Aux / IAA degron mutation conferring tolerance to synthetic auxin herbicides

Abstract

The present disclosure relates to plants comprising a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region that confers to the plant tolerance to synthetic auxin herbicides, such as dicamba.

Description

Agent Ref. No. P14663WOOOTITLE: AUX / 1AA DEGRON MUTATION CONFERRING TOLERANCE TOSYNTHETIC AUXIN HERBICIDESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional applications U.S. Serial No. 63 / 623,574, filed January 22, 2024, and U.S. Senal No. 63 / 706,865, filed October 14, 2024, which are incorporated herein by reference in their entireties.SEQUENCE LISTING XML

[0002] The instant application contains a sequence listing, which has been submitted in XML file format by electronic submission and is hereby incorporated by reference in its entirety. The XML file, created on January 21, 2025, is named P14663WOOO.xml and is 58,695 bytes in size.TECHNICAL FIELD

[0003] The present disclosure relates in general to compositions and methods for conferring plants with tolerance to herbicides.BACKGROUND

[0004] Weeds left uncontrolled can decrease the yields of several major crops by more than 50% in present North American agronomic systems. Many growers in the United States currently rely heavily on chemical means (i.e.. herbicides) to control their weed populations, but the effectiveness of this approach is steadily declining due to growing numbers of herbicide-resistant weeds. While herbicide resistance has been present in the United States since the late 1950s, the widespread adoption of herbicide-tolerant crop varieties in the mid-1990s and overreliance on one or two herbicidal modes of action contributed to an exponential increase in the number of resistant weed species over the last two decades.

[0005] Auxin, mainly as indoleacetic acid (IAA), is an important plant hormone essential for plant growth and development. It also plays a key role in the response of plants to changes in their environment. Auxins precipitate in a cascade of reactions, the best described of which is the transport inhibitor response 1 (TIRl) / auxin-binding F-box (AFB)Agent Ref. No. P14663WOOOTlRl / AFB-auxin-Aux / IAA co-receptor system that is part of the Skpl-cullin-F-box protein (SCF) E3 ubiquitin ligase complex. Auxin interacts with both TIR1 / AFB and the transcriptional repressor protein Aux / IAA. This leads to ubiquitination of the Aux / IAA protein, signaling the 26s proteasome to degrade the Aux / IAA protein resulting in derepression of auxin response factors (ARFs) and transcription of auxin-regulated genes.

[0006] A number of synthetic auxins are used as commercial herbicides, with 2,4-D first being used in 1945. These herbicides belong to several chemical families and can have varying efficacy across plant species. The herbicides bind to the TIR1 receptor and the Aux / IAA repressor as auxin does; however, with herbicides the response leads to hyperaccumulation of ethylene, abscisic acid (ABA), and the production of reactive oxygen species (ROS). The events cause stomatai closure, uncontrolled cell differentiation, and elongation visible through swelling, epinasty, leaf withering, and ultimately senescence.

[0007] Understanding how weeds deal with herbicidal compounds to avoid damage is a major goal of weed science, both to generate workarounds to combat herbicide resistance and to gain insights into plant evolution. Herbicide tolerant plants are useful in systems in which a plurality' of such plants are planted, and can produce a crop, and either prior to planting, or after planting, an herbicide is applied that would otherwise kill or harm the plants but for their tolerance to the herbicide. Undesirable plants are killed or damaged, and the tolerant plants survive. There is a need to produce such plants.SUMMARY

[0008] Compositions and methods for conferring herbicide tolerance to plants, plant parts, and plant cells are provided. Modified plants having tolerance to an herbicide are provided, the modified plants comprising a polynucleotide encoding an Aux / 1 AA polypeptide with a modified degron region. In certain embodiments, the modified degron region comprises SEQ ID NO: 5 or a conservatively modified variant thereof. In certain embodiments, the Aux / IAA polypeptide with the modified degron region comprises one or more of a serine (S) at amino acid position 123, an isoleucine (I) at amino acid position 124, a lysine (K) at amino acid position 125, a phenylalanine (F) at amino acid position 126, a proline (P) at amino acid position 127, or a threonine (T) at amino acid position 128, wherein the aminoAgent Ref. No. P14663WOOO acid position numbering corresponds to SEQ ID NO: 1. Progeny, plant seeds, plant parts, and plant cells of the modified plants are also provided.

[0009] Polynucleotides encoding Aux / IAA polypeptides with a modified degron region capable of conferring herbicide tolerance are provided. In certain embodiments, the modified degron region comprises SEQ ID NO: 5 or a conservatively modified variant thereof. In certain embodiments, the Aux / IAA polypeptide with the modified degron region comprises one or more of a serine (S) at amino acid position 123, an isoleucine (I) at amino acid position 124, a lysine (K) at amino acid position 125, a phenylalanine (F) at amino acid position 126, a proline (P) at amino acid position 127, or a threonine (T) at amino acid position 128, wherein the amino acid position numbering corresponds to SEQ ID NO: 1. In certain embodiments, the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In certain embodiments, the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region has at least 80%. at least 90%. at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. Aux / IAA polypeptides with a modified degron region are also provided.

[0010] Expression constructs, vectors, biological samples, plants, plant seeds, plant parts, and plant cells comprising the aforementioned nucleic acid molecules are also provided.

[0011] Methods for producing a plant with herbicide tolerance are provided. In certain embodiments, the methods comprise modifying an endogenous Aux / IAA gene in the plant to encode an Aux / IAA polypeptide with a modified degron region of the disclosure. In certain embodiments, the methods comprise introducing to the plant a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region of the disclosure.

[0012] Methods for controlling undesired vegetation at a plant cultivation site comprising providing at the site a plant that comprises a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region of the disclosure, wherein expression of the polynucleotide confers to the plant tolerance to an herbicide; and applying to the site an effective amount of the herbicide are provided.

[0013] Methods for controlling the growth of an herbicide resistant weed at a plant cultivation site comprising contacting the weed with a composition that reduces expression or activity of an Aux / IAA polypeptide of the disclosure; and applying to the site an effective amount of the herbicide are provided.Agent Ref. No. P14663WOOO

[0014] Commodity plant products prepared from the aforementioned modified plants, plant parts, and plant cells are provided. In certain embodiments, the product comprises the Aux / IAA polypeptide with the modified degron region or the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region. Methods for producing a commodity plant product comprising processing the aforementioned modified plants or plant parts to obtain the product are also provided.

[0015] Methods for identifying an herbicide tolerant plant are provided. In certain embodiments, the methods comprise obtaining a sample from a plant suspected of having herbicide tolerance; detecting in the sample a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region of the disclosure; and determining that the plant is herbicide tolerant based on the presence of the polynucleotide. Kits for identifying an herbicide tolerant plant are also provided.

[0016] While multiple embodiments are disclosed, still other embodiments of the inventions will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention. Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive.BRIEF DESCRIPTION OF THE FIGURES

[0017] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention 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 aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0018] FIG. 1A is a dose response showing the magnitude of resistance for the M32 population of Bassia scoparia compared to a known dicamba-sensitive population (7710) and known dicamba resistant population (9425). FIG IB is quantitative PCR results showing lack oiSAUR21 induction in M32 following dicamba treatment (six hours after treatment with 140 g dicamba ha1). Different letter represent significant differences betw een groups from a tukey’s test at alpha=0. 1. Error bars represent standard error. FIG.Agent Ref. No. P14663WOOO1C is a time course of14C dicamba absorption showing lack of herbicide absorption is not associated with dicamba resistance in the M32 or 9425 populations of Bassia scoparia. FIG. ID shows the proportion of absorbed14C dicamba retained in the treated leaf showing less dicamba exits the treated leaf in dicamba-resistant populations (M32 and 9425) compared to a dicamba-sensitive population (7710). Error bars represent standard error.

[0019] FIG. 2A shows a quantitative trait loci (QTL) genome scan from a segregating F3 population derived from a biparental cross of individuals from the M32 and 7710 populations of Bassia scoparia. Red line represents a significance threshold determined by Bonferroni-adjustment of alpha=0.05 followed by conversion to LOD as described by Nyholt (2000). FIG. 2B shows the effect of genotype within the identified QTL on chromosome 4 on visual injury in an independent segregating F3 population of Bassia scoparia. Black line represents a linear model fitted to all points with equation listed in black.

[0020] FIG. 3A shows a CLUSTAL multiple protein sequence alignment of 7710 and M32 alleles of IAA16 (SEQ ID NO: 1 and SEQ ID NO: 3). The degron domain is bolded. FIG. 3B shows the sequence of genomic and coding BsIAA16 alleles detected in a mapping population segregating for dicamba resistance showing an insertion in the beginning of exon two of BSIAA16M32 that changes the coding sequence near the degron domain (SEQ ID NOs: 24-31). FIG. 3C shows the crystal structure of IAA7 bound to TIR1 showing the glycine 127 residue superimposed over the predicted structure if this residue is substituted for either threonine or asparagine. The cofactor 2,4-D is also shown. FIG. 3D is a graphical descnption of retrotransposon insertion from IAA16 and the autonomous version found elsewhere in the genome. FIG. 3E shows a neighbor joining tree estimating the evolutionary relatedness of long termina repeats from gi_5_l 3kb and retrotransposons from Arab idops is thaliana. Numbers at each branch indicate the proportion of 1000 bootstraps that support that branch.

[0021] FIG. 4A shows the root length of Arabidopsis thaliana plants expressing either BSIAA16M32 (populations starting in M32), BSIAA16WT (populations starting in 7710), or no transgene (ColO and 02A) grown on media not containing dicamba. FIG. 4B shows the proportional root length (compared to no-dicamba control) of the same populations when grown on media containing 5 pM dicamba. FIG. 4C shows representative photos ofAgent Ref. No. P14663WOOOAr cibidopsis thaliana (genotype Col 0) seedlings expressing BsIAA16 alleles or with no transgene (02A) grow n on agarose plates with or without 5 pM dicamba. Photos were taken seven days after germination. FIG. 4D shows Arabidopsis thaliana plants (genotype Col 0) plants expressing BsIAA16 alleles (BSIAA16WT, 7710 2-6-2; BSIAA16MS2, M32 2-3- 5) or with no transgene (02A) either untreated or treated with 140 g dicamba ha’1. Photo taken 14 days after treatment. FIG. 4E-G shows expression of the BsIAA16 trans gene (FIG. 4E) and the auxin response genes AtL4A19 (FIG. 4F) and AtGH3.3 (FIG. 4G) in plants expressing BSIAA16M32 (M32), BSIAA16WT (7710), or no transgene (ColO) that were either treated (T) or untreated (U) with 140 g dicamba ha’1six hours after treatment. Different letters represent significant differences between groups from a tukey’s test at alpha=0.1. Error bars represent standard error.

[0022] FIG. 5A-D show s the effect of BsAUX IAA 16 genotype on plant height at transplanting (FIG. 5A) or maturity (FIG. 5B). dry biomass accumulation at maturity (FIG. 5C), or weight of 50 seeds (FIG. 5D) for kochia plants from an F3 population segregating for dicamba resistance. Number of mutant alleles indicate homozy gous wildty pe (0), heterozy gous (1), or homozygous mutant (2). Linear models fitted to the data are plotted in red with equations listed at the top right of each pane. Replicate number is listed at the base of each violin with either boxplots or data plotted as dots within each violin.

[0023] FIG. 6A-B shows images of soybeans that w ere treated with Dicamba at application rates of 140 g / ha (FIG. 6A) and 280 g / ha (FIG. 6B). These images reflect visual observations taken one week after application. The selected transgenic events show resistance to Dicamba, as they do not exhibit the typical dicamba injury symptoms, such as leaf epinasty7, stem twisting, and downward growth commonly observed in susceptible plants and observed in the wild-type plants.BRIEF DESCRIPTION OF THE SEQUENCES

[0024] SEQ ID NO: 1 is the IAA16 M32 resistant amino acid sequence.

[0025] SEQ ID NO: 2 is the IAA16 M32 resistant coding sequence.

[0026] SEQ ID NO: 3 is the IAA16 7710 susceptible amino acid sequence.

[0027] SEQ ID NO: 4 is the IAA16 7710 susceptible coding sequence.

[0028] SEQ ID NO: 5 is the resistant modified degron region amino acid sequence.Agent Ref. No. P14663WOOO

[0029] SEQ ID NO: 6 is the susceptible wildtype degron region amino acid sequence. SEQ ID NO: 7 through SEQ ID NO: 23 are primer sequences summarized in Table 3A-C. SEQ ID NO: 24 through SEQ ID NO: 31 are the sequences shown in FIG. 3B.

[0030] SEQ ID NO: 32 is the complete vector sequence used for soybean transformation. The transgene (Mut IAA16 CDS) is driven by the UBQ1 promotor and NOS terminator.

[0031] SEQ ID NO: 33 is the complete vector sequence used for Arabidopsis transformation. The transgene (Mut IAA16 CDS) is driven by the CaMV 35S promotor and OCS terminator.DETAILED DESCRIPTION

[0032] So that the present invention 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 invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present invention without undue experimentation; the preferred materials and methods are described herein. In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below.

[0033] 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 w ord “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.

[0034] 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 aspects of this invention 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 invention.Accordingly, the description of a range should be considered to have specifically disclosedAgent Ref. No. P14663WOOO 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. I1 / ?, and 4%. This applies regardless of the breadth of the range.

[0035] The term “about”, as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, and temperature. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations. Whether or not modified by the term “about.” the claims include equivalents to the quantities.

[0036] 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, 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).

[0037] As used herein, the term “confer” refers to providing a characteristic or trait, such as herbicide tolerance or resistance and / or other desirable traits to a plant.

[0038] As used herein, the terms “correspond,” “corresponding,” and the like, when used in the context of a nucleotide or amino acid position in any given polynucleotide or polypeptide with respect to the reference polynucleotide or polypeptide sequence refer to the position of the nucleotide or amino acid in the given sequence that has identity to the nucleotide or amino acid in the reference nucleotide sequence when the given polynucleotide is aligned to the reference polynucleotide or polypeptide sequence using a sequence alignment algorithm (e.g., Clustal Omega with default parameters).

[0039] The term “control of undesired vegetation or weeds” is to be understood as meaning the killing of weeds and / or otherwise retarding or inhibiting the normal growth of the weeds. Weeds, in the broadest sense, are understood as meaning all those plants whichAgent Ref. No. P14663WOOO grow in locations where they are undesired. The weeds of the present disclosure include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus. Ipomoea. Polygonum, Sesbania. Ambrosia, Cirsium, Carduus, Sonchus, Solanum. Rorippa. Rotala. Lindernia, Lamium, Veronica. Abutilon. Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum. Monocotyledonous weeds include, but are not limited to, weeds of the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus. Avena. Cyperus. Sorghum, Agropyron, Cynodon. Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, w Apera. In addition, the weeds of the present disclosure can include, for example, crop plants that are growing in an undesired location. For example, a volunteer maize plant that is in a field that predominantly comprises soybean plants can be considered a weed, if the maize plant is undesired in the field of soybean plants.

[0040] 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.

[0041] As used herein, the term “DNA” or “DNA molecule” refers to a double-stranded DNA molecule of genomic or synthetic origin, i.e. a polymer of deoxy ribonucleotide bases or a polynucleotide molecule, read from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA molecule.

[0042] As used herein, an “endogenous gene” or a “native copy” of a gene refers to a gene that originates from within a given organism, cell, tissue, genome, or chromosome. An “endogenous gene” or a “native copy” of a gene is a gene that w as not previously modified by human action. Similarly, an “endogenous protein” refers to a protein encoded by an endogenous gene.Agent Ref. No. P14663WOOO

[0043] As used herein, “expression7’ means the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into polypeptide chains, which are ultimately folded into proteins.

[0044] Generally, the term “herbicide” is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies the growth of plants. The preferred amount or concentration of the herbicide is an “effective amount” or “effective concentration.” By “effective amount” and “effective concentration” is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a similar, wild-type, plant, plant tissue, plant cell, or host cell, but that said amount does not kill or inhibit as severely the growth of the herbicide-resistant plants, plant tissues, plant cells, and host cells of the present disclosure. Typically, the effective amount of an herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest. Such an amount for a given herbicide is known to those of ordinary skill in the art. Herbicidal activity is exhibited by herbicides useful for the present disclosure when they are applied directly to the plant or to the locus of the plant at any stage of growth or before planting or emergence. The effect observed depends upon the plant species to be controlled, the stage of growth of the plant, the application parameters of dilution and spray drop size, the particle size of solid components, the environmental conditions at the time of use, the specific compound employed, the specific adjuvants and carriers employed, the soil type, and the like, as well as the amount of chemical applied. These and other factors can be adjusted as is known in the art to promote non-selective or selective herbicidal action. Generally, the herbicide treatments can be applied PPI (Pre Plant Incorporated), PPSA (Post plant surface applied). PRE- or POST-emergent. Postemergent treatment typically occurs to relatively immature undesirable vegetation to achieve the maximum control of weeds.

[0045] By a “herbicide tolerant” or “herbicide resistant” plant, it is intended that a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill, or inhibit the growth of, a normal or wildtype plant. Levels of herbicide that normally inhibit grow th of a non-tolerant plant are known and readily determined by those skilled in the art. Examples include the amounts recommended by manufacturers for application. The maximum rate is an example of an amount of herbicide that would normally inhibit growth of a non-tolerant plant. For the present disclosure, the terms “herbicide tolerant” andAgent Ref. No. P14663WOOO“herbicide resistant’7are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms “herbicide tolerance” and “herbicide resistance” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms “tolerant” and “resistant” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. As used herein, in regard to an herbicidal composition useful in various embodiments hereof, terms such as herbicides, and the like, refer to those agronomically acceptable herbicide active ingredients (A.I.) recognized in the art. As used herein, an “herbicide tolerance trait” is a trait imparting improved herbicide tolerance to a plant as compared to the wild-type plant.

[0046] 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.

[0047] The term “introduced” in the context of inserting a nucleic acid into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokary otic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0048] As used herein, an “isolated” nucleic acid molecule is substantially separated away from other nucleic acid sequences with which the nucleic acid is normally associated, such as. from the chromosomal or extrachromosomal DNA of a cell in which the nucleic acid naturally occurs. The term also embraces nucleic acids that are biochemically purified so as to substantially remove contaminating nucleic acids and other cellular components.

[0049] 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 using site-specific nucleases).Agent Ref. No. P14663WOOO 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 certain embodiments, a modified plant provided herein comprises no interspecies genetic material or sequences.

[0050] 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. Plant parts include, but are not limited to seeds microspores, pollen, anthers, silk, spike, ovules, ovaries, flowers, pods, cobs, embryos, stems, leaves, roots, and calli.

[0051] The term “polynucleotide” as used herein is a nucleic acid molecule comprising a plurality of polymerized nucleotides, e.g., at least about five consecutive polymerized nucleotides. A polynucleotide may be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5’ or 3' untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single-stranded or double-stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, e.g.. genomic DNA or RNA. a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can be combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). The polynucleotide can comprise a sequence in either sense or antisense orientations. “Oligonucleotide” is substantially equivalent to the terms amplimer, amplicon, primer, oligomer, element, target, and probe and in some embodiments is singlestranded.

[0052] The term “primer” as used herein encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such asAgent Ref. No. P14663WOOOPCR. Typically, primers are oligonucleotides from 10 to 30 nucleotides in length, but longer sequences may be used. Primers may be provided in single or double-stranded form. Probes may be used as primers, but are designed to bind to the target DNA or RNA and need not be used in an amplification process.

[0053] As used herein, the terms “progeny’7and “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.

[0054] As used herein “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Illustrative plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such as Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters that initiate transcription only in certain tissue are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter that is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter that is active under most environmental conditions.

[0055] As used herein, “recombinant,” when referring to nucleic acid or polypeptide, indicates that such material has been altered as a result of human application of a recombinant technique, such as by polynucleotide restriction and ligation, by polynucleotide overlap-extension, or by genomic insertion or transformation. A gene sequence open reading frame is recombinant if that nucleotide sequence has been removed from its natural context and cloned into any type of artificial nucleic acid vector. The termAgent Ref. No. P14663WOOO recombinant also can refer to an organism having a recombinant material, e.g., a plant that comprises a recombinant nucleic acid can be considered a recombinant plant.

[0056] “Regulatory7elements” refer to nucleotide sequences located upstream (5' noncoding 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. Regulatory7elements may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. Regulatory7elements 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.

[0057] A “sequence” means a sequential arrangement of nucleotides or amino acids. The boundaries of a protein-coding sequence may be determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. In certain embodiments, a protein-coding molecule may comprise a DNA sequence encoding a protein sequence. In certain embodiments, a protein-coding molecule may comprise a RNA sequence encoding a protein sequence.

[0058] As used herein, the term “percent sequence identity” or “% sequence identity” refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference (“query”) sequence (or its complementary strand) as compared to a test (“subject”) sequence (or its complementary strand) when the tw o sequences are optimally aligned (with appropriate nucleotide or amino acid insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). Optimal alignment of sequences for aligning a comparison window7are w ell known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the Sequence Analysis softw are package of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, Calif.), MEGAlign (DNAStar Inc., Madison, Wis.), and MUSCLE (version 3.6) (Edgar, “MUSCLE: multiple sequence alignment with high accuracy and high throughput” Nucleic Acids Research 32(5): 1792-7Agent Ref. No. P14663WOOO(2004)) for instance with default parameters. An "identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in the portion of the reference sequence segment being aligned, that is, the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more sequences may be to a full-length sequence or a portion thereof, or to a longer sequence.

[0059] As used herein, “vector” includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.Aux / IAA Polynucleotides and Polypeptides

[0060] The plant hormone auxin serves as a central regulator of genes involved numerous plant growth, developmental, and response pathways. The naturally occurring active auxin is indole-3-acetic acid (IAA), but many other compounds have been found to mimic the function of IAA when applied to plants. This has led to the identification and commercialization of a number of compounds that function as effective herbicides. While com and other monocotyledonous crops are naturally tolerant to low levels of synthetic auxin herbicides, dicotyledonous crops such as soybean and cotton are highly sensitive.

[0061] Auxin / Indole-3-Acetic Acid (Aux / IAA) genes encode 25 to 35 kD proteins localized to the nuclease. The proteins have four conserved domains, domains I, II, III and IV. Domain II plays a role in destabilizing IAA proteins. The domain has the residues necessary for the receptor, auxin and co-receptor complex modulating the rate of Aux / IAA turnover and contain a degron that is necessary and sufficient for auxin-induced degradation. See Leyser et al. (2018) “Auxin Signaling” Plant Physiol. 176(l):465-479 and Zenser et al. (2001) “Auxin modulates the degradation rate of Aux / IAA proteins” Proc. Natl. Acad. Sci USA 98(20): 11795-11800.

[0062] Degradation signals or degrons are a region of a protein that is involved in regulation of the rate of degradation of protein. It is a minimal element within a protein that is sufficient for targeting the protein for degradation. Degrons may lead to a change in more or less ubiquitination and may be categorized as ubiquitin dependent or may be ubiquitin independent and the presence of the degron is not necessary forAgent Ref. No. P14663WOOO polyubiquitination of the protein. A degron may be identified by measuring the increase or decrease in amount of a protein when the region is present or absent.

[0063] The degron of Aux / IAA polypeptides has a conserved core GWPPV motif which is a binding site with TIR / AFB proteins and auxin.

[0064] Auxin / Indole-3-Acetic Acid (Aux / IAA) sequences with a modified degron region are provided that confer herbicide tolerance. Such sequences include the amino acid sequence set forth in SEQ ID NO: 1, and variants thereof. Also provided are polynucleotide sequences encoding such amino acid sequences, including SEQ ID NO: 2.

[0065] Several embodiments also relate to the use of Aux / IAA polypeptides with a modified degron region or variants thereof that confer tolerance to herbicides, including synthetic auxin herbicides. “Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and / or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and / or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode Aux / IAA polypeptides with a modified degron region described above. Naturally occurring allelic variants can be identified with the use of w ell-known molecular biology techniques, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined above. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site- directed mutagenesis but which still encode an Aux / IAA polypeptide with a modified degron region conferring herbicide tolerance. Generally, variants of a particular polynucleotide will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide.

[0066] Variants of a particular polynucleotide encoding an Aux / IAA polypeptide with a modified degron region that confers herbicide tolerance are encompassed and can be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequenceAgent Ref. No. P14663WOOO alignment programs and algorithms described herein. Where any given pair of polynucleotides is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0067] Methods of alignment of sequences for comparison are well known in the art and can be accomplished using mathematical algorithms such as the algorithm of Myers and Miller (1988) CABIOS 4: 11-17; the local alignment algorithm of Smith et al. (1981 ) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; and the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, Calif); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA).

[0068] Orthologs’’ and ‘■paral°gs’’ encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogs are genes within the same species that have originated through duplication of an ancestral gene; orthologs are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.

[0069] Those skilled in the art may find further candidate Aux / IAA genes based on genome synteny and sequence similarity. In one embodiment, additional gene candidates can be obtained by hybridization or PCR using sequences based on the Aux / IAA nucleotide sequences noted above.

[0070] 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,Agent Ref. No. P14663WOOON.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).

[0071] 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.).

[0072] 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.

[0073] “Stringent hybridization conditions” and “stringent hybridization w ash 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 and Molecular 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 low er 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.Agent Ref. No. P14663WOOO

[0074] The Tmis 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 xSSC 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 2x (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.

[0075] 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 NaPCfi, 1 mM EDTA at 50 °C with washing in 2xSSC, 0. 1% SDS at 50 °C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPOr. 1 mM EDTA at 50 °C with washing in I xSSC, 0.1% SDS at 50 °C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCh, 1 mM EDTA at 50 °C with washing in 0.5xSSC, 0.1% SDS at 50 °C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPC , 1 mM EDTA at 50 °C with washing in 0. 1 xSSC, 0.1% SDS at 50 °C, moreAgent Ref. No. P14663WOOO preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCh. 1 mM EDTA at 50 °C with washing in O.l xSSC, 0.1% SDS at 65 °C.

[0076] The terms “polypeptide” and “protein” are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms “proteins” and “polypeptides” as used herein also include variants, mutants, modifications, analogous and / or derivatives of the polypeptides of the disclosure as described herein.

[0077] With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the Aux / IAA polypeptide comprises an amino acid sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%. more preferably at least 60%. more preferably at least 65%, more preferably at least 70%. more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%. more preferably at least 96%. more preferably at least 97%, more preferably at least 98%. more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to SEQ ID NO: 1.

[0078] By “variant” polypeptide is intended a polypeptide derived from the protein of SEQ ID NO: 1, by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and / or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

[0079] As used herein, a “conservatively modified variant” refers to the substitution of one or more amino acids with a chemically similar amino acid. Conservative substitution tables are well known in the art. The following six groups each contain amino acids that areAgent Ref. No. P14663WOOO conservative substitutions for one another: 1) Alanine (A). Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q): 4) Arginine (R). Lysine (K); 5) Isoleucine (T), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also, Creighton (1984) Proteins W.H. Freeman and Company.

[0080] “Derivatives” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and / or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. Thus, functional variants and fragments of the Aux / IAA polypeptides, and nucleic acid molecules encoding them, also are within the scope of the present disclosure, and unless specifically described otherwise, irrespective of the origin of the polypeptide and irrespective of whether it occurs naturally.

[0081] In addition, one of ordinary skill in the art will further appreciate that changes can be introduced by mutation into the nucleotide sequences thereby leading to changes in the amino acid sequence of the encoded proteins without altering the biological activity of the proteins. Thus, for example, an isolated polynucleotide molecule encoding an Aux / IAA polypeptide having an amino acid sequence that differs from that of SEQ ID NO: 1 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present disclosure.

[0082] A deletion refers to removal of one or more amino acids from a protein. An insertion refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and / or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues. Examples of N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two- hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag- 100 epitope, c-myc epitope, FLAG"-epitope. lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitopeAgent Ref. No. P14663WOOOVSV epitope, and fluorescent tags such a green fluorescent protein (GFP) or yellow fluorescent protein (YFP).

[0083] A substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or 0-sheet structures). Amino acid substitutions are typically of single residues but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids; insertions will usually be of the order of about 1 to 10 amino acid residues.

[0084] Amino acid substitutions, deletions and / or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.

[0085] In certain embodiments, the polypeptides include at least one amino acid substitution, insertion, or deletion so that they do not recite a naturally occurring amino acid sequence.Genome Editing

[0086] Targeted modification of plant genomes through the use of genome editing methods can be used to modify the degron region of an endogenous Aux / IAA gene through modification of plant genomic DNA. Genome editing methods can also enable targeted insertion of one or more nucleic acids of interest (e.g., a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region) into a plant genome. Methods of genome editing to modify, delete, or insert nucleic acid sequences into genomic DNA are known in the art.

[0087] The targeted DNA modification of the genomic locus may be done using any genome modification technique known in the art. In certain embodiments the targeted DNA modification is through a genome modification technique selected from aAgent Ref. No. P14663WOOO polynucleotide-guided endonuclease. CRISPR-Cas endonucleases, base editing deaminases, zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), engineered site-specific meganuclease, or Argonaute.

[0088] In certain embodiments, the genome modification may be facilitated through the induction of a double-stranded break (DSB) or single-strand break, in a defined position in the genome near the desired alteration. DSBs can be induced using any DSB-inducing agent available, including, but not limited to, TALENs, meganucleases, zinc finger nucleases, Cas9-gRNA systems (based on bacterial CRISPR-Cas systems), guided cpfl endonuclease systems, and the like. In certain embodiments, the introduction of a DSB can be combined with the introduction of a polynucleotide modification template.

[0089] A polynucleotide modification template can be introduced into a cell by any method known in the art, such as, but not limited to, transient introduction methods, transfection, electroporation, microinjection, particle mediated delivery, topical application, whiskers mediated delivery, delivery via cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct delivery.

[0090] The polynucleotide modification template can be introduced into a cell as a single stranded polynucleotide molecule, a double stranded polynucleotide molecule, or as part of a circular DNA (vector DNA). The polynucleotide modification template can also be tethered to the guide RNA and / or the Cas endonuclease. Tethered DNAs can allow for colocalizing target and template DNA, useful in genome editing and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous HR machinery is expected to be highly diminished (Mali et al. 2013 Nature Methods Vol. 10: 957-963.) The polynucleotide modification template may be present transiently in the cell or it can be introduced via a viral replicon.

[0091] A “modified nucleotide” or “edited nucleotide” refers to a nucleotide sequence of interest that comprises at least one alteration when compared to its non-modified nucleotide sequence. Such “alterations” include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0092] The term “polynucleotide modification template” includes a polynucleotide that comprises at least one nucleotide modification when compared to the nucleotide sequence to be edited. A nucleotide modification can be at least one nucleotide substitution, additionAgent Ref. No. P14663WOOO or deletion. Optionally, the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

[0093] The process for editing a genomic sequence combining DSB and modification templates generally comprises: providing to a host cell, a DSB-inducing agent, or a nucleic acid encoding a DSB-inducing agent, that recognizes a target sequence in the chromosomal sequence and is able to induce a DSB in the genomic sequence, and at least one polynucleotide modification template comprising at least one nucleotide alteration when compared to the nucleotide sequence to be edited. The polynucleotide modification template can further comprise nucleotide sequences flanking the at least one nucleotide alteration, in which the flanking sequences are substantially homologous to the chromosomal region flanking the DSB.

[0094] The endonuclease can be provided to a cell by any method known in the art. for example, but not limited to, transient introduction methods, transfection, microinjection, and / or topical application or indirectly via recombination constructs. The endonuclease can be provided as a protein or as a guided polynucleotide complex directly to a cell or indirectly via recombination constructs. The endonuclease can be introduced into a cell transiently or can be incorporated into the genome of the host cell using any method known in the art. In the case of a CRISPR-Cas system, uptake of the endonuclease and / or the guided polynucleotide into the cell can be facilitated with a Cell Penetrating Peptide (CPP) as described in WO2016073433 published May 12, 2016.

[0095] As used herein, a “genomic region” is a segment of a chromosome in the genome of a cell that is present on either side of the target site or, alternatively, also comprises a portion of the target site. The genomic region can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100. 5- 200. 5-300, 5-400, 5-500. 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100. 5-1200, 5-1300, 5- 1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800. 5-2900, 5-3000, 5-3100 or more bases such that the genomic region has sufficient homology to undergo homologous recombination with the corresponding region of homology.Agent Ref. No. P14663WOOO

[0096] 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).

[0097] 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.

[0098] 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, BbvCL 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 enzy me can be created.

[0099] Endonucleases are enzy mes that cleave the phosphodiester bond within a polynucleotide chain. Endonucleases include restriction endonucleases, which cleave DNA at specific sites without 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 alsoAgent Ref. No. P14663WOOO characterized by prefix F-, 1-. 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.

[0100] 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.

[0101] Genome editing using DSB-inducing agents, such as Cas9-gRNA complexes, has been described, for example in U.S. Patent Application US 2015-0082478 Al. published on Mar. 19, 2015, WO2015 / 026886 Al, published on Feb. 26, 2015, W02016007347, published on Jan. 14, 2016, and WO201625131, published on Feb. 18, 2016, all of which are incorporated by reference herein.

[0102] The term “Cas gene” herein refers to a gene that is generally coupled, associated or close to, or in the vicinity of flanking CRISPR loci in bacterial systems. The terms “Cas gene”, “CRISPR-associated (Cas) gene” are used interchangeably herein. The term “Cas endonuclease” herein refers to a protein encoded by a Cas gene. A Cas endonuclease herein, when in complex with a suitable polynucleotide component, is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a specific DNAAgent Ref. No. P14663WOOO target sequence. A Cas endonuclease described herein comprises one or more nuclease domains. Cas endonucleases of the disclosure includes those having a HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain. A Cas endonuclease of the disclosure include, for example a Cas9 protein, a Cas 12a protein, a Cas 12b protein, or complexes of these.

[0103] As used herein, the terms “guide polynucleotide / Cas endonuclease complex”, “guide polynucleotide / Cas endonuclease system”, “guide polynucleotide / Cas complex”, “guide polynucleotide / Cas system”, “guided Cas system” are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease that are capable of forming a complex, wherein the guide polynucleotide / Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site. A guide polynucleotide / Cas endonuclease complex herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any of the four known CRISPR systems (Horvath and Barrangou, 2010, Science 321A61- 170) such as a type I, II, or III CRISPR system. A Cas endonuclease unwinds the DNA duplex at the target sequence and optionally cleaves at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (such as. but not limited to, a crRNA or guide RNA) that is in complex with the Cas protein. Such recognition and cutting of a target sequence by a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. Alternatively, a Cas protein herein may lack DNA cleavage or nicking activity, but can still specifically bind to a DNA target sequence when complexed with a suitable RNA component. (See also U.S. Patent Application US 2015-0082478 Al, published on Mar. 19, 2015 and US 2015-0059010 Al, published on Feb. 26, 2015, both are hereby incorporated in its entirety by reference).

[0104] A guide polynucleotide / Cas endonuclease complex can cleave one or both strands of a DNA target sequence. A guide polynucleotide / Cas endonuclease complex that can cleave both strands of a DNA target sequence typically comprise a Cas protein that has all of its endonuclease domains in a functional state (e.g., wild type endonuclease domains or variants thereof retaining some or all activity in each endonuclease domain). Non-limitingAgent Ref. No. P14663WOOO examples of Cas9 nickases suitable for use herein are disclosed in U.S. Patent Appl. Publ. No. 2014 / 0189896, which is incorporated herein by reference.

[0105] Other Cas endonuclease systems have been described in PCT patent applications PCT / US 16 / 32073, filed May 12, 2016 and PCT / US 16 / 32028 filed May 12, 2016, both applications incorporated herein by reference.

[0106] ‘ ‘Cas9” (formerly referred to as Cas5, Csnl, or Csxl2) herein refers to a Cas endonuclease of a type II CRISPR system that forms a complex with a crNucleotide and a tracrNucleotide, or with a single guide polynucleotide, for specifically recognizing and cleaving all or part of a DNA target sequence. Cas9 protein comprises a RuvC nuclease domain and an HNH (H-N-H) nuclease domain, each of which can cleave a single DNA strand at a target sequence (the concerted action of both domains leads to DNA doublestrand cleavage, whereas activity7of one domain leads to a nick). In general, the RuvC domain comprises subdomains I, II and III, where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu et al. Cell 157: 1262-1278). A type II CRISPR system includes a DNA cleavage system utilizing a Cas9 endonuclease in complex with at least one polynucleotide component. For example, a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In another example, a Cas9 can be in complex with a single guide RNA.

[0107] Any guided endonuclease can be used in the methods disclosed herein. Such endonucleases include, but are not limited to Cas9, Cas 12a, and Cas 12b endonucleases. Many endonucleases have been described to date that can recognize specific PAM sequences (see for example — Jinek et al. (2012) Science 337 p 816-821. PCT patent applications PCT / US16 / 32073, filed May 12, 2016 and PCT / US 16 / 32028 filed May 12, 2016 and Zetsche B et al. 2015. Cell 163, 1013) and cleave the target DNA at a specific position. It is understood that based on the methods and embodiments described herein utilizing a guided Cas system one can now tailor these methods such that they can utilize any guided endonuclease system.

[0108] The guide polynucleotide can also be a single molecule (also referred to as single guide polynucleotide) comprising a crRNA sequence linked to a tracrRNA sequence. The single guide polynucleotide comprises a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that can hybridize to a nucleotide sequence in aAgent Ref. No. P14663WOOO target DNA and a Cas endonuclease recognition domain (CER domain), that interacts with a Cas endonuclease polypeptide. By ‘’domain” it is meant a contiguous stretch of nucleotides that can be RNA, DNA, and / or RNA-DNA-combination sequence. The VT domain and / or the CER domain of a single guide polynucleotide can comprise a RNA sequence, a DNA sequence, or a RNA-DNA-combination sequence. The single guide polynucleotide being comprised of sequences from the crRNA and the tracrRNA may be referred to as “single guide RNA” (when composed of a contiguous stretch of RNA nucleotides) or “single guide DNA” (when composed of a contiguous stretch of DNA nucleotides) or “single guide RNA-DNA” (when composed of a combination of RNA and DNA nucleotides). The single guide polynucleotide can form a complex with a Cas endonuclease, wherein the guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) can direct the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to. and optionally nick or cleave (introduce a single or double strand break) the target site. (See also U.S. Patent Application US 2015-0082478 Al, published on Mar. 19, 2015 and US 2015- 0059010 Al, published on Feb. 26, 2015, both are hereby incorporated in its entirety7by reference.)

[0109] The term “variable targeting domain” or “VT domain” is used interchangeably herein and includes a nucleotide sequence that can hybridize (is complementary) to one strand (nucleotide sequence) of a double strand DNA target site. In certain embodiments, the variable targeting domain comprises a contiguous stretch of 12 to 30 nucleotides. The variable targeting domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

[0110] The terms “single guide RNA” and “sgRNA” are used interchangeably herein and relate to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain (linked to a tracr mate sequence that hybridizes to a tracrRNA), fused to a tracrRNA (trans-activating CRISPR RNA). The single guide RNA can comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of the type II CRISPR / Cas system that can form a complex with a type II Cas endonuclease, wherein the guide RNA / Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site.Agent Ref. No. P14663WOOO

[0111] The terms "guide RNA / Cas endonuclease complex7’, "guide RNA / Cas endonuclease system”, ‘'guide RNA / Cas complex”, “guide RNA / Cas system”, “gRNA / Cas complex”, “gRNA / Cas system”, “RNA-guided endonuclease”, “RGEN” are used interchangeably herein and refer to at least one RNA component and at least one Cas endonuclease that are capable of forming a complex, wherein the guide RNA / Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site. A guide RNA / Cas endonuclease complex herein can comprise Cas protein(s) and suitable RNA component(s) of any of the four known CRISPR systems (Horvath and Barrangou, 2010, Science 327: 167-170) such as a type I, II, or III CRISPR system. A guide RNA / Cas endonuclease complex can comprise a Type II Cas9 endonuclease and at least one RNA component (e.g., a crRNA and tracrRNA, or a gRNA). (See also U.S. Patent Application US 2015-0082478 Al, published on Mar. 19. 2015 and US 2015-0059010 Al, published on Feb. 26. 2015. both are hereby incorporated in its entirety by reference).

[0112] The guide polynucleotide of the methods and compositions described herein may be any polynucleotide sequence that targets the genomic loci of a plant cell comprising a polynucleotide that encodes an amino acid sequence that has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In certain embodiments, the guide polynucleotide is a guide RNA. The guide polynucleotide may also be present in a recombinant DNA construct.

[0113] The guide polynucleotide can be introduced into a cell transiently, as single stranded polynucleotide or a double stranded polynucleotide, using any method known in the art such as, but not limited to, particle bombardment, Agrobacterium transformation or topical applications. The guide polynucleotide can also be introduced indirectly into a cell by introducing a recombinant DNA molecule (via methods such as, but not limited to. particle bombardment or Agrobacterium transformation) comprising a heterologous nucleic acid fragment encoding a guide polynucleotide, operably linked to a specific promoter that is capable of transcribing the guide RNA in the cell. The specific promoter can be, but is not limited to, a RNA polymerase III promoter, which allow for transcription of RNA with precisely defined, unmodified, 5'- and 3'-ends (DiCarlo et al., Nucleic Acids Res. 41 : 4336-4343; Ma et al., Mol. Then Nucleic Acids 3 :e!61) as described inAgent Ref. No. P14663WOOOW02016025131, published on Feb. 18. 2016, incorporated herein in its entirety by reference.

[0114] The terms “target site”, “target sequence”, “target site sequence, “target DNA”, “target locus”, “genomic target site”, “genomic target sequence”, “genomic target locus” and “protospacer”, are used interchangeably herein and refer to a polynucleotide sequence such as, but not limited to, a nucleotide sequence on a chromosome, episome, or any other DNA molecule in the genome (including chromosomal, chloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which a guide polynucleotide / Cas endonuclease complex can recognize, bind to, and optionally nick or cleave. The target site can be an endogenous site in the genome of a cell, or alternatively, the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell, or the target site can be found in a heterologous genomic location compared to where it occurs in nature. As used herein, terms “endogenous target sequence” and “native target sequence” are used interchangeable herein to refer to a target sequence that is endogenous or native to the genome of a cell and is at the endogenous or native position of that target sequence in the genome of the cell. Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cells as well as plants and seeds produced by the methods described herein. An “artificial target site” or “artificial target sequence” are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence can be identical in sequence to an endogenous or native target sequence in the genome of a cell but be located in a different position (i.e., a non-endogenous or non-native position) in the genome of a cell.

[0115] An “altered target site”, “altered target sequence”, “modified target site”, “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence. Such “alterations” include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (hi) an insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0116] Methods for “modifying a target site” and “altering a target site” are used interchangeably herein and refer to methods for producing an altered target site.

[0117] The length of the target DNA sequence (target site) can vary, and includes, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,Agent Ref. No. P14663WOOO26, 27, 28. 29. 30 or more nucleotides in length. It is further possible that the target site can be palindromic, that is, the sequence on one strand reads the same in the opposite direction on the complementary' strand. The nick / cleavage site can be within the target sequence or the nick / cleavage site could be outside of the target sequence. In another variation, the cleavage could occur at nucleotide positions immediately opposite each other to produce a blunt end cut or, in other cases, the incisions could be staggered to produce single-stranded overhangs, also called “sticky7ends”, which can be either 5' overhangs, or 3' overhangs. Active variants of genomic target sites can also be used. Such active variants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the given target site, wherein the active variants retain biological activity7and hence are capable of being recognized and cleaved by a Cas endonuclease. Assays to measure the single or double-strand break of a target site by an endonuclease are known in the art and generally measure the overall activity and specificity of the agent on DNA substrates containing recognition sites.

[0118] A “protospacer adjacent motif’ (PAM) herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide / Cas endonuclease system described herein. The Cas endonuclease may not successfully recognize a target DNA sequence if the target DNA sequence is not followed by a PAM sequence. The sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used.

[0119] The terms “targeting”, “gene targeting” and “DNA targeting” are used interchangeably herein. DNA targeting herein may be the specific introduction of a knockout, edit, or knock-in at a particular DNA sequence, such as in a chromosome or plasmid of a cell. In general, DNA targeting can be performed herein by7cleaving one or both strands at a specific DNA sequence in a cell wi th an endonuclease associated with a suitable polynucleotide component. Such DNA cleavage, if a double-strand break (DSB), can prompt NHEJ or HDR processes which can lead to modifications at the target site.

[0120] A targeting method herein can be performed in such a way that two or more DNA target sites are targeted in the method, for example. Such a method can optionally be characterized as a multiplex method. Two, three, four, five, six, seven, eight, nine, ten, or more target sites can be targeted at the same time in certain embodiments. A multiplex method is typically performed by a targeting method herein in which multiple differentAgent Ref. No. P14663WOOORNA components are provided, each designed to direct a guide polynucleotide / Cas endonuclease complex to a unique DNA target site.

[0121] The guide polynucleotide / Cas endonuclease system can be used in combination with a co-delivered polynucleotide modification template to allow for editing (modification) of a genomic nucleotide sequence of interest. (See also U.S. Patent Application US 2015-0082478 Al, published on Mar. 19, 2015 and WO2015 / 026886 Al, published on Feb. 26, 2015, both are hereby incorporated in its entirety by reference.)

[0122] Various methods and compositions can be employed to obtain a cell or organism having a polynucleotide of interest inserted in a target site. Such methods can employ homologous recombination to provide integration of the polynucleotide of Interest at the target site. In one method provided, a polynucleotide of interest is provided to the organism cell in a donor DNA construct. As used herein, “donor DNA” is a DNA construct that comprises a polynucleotide of Interest to be inserted into the target site. The donor DNA construct further comprises a first and a second region of homology that flank the polynucleotide of Interest. The first and second regions of homology of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the target site of the cell or organism genome. By “homology” is meant DNA sequences that are similar. For example, a “region of homology to a genomic region” that is found on the donor DNA is a region of DNA that has a similar sequence to a given “genomic region” in the cell or organism genome. A region of homology can be of any length that is sufficient to promote homologous recombination at the cleaved target site. For example, the region of homology can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5- 45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100. 5-200, 5-300, 5-400. 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, 5-1400, 5-1500, 5- 1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000, 5-3100 or more bases in length such that the region of homology has sufficient homology to undergo homologous recombination with the corresponding genomic region. “Sufficient homology” indicates that two polynucleotide sequences have sufficient structural similarity to act as substrates for a homologous recombination reaction. The structural similarity includes overall length of each polynucleotide fragment, as well as the sequence similarity of the polynucleotides.Sequence similarity can be described by the percent sequence identity over the wholeAgent Ref. No. P14663WOOO length of the sequences, and / or by conserved regions comprising localized similarities such as contiguous nucleotides having 100% sequence identity, and percent sequence identity over a portion of the length of the sequences.

[0123] The amount of sequence identity' shared by a target and a donor polynucleotide can vary and includes total lengths and / or regions having unit integral values in the ranges of about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250- 500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700- 1500 bp. 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the target site. These ranges include every integer within the range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps. The amount of homology can also be described by percent sequence identity over the full aligned length of the two polynucleotides which includes percent sequence identity of about at least 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 100%. Sufficient homology includes any combination of polynucleotide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus. Sufficient homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high stringency conditions, see, for example, Sambrook et al.. (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology. Ausubel et al.. Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen (1993) Laboratory' Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0124] The structural similarity between a given genomic region and the corresponding region of homology found on the donor DNA can be any degree of sequence identity that allows for homologous recombination to occur. For example, the amount of homology or sequence identity shared by the “region of homology” of the donor DNA and the “genomic region” of the organism genome can be at least 50%. 55%, 60%. 65%. 70%. 75%. 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,Agent Ref. No. P14663WOOO96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo homologous recombination.

[0125] The region of homology on the donor DNA can have homology7to any sequence flanking the target site. While in certain embodiments the regions of homology share significant sequence homology to the genomic sequence immediately flanking the target site, it is recognized that the regions of homology7can be designed to have sufficient homology7to regions that may be further 5' or 3' to the target site. In still other embodiments, the regions of homology can also have homology with a fragment of the target site along with downstream genomic regions. In certain embodiments, the first region of homology further comprises a first fragment of the target site and the second region of homology7comprises a second fragment of the target site, wherein the first and second fragments are dissimilar.

[0126] As used herein, "‘homologous recombination” includes the exchange of DNA fragments between two DNA molecules at the sites of homology.

[0127] Further uses for guide RNA / Cas endonuclease systems have been described (See U.S. Patent Application US 2015-0082478 Al, published on Mar. 19, 2015, WO2015 / 026886 Al, published on Feb. 26, 2015, US 2015-0059010 Al, published on Feb. 26, 2015. U.S. application 62 / 023.246, filed on Jul. 7. 2014, and U.S. application 62 / 036,652, filed on Aug. 13, 2014, all of which are incorporated by reference herein) and include but are not limited to modifying or replacing nucleotide sequences of interest, insertion of polynucleotides of interest, gene knock-out, gene-knock in, modification of splicing sites and / or introducing alternate splicing sites, and modifications of nucleotide sequences encoding a protein of interest.Expression Constructs

[0128] Polynucleotides as described herein can be provided in an expression construct. Expression constructs generally7include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed. Thus, a person of ordinary skill in the art can select regulatory7elements for use in bacterial host cells, yeast host cells, plant host cells, insect host cells, and mammalian host cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. As used herein, the term “expression construct” refers to a combination of nucleic acid sequences that provides for transcriptionAgent Ref. No. P14663WOOO of an operably linked nucleic acid sequence. As used herein, “operably linked” means two DNA molecules linked in manner so that one may affect the function of the other.Operably -linked DNA molecules may be part of a single contiguous molecule and may or may not be adjacent. For example, a promoter is operably linked with a polypeptide- encoding DNA molecule in a DNA construct where the two DNA molecules are so arranged that the promoter may affect the expression of the DNA molecule.

[0129] As used herein, the term “heterologous” refers to the relationship between two or more items derived from different sources and thus not normally associated in nature. For example, a protein-coding recombinant DNA molecule is heterologous with respect to an operably linked promoter if such a combination is not normally found in nature. In addition, a particular recombinant DNA molecule may be heterologous with respect to a cell, seed, or organism into which it is inserted when it would not naturally occur in that particular cell. seed, or organism.

[0130] An expression construct can comprise a promoter sequence operably linked to a polynucleotide sequence encoding an Aux / IAA polypeptide 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 construct as described herein. In certain embodiments, a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without a substantial decrease in promoter activity. A transcription start site is typically included in the expression construct.

[0131] Embodiments relate to a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region, wherein the polynucleotide is further defined as operably linked to a heterologous regulatory element. In certain embodiments, the heterologous regulatory' element is a promoter functional in a plant cell.

[0132] If the expression construct is to be provided in or introduced into a plant cell, then plant viral promoters, such as, for example, a cauliflower mosaic virus (CaMV) 35S (including the enhanced CaMV 35S promoter (see, for example U.S. Pat. No. 5,106,739)) or a CaMV 19S promoter or a cassava vein mosaic can be used. Other promoters that can be used for expression constructs in plants include, for example, zein promoters including maize zein promoters, proh fera promoter, Ap3 promoter, heat shock promoters, T-DNA 1'-Agent Ref. No. P14663WOOO or 2'-promoter of tumefaciens . polygalacturonase promoter, chaicone synthase A (CHS- A) promoter from petunia, tobacco PR-la promoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2 promoter (Xu et al., 1993), maize Wipl promoter, maize trpA gene promoter (U.S. Pat. No. 5,625,136), maize CDPK gene promoter, and RUBISCO SSU promoter (U.S. Pat. No. 5.034,322) can also be used. Constitutive promoters (such as the CaMV, ubiquitin, actin, or NOS promoter), developmentally regulated promoters, and inducible promoters (such as those promoters than can be induced by heat, light, hormones, or chemicals) are also contemplated for use with polynucleotide expression constructs described herein.

[0133] Expression constructs may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and / or enhancer elements. Transcription termination regions can ty pically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination. A signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular environment. Targeting gene products to an intended cellular and / or extracellular destination through the use of an operably linked signal peptide sequence is contemplated for use with the polypeptides described herein. Classical enhancers are cis-acting elements that increase gene transcription and can also be included in the expression construct. Classical enhancer elements are known in the art. and include, but are not limited to, the CaMV 35 S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element. Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation dependent. Examples include the maize shrunken- 1 enhancer element (Clancy and Hannah, 2002).

[0134] Optionally the polynucleotide encoding the Aux / IAA polypeptide is 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;Agent Ref. No. P14663WOOOEP 80385962; EP 0431829; and Perlak et al. (1991) PNAS USA 88:3324-3328; all of which are herein incorporated by reference).

[0135] In certain embodiments, the polynucleotides include at least one nucleotide substitution, insertion, or deletion so that they do not recite a naturally occurring nucleic acid sequence.Transformation Methods

[0136] 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 or an RNA is transiently provided to a plant cell) and are well known in the art. Tw o effective methods for cell transformation are Agrobacierium-mc .\a c 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-meAialeA 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.

[0137] 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. Any of the herbicides to which plants of this disclosure can be resistant is an agent for selective markers. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells are those cells where, generally, the resistanceconferring gene is integrated and expressed at sufficient levels to permit cell survival. CellsAgent Ref. No. P14663WOOO 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 (DMO) 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.Synthetic Auxin Herbicides and Other Herbicides

[0138] Synthetic auxin herbicides are also called auxinic, growth regulator herbicides, or Group O or Group 4 herbicides, based on their mode of action. The mode of action of the synthetic auxin herbicides is that they appear to affect cell wall plasticity and nucleic acid metabolism, which can lead to uncontrolled cell division and growth. The group of synthetic auxin herbicides includes four chemical families: phenoxy. carboxylic acid (or pyridine), benzoic acid, and the newest family quinoline carboxylic acids.

[0139] The phenoxy herbicides are most common and have been used as herbicides since the 1940s when (2,4-dichlorophenoxy) acetic acid (2,4-D) was discovered. Other examples include 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB), 2-(2,4-dichlorophenoxy) propanoic acid (2, 4-DP), (2.4.5-trichlorophenoxy)acetic acid (2,4,5-T), 2-(2,4,5-Trichlorophenoxy) Propionic Acid (2,4,5-TP), 2-(2,4-dichloro-3-methylphenoxy)-N-phenylpropanamide (clomeprop), (4-chloro-2 -methylphenoxy) acetic acid (MCPA), 4-(4-chloro-o-tolyloxy) butyric acid (MCPB), and 2-(4-chloro-2-methylphenoxy) propanoic acid (MCPP).

[0140] The next largest chemical family is the carboxylic acid herbicides, also called pyridine herbicides. Examples include 3,6-dichloro-2-pyridinecarboxylic acid (Clopyralid). 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram), (2,4,5 -tri chlorophenoxy) acetic acid (triclopyr), and 4-amino-3,5-dichloro-6-fluoro-2-pyridyloxyacetic acid (fluroxypyr).

[0141] The third chemical family is the benzoic acids, examples of which include 3,6- dichloro-o-anisic acid (dicamba) and 3-amino-2,5-dichlorobenzoic acid (choramben).Agent Ref. No. P14663WOOO

[0142] The fourth and newest chemical family of the auxinic herbicides is the quinahne carboxylic acid family, which includes 7-chloro-3-methyl-8-quinolinecarboxylic acid (quinmerac) and 3,7-dichloro-8-quinolinecarboxylic acid (quinclorac). This latter is unique in that it also will control some grass weeds, unlike the other auxin-like herbicides which essentially control only broadleaf or dicotyledonous plants.

[0143] Synthetic auxin herbicides or other herbicides may be applied to a plant growth area comprising the plants and seeds provided by the compositions and methods described herein as a method for controlling weeds. Plants and seeds provided by the compositions and methods described herein comprise a synthetic auxin herbicide tolerance trait and as such are tolerant to the application of one or more auxin herbicides. The herbicide application may be the recommended commercial rate (1 x) or any fraction or multiple thereof, such as twice the recommended commercial rate (2*). Auxin herbicide rates maybe expressed as acid equivalent per pound per acre (lb ae / acre) or acid equivalent per gram per hectare (g ae / ha) or as pounds active ingredient per acre (lb ai / acre) or grams active ingredient per hectare (g ai / ha), depending on the herbicide and the formulation. The plant grow th area may or may not comprise weed plants at the time of herbicide application.

[0144] Herbicide applications may be sequentially or tank mixed with one, two, or a combination of several auxin herbicides or any other compatible herbicide. Multiple applications of one herbicide or of two or more herbicides, in combination or alone, may be used over a growing season to areas comprising plants expressing an Aux / IAA polypeptide with a modified degron region as described herein for the control of a broad spectrum of dicot weeds, monocot weeds, or both, for example, two applications (such as a pre-planting application and a post-emergence application or a pre-emergence application and a post-emergence application) or three applications (such as a pre-planting application, a pre-emergence application, and a post-emergence application or a pre-emergence application and two post-emergence applications).Plants with Herbicide Tolerance

[0145] Several embodiments relate to plant cells, plant tissues, plants, and seeds that comprise a polynucleotide encoding an Aux / IAA polypeptide with the modified degron region, wherein expression of the polynucleotide confers tolerance to a synthetic auxin herbicide. Plants may be monocots or dicots, and may include, for example, rice, wheat.Agent Ref. No. P14663WOOO barley, oats, rye, sorghum, maize, grape, tomato, potato, lettuce, broccoli, cucumber, peanut, melon, pepper, carrot, squash, onion, soybean, alfalfa, sunflower, cotton, canola, sugar cane, and sugar beet plants.

[0146] Plants that are particularly useful in the methods of the present disclosure include all plants which belong to the superfamily Viri diplantae, 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 saliva, Avena fatua, Avena byzantina, Avena fatua var. saliva, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis. Canna indica, Cannabis saliva, Capsicum spp.. Carex elala, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp.. Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp.. Daucus carota, 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), Hemer ocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp.. Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp.. Ma / us 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 latij lia), PanicumAgent Ref. No. P14663WOOO miliaceum, Panicum virgatum, Passiflora edulis. Pastinaca sativa. Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, 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 odorata, 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 soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato, or tobacco.

[0147] Also provided are a progeny or a descendant of an herbicide-tolerant plant as well as seeds derived from the herbicide-tolerant plants and cells derived from the herbicide- tolerant plants as described herein.

[0148] The present disclosure also provides a progeny or descendant plant derived from a plant comprising in at least some of its cells a polynucleotide encoding an Aux / lAA polypeptide with a modified degron region, the expression of the Aux / IAA polypeptide conferring to the progeny or descendant plant tolerance to the herbicide.

[0149] In certain embodiments, seeds of the present disclosure comprise the herbicidetolerance characteristics of the herbicide-tolerant plant. In certain embodiments, a seed is capable of germination into a plant comprising in at least some of its cells a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region, the expression of the Aux / IAA polypeptide conferring to the progeny or descendant plant tolerance to the herbicide.Agent Ref. No. P14663WOOO

[0150] 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.

[0151] Several embodiments provide a commodity plant product prepared from the herbicide-tolerant plants. Examples of commodity plant products include, without limitation, grain, oil, and meal. In one embodiment, a 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). For example, the commodity plant product can comprise fodder, seed meal, oil, milk (e.g., soy milk), flour (e.g., soy flour), grits, protein (e.g., protein concentrate, hydrolyzed vegetable protein, textured vegetable protein), tofu, miso, tempeh, fiber, starch, bio-composite building materials (e.g., particleboard, laminated plywood, or lumber products), or seed-treatment- coated seeds. The commodity plant products can compnse the Aux / IAA polypeptide with the modified degron region or the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region.

[0152] A commodity plant product prepared from a plant or plant part is provided, wherein the plant or plant part comprises in at least some of its cells a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region, the expression of Aux / IAA polypeptide conferring to the plant or plant part tolerance to the herbicide.

[0153] The commodity plant 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 produce the 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 mayAgent Ref. No. P14663WOOO be performed with an overlap in time, even simultaneously to a large extent or sequentially. Generally, the plants are grown for some time before the product is produced.Gene Stacking

[0154] The Aux / IAA genes of the disclosure can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. For example, the Aux / IAA genes may be stacked with one or more additional herbicide tolerance genes, including one or more additional synthetic auxin herbicide tolerance genes.

[0155] By way of example, polynucleotides that may be stacked ith the Aux / IAA genes include nucleic acids encoding polypeptides conferring resistance to pests / pathogens such as viruses, nematodes, insects or fungi, and the like. Illustrative polynucleotides that may be stacked with the Aux / IAA genes of the disclosure include polynucleotides encoding: polypeptides having pesticidal and / or insecticidal activity, such as Bacillus thuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al., (1986) Gene 48: 109), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825, pentin (described in U.S. Pat. No. 5,981,722), and the like; traits desirable for disease or herbicide resistance (e.g.. fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al., (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and / or Hra mutations; glyphosate resistance (e.g., 5-enol-pyrovyl-shikimate-3- phosphate-synthase (EPSPS) gene, described in U.S. Pat. Nos. 4,940,935 and 5.188.642; or the glyphosate N-acetyltransferase (GAT) gene, described in Castle et al. (2004) Science, 304:1151-1154; and in U.S. Patent App. Pub. Nos. 20070004912, 20050246798, and 20050060767)); glufosinate resistance (e.g. phosphinothricin acetyl transferase genes PAT and BAR, described in U.S. Pat. Nos. 5,561,236 and 5.276,268); resistance to herbicides including sulfonyl urea, DHT (2,4D), and PPO herbicides (e.g., glyphosate acetyl transferase, aryloxy alkanoate dioxygenase, acetolactate synthase, and protoporphyrinogen oxidase); other cytochrome P450s that confer herbicide tolerance (U.S. patent application Ser. No. 12 / 156.247; U.S. Pat. Nos. 6,380,465; 6,121,512; 5.349,127; 6,649,814; and 6,300,544; and PCT Patent App. Pub. No. W02007000077); and traits desirable forAgent Ref. No. P14663WOOO processing or process products such as high oil (e.g.. U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94 / 11516)); and modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE)); the disclosures of which are herein incorporated by reference.

[0156] In certain embodiments, the plant comprises at least one additional herbicide- tolerant trait selected, for example, from 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), phosphinothricin acetyltransferase (PAT), Acetohydroxyacid synthase (AHAS; EC 4. 1.3. 18, also known as acetolactate synthase or ALS), hydroxyphenyl pyruvate dioxygenase (HPPD), Phytoene desaturase (PD) and dicamba degrading enzymes as disclosed in WO 02 / 068607, or phenoxyacetic acid- and phenoxypropionic acid-derivative degrading enzy mes as disclosed in WO 2008141154 or WO 2005107437.Use in Breeding Methods

[0157] 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 hy brid, 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 and ear 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 phenoty pe as described herein.

[0158] 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.

[0159] 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 ofAgent Ref. No. P14663WOOO 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.

[0160] 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.

[0161] 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 homozy gosity 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.[0162 Plants of the present disclosure may be used to produce, e g., a single cross hybrid, a three-w ay 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-w-ay 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.Agent Ref. No. P14663WOOOHerbicide Resistant Weed Control

[0163] Several embodiments provide compositions and methods for controlling the growth of an herbicide resistant weed at a plant cultivation site by contacting the weed with a composition that reduces expression or activity of an Aux / IAA polypeptide with a modified degron region.

[0164] In certain embodiments, the compositions comprise a polynucleotide that reduces expression or activity of the Aux / IAA polypeptide with the modified degron region. Systemic regulation (e.g., systemic suppression or silencing) of a target Aux / IAA gene in a plant can be by topical application to the plant of a polynucleotide molecule with a segment in a nucleotide sequence essentially identical to, or essentially complementary to, a sequence of 18 or more contiguous nucleotides in either the target Aux / IAA gene or RNA transcribed from the target Aux / IAA gene, whereby the composition permeates the interior of the plant and induces systemic regulation of the target Aux / IAA gene by the action of single-stranded RNA that hybndizes to the transcribed RNA. e.g., messenger RNA

[0165] The polynucleotides are designed to induce systemic regulation or suppression of an endogenous gene in a plant and are designed to have a sequence essentially identical or essentially complementary to the sequence (which can be coding sequence or non-coding sequence) of an endogenous Aux / IAA gene of a resistant plant or to the sequence of RNA transcribed from an endogenous Aux / IAA gene of a resistant plant. By “essentially identical” or “essentially complementary” is meant that the polynucleotides (or at least one strand of a double-stranded polynucleotide) are designed to hybridize under physiological conditions in cells of the plant to the endogenous gene or to RNA transcribed from the endogenous gene to effect regulation or suppression of the endogenous gene.

[0166] In certain embodiments, the compositions and methods can comprise permeabilityenhancing agents and treatments to condition the surface of plant tissue, e.g., leaves, stems, roots, flowers, or fruits, to permeation by the polynucleotides into plant cells. The transfer of polynucleotides into plant cells can be facilitated by the prior or contemporaneous application of a polynucleotide to the plant tissue. In some embodiments the permeabilityenhancing agent is applied subsequent to the application of the polynucleotide composition. The permeability -enhancing agent enables a pathway for polynucleotides through cuticle wax barriers, stomata and / or cell wall or membrane barriers and into plantAgent Ref. No. P14663WOOO cells. Suitable agents to facilitate transfer of the composition into a plant cell include agents that increase permeability of the exterior of the plant or that increase permeability of plant cells to oligonucleotides or polynucleotides. Such agents to facilitate transfer of the composition into a plant cell include a chemical agent, or a physical agent, or combinations thereof.

[0167] Chemical agents for conditioning include (a) surfactants, (b) an organic solvent or an aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (e) acids, (f) bases, (g) oils, (h) enzy mes, or combinations thereof. Embodiments of the method can optionally include an incubation step, a neutralization step (e.g.. to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof. Such agents for conditioning of a plant to permeation are applied to the plant by any convenient method, e.g., spraying or coating with a powder, emulsion, suspension, or solution; similarly, the polynucleotide molecules are applied to the plant by any convenient method, e.g., spraying or wiping a solution, emulsion, or suspension.Detection Tools

[0168] Several embodiments provide a method for identifying an herbicide tolerant plant, or cells or tissues thereof. In certain embodiments, the method includes using primers or probes which specifically recognize a portion of the sequence of the Aux / IAA gene of the disclosure. In certain embodiments, the identification is performed using polymerase chain reaction. Several embodiments provide kits for identifying herbicide tolerant plants.

[0169] Probes and primers are provided which are of sufficient nucleotide length to bind specifically to the target DNA sequence under the reaction or hybridization conditions. Suitable probes and primers are at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, and less than 35, 34, 33, 32, 31, 30, 29, 28, 27. 26. 2, 5 2, 4 23, 22, 21, 20, 19, 18, 17. 16. 15, 14, 13, or 12 nucleotides in length. Such probes and primers can hybridize specifically to a target sequence under high stringency hybridization conditions. In certain embodiments, probes and primers have complete or 100% DNA sequence similarity of contiguous nucleotides with the target sequence, although probes which differ from the target DNA sequence but retain the ability to hybridize to target DNA sequence may also be used. Reverse complements of theAgent Ref. No. P14663WOOO primers and probes disclosed herein are also provided and can be used in the methods and compositions described herein.

[0170] The methods, kits, and primers can be used for different purposes including, but not limited to the following: identifying the presence or absence of herbicide resistance in plants, plant material such as seeds or cuttings; determining the presence of herbicideresistant weeds in crop fields; and tailoring an herbicide regime to effectively and economically manage weeds affecting agricultural crops.Embodiments

[0171] The following numbered embodiments also form part of the present disclosure:

[0172] 1. A modified plant, or a progeny, plant seed, plant part, or plant cell thereof, having tolerance to a synthetic auxin herbicide, the modified plant comprising a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region.

[0173] 2. The modified plant, progeny, plant seed, plant part, or plant cell of embodiment 1, wherein the Aux / IAA polypeptide with the modified degron region comprises SEQ ID NO: 5 or a conservatively modified variant thereof.

[0174] 3. The modified plant, progeny, plant seed, plant part, or plant cell of embodiment 1 or embodiment 2, wherein the Aux / IAA polypeptide with the modified degron region comprises one or more of a serine (S) at amino acid position 123, an isoleucine (I) at amino acid position 124, a lysine (K) at amino acid position 125, a phenylalanine (F) at amino acid position 126, a proline (P) at amino acid position 127, or a threonine (T) at amino acid position 128, wherein the amino acid position numbering corresponds to SEQ ID NO: 1.

[0175] 4. The modified plant, progeny, plant seed, plant part, or plant cell of any one of embodiments 1-3, wherein the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

[0176] 5. The modified plant, progeny, plant seed, plant part, or plant cell of any one of embodiments 1-4, wherein the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identify to SEQ ID NO: 2.

[0177] 6. The modified plant, progeny, plant seed, plant part, or plant cell of any one of embodiments 1-5, wherein the polynucleotide encoding the Aux / IAA polypeptide with theAgent Ref. No. P14663WOOO modified degron region is operably linked to a heterologous promoter functional in a plant cell.

[0178] 7. The modified plant, progeny, plant seed, plant part, or plant cell of any one of embodiments 1 -6, wherein the synthetic auxin herbicide is dicamba.

[0179] 8. The modified plant, progeny, plant seed, plant part, or plant cell of any one of embodiments 1-7, wherein the plant is a dicotyledonous or monocoty ledonous plant.

[0180] 9. The modified plant, progeny, plant seed, plant part, or plant cell of any one of embodiments 1-8, wherein the plant is a maize, sorghum, wheat, sunflower, rice, soybean, cotton, canola, tobacco, tomato, potato, pepper, barley, alfalfa, sugar cane, or sugar beet plant.

[0181] 10. The modified plant, progeny, plant seed, plant part, or plant cell of any one of embodiments 1 -9, wherein the plant is not a Bassia scoparia plant.

[0182] 11. The modified plant, progeny, plant seed, plant part, or plant cell of any one of embodiments 1-10, wherein the modified plant further comprises a second herbicide- tolerant trait.

[0183] 12. The modified plant, progeny, plant seed, plant part, or plant cell of any one of embodiments 1-11, wherein the plant, progeny, plant seed, plant part, or plant cell is non- viable and / or non-regenerable.

[0184] 13. A polynucleotide encoding an Aux / IAA polypeptide with a modified degron region.

[0185] 14. The polynucleotide of embodiment 13, wherein the Aux / IAA polypeptide with the modified degron region comprises SEQ ID NO: 5 or a conservatively modified variant thereof.

[0186] 15. The polynucleotide of embodiment 13 or embodiment 14, wherein the Aux / IAA polypeptide with the modified degron region comprising one or more of a serine (S) at amino acid position 123, an isoleucine (I) at amino acid position 124, a lysine (K) at amino acid position 125, a phenylalanine (F) at amino acid position 126, a proline (P) at amino acid position 127, or a threonine (T) at amino acid position 128, wherein the amino acid position numbering corresponds to SEQ ID NO: 1.

[0187] 16. The polynucleotide of any one of embodiments 13-15, wherein the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.Agent Ref. No. P14663WOOO

[0188] 17. The polynucleotide of any one of embodiments 13-16, wherein the polynucleotide encodes an Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

[0189] 18. The polynucleotide of any one of embodiments 13-17, wherein the polynucleotide is an isolated, synthetic, or recombinant polynucleotide.

[0190] 19. An expression construct comprising the polynucleotide of any one of embodiments 13-18 operably linked to a heterologous promoter functional in a plant cell.

[0191] 20. A vector comprising the polynucleotide of any one of embodiments 13-18 or the expression construct of embodiment 19.

[0192] 21. A biological sample comprising the polynucleotide of any one of embodiments 13-18, the expression construct of embodiment 19, or the vector of embodiment 20.

[0193] 22. A plant, plant part, plant seed, or plant cell comprising the polynucleotide of any one of embodiments 13-18. the expression construct of embodiment 19. or the vector of embodiment 20.

[0194] 23. An Aux / IAA polypeptide with a modified degron region comprising SEQ ID NO: 5 or a conservatively modified variant thereof, optionally wherein the Aux / IAA polypeptide with the modified degron region comprises one or more of a serine (S) at amino acid position 123, an isoleucine (I) at amino acid position 124, a lysine (K) at amino acid position 125, a phenylalanine (F) at amino acid position 126, a proline (P) at amino acid position 127, or a threonine (T) at amino acid position 128, wherein the amino acid position numbering corresponds to SEQ ID NO: 1.

[0195] 24. The Aux / IAA polypeptide of embodiment 23. wherein the Aux / IAA polypeptide with the modified degron region comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

[0196] 25. A method for producing a plant with tolerance to a synthetic auxin herbicide, the method comprising: modifying an endogenous Aux / IAA gene in the plant to encode an Aux / IAA polypeptide with a modified degron region.

[0197] 26. The method of embodiment 25, wherein the Aux / IAA polypeptide with the modified degron region comprises SEQ ID NO: 5 or a conservatively modified variant thereof.Agent Ref. No. P14663WOOO

[0198] 27. The method of embodiment 25 or embodiment 26, wherein the Aux / IAA polypeptide with the modified degron region comprises one or more of a serine (S) at amino acid position 123, an isoleucine (I) at amino acid position 124, a lysine (K) at amino acid position 125, a phenylalanine (F) at amino acid position 126, a proline (P) at amino acid position 127, or a threonine (T) at amino acid position 128, wherein the amino acid position numbering corresponds to SEQ ID NO: 1.

[0199] 28. The method of any one of embodiments 25-27, wherein the method comprises introducing a genome editing system that targets the endogenous Aux / IAA gene.

[0200] 29. The method of embodiment 28, wherein the genome editing system comprises a CRISPR / Cas system, a TALEN, or a zinc finger nuclease.

[0201] 30. The method of any one of embodiments 25-29, wherein the Aux / IAA polypeptide with the modified degron region comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

[0202] 31. The method of any one of embodiments 25-30, wherein the endogenous Aux / IAA gene has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4.

[0203] 32. The method of any one of embodiments 25-31, wherein the synthetic auxin herbicide is dicamba.

[0204] 33. The method of any one of embodiments 25-32, wherein the plant is a dicotyledonous or monocotj ledonous plant.

[0205] 34. The method of any one of embodiments 25-33, wherein the plant is a maize, sorghum, wheat, sunflower, rice, soybean, cotton, canola, tobacco, tomato, potato, pepper, barley, alfalfa, sugar cane, or sugar beet plant.

[0206] 35. A method for producing a plant with tolerance to a synthetic auxin herbicide, the method comprising: introducing to the plant a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region.

[0207] 36. The method of embodiment 35, wherein the Aux / IAA polypeptide with the modified degron region comprises SEQ ID NO: 5 or a conservatively modified variant thereof.

[0208] 37. The method of embodiment 35 or embodiment 36, wherein the Aux / IAA polypeptide with the modified degron region compnses one or more of a serine (S) atAgent Ref. No. P14663WOOO amino acid position 123, an isoleucine (1) at amino acid position 124, a lysine (K) at amino acid position 125, a phenylalanine (F) at amino acid position 126, a proline (P) at amino acid position 127, or a threonine (T) at amino acid position 128, wherein the amino acid position numbering corresponds to SEQ ID NO: 1

[0209] 38. The method of any one of embodiments 35-37, wherein the polynucleotide is operably linked to a heterologous promoter functional in a plant cell.

[0210] 39. The method of any one of embodiments 35-38, wherein the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% sequence identity to SEQ ID NO: 1.

[0211] 40. The method of any one of embodiments 35-39, wherein the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

[0212] 41. The method of any one of embodiments 35-40, wherein the synthetic auxin herbicide is dicamba.

[0213] 42. The method of any one of embodiments 35-41, wherein the plant is a dicotyledonous or monocotyledonous plant.

[0214] 43. The method of any one of embodiments 35-42, wherein the plant is a maize, sorghum, wheat, sunflower, rice, soybean, cotton, canola, tobacco, tomato, potato, pepper, barley, alfalfa, sugar cane, or sugar beet plant.

[0215] 44. A method for controlling undesired vegetation at a plant cultivation site, the method comprising: providing at the site a plant that comprises a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region, wherein the polynucleotide confers to the plant tolerance to a synthetic auxin herbicide; and applying to the site an effective amount of the synthetic auxin herbicide.

[0216] 45. The method of embodiment 44, wherein the Aux / IAA polypeptide with the modified degron region comprises SEQ ID NO: 5 or a conservatively modified variant thereof.

[0217] 46. The method of embodiment 44 or embodiment 45, wherein the Aux / IAA polypeptide with the modified degron region comprises one or more of a serine (S) at amino acid position 123, an isoleucine (I) at amino acid position 124, a lysine (K) at amino acid position 125, a phenylalanine (F) at ammo acid position 126, a proline (P) at aminoAgent Ref. No. P14663WOOO acid position 127. or a threonine (T) at amino acid position 128, wherein the amino acid position numbering corresponds to SEQ ID NO: 1.

[0218] 47. The method of any one of embodiments 44-46, wherein the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% sequence identity to SEQ ID NO: 1.

[0219] 48. The method of any one of embodiments 44-47, wherein the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

[0220] 49. The method of any one of embodiments 44-48, wherein the synthetic auxin herbicide is dicamba.

[0221] 50. The method of any one of embodiments 44-49, wherein the plant is a dicotyledonous or monocotyledonous plant.

[0222] 51. The method of any one of embodiments 44-50, wherein the plant is a maize, sorghum, wheat, sunflower, rice, soybean, cotton, canola, tobacco, tomato, potato, pepper, barley, alfalfa, sugar cane, or sugar beet plant.

[0223] 52. A method for controlling the grow th of an herbicide resistant weed at a plant cultivation site, the method comprising: contacting the weed with a composition that reduces expression or activity of an Aux / IAA polypeptide with a degron region; and applying to the site an effective amount of the herbicide.

[0224] 53. The method of embodiment 52, wherein the Aux / IAA polypeptide with the modified degron region comprises SEQ ID NO: 5 or a conservatively modified variant thereof.

[0225] 54. The method of embodiment 52 or embodiment 53, wherein the Aux / IAA polypeptide with the modified degron region comprises one or more of a serine (S) at amino acid position 123, an isoleucine (I) at amino acid position 124, a lysine (K) at amino acid position 125, a pheny lalanine (F) at amino acid position 126, a proline (P) at amino acid position 127, or a threonine (T) at amino acid position 128, wherein the amino acid position numbering corresponds to SEQ ID NO: 1.

[0226] 55. The method of any one of embodiments 52-54, wherein the composition comprises a polynucleotide that reduces expression or activity of the Aux / IAA polypeptide.Agent Ref. No. P14663WOOO

[0227] 56. The method of any one of embodiments 52-55, wherein the polynucleotide is a double-stranded RNA, a single-stranded RNA, or a double-stranded DNA / RNA hybrid polynucleotide.

[0228] 57. The method of any one of embodiments 52-56, wherein the polynucleotide comprises a sequence essentially identical or essentially complementary to at least 18 or more contiguous nucleotides of SEQ ID NO: 2.

[0229] 58. The method of any one of embodiments 52-57, wherein the polynucleotide has a length of 26-60 nucleotides.

[0230] 59. The method of any one of embodiments 52-58, wherein the composition comprises a chemical inhibitor that reduces expression or activity of the Aux / IAA polypeptide.

[0231] 60. The method of any one of embodiments 52-59, wherein the synthetic auxin herbicide is dicamba.

[0232] 61. The method of any one of embodiments 52-60, wherein the weed is aBassia scoparia plant.

[0233] 62. The method of any one of embodiments 52-61, wherein the composition comprises a permeability -enhancing agent.

[0234] 63. A commodity plant product prepared from the plant, plant part, plant seed, or plant cell of any one of embodiments 1-12.

[0235] 64. The commodity plant product of embodiment 63, wherein the product comprises the Aux / IAA polypeptide with the modified degron region or the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region.

[0236] 65. The commodity plant product of embodiment 63 of embodiment 64, wherein the product comprises fodder, seed meal, oil, milk, flour, grits, protein, tofu, miso, tempeh, fiber, starch, bio-composite building materials or seed-treatment-coated seed.

[0237] 66. A method for producing a commodity plant product, the method comprising processing the plant, plant part, plant seed, or plant cell of any one of embodiments 1-12 to obtain the product.

[0238] 67. The method of embodiment 66, wherein the product comprises the Aux / IAA polypeptide with the modified degron region or the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region.Agent Ref. No. P14663WOOO

[0239] 68. The method of embodiment 66 or embodiment 67, wherein the plant product comprises fodder, seed meal, oil, milk, flour, grits, protein, tofu, miso, tempeh, fiber, starch, bio-composite building materials, or seed-treatment-coated seeds.

[0240] 69. A method for identifying a plant having tolerance to a synthetic auxin herbicide, the method comprising: obtaining a nucleic acid sample from a plant suspected of having the herbicide tolerance; detecting in the sample a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region; and determining that the plant is herbicide tolerant based on the presence of the polynucleotide.

[0241] 70. The method of embodiment 69, wherein the Aux / IAA polypeptide with the modified degron region comprises SEQ ID NO: 5 or a conservatively modified variant thereof.

[0242] 71. The method of embodiment 69 or embodiment 70, wherein the Aux / IAA polypeptide with the modified degron region comprises one or more of a serine (S) at amino acid position 123, an isoleucine (I) at amino acid position 124, a lysine (K) at amino acid position 125, a phenylalanine (F) at amino acid position 126, a proline (P) at amino acid position 127, or a threonine (T) at amino acid position 128, wherein the amino acid position numbering corresponds to SEQ ID NO: 1.

[0243] 72. The method of any one of embodiments 69-71, wherein the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

[0244] 73. The method of any one of embodiments 69-72, wherein the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%. at least 95%. at least 98%. at least 99%. or 100% sequence identity to SEQ ID NO: 2.

[0245] 74. The method of any one of embodiments 69-73, wherein the detecting comprises amplifying the polynucleotide using at least two primers.

[0246] 75. The method of any one of embodiments 69-74, wherein the plant is aBassia scoparia plant.

[0247] 76. The method of any one of embodiments 69-75, wherein the plant is a maize, sorghum, wheat, sunflower, rice, soybean, cotton, canola, tobacco, tomato, potato, pepper, barley, alfalfa, sugar cane, or sugar beet plant.Agent Ref. No. P14663WOOO

[0248] 77. A kit for identifying a plant having tolerance to a synthetic auxin herbicide, the kit comprising at least two primers, wherein the at least two primers recognize a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region.

[0249] 78. The method of embodiment 77, wherein the Aux / IAA polypeptide with the modified degron region comprises SEQ ID NO: 5 or a conservatively modified variant thereof.

[0250] The method of embodiment 77 or embodiment 78, wherein the Aux / IAA polypeptide with the modified degron region comprises one or more of a serine (S) at amino acid position 123, an isoleucine (I) at amino acid position 124, a lysine (K) at amino acid position 125, a phenylalanine (F) at ammo acid position 126, a proline (P) at amino acid position 127, or a threonine (T) at amino acid position 128, wherein the amino acid position numbering corresponds to SEQ ID NO: 1.

[0251] 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.

[0252] 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.

[0253] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1: Characterization of dicamba resistance in M32

[0254] A dose response experiment was conducted to confirm and quantify the level of dicamba resistance in the M32 population of kochia (FIG. 1A). Parameter estimates for a three-parameter log-logistic model for each population are shown in Table 1. Populations M32 and 9425 (a known dicamba-resistant kochia population characterized by LeClere et al., 2018 and Pettinga et al., 2018) had ED50 (the dose of dicamba needed to cause 50% injury) estimates 5.6 and 8.1 times greater than 7710 (an herbicide-sensitive kochia population). These estimates were significantly different from 7710 and from each other, suggesting dicamba resistance in M32 is not as strong as in 9425.Agent Ref. No. P14663WOOO

[0255] Reanalysis of RNA sequencing data from Petinga et al. (2018) using the reference genome assembled by Patterson et al. (2019) identified Bs.00g297840.m01, a gene annotated as SMALL AUXIN UP RNA 21 (SAUR21), as a highly differentially expressed gene following dicamba treatment. SA URs are early auxin-response genes that are transcriptionally induced within minutes of auxin application and help regulate a wide array of cellular processes (Ren and Gray, 2015). As such, they can be used as molecular markers for auxin perception. Quantitative PCR (qPCR) performed on M32 and 7710 plants before and after dicamba treatment showed that there was no significant difference between the two populations before treatment. However, six hours after treatment, SAUR21 was up regulated in 7710, but not in M32 (FIG. IB). This lack of induction of an auxin response gene suggests that M32 plants do not show an early response to dicamba, possibly through very rapid dicamba inactivation (Behrens et al., 2007) or target-site mutation(s) that reduce interaction of dicamba with its molecular target(s) (LeClere et al., 2018; de Figueiredo et al., 2022).

[0256] Radiolabeled dicamba was used to measure dicamba absorption / translocation in 7710, 9425, and M32 plants (similar to experiments by Pettinga et al., 2018 and de Figueiredo et al., 2022). Significantly less dicamba was absorbed by 7710 plants over 192 hours, but rate of absorption (time required for 90% absorption) was not significantly different between populations (FIG. 1C, Table 2), indicating that reduced dicamba absorption is not responsible for resistance (in agreeance with Pettinga et al., 2018). Data for the translocation of14C dicamba out of the treated leaf did not fit well to any tested models, so t-tests were used to determine if means differed between M32 and 7710 at all time points (FIG. ID). Again, as seen by Pettinga et al. (2018), resistant plants translocated less14C dicamba out of the treated leaf compared to the sensitive plants. These results suggested the dicamba resistance mechanism in the M32 population is physiologically similar to the one described by Pettinga et al. (2018) and LeClere et al. (2018).

[0257] TABLE 1. Parameter estimates for 3-parameter log-logistic dose response curves for M32, 9425, and 7710 kochia populations. The equation fitted for each population was where y is predicted visual injure 21 days after dicamba treatment, x is the rate of dicamba in g ha'1, d is the upper asymptote, ED50 is the rate of dicamba required to cause a visual injury rating 50% of the upper asymptote, and b is the slope of the curve at x=ED50.Agent Ref. No. P14663WOOO

[0258] TABLE 2. Parameter estimates for rectangular hyperbolic models fitted to herbicide absorption data for M32, 9425, and 7710 kochia populations. The equation fitted for each population was where y is percent of applied herbicide that was absorbed, t is time after herbicide application (in hours), t90 is the time required for 90% herbicide absorption, and Amctx is the maximum amount of herbicide absorbed.Example 2: A single locus on chromosome 4 is associated with dicamba resistance

[0259] A quantitative trait loci (QTL) mapping approach was taken to identify regions of the genome associated with dicamba resistance in M32 plants. Segregating F3 populations were developed from multiple independent biparental crosses using a dicamba-resistant M32 plant as the male parent and a dicamba-sensitive 7710 plant as the female parent. The ratio of alive: dead plants (200:85) did not significantly differ from the expected ratio of a dominant trait caused by a single locus (3: 1; p = 0.06). However, it was hypothesized that the relative abundance of dead plants was because the rate of dicamba used for theAgent Ref. No. P14663WOOO screening (560 g ha'1) was high enough to damage some "resistant7’ plants. A novel doubledigest restriction site associate DNA sequencing (ddRADseq) protocol was used to determine genotypes of 103 segregating plants for 1,592 variant loci across the genome. A genome scan associating this genotype information to visual injury rating identified one significant QTL centered at 88Mbp on chromosome 4 (FIG. 2A). This interval contains three isoforms of IAA including IAA 16 at 87.4Mbp. Genotype within this QTL of plants from an independent segregating Fs line explained 43% of variation in visual injury with their injury' following dicamba treatment (FIG. 2B). While survival at the used rate of dicamba may be more dominant, visual injury is incompletely dominant (FIG. 2B) with a calculated degree of dominance of -0.58 (Falconer, 1964).Example 3: A transposable element insertion in BSIAA16M32 causes a change in exon splicing

[0260] Sequencing transcripts of BsIAA16 from plants homozygous for either the M32 or 7710 allele of the QTL discovered on chromosome 4 revealed near identical protein sequences except for the region around the degron domain (FIG. 3A). The degron domain interacts with ARF / TIR1 in the presence of auxin. This interaction leads to the ubiquitination of IAA and subsequent degradation. In kochia, the degron of IAA16 is located near the splice junction of exons one and two. To understand the sequence of genomic DNA around BsIAA16, a plant homozygous for the M32 allele of BsIAA16 was used for PacBio HiFi sequencing. After de novo assembly, a ~3.5Kbp insertion was discovered at the beginning of exon two of BSIAA16M32 (FIG. 3B). The insertion disrupts splicing of BSIAA16M32. replacing 16 bases at the beginning of exon two with the final 19 bases of the insertion (FIG. 3C). This results in the addition of one codon and the substitution of four amino acids (including the glycine of the degron domain (FIG. 3A). Given that LeClere et al. (2018) showed that a substitution of this glycine to asparagine in IAA16 is sufficient for dicamba resistance in kochia. this disruption (including a substitution of the same glycine for a threonine) is likely to also cause resistance.Additionally, a proline residue is introduced near the degron that may limit flexibility of the domain. Protein modeling shows that the larger side group of either asparagine or threonine contacts the surface of TIR1 and likely causes steric hindrance of the IAA / TIR1 interaction (FIG. 3C). Because the insertion is hypothesized to reduce this interaction andAgent Ref. No. P14663WOOO interfere with natural auxin signaling as shown by LeClere et al (2018) and de Figuieredo et al. (2022), this change is expected to also confer a fitness penalty in the absence of synthetic auxin application.

[0261] The insertion in BSIAA16M32 has 5 bp target site duplications flanking 429 bp long terminal repeats (LTRs), identifying it as a Class 2 retro element. However, no protein coding sequences were found within this insertion, suggesting this element is non- autonomous and relies on another transposable element for transposition. Aligning the insertion to the de novo assembly of M32 and the reference genome of 7710 identifies a near perfect match (99.3% homology) with a site 1 Mbp away on chromosome 4. Because this site is the most similar and retrotransposons use a ‘'copy and paste’’ mechanism of transposition, this was hypothesized to be the donor location for this new event.

[0262] The LTR sequence was aligned to the 7710 reference genome (Patterson et al. 2018) and putative matches filtered to identify possible autonomous version(s) of this element. This process led to the identification of a single site (gi_5_13kb) with matching LTRs and containing all the elements necessary for transposition (FIG. 3D). A phylogenetic tree built from the LTRs of know n classes of retrotransposons suggest this to be a Ty f / Copia element (FIG. 3E), which was named Outlaw.Example 4: BSIAA16M32 expression is sufficient for dicamba resistance in Arabidopsis thaliana

[0263] Stable transgenic lines of Arabidopsis thaliana expressing BSIAA16WT or BSIAA16MS2 were generated through the floral dip method. When seeds were grown on agarose plates without herbicide, Arabidopsis expressing BsIAAlbw grew shorter roots compared to those expressing BSIAA16WT or with no transgene (FIG. 4A; FIG. 4C). This is further evidence of a likely fitness cost associated with disrupting normal auxin signaling in roots as seen by de Figuieredo et al. (2022) and LeClere et al. (2018). When grown on media containing dicamba, only seedlings expressing BSIAA16M32 were able to grow and had significantly longer roots w hen normalized to the length of roots grown on dicamba- free media (FIG. 4B; FIG. 4C). In fact, all populations expressing BsIAAl 6M32 had longer average normalized root lengths than lines expressing BSIAA16WT or with no transgene (FIG. 4B). Giving strong evidence to the claim that expression of BSIAA16M32 is sufficientAgent Ref. No. P14663WOOO for dicamba resistance, no seeds grew roots longer than 1 mm when grown on media containing 5 pM 2,4-D or 0.5 pM Indole-3 acetic acid (IAA).

[0264] To confirm the results of root growth assays that indicate BsIAA l 6'232 is sufficient for dicamba resistance, plants of select T3 lines either expressing BsIAA16wr, BsIAAl 6M32, or no transgene were grown and treated with 140 g dicamba ha-1. Only one of the lines expressing BSIAA16MS2 (M32 2-3-5) showed less visual injury than the other lines tested and appeared stunted and darker green than the other lines even when no dicamba treatment was administered (FIG. 4D). Quantitative PCR indicated similar expression levels of BsIAA16 between M32 2-3-5 and a tested line expressing BSIAA16WT (FIG. 4E). However, M32 2-3-5 plants had no detected induction of auxin response genes AUAA19 or AXGH3.3 six hours after dicamba treatment (FIG. 4F and FIG. 4G). These genes are known to have increased expression following exposure to natural or synthetic auxins. Differences between root and whole plant assays may be caused by differences in expression of the transgene between root and above ground tissue.Methods of Examples 1-4Plant growth and herbicide treatment conditions

[0265] Unless otherwise noted, all plants were grown in greenhouses at Colorado State University in 3.8-cm by 3.8-cm by 5.8-cm pots containing fine-grade potting mix (Fafard #2-SV; American Clay Works, Denver, CO). Temperatures were held between 22°C and 24°C, and supplemental light was provided by liquid halogen grow' lights to ensure a photoperiod of 14h / 10h. Herbicide applications were made using a moving overhead single-nozzle sprayer calibrated to deliver 187 L ha Enginia (BASF) was used for dicamba applications, and Clean Amine (Loveland Products, Inc.) was used for 2,4-D applications. For kochia, herbicide applications took place when plants w ere 10-15 cm tall, and for Arabidopsis, plants were sprayed when they had 10-12 leaves and before bolting. Plant material

[0266] The M32 population of kochia was collected near Akron, Colorado as part of an herbicide resistance survey conducted between 2012 and 2014. Seeds from this field collection were grown and the resulting plants were treated with 560 g dicamba ha'1. Survivors were open-pollenated, and their seed was combined to form a composite resistant seed lot. Seed from this combined lot were grown and again treated with 560Agent Ref. No. P14663WOOO dicamba ha’1. The individual with the least herbicide injury was used as a male parent in a biparental cross with an individual from the population 7710, an inbred, herbicide- susceptible population that was used to generate the reference genome assembly for kochia. For this, immature flowers of the dicamba-sensitive plant were emasculated using forceps under a microscope, then the two plants were grown to maturity together in a pollen-exclusion tent. Seeds from the dicamba-sensitive plant were collected and grown; the resulting plants were treated with 560 g dicamba ha’1to identify hybrid plants. Several hybrid plants were grown in pollen exclusion tents and self-pollenated to create several F2 families. Plants from two F2 families were grown, but because of low seed production in the Fi generation, plants were allowed to self-pollenate to produce F3 families.Dose response

[0267] Plants were grown from the composite resistant seed lot and treated with several rates of dicamba. These rates included 8.75, 17.5, 35, 70. 140, 280, 560, and 1120 g dicamba ha’1for the 7710 population and 70. 140, 280. 560, 1120. 2240. and 4480 g dicamba ha’1for the M32 and 9425 populations. Six plants were used for each population at each herbicide rate. Visual injury' and survival were rated 21 days after treatment (DAT). The drc package (v3.0-l) in R (v4.0.2) was used to fit a 3-parameter log-logistic dose response curve, and the results were plotted using the plot function. Significant difference is in the ED50 parameter were determined using the compParm function from the drc package.SAUR21 expression

[0268] Young leaf tissue of M32 and 7710 plants either untreated or treated with 140 g dicamba ha’1was sampled six hours after treatment. Total RNA was extracted using the Direct-zol RNA Microprep kit (Zymo Research), and cDNA libraries were generated using the ProtoScript® II First Strand cDNA Synthesis Kit (New England Biolabs). The PerfeCTa SYBR® Green FastMix kit (QuantaBio) was used to quantify the expression of SAUR21 using actin as a reference gene (primer sequences listed in Table 3 A). ANOVA followed by Tukey’s test was used to detect significant differences between groups, and results were plotted with ggplot2 in R.

[0269] TABLE 3A. Quantitative PCR primers to quantify expression of various Arabidops is thaliana and Bassia scoparia genes. Thermocycler conditions for all primer sets are 95C for 3 minutes; 40 cycles of 95C for 15 seconds, 60C for 30 seconds, quantifying SYBR fluorescence after each round at 60C.Agent Ref. No. P14663WOOO

[0270] TABLE 3B. KASP primer set for identification of M32 allele of IAA16. The primer that binds to the wildtype has a FAM tail (bold) and the primer that binds the M32 allele has a HEX tail (underlined). Thermocycler conditions are 94C for 15 min; 10 cycles of 94C for 20 seconds, 61 C for 60 seconds and decreasing 0.6 C each cycle; 40 cycles of 94C for 20 seconds, 55 C for 60 seconds, quantifying FAM and HEX fluorescence at each round at 30C.

[0271] TABLE 3C. Polymerase chain reaction primers used to amplify BsIAA16 from cDNA libraries. Thermocycler conditions were 95 C for 5 minutes; 35 cycles of 95 C for 15 seconds, 60 C for 15 seconds, 72 C for 1 minute; 72 C for 5 minutes.Agent Ref. No. P14663WOOOHerbicide absorption and translocation profiles

[0272] Kochia plants from M32, 7710, and 9425 were germinated in a grow th chamber (60% relative humidity, 21 / 18°C, and 16 / 8 h photoperiod) in potting soil and transplanted to fine sand when they reached ~3 true leaves. The plants were irrigated with fertilizer until the plants reached 10 cm in height. At this point, aluminum foil was used to cover the second youngest fully expanded leaf while the plants were sprayed with 560 g dicamba ha' '. After spraying, the aluminum foil was removed, the leaf was then marked and treated with 10 uL of a14C -labeled dicamba solution (total radioactivity of 3.33 KBq or 200,000 dpm per plant). Plants were returned to the growth chamber until sampling at 3, 6. 12. 24, 48, 96, and 192 hours after treatment. The treated leaf, remaining above ground tissue, and below ground tissue of three biological replicates were separated for each population at each time point. The treated leaf was w ashed in 10% methanol and 1% NIS, and radioactivity in this wash solution was quantified in 10 mL of scintillation mixture (Ecoscint XR. National Diagnostics) using liquid scintillation spectrometry (Packard Tricarb 2300TR, Packard Instrument Co.). Plant tissue was dried in an oven at 60°C for at least 14 days before oxidation in a biological oxidizer (0X500; RJ Harvey Instrument Co.) followed by radioactivity measurement by liquid scintillation spectrometry. Absorption time series data was fitted to a rectangular hyperbolic model, and the parameters "absorption max” (A max) and “time to 90 percent absorption” (t90) were calculated and compared in R.QTL mapping

[0273] Plants from several F3 families derived from a biparental cross describe above were grown and treated with 560 g dicamba ha-1. Two families that segregated for survival following dicamba treatment (4-1-1 and 4-5-10, each from a different Fi hybridization event) were used for QTL mapping. Approximately 300 plants from each Fs family were grown, and a single young leaf was sampled and frozen in liquid nitrogen before treatment with 560 g dicamba ha'1. Visual injury was rated for each plant 21 DAT. and DNA was extracted from each plant (including the original parents of the cross) using the CTAB method (Doyle and Doyle, 1990). The concentration of each DNA sample was quantified using the Qubit™ dsDNA BR kit (Thermo Scientific) and diluted to 20 ng / ul. A doubledigest restriction-site associated DNA sequencing (ddRADseq) protocol was developed in conjunction with the University of Minnesota Genomics Center (Minneapolis, MN) byAgent Ref. No. P14663WOOO estimating the number of RAD sites across the reference genome for several enzyme combinations and then empirically testing the Btgl-TaqI combination on several samples at multiple sequencing depths. DNA from the two original parents and 103 plants from the 4- 5-10 F3 family were used for ddRADseq with Btgl and TaqI as the restriction enz mes with a target of 4 million 150bp single-end reads per sample. Library prep and sequencing was completed by the University of Minnesota Genomics Center.

[0274] Reads from each sample were passed through a variant calling pipeline that can be found at github.com / JMontl2 / dicamba_kochia. Briefly, raw reads were trimmed with Trimmomatic (v0.36) and aligned to the reference genome of kochia using the burrows- wheeler aligner (vO.7.17). The alignments were processed with samtools (vl . 15) and passed to GATK (v4.2.0) to call variants and filter them based on depth, quality, and strand bias. Variants were further filtered using custom scripts to only include biallelic single nucleotide polymorphisms that were homozygous and different between the two original parents. Filtered variants and phenotype data were used to conduct a genome scan within the qtl2 package in R (v0.32). Because a permutation test for significance was not possible, a significance threshold was established through a bonferroni correction of alpha=0.05 considering the number of tests to equal the number of filtered markers used in the scan then converting the adjusted p-value to LOD. The genotype around the QTL discovered on chr4 was determined for 287 plants from another F3 family (4-1-1) and 11 1 additional plants from 4-5-10 using a KASP assay developed for the IAA16 gene, which starts near basepair 87,385,000 on chr4 using the KASP-TF Standard ROX kit (LGC). Primer sequences are listed in Table 3B. A simple linear model was fitted using visual injury 21 DAT as the dependent variable and IAA16 genotype as the independent variable and plotted using ggplot2 (v3.4.4) in R.BSIAA16M32 sequencing

[0275] Total RNA was extracted using the Direct-zol RNA Microprep kit (Zymo Research) from 4-5-10 individuals that were homozygous for either the 7710 or M32 allele of IAA3 based on the results of KASP testing. This RNA was used to generate cDNA libraries for each sample using the PROTOSCRIPT® II First Strand cDNA Synthesis Kit (New England Biolabs). Primers that bind to the beginning and end of the BsIAA16 gene (sequences listed in Table 3C) were used with the EconoTaq PLUS Green kit (LGC) to amplify the full-length coding sequence. The product of each PCR reaction was run on aAgent Ref. No. P14663WOOO1% agarose gel to ensure a single product, then sent to Azenta for sanger sequencing from the forward and reverse primers.

[0276] To determine the genomic sequence around the M32 allele of BsIAA16, 2 grams of dark-treated leaf tissue from a plant that was homozygous for the M32 allele of IAA3 was sampled and flash frozen in liquid nitrogen. This tissue was sent to Corteva Agriscience Center for Genome Excellence for whole genome PacBio HiFi sequencing to a depth of ~30X. The resulting reads were assembled with HiFiasm, and BsIAA16 was located by BLAST.Characterization of transposable element insertion

[0277] To find similar elements to the one inserted in BSIAA16M32, the sequence of the 429 bp long terminal repeats (LTRs) of the insertion were aligned to the kochia reference genome assembly and to the newly created assembly described above using BLAST. Filters to identify the autonomous version of the element included: two alignments of >85% identify and >100bp length must be <25 kb, but >4 kb apart and in the direct orientation and containing all required retrotransposon domains. Necessary retrotransposon domains were identified manually and with TESorter. To classify the retro element, the autonomous version of the element was included in a phylogenetic tree with 25 complete Arabidopsis thaliana LTRs, representing each domain.Development of transgenic Arabidopsis thaliana lines

[0278] The coding sequence of the M32 and 7710 alleles of BsIAA16 were amplified from cDNA libraries discussed above using the PrimeSTAR MAX DNA Polymerase kit (Takara Bio USA, Inc.) and primers with tails that complement restriction sites in the expression vector p 'GC5941. Empty pb'GC5941 was digested with Asci and BamHl (New England Biolabs), and the BsIAA16 amplicons were ligated into the digested vector using the InFusion Cloning kit (Takara Bio USA, Inc.). Plasmid sequencing ensured correct assembly of expression vectors (Plasmidsaurus). Vectors containing the 7710 and M32 alleles of IAAJ6 were transformed into the GV3101 strain of Agrobacterium tumefaciens, and subsequently transfected into the Columbia genotype of Arabidopsis thaliana using the floral dip method. Transformants were selected by spraying Ti seedlings with BASTA herbicide (BASF) and confirmed with PCR for the transgene. Confirmed transformants were inbred for two generations, and Ts populations fixed for the transgene were identified by spraying seedlings with BASTA herbicide.Agent Ref. No. P14663WOOOEffect of dicamba on transgenic Arabidopsis lines

[0279] Seeds of Columbia and of fixed T3 lines of Arabidopsis, each from a different transformation event, were gas sterilized and plated on media plates containing synthetic or natural auxin. Ten seeds were used per population for each treatment. Root growth was measured seven days after moving plates to growing conditions, and ANOVA followed by Tukey’s test w as used to detect significant differences between groups. Results were plotted with ggplot2 in R.

[0280] Plants from select T3 lines w ere grown and either untreated or treated with 140 g dicamba ha1. Four plants of each population were used at each treatment level. Photos were taken 21 DAT to illustrate visual injury. Young leaf tissue was collected from treated and untreated plants six hours after treatment. Total RNA was extracted from these leaf samples using the Direct-zol RNA Microprep kit (Zymo Research), and cDNA libraries were generated using the PROTOSCRIPT® II First Strand cDNA Synthesis Kit (New England Biolabs). Relative expression was quantified for the BsIAA16 transgene and the auxin response genes AtlAAlQ and AtGH3.3 using AtCyclophil in as a reference gene (primer sequences listed in Table 3A). ANOVA followed by Tukey’s test was used to detect significant differences between groups, and results were plotted with ggplot2 in R. In silico docking ofIAA16 variants with TIR1

[0281] The crystal structure of the degron of IAA7 bound to TIR1 in the presence of 2,4-D (2pln) was downloaded from the RCSB Protein Data Bank. Mutagenesis of the G127 residue was conducted and visualized in Pymol.Example 5: Reduced binding of BsAUX / IAA16Mut to auxin receptor protein T1R1 provides a molecular explanation of resistance

[0282] To provide a molecular explanation of the role of BSAUX / IAA16MUI in dicamba resistance, binding of the Arabidopsis auxin receptor protein AtTIRl to the WT and mutant BsAUX / IAA16 degron sequences was measured using surface plasmon resonance (SPR). Binding w as auxin-dependent. In the presence of indole-3 acetic acid (IAA), binding of the WT peptide w as strong and persistent (dissociation of the co-receptor complex w as slow ). Binding of the WT peptide in the presence of 2,4-D and dicamba w as somewhat low er than with IAA with binding amplitudes at the end of the binding phase 56% and 21% of the IAA value, respectively. Binding to the mutant degron peptide was poorer in all respects.Agent Ref. No. P14663WOOOThe binding amplitudes were lower than for the WT degron (1AA = 37%, 2.4-D 18% and dicamba 5%), and the complexes dissociated more rapidly. In summary, the assembly of the co-receptor complex with the mutant degron was greatly reduced, and when formed, its lifetime was shorter. This would likely lead to far less ubiquitination, higher concentrations of BsAux / IAA16Mut remaining in the cell and reduced auxin signal strength. The greatly- reduced signals in the presence of the synthetic auxins 2,4-D and dicamba probably account for resistance as recorded in this line.Example 6: The BSIAA16M ut allele is associated with a reduction in fitness

[0283] Because lower binding of BsAUX IAA16iiut with TIR1 was observed in the presence of natural auxin, this change was expected to also confer a fitness penalty in the absence of synthetic auxin application. To formally test this, kochia plants w ere grown from an F3 family that is segregating for the BsAUX / IAA16 locus and fitness traits were measured without the application of auxin. The ratio of genotypes significantly differed from the expected 1:2: 1 (p<0.0001), with fewer than expected plants with the BsA UX / IAA 16uut allele (FIG. 5). All seedlings that emerged were used in the experiment, so we hypothesize that BSAUX / IAA16MUI causes reduced germination. Further, BSAU 1AA16MUI was associated with shorter plants, both at transplanting and at maturity- (FIG. 5A-B).However, BsAUX IAA16 did not have a significant effect on above ground biomass (FIG. 5C) or average seed weight (FIG. 5D). Thus, while the BsAUX / IAA16 ut allele is associated with reduced plant height, plants with this allele accumulate similar biomass and produce similarly sized seeds. Taken together, these results suggest this herbicide resistance mechanism does slightly reduce plant fitness under normal (non-selective) conditions, but not enough to cause it to be completely purged from the population.Nevertheless, evidence of a fitness cost associated with herbicide resistance has implications for kochia management. For instance, if dicamba is not used for several years, the frequency of BsAUX / IAA16uut is expected to decrease.Example 7 : Dicamba herbicide tolerance in soybean

[0284] For soybean testing, the mutIAA16 transgene w as synthesized by GeneScript that contained flanking sequence that complemented parts of a vector used by the Wisconsin Plant Innovation Center. Assembly of the final vector (RC9042, SEQ ID NO: 32) andAgent Ref. No. P14663WOOO transformation into soybean (genotype: Williams 82) was completed at the Wisconsin Plant Innovation Center. The transgene (Mut IAA16 CDS) was driven by the UBQ1 promotor and NOS terminator. Transgenic seed from 8 independent transformation events were shipped to Colorado State University, where seedlings were grown from each event and treated with a variety of dicamba treatments. These treatments included rates of 140 and 280 g dicamba ha-1 and used a earner volume of 20 gallons per acre. Plants were treated when the first trifoliate was fully developed (VI). Plants were visually inspected for injury and photos were taken seven days after treatment.

[0285] Several transgenic soybean lines showed little to no injury following dicamba treatment (FIG. 6). Expected symptoms in sensitive plants include twisting, swelling of stems, arrested growth, and downward turning of leaves. These were all observed in the wildtype line, while less severe or not present in several transgenic events. The events with the least injury include events 008, 011, and 013.References

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Claims

Agent Ref. No. P14663WOOOWhat is claimed is:

1. A modified plant, or a progeny, plant seed, plant part, or plant cell thereof, having tolerance to a synthetic auxin herbicide, the modified plant comprising a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region comprising SEQ ID NO: 5 or a conservatively modified variant thereof.

2. The modified plant, progeny, plant seed, plant part, or plant cell of claim 1, wherein the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

3. The modified plant, progeny, plant seed, plant part, or plant cell of claim 1, wherein the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%. at least 99%. or 100% sequence identity to SEQ ID NO: 2.

4. The modified plant, progeny, plant seed, plant part, or plant cell of claim 1, wherein the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region is operably linked to a heterologous promoter functional in a plant cell.

5. The modified plant, progeny, plant seed, plant part, or plant cell of claim 1, wherein the synthetic auxin herbicide is dicamba.

6. The modified plant, progeny, plant seed, plant part, or plant cell of claim 1, wherein the plant is a dicotyledonous or monocotyledonous plant.

7. The modified plant, progeny, plant seed, plant part, or plant cell of claim 1, wherein the plant is a maize, sorghum, wheat, sunflower, rice, soybean, cotton, canola, tobacco, tomato, potato, pepper, barley, alfalfa, sugar cane, or sugar beet plant.

8. The modified plant, progeny, plant seed, plant part, or plant cell of claim 1, wherein the plant is not a Bassia scoparia plant.Agent Ref. No. P14663WOOO9. The modified plant, progeny, plant seed, plant part, or plant cell of claim 1, wherein the modified plant further comprises a second herbicide-tolerant trait.

10. The modified plant, progeny, plant seed, plant part, or plant cell of claim 1, wherein the plant, progeny, plant seed, plant part, or plant cell is non- viable and / or non-regenerable.

11. A polynucleotide encoding an Aux / IAA polypeptide with a modified degron region comprising SEQ ID NO: 5 or a conservatively modified variant thereof.

12. The polynucleotide of claim 11, wherein the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

13. The polynucleotide of claim 11. wherein the polynucleotide encodes an Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

14. The polynucleotide of claim 11. wherein the polynucleotide is an isolated, synthetic, or recombinant polynucleotide.

15. An expression construct comprising the polynucleotide of claim 11 operably linked to a heterologous promoter functional in a plant cell.

16. A vector comprising the polynucleotide of claim 11.

17. A biological sample comprising the polynucleotide of claim 11.

18. A plant, plant part, plant seed, or plant cell comprising the polynucleotide of claim 11.

19. An Aux / IAA polypeptide with a modified degron region comprising SEQ ID NO: 5 or a conservatively modified variant thereof.Agent Ref. No. P14663WOOO20. The Aux / IAA polypeptide of claim 19, wherein the Aux / IAA polypeptide with the modified degron region comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.21 . A method for producing a plant with tolerance to a synthetic auxin herbicide, the method comprising: modifying an endogenous Aux / IAA gene in the plant to encode an Aux / IAA polypeptide with a modified degron region comprising SEQ ID NO: 5 or a conservatively modified variant thereof.

22. The method of claim 21, wherein the method comprises introducing a genome editing system that targets the endogenous Aux / IAA gene.

23. The method of claim 22, wherein the genome editing system comprises a CRISPR / Cas system, a TALEN, or a zinc finger nuclease.

24. The method of claim 21, wherein the Aux / IAA polypeptide with the modified degron region comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

25. The method of claim 21, wherein the endogenous Aux / IAA gene has at least 80%. at least 90%, at least 95%, at least 98%, at least 99%. or 100% sequence identity to SEQ ID NO: 4.

26. The method of claim 21, wherein the synthetic auxin herbicide is dicamba.

27. The method of claim 21, wherein the plant is a dicoty ledonous or monocotyledonous plant.Agent Ref. No. P14663WOOO28. The method of claim 21, wherein the plant is a maize, sorghum, wheat, sunflower, rice, soybean, cotton, canola, tobacco, tomato, potato, pepper, barley, alfalfa, sugar cane, or sugar beet plant.

29. A method for producing a plant with tolerance to a synthetic auxin herbicide, the method comprising: introducing to the plant a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region comprising SEQ ID NO: 5 or a conservatively modified variant thereof.

30. The method of claim 29, wherein the polynucleotide is operably linked to a heterologous promoter functional in a plant cell.

31. The method of claim 30 wherein said promote is an auxin induced promoter.

32. The method of claim 29, wherein the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

33. The method of claim 29, wherein the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%. at least 99%, or 100% sequence identity to SEQ ID NO: 2.

34. The method of claim 29, wherein the synthetic auxin herbicide is dicamba.

35. The method of claim 29, wherein the plant is a dicotyledonous or monocotyledonous plant.

36. The method of claim 29, wherein the plant is a maize, sorghum, wheat, sunflower, rice, soybean, cotton, canola, tobacco, tomato, potato, pepper, barley, alfalfa, sugar cane, or sugar beet plant.Agent Ref. No. P14663WOOO37. A method for controlling undesired vegetation at a plant cultivation site, the method comprising: providing at the site a plant that comprises a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region comprising SEQ ID NO: 5 or a conservatively modified variant thereof, wherein the polynucleotide confers to the plant tolerance to a synthetic auxin herbicide; and applying to the site an effective amount of the synthetic auxin herbicide.

38. The method of claim 37, wherein the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

39. The method of claim 37, wherein the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

40. The method of claim 37, wherein the synthetic auxin herbicide is dicamba.41 . The method of claim 37, wherein the plant is a dicotyledonous or monocotyledonous plant.

42. The method of claim 37, wherein the plant is a maize, sorghum, wheat, sunflower, rice, soybean, cotton, canola, tobacco, tomato, potato, pepper, barley, alfalfa, sugar cane, or sugar beet plant.

43. A method for controlling the growth of an herbicide resistant weed at a plant cultivation site, the method comprising: contacting the weed with a composition that reduces expression or activity of an Aux / IAA polypeptide with a degron region comprising SEQ ID NO: 5 or a conservatively modified variant thereof; and applying to the site an effective amount of the herbicide.Agent Ref. No. P14663WOOO44. The method of claim 43, wherein the composition comprises a polynucleotide that reduces expression or activity of the Aux / IAA polypeptide.

45. The method of claim 44, wherein the polynucleotide is a double-stranded RNA, a single-stranded RNA. or a double-stranded DNA / RNA hybrid polynucleotide.

46. The method of claim 44, wherein the polynucleotide comprises a sequence essentially identical or essentially complementary to at least 18 or more contiguous nucleotides of SEQ ID NO: 2.

47. The method of claim 44, wherein the polynucleotide has a length of 26-60 nucleotides.

48. The method of claim 43, wherein the composition comprises a chemical inhibitor that reduces expression or activity' of the Aux / IAA polypeptide.

49. The method of claim 43, wherein the synthetic auxin herbicide is dicamba.

50. The method of claim 43, wherein the weed is a Bassia scoparia plant.

51. The method of claim 43, wherein the composition comprises a permeability enhancing agent.

52. A commodity plant product prepared from the plant, plant part, plant seed, or plant cell of claim 1.

53. The commodity plant product of claim 52, wherein the product comprises the Aux / IAA polypeptide with the modified degron region or the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region.Agent Ref. No. P14663WOOO54. The commodity plant product of claim 52, wherein the product comprises fodder, seed meal, oil, milk, flour, grits, protein, tofu, miso, tempeh, fiber, starch, bio-composite building materials or seed-treatment-coated seed.

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

56. The method of claim 55, wherein the product comprises the Aux / IAA polypeptide with the modified degron region or the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region.

57. The method of claim 55, wherein the plant product comprises fodder, seed meal, oil, milk, flour, grits, protein, tofu, miso, tempeh, fiber, starch, bio-composite building materials, or seed-treatment-coated seeds.

58. A method for identifying a plant having tolerance to a synthetic auxin herbicide, the method comprising: obtaining a nucleic acid sample from a plant suspected of having the herbicide tolerance; detecting in the sample a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region comprising SEQ ID NO: 5 or a conservatively modified variant thereof; and determining that the plant is herbicide tolerant based on the presence of the polynucleotide.

59. The method of claim 58, wherein the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

60. The method of claim 58, wherein the polynucleotide encoding the Aux / IAA polypeptide with the modified degron region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.Agent Ref. No. P14663WOOO61. The method of claim 57, wherein the detecting comprises amplifying the polynucleotide using at least two primers.

62. The method of claim 57, wherein the plant is a Bassia scoparia plant.

63. The method of claim 57, wherein the plant is a maize, sorghum, wheat, sunflower, rice, soybean, cotton, canola, tobacco, tomato, potato, pepper, barley, alfalfa, sugar cane, or sugar beet plant.

64. A kit for identifying a plant having tolerance to a synthetic auxin herbicide, the kit comprising at least two primers, wherein the at least two primers recognize a polynucleotide encoding an Aux / IAA polypeptide with a modified degron region comprising SEQ ID NO: 5 or a conservatively modified variant thereof.

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