettes may be used for the sense and antisense sequences. Multiple plantlines transformed with the dsRNA interference expression cassette or expression cassettes are then screened to identify plant lines that show the desired degree of inhibition of polypeptide expression. Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in Waterhouse et al., (1998) Proc. Natl. Acad. Sci. USA 95: 13959-64, Liu et al., (2002) Plant Physiol. 129: 1732-43 and WO 1999 / 49029, WO 1999 / 53050, WO 1999 / 61631 and WO 2000 / 49035, each of which is herein incorporated by reference.

[0172] In certain embodiments, decreasing the expression of the target S-type anion channel gene (e.g., ZmSLAH2 / 3) is obtained by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference. These methods are highly efficient at inhibiting the expression of endogenous genes. See, Waterhouse & Helliwell, (2003) Nat. Rev. Genet. 4:29-38, and the references cited therein.

[0173] For hpRNA interference, the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem. The base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence. Alternatively, the base-paired stem region may correspond to a portion of a promoter sequence controlling expression of the gene whose expression is to be inhibited. Thus, the base-paired stem region of the molecule generally determines the specificity of the RNA interference. hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes and the RNA interference they induce is inherited by subsequent generations of plants. See, e.g., Chuang & Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-90;Stoutjesdijk et al., (2002) Plant Physiol. 129: 1723-31; and Waterhouse & Helliwell, (2003) Nat. Rev. Genet. 4:29-38. Methods for using hpRNA interference to inhibit or silence the expression of genes are described, e.g., in Chuang & Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-90; Stoutjesdijk et al., (2002) Plant Physiol. 129: 1723-31; Waterhouse &d Helliwell, (2003) Nat. Rev. Genet. 4:29-38; Pandolfini et al., BMC Biotechnology 3:7; and US Patent Application Publication No. 2003 / 0175965, each of which is herein incorporated by reference. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga et al., (2003) Mol. Biol. Rep. 30: 135-40, herein incorporated by reference.

[0174] For ihpRNA, the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron that is capable of being spliced in thecell in which the ihpRNA is expressed. The use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, e.g., Smith et al., (2000) Nature 407:319-20. In fact, Smith et al., show 100% suppression of endogenous gene expression using ihpRNA-mediated interference. Methods for using ihpRNA interference to inhibit the expression of endogenous plant genes are described, for example, in Smith et al., (2000) Nature 407:319-20; Wesley et al., (2001) Plant J. 27:581-90; Wang & Waterhouse, (2001) Curr. Opin. Plant Biol. 5: 146-50; Waterhouse & Helliwell, (2003) Nat. Rev. Genet. 4:29-38; Helliwell & Waterhouse, (2003) Methods 30:289-95; and US Patent Application Publication No. 2003 / 0180945, each of which is herein incorporated by reference.

[0175] Any region of the target polynucleotide can be used to design the domain of the silencing element that shares sufficient sequence identity to allow expression of the hairpin transcript to decrease the level of the target polynucleotide. For instance, the domain can be designed to share sequence identity to the 5' untranslated region of the target polynucleotide(s), the 3' untranslated region of the target polynucleotide(s), exonic regions of the target polynucleotide(s), intronic regions of the target polynucleotide(s), and any combination thereof. In specific embodiments, a domain of the silencing element shares sufficient homology to at least about 15, 16, 17, 18, 19, 20, 22, 25 or 30 consecutive nucleotides from about nucleotides 1-50, 25-75, 75-125, 50-100,125-175, 175-225, 100-150, 150-200, 200-250, 225-275, 275-325, 250-300, 325-375, 375-425,300-350, 350-400, 425-475, 400-450, 475-525, 450-500, 525-575, 575-625, 550-600, 625-675,675-725, 600-650, 625-675, 675-725, 650-700, 725-825, 825-875, 750-800, 875-925, 925-975,850-900, 925-975, 975-1025, 950-1000, 1000-1050, 1025-1075, 1075-1125, 1050-1100, 1125-1175, 1100-1200, 1175-1225, 1225-1275, 1200-1300, 1325-1375, 1375-1425, 1300-1400, 1425- 1475, 1475-1525, 1400-1500, 1525-1575, 1575-1625, 1625-1675, 1675-1725, 1725-1775, 1775- 1825, 1825-1875, 1875-1925, 1925-1975, 1975-2025, 2025-2075, 2075-2125, 2125-2175, 2175- 2225, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000 of the target sequence. In certain embodiments, to optimize the siRNA sequences employed in the hairpin, the synthetic oligodeoxyribonucleotide / RNAse H method can be used to determine sites on the target mRNA that are in a conformation that is susceptible to RNA silencing. See, e.g., Vickers et al., (2003) J.Biol. Chem. 278:7108-18 and Yang et al., (2002) Proc. Natl. Acad. Sci. USA 99:9442-47, herein incorporated by reference. These studies indicate that there is a significant correlation between the RNase-H-sensitive sites and sites that promote efficient siRNA-directed mRNA degradation.

[0176] The expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA. In thisembodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. Thus, it is the loop region that determines the specificity of the RNA interference. See, e.g., WO 2002 / 00904; Mette et al., (2000) EMBO J 19:5194-201; Matzke et al., (2001) Curr. Opin. Genet. Devel. 11 :221-27; Scheid et al., (2002) Proc. Natl. Acad. Sci. USA 99: 13659-62; Aufsaftz et al., (2002) Proc. Natl. Acad. Sci. 99: 16499-506; Sijen et al., Curr. Biol. (2001) 11 :436-40, herein incorporated by reference.

[0177] In addition, transcriptional gene silencing (TGS) may be accomplished through use of a hairpin suppression element where the inverted repeat of the hairpin shares sequence identity with the promoter region of a target polynucleotide to be silenced. See, for example, Aufsatz et al., (2002) Proc. Natl. Acad. Sci. 99: 16499-506 and Mette et al., (2000) EMBO J. 19:5194-201.

[0178] In certain embodiments, decreasing the expression of the target S-type anion channel gene (e.g., ZmSLAH2 / 3) is obtained by amplicon-mediated interference. Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus. The viral sequences present in the transcription product of the expression cassette allow the transcription product to direct its own replication. The transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e., the messenger RNA for the polypeptide). Methods of using amplicons to inhibit the expression of endogenous plant genes are described, for example, in Angell and Baulcombe, (1997) EMBO J. 16:3675-3684; Angell and Baulcombe, (1999) Plant 20:357-362; and U.S. Pat. No. 6,646,805, each of which is herein incorporated by reference.

[0179] In certain embodiments, the polynucleotide expressed in the plant is a catalytic RNA or has ribozyme activity specific for the messenger RNA of the polypeptide. Thus, the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in decreased expression of the polypeptide. This method is described, e.g., in U.S. Pat. No. 4,987,071, herein incorporated by reference.

[0180] In certain embodiments, decreasing the expression of the target S-type anion channel gene (e.g., ZmSLAH2 / 3) is obtained by RNA interference by expression of a gene encoding a micro RNA (miRNA) or short-interfering RNA (siRNA) (Meister & Tuschl (2004) Nature 431 :343-49 and Bonetta et al., (2004) Nature Methods 1 :79-86). miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNAs are highly efficient at inhibiting the expression of endogenous genes. See, e.g., Palatnik et al., (2003) Nature 425:257-63, herein incorporatedby reference. The miRNA can be an “artificial miRNA” or “amiRNA” which comprises a miRNA sequence that is synthetically designed to silence a target sequence.

[0181] For miRNA interference, the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene. For example, the miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence). In certain embodiments, the 22- nucleotide sequence is selected from a transcript sequence from the target gene (e.g., SnRKl and / or SnRK2) and contains 22 nucleotides of the target gene in sense orientation and 21 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence. In certain embodiments, in addition to targeting the target gene directly, genes involved in recombination may also be targeted. Accordingly, in certain embodiments, the 22-nucleotide sequence is selected from a transcript sequence from a gene involved in recombination and contains 22 nucleotides of the gene involved in recombination in sense orientation and 21 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence. miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants.

[0182] The heterologous polynucleotide being expressed need not form the dsRNA by itself, but can interact with other sequences in the plant cell to allow the formation of the dsRNA. For example, a chimeric polynucleotide that can selectively silence the target polynucleotide can be generated by expressing a chimeric construct comprising the target sequence for a miRNA or siRNA to a sequence corresponding to all or part of the gene or genes to be silenced. In this embodiment, the dsRNA is “formed” when the target for the miRNA or siRNA interacts with the miRNA present in the cell. The resulting dsRNA can then decrease the level of expression of the gene or genes to be silenced. See, for example, US Application Publication 2007-0130653, entitled “Methods and Compositions for Gene Silencing”, herein incorporated by reference. The construct can be designed to have a target for an endogenous miRNA or, alternatively, a target for a heterologous and / or synthetic miRNA can be employed in the construct. If a heterologous and / or synthetic miRNA is employed, it can be introduced into the cell on the same nucleotide construct as the chimeric polynucleotide or on a separate construct. As discussed elsewhere herein, any method can be used to introduce the construct comprising the heterologous miRNA.Expression Cassettes

[0183] As used herein, the term “expression cassette” refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence. S-typeanion channel polynucleotides as described herein can be provided in an expression cassette. It is further recognized that various expression cassettes other than S-type anion channel polynucleotides constructs are also described herein. For example, expression cassette encoding an RNA-guided endonuclease, other genome editing molecules, or a silencing element are described herein. One of skill in the art will understand how to apply the disclosure to any expression cassette.

[0184] Expression cassettes generally include regulatory elements that are functional in the intended host cell in which the expression cassette is to be expressed. Thus, a person of ordinary skill in the art can select regulatory elements for use in bacterial host cells, yeast host cells, plant host cells, insect host cells, mammalian host cells, and human host cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.

[0185] An expression cassette can comprise a promoter sequence operably linked to a polynucleotide sequence as described herein. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression cassette as described herein. In certain embodiments, a promoter can be positioned about the same distance from the transcription start site in the expression cassette as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression cassette.

[0186] A “promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence) that is operably associated with the promoter. The coding sequence controlled or regulated by a promoter may encode a polypeptide and / or a functional RNA. Typically, a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. In general, promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence. A promoter may comprise other elements that act as regulators of gene expression; e.g., a promoter region. These include a TATA box consensus sequence, and often a CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50:349). In plants, the CAAT box may be substituted by the AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211- 227).

[0187] Promoters useful with this disclosure can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and / or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules. These various types of promoters are known in the art.

[0188] The choice of promoter may vary depending on the temporal and spatial requirements for expression, and also may vary based on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms and such knowledge can be readily accessed and implemented in other systems as appropriate.

[0189] In certain embodiments, a promoter functional in a plant may be used with the constructs of this disclosure. Non-limiting examples of a promoter useful for driving expression in a plant include the promoter of the RubisCo small subunit gene 1 (PrbcSl), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdcal) (See, Walker et al. Plant Cell Rep. 23:727-735 (2005); Li et al. Gene 403: 132-142 (2007); Li et al. Mol Biol. Rep. 37: 1143-1154 (2010)). PrbcSl and Pactin are constitutive promoters and Pnr and Pdcal are inducible promoters. Pnr is induced by nitrate and repressed by ammonium (Li et al. Gene 403: 132-142 (2007)) and Pdcal is induced by salt (Li et al. Mol Biol. Rep. 37: 1143-1154 (2010)). In certain embodiments, a promoter useful with this disclosure is RNA polymerase II (Pol II) promoter. In certain embodiments, a U6 promoter or a 7SL promoter from Zea mays may be useful with constructs of this disclosure. In certain embodiments, the U6c promoter and / or 7SL promoter from Zea mays may be useful for driving expression of a guide nucleic acid. In certain embodiments, a U6c promoter, U6i promoter and / or 7SL promoter from Glycine max may be useful with constructs of this disclosure. In certain embodiments, the U6c promoter, U6i promoter and / or 7SL promoter from Glycine max may be useful for driving expression of a guide nucleic acid.

[0190] Examples of constitutive promoters useful for plants include, but are not limited to, cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well as U.S. Pat. No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA 84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:6624-6629), sucrose synthasepromoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148), and the ubiquitin promoter. The constitutive promoter derived from ubiquitin accumulates in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al., 1991. Plant Science 79: 87-94), maize (Christensen et al., 1989. Plant Molec. Biol. 12: 619-632), and Arabidopsis (Norris et al. 1993. Plant Molec. Biol. 21 :895-906). The maize ubiquitin promoter (UbiP) has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926. The ubiquitin promoter is suitable for the expression of the nucleotide sequences of the disclosure in transgenic plants, especially monocotyledons. Further, the promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231 : 150-160 (1991)) can be easily modified for the expression of the nucleotide sequences of the disclosure and are particularly suitable for use in monocotyledonous hosts.

[0191] In certain embodiments, tissue specific / tissue preferred promoters can be used for expression of a heterologous polynucleotide in a plant cell. Tissue specific or preferred expression patterns include, but are not limited to, green tissue specific or preferred, root specific or preferred, stem specific or preferred, flower specific or preferred or pollen specific or preferred. Promoters suitable for expression in green tissue include many that regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. In one embodiment, a promoter useful with the disclosure is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:579-589 (1989)). Non-limiting examples of tissue-specific promoters include those associated with genes encoding the seed storage proteins (such as P-conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g., Kridl et al. (1991) Seed Sci. Res. 1:209-219; as well as EP Patent No. 255378). Tissue-specific or tissue-preferential promoters useful for the expression of the nucleotide sequences of the disclosure in plants, particularly maize, include but are not limited to those that direct expression in root, pith, leaf or pollen. Such promoters are disclosed, for example, in WO 93 / 07278, herein incorporated by reference in its entirety. Other non-limiting examples of tissue specific or tissue preferred promoters useful with the disclosure the cotton rubisco promoter disclosed in U.S. Pat. No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Pat. No. 5,604,121; the root specific promoter described by de Framond (FEBS 290: 103-106 (1991);EP 0 452 269 to Ciba-Geigy); the stem specific promoter described in U.S. Pat. No. 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene; the cestrum yellow leaf curling virus promoter disclosed in WO 01 / 73087; and pollen specific or preferred promoters including, but not limited to, ProOsLPSlO and ProOsLPSl 1 from rice (Nguyen et al. Plant Biotechnol. Reports 9(5):297-306 (2015)), ZmSTK2_USP from maize (Wang et al. Genome 60(6):485-495 (2017)), LAT52 and LAT59 from tomato (Twell et al. Development 109(3):705- 713 (1990)), Zml3 (U.S. Pat. No. 10,421,972), PLA2-8 promoter from arabidopsis (U.S. Pat. No. 7,141,424), and / or the ZmC5 promoter from maize (International PCT Publication No. WO1999 / 042587.

[0192] Additional examples of plant tissue-specific / tissue preferred promoters include, but are not limited to, the root hair-specific cis-elements (RHEs) (Kim et al. The Plant Cell 18:2958- 2970 (2006)), the root-specific promoters RCc3 (Jeong et al. Plant Physiol. 153: 185-197 (2010)) and RB7 (U.S. Pat. No. 5,459,252), the lectin promoter (Lindstrom et al. (1990) Der. Genet.11 : 160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), S adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8): 1108- 1115), corn light harvesting complex promoter (Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658), corn heat shock protein promoter (O'Dell et al. (1985) EMBO J. 5:451-458; and Rochester et al. (1986) EA7BO J. 5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore, “Nuclear genes encoding the small subunit of ribulose- 1,5-bisphosphate carboxylase” pp. 29-39 In: Genetic Engineering of Plants (Hollaender ed., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205: 193-200), Ti plasmid mannopine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid nopaline synthase promoter (Langridge et al. (1989), supra), petunia chaicone isomerase promoter (van Tunen et al. (1988) EA7BO J. 7: 1257-1263), bean glycine rich protein 1 promoter (Keller et al. (1989) Genes Dev. 3: 1639-1646), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature 313:810-812), potato patatin promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347-354), root cell promoter (Yamamoto et al. 99 Nucleic Acids Res. 18:7449), maize zein promoter (Kriz et al. (1987) Mol. Gen. Genet. 207:90-98; Langridge et al. (1983) Cell 34: 1015-1022; Reina et al. ( \ 99Q) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic Acids Res. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872), a-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet. 215:431-440), PEPCase promoter (Hudspeth & Grula (1989) Plant Mol. Biol.12:579-589), R gene complex-associated promoters (Chandler et al. (1989) Plant Cell 1 : 1175- 1183), and chaicone synthase promoters (Franken et al. (1991) EMBO J. 10:2605-2612).

[0193] Useful for seed-specific expression is the pea vicilin promoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; as well as the seed-specific promoters disclosed in U.S. Pat. No. 5,625,136. Useful promoters for expression in mature leaves are those that are switched at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al. (1995) Science 270: 1986-1988).

[0194] In addition, promoters functional in chloroplasts can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5' UTR and other promoters disclosed in U.S. Pat. No. 7,579,516. Other promoters useful with the disclosure include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0195] Additional regulatory elements useful with this disclosure include, but are not limited to, introns, enhancers, termination sequences and / or 5' and 3' untranslated regions.

[0196] An intron useful with this disclosure can be an intron identified in and isolated from a plant and then inserted into an expression cassette to be used in transformation of a plant. As would be understood by those of skill in the art, introns can comprise the sequences required for self-excision and are incorporated into nucleic acid constructs / expression cassettes in frame. An intron can be used either as a spacer to separate multiple protein-coding sequences in one nucleic acid construct, or an intron can be used inside one protein-coding sequence to, for example, stabilize the mRNA. If they are used within a protein-coding sequence, they are inserted “in-frame” with the excision sites included. Introns may also be associated with promoters to improve or modify expression.

[0197] Non-limiting examples of introns useful with the present disclosure include introns from the ADHI gene (e.g., Adhl-S introns 1, 2 and 6), the ubiquitin gene (Ubil), the RuBisCO small subunit (rbcS) gene, the RuBisCO large subunit (rbcL) gene, the actin gene (e.g., actin- 1 intron), the pyruvate dehydrogenase kinase gene (pdk), the nitrate reductase gene (nr), the duplicated carbonic anhydrase gene 1 (Tdcal), the psbA gene, the atpA gene, or any combination thereof.

[0198] An expression cassette can optionally include a transcriptional and / or translational termination region (i.e., termination region) and / or an enhancer region that is functional in the selected host cell. A variety of transcriptional terminators and enhancers are known in the art and are available for use in expression cassettes. Transcriptional terminators are responsible for the termination of transcription and correct mRNA polyadenylation. A termination region and / orthe enhancer region may be native to the transcriptional initiation region, may be native to, for example, a gene encoding a sequence-specific nucleic acid binding protein, a gene encoding a nuclease, a gene encoding a reverse transcriptase, a gene encoding a deaminase, and the like, or may be native to a host cell, or may be native to another source (e.g., foreign or heterologous to, for example, to a promoter, to a gene encoding a sequence-specific nucleic acid binding protein, a gene encoding a nuclease, a gene encoding a deaminase, and the like, or to the host cell, or any combination thereof).

[0199] An expression cassette of the disclosure also can include a polynucleotide encoding a selectable marker, which can be used to select a transformed host cell. As used herein, “selectable marker” means a polynucleotide sequence that when expressed imparts a distinct phenotype to the host cell expressing the marker and thus allows such transformed cells to be distinguished from those that do not have the marker. Such a polynucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic and the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., fluorescence). Many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0200] Examples of selectable markers include, but are not limited to, a nucleotide sequence encoding neo or nptll, which confers resistance to kanamycin, G418, and the like (Potrykus et al. (1985) Mol. Gen. Genet. 199: 183-188); a nucleotide sequence encoding bar, which confers resistance to phosphinothricin; a nucleotide sequence encoding an altered 5- enolpyruvylshikimate-3-phosphate (EPSP) synthase, which confers resistance to glyphosate (Hinchee et al. (1988) Biotech. 6:915-922); a nucleotide sequence encoding a nitrilase such as bxn from Klebsiella ozaenae that confers resistance to bromoxynil (Stalker et al. (1988) Science 242:419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP Patent Application No. 154204); a nucleotide sequence encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. 263: 12500-12508); a nucleotide sequence encoding a dalapon dehalogenase that confers resistance to dalapon; a nucleotide sequence encoding a mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI)) that confers an ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5- methyl tryptophan; and / or a nucleotide sequence encoding hph that confers resistance tohygromycin. One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette.

[0201] Additional selectable markers include, but are not limited to, a nucleotide sequence encoding P-glucuronidase or uidA (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus nucleotide sequence that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., “Molecular cloning of the maize R-nj allele by transposon-tagging with Ac,” pp. 263-282 In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); a nucleotide sequence encoding P- lactamase, an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737-3741); a nucleotide sequence encoding xylE that encodes a catechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA 80: 1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding P-galactosidase, an enzyme for which there are chromogenic substrates; a nucleotide sequence encoding luciferase (lux) that allows for bioluminescence detection (Ow et al. (1986) Science 234:856-859); a nucleotide sequence encoding aequorin, which may be employed in calcium-sensitive bioluminescence detection (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126: 1259-1268); or a nucleotide sequence encoding green fluorescent protein (Niedz et al. (1995) Plant Cell Reports 14:403-406). One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette.

[0202] In addition to expression cassettes, the nucleic acid molecules / constructs and polynucleotide sequences described herein can be used in connection with vectors. The term “vector” refers to a composition for transferring, delivering or introducing a nucleic acid (or nucleic acids) into a cell. A vector comprises a nucleic acid construct (e.g., expression cassette(s)) comprising the nucleotide sequence(s) to be transferred, delivered or introduced. Vectors for use in transformation of host organisms are well known in the art. Non-limiting examples of general classes of vectors include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, bacteriophages, artificial chromosomes, mini circles, or Agrobacterium binary vectors in double or single stranded linear or circular form which may or may not be self-transmissible or mobilizable. In certain embodiments, a viral vector can include, but is not limited, to a retroviral, lentiviral, adenoviral, adeno-associated, orherpes simplex viral vector. A vector as defined herein can transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). Additionally included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g., higher plant, mammalian, yeast or fungal cells). In certain embodiments, the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter and / or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter and / or other regulatory elements for expression in the host cell. Accordingly, a nucleic acid or polynucleotide of this disclosure and / or expression cassettes comprising the same may be comprised in vectors as described herein and as known in the art.

[0203] In certain embodiments, the polynucleotide and / or recombinant nucleic acid construct of this disclosure (e.g., expression cassettes and / or vectors) may be codon optimized to remove features inimical to expression and codon usage is optimized for expression in the particular crop (see, for example, U.S. Pat. No. 6,051,760; EP 0359472; EP 80385962; EP 0431829; and Perlak et al. (1991) PNAS USA 88:3324-3328; all of which are herein incorporated by reference). In certain embodiments, the codon optimized nucleic acids, polynucleotides, expression cassettes, and / or vectors of the disclosure have about 70% to about 99.9% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%. 99.9% or 100%) identity or more to the reference nucleic acids, polynucleotides, expression cassettes, and / or vectors that have not been codon optimized.Transformation Methods

[0204] Several embodiments relate to plant cells, plant tissues, plants, and seeds that comprise a recombinant DNA (e.g., an S-type anion channel polynucleotide, a genome editing molecule, a silencing element) as described herein.

[0205] Suitable methods for transformation of host plant cells include virtually any method by which DNA or RNA can be introduced into a cell (for example, where a recombinant DNA construct is stably integrated into a plant chromosome or where a recombinant DNA construct oran RNA is transiently provided to a plant cell) and are well known in the art. Two effective methods for cell transformation are ^grotocterzwm-mediated transformation and microprojectile bombardment-mediated transformation. Microprojectile bombardment methods are illustrated, for example, in U.S. Pat. Nos. 5,550,318; 5,538,880; 6,160,208; and 6,399,861. Agrobacterium- mediated transformation methods are described, for example in U.S. Pat. No. 5,591,616, which is incorporated herein by reference in its entirety. Transformation of plant material is practiced in tissue culture on nutrient media, for example a mixture of nutrients that allow cells to grow in vitro. Recipient cell targets include, but are not limited to, meristem cells, shoot tips, hypocotyls, calli, immature or mature embryos, and gametic cells such as microspores and pollen. Callus can be initiated from tissue sources including, but not limited to, immature or mature embryos, hypocotyls, seedling apical meristems, microspores and the like. Cells containing a transgenic nucleus are grown into transgenic plants.

[0206] In transformation, DNA is typically introduced into only a small percentage of target plant cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a recombinant DNA molecule into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or an herbicide. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells are those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells can be tested further to confirm stable integration of the exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin (nptll), hygromycin B (aph IV), spectinomycin (aadA) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat), dicamba (DM0) and glyphosate (aroA or EPSPS). Examples of such selectable markers are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047. Markers which provide an ability to visually screen transformants can also be employed, for example, a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.

[0207] Transformation of a cell may be stable or transient. Thus, in certain embodiments, a plant cell is stably transformed with a nucleic acid molecule. In other embodiments, a plant is transiently transformed with a nucleic acid molecule. “Transient transformation” in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrateinto the genome of the cell. By “stably introducing” or “stably introduced” in the context of a polynucleotide introduced into a cell is intended the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.

[0208] “ Stable transformation” or “stably transformed” as used herein means that a nucleic acid is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. “Genome” as used herein also includes the nuclear and the plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast genome. Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome.

[0209] Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a plant). Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a plant or other organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0210] In certain embodiments, transformation of a cell comprises nuclear transformation. In other embodiments, transformation of a cell comprises plastid transformation (e.g., chloroplast transformation).

[0211] Procedures for transforming plants are well known and routine in the art and are described throughout the literature. Non-limiting examples of methods for transformation of plants include transformation via bacterial-mediated nucleic acid delivery (e.g., via Agrobacteria), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker- mediated nucleic acid delivery, liposome mediated nucleic acid delivery, microinjection,microparticle bombardment, calcium-phosphate-mediated transformation, cyclodextrin- mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, as well as any other electrical, chemical, physical (mechanical) and / or biological mechanism that results in the introduction of nucleic acid into the plant cell, including any combination thereof. General guides to various plant transformation methods known in the art include Miki et al. (“Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0212] Agrobacterium-mediated transformation is a commonly used method for transforming plants, in particular, dicot plants, because of its high efficiency of transformation and because of its broad utility with many different species. Agrobacterium-mediated transformation typically involves transfer of the binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain that may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (Uknes et al. (1993) Plant Cell 5: 159-169). The transfer of the recombinant binary vector to Agrobacterium can be accomplished by a triparental mating procedure using Escherichia coli carrying the recombinant binary vector, a helper E. coli strain that carries a plasmid that is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation (Hbfgen & Willmitzer (1988) Nucleic Acids Res. 16:9877).

[0213] Transformation of a plant by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissue is regenerated on selection medium carrying an antibiotic or herbicide resistance marker between the binary plasmid T-DNA borders.

[0214] Another method for transforming plants, plant parts and / or plant cells involves propelling inert or biologically active particles at plant tissues and cells. See, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006 and 5,100,792. Generally, this method involves propelling inert or biologically active particles at the plant cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the nucleic acid of interest. Alternatively, a cell or cells can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically activeparticles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing one or more nucleic acids sought to be introduced) also can be propelled into plant tissue.

[0215] Thus, in particular embodiments, a plant cell can be transformed by any method known in the art and as described herein and intact plants can be regenerated from these transformed cells using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue culture and / or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983)); and Vasil I. R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)). Methods of selecting for transformed transgenic plants, plant cells and / or plant tissue culture are routine in the art and can be employed in the methods provided herein.

[0216] Likewise, the genetic properties engineered into the transgenic seeds and plants, plant parts, and / or plant cells described above can be passed on by sexual reproduction or vegetative growth and therefore can be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as harvesting, sowing or tilling.

[0217] A nucleotide sequence therefore can be introduced into the plant, plant part and / or plant cell in any number of ways that are well known in the art. The methods do not depend on a particular method for introducing one or more nucleotide sequences into a plant, only that they gain access to the interior of at least one cell of the plant. Where more than one nucleotide sequence is to be introduced, they can be assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, the nucleotide sequences can be introduced into the cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol.Plants with Modified Root Architecture

[0218] Several embodiments relate to plants, and plant cells, plant tissues, and seeds thereof, having modified root architecture. In certain embodiments, a plant of the present disclosure comprises modified root architecture, wherein the modified root architecture is characterized by increased root biomass, steeper root angle, increased lateral root branching, longer roots, or a combination thereof. In certain embodiments, the plant having modified root architecture exhibits improved yield traits and / or or increased tolerance to abiotic stress, optionally wherein the abiotic stress may be drought stress or limited nitrogen.

[0219] In certain embodiments, a plant of the present disclosure comprises increased root biomass. In certain embodiments, an increase in root biomass may refer to an increase of about 5% to 150% in root biomass (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 125, 130, 135, 140, 145, or 150%, or any range or value therein) as compared to a control plant.

[0220] In certain embodiments, a plant of the present disclosure comprises a steeper root angle. In certain embodiments, a steeper root angle may refer to root angle is more vertical by at least 5 degrees with a maximum of 90 degrees (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 degrees, or any value or range therein) as compared to a control plant.

[0221] In certain embodiments, a plant of the present disclosure comprises an increase in lateral root branching. In certain embodiments, an increase in lateral root branching may refer to an increase of about 30% to about 200% increase in lateral root branching (e.g., about 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200%, or any range or value therein) as compared to a control plant.

[0222] In certain embodiments, a plant of the present disclosure comprises longer roots. In certain embodiments, longer roots may refer to roots having an increase in length by about 15% to about 100% (e.g., about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any range or value therein) as compared to a control plant.

[0223] As used herein, “improved yield traits” refers to any plant trait associated with growth, for example, biomass, yield, nitrogen use efficiency (NUE), inflorescence size / weight, fruit yield, fruit quality, fruit size, seed size, seed number, foliar tissue weight, nodulation number,nodulation mass, nodulation activity, number of seed heads, number of tillers, number of branches, number of flowers, number of tubers, tuber mass, bulb mass, number of seeds, total seed mass, rate of leaf emergence, rate of tiller / branch emergence, rate of seedling emergence, or modified root architecture, including but not limited to length of roots, number of roots, size or weight of root mass (root biomass), steeper root angle, or increased lateral root branching, or any combination thereof. In certain embodiments, “improved yield traits” may include, but is not limited to, increased tolerance to abiotic stress, optionally wherein the abiotic stress is drought stress or limited nitrogen, increased inflorescence production, increased fruit production (e.g., increased number, weight and / or size of fruit; e.g., increase number, weight, and / or size of ears for, e.g., maize), increased fruit quality, increased number, size and / or weight of roots, increased meristem size, increased seed size, increased biomass, increased leaf size, increased nitrogen use efficiency, increased height, increased internode number and / or increased internode length as compared to a control plant or part thereof. Improved yield traits can also result from increased planting density of plants of the disclosure. Thus, in certain embodiments, a plant of the disclosure is capable of being planted at an increased density (as a consequence of altered plant architecture resulting from the mutation), which results in improved yield traits as compared to a control plant that is planted at the same density.

[0224] As used herein, “drought stress” refers to a period of dryness (acute or chronic / prolonged) that results in water deficit and subjects plants to stress and / or damage to plant tissues and / or negatively affects grain / crop yield. As used herein “increased drought tolerance” refers to the ability of plants to grow, develop, or yield normally, or grow, develop, or yield faster or better than normal when subjected to reduced amounts of available / applied water and / or under conditions of acute or chronic drought.

[0225] As used herein the “increased nitrogen stress tolerance” refers to the ability of plants to grow, develop, or yield normally, or grow, develop, or yield faster or better when subjected to less than optimal amounts of available / applied nitrogen, or under nitrogen limiting conditions.

[0226] Increased plant nitrogen use efficiency can be translated in the field into either harvesting similar quantities of yield, while supplying less nitrogen, or increased yield gained by supplying optimal / sufficient amounts of nitrogen. The increased nitrogen use efficiency can improve plant nitrogen stress tolerance and can also improve crop quality and biochemical constituents of the seed such as protein yield and oil yield. The terms “increased nitrogen use efficiency”, “enhanced nitrogen use efficiency”, and “nitrogen stress tolerance” are used inter-changeably inthe present disclosure to refer to plants with improved productivity under nitrogen limiting conditions.

[0227] Plants of the disclosure may be monocots or dicots, and may include, for example, rice, wheat, barley, oats, rye, sorghum, maize, grape, tomato, potato, lettuce, broccoli, cucumber, peanut, melon, pepper, carrot, squash, onion, soybean, alfalfa, sunflower, cotton, canola, and sugar beet plants.

[0228] Plants that are useful in the methods of the present disclosure include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa [canola, oilseed rape, turnip rape]), Cadabafarinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandr a, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Cor chorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus car ota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Er agrostis tef Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgar e), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luff a acutangula, Lupinus spp., Luzula sylvatica, Ly coper sicon spp. (e.g. Ly coper sicon esculentum, Lycopersicon lycopersicum, Ly coper sicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza lalifolia , Panicummiliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Per sea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia ver a, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odor ata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugar cane, sunflower, tomato, squash, tea and algae, amongst others. In certain embodiments, the plant is a crop plant. Examples of crop plants include inter alia maize, soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato, or tobacco. In certain embodiments, the plant is a maize plant.

[0229] Certain embodiments encompass a progeny or a descendant of a plant with modified root architecture as well as seeds derived from the plants with modified root architecture and cells derived from the plants with modified root architecture as described herein.

[0230] In certain embodiments, plant cells of the present disclosure are capable of regenerating a plant or plant part. In certain embodiments, plant cells are not capable of regenerating a plant or plant part. Examples of cells not capable of regenerating a plant include, but are not limited to, endosperm, seed coat (testa and pericarp), and root cap.

[0231] Several embodiments provide a commodity plant product prepared from the plants with modified root architecture. In certain embodiments, examples of plant products include, without limitation, grain, oil, and meal. In certain embodiments, a commodity plant product is plant grain (e.g., grain suitable for use as feed or for processing), plant oil (e.g., oil suitable for use as food or biodiesel), or plant meal (e.g., meal suitable for use as feed). A preferred commodity plant product is fodder, seed meal, oil, or seed-treatment-coated seeds.

[0232] The product may be produced at the site where the plant has been grown, the plants and / or parts thereof may be removed from the site where the plants have been grown to producethe product. Typically, the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant. The step of growing the plant may be performed only once each time the method is performed, while allowing repeated times the steps of product production e.g. by repeated removal of harvestable parts of the plants of the disclosure and if necessary further processing of these parts to arrive at the product. It is also possible that the step of growing the plants is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts. Also, the steps of growing the plants and producing the product may be performed with an overlap in time, even simultaneously to a large extend or sequentially. Generally, the plants are grown for some time before the product is produced.

[0233] In certain embodiments, the maize plant is a non-naturally occurring variety of maize. In certain embodiments, the maize plant is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identical to that of an elite variety of maize.

[0234] The plants of the disclosure may be used in a plant breeding program. The goal of plant breeding is to combine, in a single variety or hybrid, various desirable traits. For field crops, these traits may include, for example, resistance to diseases and insects, tolerance to heat and drought, tolerance to chilling or freezing, reduced time to crop maturity, greater yield and better agronomic quality. With mechanical harvesting of many crops, uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and plant height is desirable. Traditional plant breeding is an important tool in developing new and improved commercial crops. This disclosure encompasses methods for producing a plant by crossing a first parent plant with a second parent plant wherein one or both of the parent plants is a plant displaying a phenotype as described herein.

[0235] Plant breeding techniques known in the art and used in a plant breeding program include, but are not limited to, recurrent selection, bulk selection, mass selection, backcrossing, pedigree breeding, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, doubled haploids and transformation. Often combinations of these techniques are used.

[0236] The development of hybrids in a plant breeding program requires, in general, the development of homozygous inbred lines, the crossing of these lines and the evaluation of the crosses. There are many analytical methods available to evaluate the result of a cross. The oldest and most traditional method of analysis is the observation of phenotypic traits. Alternatively, the genotype of a plant can be examined.

[0237] A genetic trait which has been engineered into a particular plant using transformation techniques can be moved into another line using traditional breeding techniques that are well known in the plant breeding arts. For example, a backcrossing approach is commonly used to move a transgene from a transformed plant to an elite inbred line and the resulting progeny would then comprise the transgene(s). Also, if an inbred line was used for the transformation, then the transgenic plants could be crossed to a different inbred in order to produce a transgenic hybrid plant. As used herein, "crossing" can refer to a simple X by Y cross or the process of backcrossing, depending on the context.

[0238] The development of a hybrid in a plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, while different from each other, breed true and are highly homozygous and (3) crossing the selected inbred lines with different inbred lines to produce the hybrids. During the inbreeding process, the vigor of the lines decreases. Vigor is restored when two different inbred lines are crossed to produce the hybrid. An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid created by crossing a defined pair of inbreds will always be the same. Once the inbreds that give a superior hybrid have been identified, the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parents is maintained.

[0239] Plants of the present disclosure may be used to produce, e.g., a single cross hybrid, a three-way hybrid or a double cross hybrid. A single cross hybrid is produced when two inbred lines are crossed to produce the Fl progeny. A double cross hybrid is produced from four inbred lines crossed in pairs (A x B and C x D) and then the two Fl hybrids are crossed again (A x B) times (C x D). A three-way cross hybrid is produced from three inbred lines where two of the inbred lines are crossed (A x B) and then the resulting Fl hybrid is crossed with the third inbred (A x B) x C. Much of the hybrid vigor and uniformity exhibited by Fl hybrids is lost in the next generation (F2). Consequently, seed produced by hybrids is consumed rather than planted.Genetic Mapping

[0240] It has been recognized for quite some time that specific genetic loci correlating with particular traits can be mapped in an organism’s genome. The plant breeder can advantageously use molecular markers to identify desired individuals by detecting marker alleles that show a statistically significant probability of co- segregation with a desired phenotype, manifested aslinkage disequilibrium. By identifying a molecular marker or clusters of molecular markers that co-segregate with a trait of interest, the breeder is able to rapidly select a desired phenotype by selecting for the proper molecular marker allele (a process called marker-assisted selection).

[0241] A variety of methods well known in the art are available for detecting molecular markers or clusters of molecular markers that co-segregate with a trait of interest, such as modified root architecture in maize. The basic idea underlying these methods is the detection of markers, for which alternative genotypes (or alleles) have significantly different average phenotypes. Thus, one makes a comparison among marker loci of the magnitude of difference among alternative genotypes (or alleles) or the level of significance of that difference. Trait genes are inferred to be located nearest the marker(s) that have the greatest associated genotypic difference. Two such methods used to detect trait loci of interest are: 1) Population-based association analysis and 2) Traditional linkage analysis.

[0242] In a population-based association analysis, lines are obtained from pre-existing populations with multiple founders, e.g. elite breeding lines. Population-based association analyses rely on linkage disequilibrium (LD) and the idea that in an unstructured population, only correlations between genes controlling a trait of interest and markers closely linked to those genes will remain after so many generations of random mating. In reality, most pre-existing populations have population substructure. Thus, the use of a structured association approach helps to control population structure by allocating individuals to populations using data obtained from markers randomly distributed across the genome, thereby minimizing disequilibrium due to population structure within the individual populations (also called subpopulations). The phenotypic values are compared to the genotypes (alleles) at each marker locus for each line in the subpopulation. A significant marker-trait association indicates the close proximity between the marker locus and one or more genetic loci that are involved in the expression of that trait.

[0243] The same principles underlie traditional linkage analysis; however, linkage disequilibrium is generated by creating a population from a small number of founders. The founders are selected to maximize the level of polymorphism within the constructed population, and polymorphic sites are assessed for their level of co-segregation with a given phenotype. A number of statistical methods have been used to identify significant marker-trait associations. One such method is an interval mapping approach (Lander and Botstein, Genetics 121 : 185-199 (1989), in which each of many positions along a genetic map (say at 1 cM intervals) is tested for the likelihood that a gene controlling a trait of interest is located at that position. The genotype / phenotype data are used to calculate for each test position a LOD score (log oflikelihood ratio). When the LOD score exceeds a threshold value, there is significant evidence for the location of a gene controlling the trait of interest at that position on the genetic map (which will fall between two particular marker loci).Markers and Linkage Relationships

[0244] A common measure of linkage is the frequency with which traits co-segregate. This can be expressed as a percentage of co-segregation (recombination frequency) or in centiMorgans (cM). The cM is a unit of measure of genetic recombination frequency. One cM is equal to a 1% chance that a trait at one genetic locus will be separated from a trait at another locus due to crossing over in a single generation (meaning the traits segregate together 99% of the time). Because chromosomal distance is approximately proportional to the frequency of crossing over events between traits, there is an approximate physical distance that correlates with recombination frequency.

[0245] Marker loci are themselves traits and can be assessed according to standard linkage analysis by tracking the marker loci during segregation. Thus, one cM is equal to a 1% chance that a marker locus will be separated from another locus, due to crossing over in a single generation.

[0246] The closer a marker is to a gene controlling a trait of interest, the more effective and advantageous that marker is as an indicator for the desired trait. Closely linked loci display an inter-locus cross-over frequency of about 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci (e.g., a marker locus and a target locus) display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or less apart. Put another way, two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be “proximal to” each other.

[0247] Although particular marker alleles can co-segregate with modified root architecture, it is important to note that the marker locus is not necessarily responsible for the expression of the phenotype. For example, it is not a requirement that the marker polynucleotide sequence be partof a gene that is responsible for the phenotype (for example, is part of the gene open reading frame). The association between a specific marker allele and a trait is due to the original “coupling” linkage phase between the marker allele and the allele in the ancestral maize line from which the allele originated. Eventually, with repeated recombination, crossing over events between the marker and genetic locus can change this orientation. For this reason, the favorable marker allele may change depending on the linkage phase that exists within the parent having the favorable trait that is used to create segregating populations. This does not change the fact that the marker can be used to monitor segregation of the phenotype. It only changes which marker allele is considered favorable in a given segregating population.

[0248] Methods presented herein include detecting the presence of one or more marker alleles associated with modified root architecture in a plant and then identifying or selecting plants that have favorable alleles at those marker loci. Markers associated with modified root architecture in maize can be used to identify and select maize plants having modified root architecture. Any marker within 20 cM, 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.9 cM, 0.8 cM, 0.7 cM, 0.6 cM, 0.5 cM, 0.4 cM, 0.3 cM, 0.2 cM, 0.1 cM or less of the endogenous S-type anion channel gene or any of the markers identified herein could also be used to identify and select maize plants with modified root architecture. Any marker allele linked to and associated with the favorable alleles of the markers can be used for detection purposes in the identification and / or selection of plants with modified root architecture.

[0249] The markers can be used for detecting the presence of an allele associated with modified root architecture in a maize plant or germplasm, and can therefore be used in methods involving marker-assisted breeding and selection of, for example, maize plants exhibiting increased tolerance to drought stress or limited nitrogen having an allele associated with modified root architecture.Marker Assisted Selection

[0250] Molecular markers can be used in a variety of plant breeding applications (e.g. see Staub et al. (1996) Hortscience 31 : 729-741; Tanksley (1983) Plant Molecular Biology Reporter. 1 : 3- 8). One of the main areas of interest is to increase the efficiency of backcrossing and introgressing genes using marker-assisted selection. A molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay. Since DNA marker assays are less laborious and take up less physical space than fieldphenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Having flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed. The ideal situation is to have a marker in the gene itself, so that recombination cannot occur between the marker and the gene. Such a marker is called a ‘perfect marker’ .

[0251] When a gene is introgressed by marker assisted selection, it is not only the gene that is introduced but also the flanking regions (Gepts. (2002). Crop Sci; 42: 1780-1790). This is referred to as “linkage drag.” In the case where the donor plant is highly unrelated to the recipient plant, these flanking regions carry additional genes that may code for agronomically undesirable traits. This “linkage drag” may also result in reduced yield or other negative agronomic characteristics even after multiple cycles of backcrossing into the elite maize line. This is also sometimes referred to as “yield drag.” The size of the flanking region can be decreased by additional backcrossing, although this is not always successful, as breeders do not have control over the size of the region or the recombination breakpoints (Young et al. (1998) Genetics 120:579-585). In classical breeding it is usually only by chance that recombination events are selected that contribute to a reduction in the size of the donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264). Even after 20 backcrosses in backcrosses of this type, one may expect to find a sizeable piece of the donor chromosome still linked to the gene being selected. With markers however, it is possible to select those rare individuals that have experienced recombination near the gene of interest. In 150 backcross plants, there is a 95% chance that at least one plant will have experienced a crossover within 1 cM of the gene. Markers will allow unequivocal identification of those individuals. With one additional backcross of 300 plants, there would be a 95% chance of a crossover within 1 cM of the other side of the gene, generating a segment around the target gene of less than 2 cM. This can be accomplished in two generations with markers, while it would have required on average 100 generations without markers (See Tanksley et al., supra). When the exact location of a gene is known, flanking markers surrounding the gene can be utilized to select for recombination events in different population sizes. For example, in smaller population sizes, recombination events may be expected further away from the gene, so more distal flanking markers would be required to detect the recombination.

[0252] The key components to the implementation of marker assisted selection are: (i) defining the population within which the marker-trait association will be determined, which can be a segregating population, or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers relative to the trait, and determining linkage or association using statistical methods; (iii) defining a set of desirable markers based on the results of the statistical analysis, and (iv) the use and / or extrapolation of this information to the current set of breeding germ plasm to enable marker-based selection decisions to be made. The markers described in this disclosure, as well as other marker types such as SSRs and FLPs, can be used in marker assisted selection protocols.

[0253] SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17: 6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88: 1-6). Polymorphisms arise due to variation in the number of repeat units, probably caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol 4: 203-221). The variation in repeat length may be detected by designing PCR primers to the conserved non-repetitive flanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396). SSRs are highly suited to mapping and marker assisted selection as they are multi- allelic, codominant, reproducible and amenable to high throughput automation (Rafalski et al. (1996) Generating and using DNA markers in plants. In: Non-mammalian genomic analysis: a practical guide. Academic press, pp 75-135).

[0254] Various types of SSR markers can be generated, and SSR profiles can be obtained by gel electrophoresis of the amplification products. Scoring of marker genotype is based on the size of the amplified fragment. Various types of FLP markers can also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are in many ways similar to SSR markers, except that the region amplified by the primers is not typically a highly repetitive region. Still, the amplified region, or amplicon, will have sufficient variability among germplasm, often due to insertions or deletions, such that the fragments generated by the amplification primers can be distinguished among polymorphic individuals.

[0255] SNP markers detect single base pair nucleotide substitutions. Of all the molecular marker types, SNPs are the most abundant, thus having the potential to provide the highest genetic map resolution (PLos One (2013). 8 (11): e79192). SNPs can be assayed at an even higher level of throughput than SSRs, in a so-called ‘ultra-high-throughput’ fashion, as they do not require large amounts of DNA and automation of the assay may be straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highlyattractive for use in marker assisted selection. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, mini sequencing and coded spheres. Such methods have been reviewed in: Gut (2001) Hum Mutat 17 pp. 475-492; Shi (2001) Clin Chem 47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp. 95-100; and Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: R. J. Henry, Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs including Masscode™ (Qiagen), INVADER®. (Third Wave Technologies) and Invader PLUS®, SNAPSHOT®. (Applied Biosystems), TAQMAN®. (Applied Biosystems) and BEADARRAYS®. (Illumina).

[0256] A number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype for any particular genotype (Ching et al. (2002), BMC Genet. 3: 19 pp Gupta et al. 2001, Rafalski (2002b), Plant Science 162:329-333). Haplotypes can be more informative than single SNPs and can be more descriptive of any particular genotype. Once a unique haplotype has been assigned to a donor chromosomal region, that haplotype can be used in that population or any subset thereof to determine whether an individual has a particular gene. See, for example, W02003054229. Using automated high throughput marker detection platforms known to those of ordinary skill in the art makes this process highly efficient and effective.

[0257] In addition to SSRs, FLPs and SNPs, as described above, other types of molecular markers are also widely used, including but not limited to expressed sequence tags (ESTs), SSR markers derived from EST sequences, randomly amplified polymorphic DNA (RAPD), and other nucleic acid-based markers.

[0258] Isozyme profiles and linked morphological characteristics can, in some cases, also be indirectly used as markers. Even though they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are far more numerous and polymorphic than isozyme or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter 1 :3-8).

[0259] Sequence alignments or contigs may also be used to find sequences upstream or downstream of the specific markers listed herein. These new sequences, close to the markers described herein, are then used to discover and develop functionally equivalent markers. For example, different physical and / or genetic maps are aligned to locate equivalent markers notdescribed within this disclosure but that are within similar regions. These maps may be within the maize species, or even across other species that have been genetically or physically aligned with maize, such as soybean, rice, wheat, or barley.

[0260] In general, marker assisted selection uses polymorphic markers that have been identified as having a significant likelihood of co-segregation with a phenotype, such as modified root architecture in maize. Such markers map near the gene that confers modified root architecture in a maize plant, and are considered indicators for the desired trait, or markers. Plants are tested for the presence of a desired allele in the marker, and plants containing a desired genotype at one or more loci are expected to transfer the desired genotype, along with a desired phenotype, to their progeny. Thus, maize plants with modified root architecture can be selected for by detecting one or more marker alleles, and in addition, progeny plants derived from those plants can also be selected. Hence, a plant containing a desired genotype in a given chromosomal region is obtained and then crossed to another plant. The progeny of such a cross would then be evaluated genotypically using one or more markers and the progeny plants with the same genotype in a given chromosomal region would then be selected as exhibiting modified root architecture.

[0261] The SNPs identified herein could be used alone or in combination (i.e. a SNP haplotype) to select for plants having a favorable QTL allele (i.e. associated with modified root architecture). The skilled artisan would expect that there might be additional polymorphic sites at marker loci in and around the markers identified herein, wherein one or more polymorphic sites is in linkage disequilibrium with an allele at one or more of the polymorphic sites in the haplotype and thus could be used in a marker assisted selection program to introgress a QTL allele of interest. Two particular alleles at different polymorphic sites are said to be in linkage disequilibrium if the presence of the allele at one of the sites tends to predict the presence of the allele at the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)).

[0262] The skilled artisan would understand that allelic frequency (and hence, haplotype frequency) can differ from one germplasm pool to another. Germplasm pools vary due to maturity differences, heterotic groupings, geographical distribution, etc. As a result, SNPs and other polymorphisms may not be informative in some germplasm pools.Embodiments

[0263] The following numbered embodiments also form part of the present disclosure:

[0264] 1. A plant, or a progeny, a plant part, or a plant cell thereof, having modified root architecture, the plant comprising one or more mutations in an endogenous S-type anion channel gene.

[0265] 2. The plant of embodiment 1, wherein the endogenous S-type anion channel gene encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3 or 6.

[0266] 3 The plant of embodiment 1 or embodiment 2, wherein the endogenous S-type anion channel gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5.

[0267] 4. The plant of any one of embodiments 1-3, wherein the one or more mutations reduce or eliminate expression of the endogenous S-type anion channel gene.

[0268] 5. The plant of any one of embodiments 1-4, wherein one or more mutations are present within the coding region, non-coding region, regulatory sequence, or untranslated region of the endogenous S-type anion channel gene.

[0269] 6. The plant of any one of embodiments 1-5, wherein the one or more mutations reduce expression of the endogenous S-type anion channel gene, reduce transcriptional activity of the polypeptide encoded by the endogenous S-type anion channel gene, generate one or more alternative spliced variants of the endogenous S-type anion channel gene, introduce a frameshift mutation in one or more exons of the endogenous S-type anion channel gene, delete a substantial portion of the endogenous S-type anion channel gene, delete a full-length open reading frame of the endogenous S-type anion channel gene, repress an enhancer motif present within a regulatory region encoding the endogenous S-type anion channel gene, modify one or more nucleotides of a regulatory element operably linked to the endogenous S-type anion channel gene, or any combination thereof.

[0270] 7. The plant of any one of embodiments 1-6, wherein the plant comprises in its genome an introgressed genetic locus comprising the one or more mutations in the endogenous S-type anion channel gene.

[0271] 8. The plant of any one of embodiments 1-7, wherein the introgressed genetic locus is derived from the UFmu-01876 maize mutant line.

[0272] 9. The plant of any one of embodiments 1-8, wherein the plant is a maize plant.

[0273] 10. The plant of any one of embodiments 1-9, wherein the plant comprises elite germplasm.

[0274] 11. The plant of any one of embodiments 1-10, wherein the modified root architecture comprises increased root biomass, steeper root angle, increased lateral root branching, longer roots, or a combination thereof.

[0275] 12. The plant of any one of embodiments 1-11, wherein the plant exhibits increased tolerance to drought stress or limited nitrogen relative to a control plant without the one or more mutations in the endogenous S-type anion channel gene.

[0276] 13. A seed or an asexual propagate of the plant of any one of embodiments 1-12.

[0277] 14. A method for modifying the root architecture of a plant, the method comprising: introducing one or more mutations in an endogenous S-type anion channel gene of the plant and / or reducing expression or activity of the endogenous S-type anion channel gene.

[0278] 15. The method of embodiment 14, wherein the endogenous S-type anion channel gene encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3 or 6.

[0279] 16. The method of embodiment 14 of embodiment 15, wherein the endogenous S-type anion channel gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5.

[0280] 17. The method of any one of embodiments 14-16, wherein the one or more mutations are introduced through targeted DNA modification.

[0281] 18. The method of any one of embodiments 14-17, wherein the one or more mutations are introduced through use of an RNA-guided endonuclease and a guide RNA.

[0282] 19. The method of any one of embodiments 14-18, wherein the one or more mutations are introduced within the coding region, non-coding region, regulatory sequence, or untranslated region of the endogenous S-type anion channel gene.

[0283] 20. The method of any one of embodiments 14-19, wherein the one or more mutations reduce expression of the endogenous S-type anion channel gene, reduce transcriptional activity of the polypeptide encoded by the endogenous S-type anion channel gene, generate one or more alternative spliced variants of the endogenous S-type anion channel gene, introduce a frameshift mutation in one or more exons of the endogenous S-type anion channel gene, delete a substantial portion of the endogenous S-type anion channel gene, delete a full-length open reading frame of the endogenous S-type anion channel gene, repress an enhancer motif present within a regulatory region encoding the endogenous S-type anion channel gene, modify one or more nucleotides of a regulatory element operably linked to the endogenous S-type anion channel gene, or any combination thereof.

[0284] 21. The method of any one of embodiments 14-20, wherein the plant is a maize plant.

[0285] 22. The method of any one of embodiments 14-21, wherein the modified root architecture comprises increased root biomass, steeper root angle, increased lateral root branching, longer roots, or a combination thereof.

[0286] 23. The method of any one of embodiments 14-22, wherein the plant exhibits increased tolerance to drought stress or limited nitrogen relative to a control plant without the one or more mutations in the endogenous S-type anion channel gene.

[0287] 24. A method of producing a plant having modified root architecture, the method comprising: (a) crossing the plant of any one of embodiments 1-12 with itself or another plant to produce seed; and (b) growing a progeny plant from the seed to produce a plant having modified root architecture.

[0288] 25. The method of embodiment 24, further comprising: (c) crossing the progeny plant with itself or another plant; and (d) repeating steps (b) and (c) for an additional 0-7 generations to produce a plant having modified root architecture.

[0289] 26. A crop comprising a plurality of the plants of any one of embodiments 1-12 planted together in an agricultural field.

[0290] 27. A method of producing seeds from a crop, the method comprising:

[0291] cultivating a plurality of the plants of any one of embodiments 1-12 as a crop, and harvesting seeds from the crop, wherein the plants have modified root architecture relative to a corresponding control plant without the mutation.

[0292] 28. A commodity plant product prepared from the plant or plant part of any one of embodiments 1-12.

[0293] 29. The commodity plant product of embodiment 28, wherein the commodity plant product comprises the one or more mutations in an endogenous S-type anion channel gene.

[0294] 30. The commodity plant product of embodiment 28 or embodiment 29, wherein the product is fodder, seed meal, oil, or seed-treatment-coated seed.

[0295] 31. A method for producing a commodity plant product, the method comprising processing the plant or plant part of any one of embodiments 1-12 to obtain the product.

[0296] 32. The method of embodiment 31, wherein the commodity plant product is fodder, seed meal, oil, or seed-treatment-coated seeds.

[0297] 33. A method of introgressing a modified root architecture locus into a plant, the method comprising: providing a first plant with the modified root architecture locus, wherein the locus comprises a one or more mutations in an endogenous S-type anion channel gene; providing asecond plant; crossing the first plant with the second plant to produce a population of progeny plants; selecting from the population at least one plant having the modified root architecture locus.

[0298] 34. The method of embodiment 33, wherein the endogenous S-type anion channel gene encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3 or 6.

[0299] 35. The method of embodiment 33 or embodiment 34, wherein the endogenous S-type anion channel gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5.

[0300] 36. The method of any one of embodiments 33-35, further comprising performing backcrosses and selections.

[0301] 37. The method of any one of embodiments 33-36, wherein the introgressing comprises marker assisted selection.

[0302] 38. The method of any one of embodiments 33-37, wherein the plant is a maize plant.

[0303] 39. The method of any one of embodiments 33-39, wherein the first plant is the UFmu- 01876 maize mutant line, or a progeny thereof.

[0304] 40. A method of identifying or selecting a plant comprising a modified root architecture locus, the method comprising: genotyping at least one plant for the presence of one or more mutations in an endogenous S-type anion channel gene or a marker in linkage disequilibrium therewith.

[0305] 41. The method of embodiment 40, wherein the endogenous S-type anion channel gene encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3 or 6.

[0306] 42. The method of embodiment 40 or embodiment 41, wherein the endogenous S-type anion channel gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5.

[0307] 43. The method of any one of embodiments 40-42, wherein the marker is located within 10 cM, 5 cM, 1 cM, or 0.5 cM of the endogenous S-type anion channel gene.

[0308] 44. The method of any one of embodiments 40-43, wherein the at least one plant from a population generated by a cross.

[0309] 45. The method of any one of embodiments 40-44, further comprising selecting from the population at least one plant comprising the modified root architecture based on the genotyping.

[0310] 46. The method of any one of embodiments 40-45, further comprising the step of assaying the selected plant for modified root architecture.

[0311] 47. The method of any one of embodiments 40-46, wherein the modified root architecture comprises increased root biomass, steeper root angle, increased lateral root branching, longer roots, or a combination thereof.

[0312] 48. The method of any one of embodiments 40-47, further comprising the step of crossing the plant identified with a second plant to produce a population of progeny plants.

[0313] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0314] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

[0315] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1: Maize SLAH2 / 3 gene regulating root system growth in the field

[0316] In this study, it was posited that the alternative states of the ZmSLAH2 / 3 gene lead to varying signals of nutrient deprivation and supplementation, which would subsequently influence distinct root growth patterns in field-grown maize. The goal of this study was to provide functional evidence for ZmSLAH2 / 3 as a gene regulator of root system size and nitrogen capture in field-grown maize.Materials and MethodsPlant materials

[0317] Seed of sib-pollinated F3 plants for the UFmu-01876 line, from the UniformMu transposon insertion population, was obtained from the Maize Genetics Cooperation Stock Center (MGCSC). Plants were backcrossed to the W22 wild type (MGCSC stock number X17EA), and resulting plants were selfed to develop a BCF3 population of lines segregating the insertion mul014502.Characterization of insertion location

[0318] Genomic DNA was extracted from lyophilized leaf tissue of individual plants across generations to determine the presence and insertion locations of Mu elements within the ZmSLAH2 / 3 gene. PCR was conducted using gene-specific primers (5’-GTCTCTATACCCCTGCGTGC-3’; SEQ ID NO: 7) and (5’-AGATCTTGGAGTCCGGGGAT- 3’; SEQ ID NO: 8), in combination with the Mu terminal inverted repeat specific primer TIR6. Sanger sequencing of the amplicons was performed to determine location of the insertion.Field experiment

[0319] Maize mutant and wild-type allele lines were evaluated at the Colorado State University Agricultural Research Development and Education Center in Fort Collins, CO, USA (40.653 N, -104.993 W) over two consecutive years (2023-2024). Each year, the seeds were sown in midMay at a 22-cm spacing in replicated single row, 6.1 -meter plots with a row spacing of 76 cm. Throughout the growing season, the plots received approximately 2.5 cm of weekly irrigation and were regularly hand-weeded.

[0320] A preliminary field trial was conducted in the summer of 2023 to evaluate the effects of genotype on maize RSA under normal conditions. The field was fertilized according to recommendations for a targeted yield of 200 bu / ac, amounting to 170 Ib / ac N prior to planting. Plants were harvested and phenotyped during the mid-flowering stage, approximately 14-weeks after planting.

[0321] A replicated nitrogen stress experiment was conducted in the summer of 2024 with three field replicates of mutant and wild-type genotypes per treatment (high N and low N). Plots in the high N treatment were fertilized at the same rate as in the previous year, receiving 170 Ib / ac N prior to planting. For the low N treatment, fertilizer application was withheld for the duration of the experiment. Plants were harvested and phenotyped during the mid-flowering stage, approximately 12-weeks after planting.Mutant Phenotyping

[0322] Intact whole-root systems of mutant and wild-type maize genotypes were extracted from the soil using a combination of nondestructive excavation methods. In the 2023 field study, a custom-built specialized root pulling device was affixed to a high-clearance tractor was used. This mechanical pulling device used a camera to identify maize stalks and fastened on to a stalk by closing a gripper around it. Subsequently, a motor vertically extracted the plants from the soil. A load cell connected to the motor measured the vertical force applied as a function of the vertical displacement of the gripper. The maximum force recorded during root extraction wasdenoted as the RPF (Root Pulling Force), akin to RPF measurements with hand-held force gauges. Concurrently, additional roots were extracted manually by shoveling. Following excavation, root crowns collected from both studies were hand-washed with water and air-dried by hanging. After air-drying to a constant weight, shoot and root biomass were recorded in grams.

[0323] Image-based RSA phenotyping was performed on excavated maize root crowns to obtain a comprehensive dataset of 154 RSA measurements. In both years, two-dimensional (2D) root imaging was conducted by vertically suspending the root samples in front of a black backdrop and capturing images using a DSLR camera. These images were processed and analyzed for RSA features using 2D DIRT image analysis software. In the following field season, a 3D root imaging and analysis pipeline using XRT and RCAP was introduced to analyze RSA variation in mutant and wild-type maize lines.

[0324] Nitrogen elemental analysis was performed to determine the effect of genotype and environmental nitrogen on leaf nitrogen allocation across individuals during mid-flowering in the 2024 experiment. Leaf samples were collected from three representative plants in each plot. A tissue punch was used to obtain 10 individual A-inch disks from the topmost fully expanded leaf of each plant. These leaf samples were placed in 2-ml microcentrifuge tubes and transported on ice, where they were immediately lyophilized for 48 h. Following lyophilization, the samples were pulverized, and the nitrogen concentration was measured using an elemental analyzer (LECO TruSpec CN).Statistical analysis

[0325] Statistical analyses were performed using R version 4.3.0. Phenotypic data were collected from genotyped individuals across homozygous wild-type and mutant genotypes, two nitrogen treatments (high N and low N), and two field seasons (2023 and 2024). For each year, trait outliers were identified and excluded based on visual comparison of the mean and median values within each treatment / genotype combination. In the 2023 field study, average genotype values were estimated across replicates. Statistical comparisons among average genotype values were performed using two-sample t-tests at a significance threshold of 0.05 in R / car. In the 2024 experiment, a type III two-way ANOVA was performed to test the effects of genotype, treatment, and their interactions on average trait values using a significance threshold of 0.05 in R / car. The data structure was assessed for normality using diagnostic plots to ensure that it met the ANOVA assumptions before testing. For each model containing significant fixed effects,post hoc Tukey’s tests were performed to assess the significance of differences among the genotypes at each treatment level.ResultsMu transposon induces loss-of-function mutation in maize SLACl-like domain

[0326] Based on field-based measurements of root pulling force (RPF), the gene ZmOOOOl eb 159490 was previously identified as a candidate for regulating root system size. This RPF-associated polymorphism was a synonymous polymorphism (C / T) located within an exon of the v2 (chr3:217,665,275) and v5 (chr3:223, 191,889) B73 gene models. Further evaluation of this gene candidate was conducted using the W22 genetic background (Zm00004b019600), which differs from the B73 reference. This candidate gene was one of 14 W22 maize paralogs and showed protein sequence similarities to Arabidopsis [Arabidopsis thaliana (L.) Heynh.] genes AtSLAH2 and AtSLAH3, at 57.54% and 60.91%, respectively. Protein sequence analysis predicted that this region in maize is a highly conserved Tellurite-resistance / Dicarboxylate Transporter (TDT) SLACl-like domain (residues 256-558; cd09323) within the SLAC / SLAH protein family (residues 173-584; IPR030183

[0327] A maize mutant line (UFmu-01876) using a Mu transposon insertional mutation in Zm00004b019600 was obtained to generate a population of maize mutant and wild-type lines homozygous for ZmSLAH2 / 3. This population was used to study the effects of the insertion on root development and its biological function in relation to its Arabidopsis homologs under field conditions. The presence of the insertion was confirmed by PCR and subsequently verified by Sanger sequencing. The size and position of the insertion were found to be approximately 3,000 bp in length, located within the first exon of the v2 W22 genome (chr3:226,299,935 - chr3:226,299,965; FIG. 1) and within the second exon of the v5 B73 genome (chr3:223,191,719 - chr3:223, 191,749). Genotyping and selection of homozygous ZmSLAH2 / 3 lines were performed in each generation until field testing. Measurements of RPF at flowering demonstrated that the mutant line exhibited significantly higher RPF than the wild-type line (P<0.05, FIG. 2).ZmSLAH2 / 3 affects whole-plant-level phenotypic plasticity in response to environmental nitrogen

[0328] The phenotypes of maize homozygous mutant and wild-type allele lines were evaluated in a replicated nitrogen stress experiment under field conditions. Variance analysis revealed significant genotype effects on leaf nitrogen content (p < 0.05) and a nearly significant genotype-by-environment (G x E) interaction [F(3, 68) = 3.42, / ? = 0.069], In the low Ntreatment, wild-type plants had a 7.61% higher leaf nitrogen content than mutant plants (FIG. 3A). Additionally, significant genotype effects were observed for both root and shoot biomass across nitrogen treatments (p < 0.05). On average, wild-type plants exhibited approximately 25.2% greater root biomass and 21.3% greater shoot biomass than mutant plants across treatments (FIG. 3C-D). However, both genotypes showed a non-significant reduction in root- mass-ratio (RMR) when shifting from high to low N conditions (FIG. 3B). Tukey’s post hoc multiple comparisons confirmed that the genotypes differed significantly for root and shoot biomass traits (p < 0.05). These results suggest that ZmSLAH2 / 3 contributes to systemic differences in biomass accumulation and nitrogen capture, particularly under low N conditions.

[0329] Measurements of RS A in wild-type and mutant plants revealed contrasting root growth patterns under high and low N conditions (FIG. 4A). Mutant plants had higher global RSA trait values than wild-type plants under high N conditions. These results aligned with preliminary data from the previous field season, in which mutant plants showed higher average RPF (mean = 137 kg, SD = 31 kg) than wild-type plants (mean = 101 kg, SD = 32.3 kg) in the same environment. This variation was reflected in the 2D and 3D measurements of the RSA, where the mutant plants under high N conditions exhibited on average increased root width and number of root tips compared to the wild-type plants (TABLE 1). A significant genotype effect (p < 0.05) was detected for 2D root tip count, but this finding was not corroborated by 3D imaging data. The limited sample size and unequal genotype representation in the preliminary study likely confounded these results. Phenotypic variation in RSA between genotypes was larger in the low N treatment compared with the high N treatment. Mutant plants demonstrated a consistent reduction in standardized average trait values (Z-scores) from high to low N conditions, whereas wild-type plants displayed a consistent increase in trait values, with less variability across treatments (FIG. 4B). Although variance analyses did not identify statistically significant differences in genotype or G x E effects (p > 0.05), they were indicative of root system plasticity in response to nitrogen stress.

[0330] TABLE 1. Genotypic differences in root system architecture (RS A) traits betweenZmSLAH2 / 3 maize mutant and wild-type lines grown under normal conditions in the 2023 preliminary field trial.References

[0331] McCarty DR, Settles AM, Suzuki M, Tan BC, S Latshaw, et al. (2005) Steady-state transposon mutagenesis in inbred maize. Plant J 44: 52-61.

[0332] Settles AM, Latshaw S, McCarty DR (2004) Molecular analysis of high-copy insertion sites in maize. Nucleic Acids Res 32: e54.

[0333] Woods P, Lehner KR, Hein K, Mullen JL, McKay JK (2022) Root pulling force across drought in maize reveals genotype by environment interactions and candidate genes. Frontiers in Plant Science 13:883209.

Claims

What is claimed is:

1. A plant, or a progeny, a plant part, or a plant cell thereof, having modified root architecture, the plant comprising one or more mutations in an endogenous S-type anion channel gene.

2. The plant of claim 1, wherein the endogenous S-type anion channel gene encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3 or 6.

3. The plant of claim 1, wherein the endogenous S-type anion channel gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5.

4. The plant of claim 1, wherein the one or more mutations reduce or eliminate expression of the endogenous S-type anion channel gene.

5. The plant of claim 1, wherein one or more mutations are present within the coding region, non-coding region, regulatory sequence, or untranslated region of the endogenous S-type anion channel gene.

6. The plant of claim 1, wherein the one or more mutations reduce expression of the endogenous S-type anion channel gene, reduce transcriptional activity of the polypeptide encoded by the endogenous S-type anion channel gene, generate one or more alternative spliced variants of the endogenous S-type anion channel gene, introduce a frameshift mutation in one or more exons of the endogenous S-type anion channel gene, delete a substantial portion of the endogenous S-type anion channel gene, delete a full-length open reading frame of the endogenous S-type anion channel gene, repress an enhancer motif present within a regulatory region encoding the endogenous S-type anion channel gene, modify one or more nucleotides of a regulatory element operably linked to the endogenous S-type anion channel gene, or any combination thereof.

7. The plant of claim 1, wherein the plant comprises in its genome an introgressed genetic locus comprising the one or more mutations in the endogenous S-type anion channel gene.

8. The plant of claim 7, wherein the introgressed genetic locus is derived from the UFmu- 01876 maize mutant line.

9. The plant of claim 1, wherein the plant is a maize plant.

10. The plant of claim 1, wherein the plant comprises elite germplasm.

11. The plant of claim 1, wherein the modified root architecture comprises increased root biomass, steeper root angle, increased lateral root branching, longer roots, or a combination thereof.

12. The plant of claim 1, wherein the plant exhibits increased tolerance to drought stress or limited nitrogen relative to a control plant without the one or more mutations in the endogenous S-type anion channel gene.

13. A seed or an asexual propagate of the plant of claim 1.

14. A method for modifying the root architecture of a plant, the method comprising: introducing one or more mutations in an endogenous S-type anion channel gene of the plant and / or reducing expression or activity of the endogenous S-type anion channel gene.

15. The method of claim 14, wherein the endogenous S-type anion channel gene encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3 or 6.

16. The method of claim 14, wherein the endogenous S-type anion channel gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5.

17. The method of claim 14, wherein the one or more mutations are introduced through targeted DNA modification.

18. The method of claim 14, wherein the one or more mutations are introduced through use of an RNA-guided endonuclease and a guide RNA.

19. The method of claim 14, wherein the one or more mutations are introduced within the coding region, non-coding region, regulatory sequence, or untranslated region of the endogenous S-type anion channel gene.

20. The method of claim 14, wherein the one or more mutations reduce expression of the endogenous S-type anion channel gene, reduce transcriptional activity of the polypeptide encoded by the endogenous S-type anion channel gene, generate one or more alternative spliced variants of the endogenous S-type anion channel gene, introduce a frameshift mutation in one or more exons of the endogenous S-type anion channel gene, delete a substantial portion of the endogenous S-type anion channel gene, delete a full-length open reading frame of the endogenous S-type anion channel gene, repress an enhancer motif present within a regulatory region encoding the endogenous S-type anion channel gene, modify one or more nucleotides of a regulatory element operably linked to the endogenous S-type anion channel gene, or any combination thereof.

21. The method of claim 14, wherein the plant is a maize plant.

22. The method of claim 14, wherein the modified root architecture comprises increased root biomass, steeper root angle, increased lateral root branching, longer roots, or a combination thereof.

23. The method of claim 14, wherein the plant exhibits increased tolerance to drought stress or limited nitrogen relative to a control plant without the one or more mutations in the endogenous S-type anion channel gene.

24. A method of producing a plant having modified root architecture, the method comprising:(a) crossing the plant of claim 1 with itself or another plant to produce seed; and(b) growing a progeny plant from the seed to produce a plant having modified root architecture.

25. The method of claim 24, further comprising:(c) crossing the progeny plant with itself or another plant; and(d) repeating steps (b) and (c) for an additional 0-7 generations to produce a plant having modified root architecture.

26. A crop comprising a plurality of the plants of claim 1 planted together in an agricultural field.

27. A method of producing seeds from a crop, the method comprising: cultivating a plurality of the plants of claim 1 as a crop, and harvesting seeds from the crop, wherein the plants have modified root architecture relative to a corresponding control plant without the mutation.

28. A commodity plant product prepared from the plant or plant part of claim 1.

29. The commodity plant product of claim 28, wherein the commodity plant product comprises the one or more mutations in an endogenous S-type anion channel gene.

30. The commodity plant product of claim 28, wherein the product is fodder, seed meal, oil, or seed-treatment-coated seed.

31. A method for producing a commodity plant product, the method comprising processing the plant or plant part of claim 1 to obtain the product.

32. The method of claim 31, wherein the commodity plant product is fodder, seed meal, oil, or seed-treatment-coated seeds.

33. A method of introgressing a modified root architecture locus into a plant, the method comprising: providing a first plant with the modified root architecture locus, wherein the locus comprises a one or more mutations in an endogenous S-type anion channel gene; providing a second plant; crossing the first plant with the second plant to produce a population of progeny plants; selecting from the population at least one plant having the modified root architecture locus.

34. The method of claim 33, wherein the endogenous S-type anion channel gene encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3 or 6.

35. The method of claim 33, wherein the endogenous S-type anion channel gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5.

36. The method of claim 33, further comprising performing backcrosses and selections.

37. The method of claim 33, wherein the introgressing comprises marker assisted selection.

38. The method of claim 33, wherein the plant is a maize plant.

39. The method of claim 33, wherein the first plant is the UFmu-01876 maize mutant line, or a progeny thereof.

40. A method of identifying or selecting a plant comprising a modified root architecture locus, the method comprising: genotyping at least one plant for the presence of one or more mutations in an endogenous S-type anion channel gene or a marker in linkage disequilibrium therewith.

41. The method of claim 40, wherein the endogenous S-type anion channel gene encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3 or 6.

42. The method of claim 40, wherein the endogenous S-type anion channel gene comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5.

43. The method of claim 40, wherein the marker is located within 10 cM, 5 cM, 1 cM, or 0.5 cM of the endogenous S-type anion channel gene.

44. The method of claim 40, wherein the at least one plant from a population generated by a cross.

45. The method of claim 40, further comprising selecting from the population at least one plant comprising the modified root architecture based on the genotyping.

46. The method of claim 40, further comprising the step of assaying the selected plant for modified root architecture.

47. The method of claim 40, wherein the modified root architecture comprises increased root biomass, steeper root angle, increased lateral root branching, longer roots, or a combination thereof.

48. The method of claim 40, further comprising the step of crossing the plant identified with a second plant to produce a population of progeny plants.

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