Methods and compositions for herbicide tolerance in plants

Abstract

Provided herein are novel methods and compositions for conferring auxin herbicide tolerance to plants. Also provided are herbicide-tolerant plants, seeds, cells, and plant parts comprising modified AUX / IAA proteins, and methods for their production.

Description

[0001] Citation of relevant applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 485,751, filed April 14, 2017, which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to the field of biotechnology. More specifically, it relates to compositions and methods for producing crop plants resistant to auxin herbicides. In particular, it relates to plants, plant tissues, and plant seeds containing modified KsIAA16 genes and proteins, said modified KsIAA16 genes and proteins providing resistance to auxin herbicides compared to unmodified KsIAA16 genes and proteins.

[0004] Merging of sequence lists

[0005] The file named "MONS406WO_ST25.txt" is located in... The 468-kilobyte file created on April 12, 2018, contains a sequence listing of 292 nucleotide sequences, which is hereby submitted electronically and incorporated in full by reference. Background Technology

[0006] Weeds are plants that compete with cultivated plants in agricultural environments, causing farmers billions of dollars in crop losses annually and requiring significant effort to control them. Weeds also serve as hosts for crop diseases and pests. Losses caused by weeds in agricultural production environments include: reduced crop yields, decreased crop quality, increased irrigation costs, increased harvesting costs, reduced land value, livestock damage, and damage to crops from insects and diseases that weeds harbor. The main ways weeds cause these effects are: 1) competing with crop plants for water, nutrients, sunlight, and other essential resources for growth and development; 2) producing toxic or irritating chemicals that cause health problems for humans or animals; 3) producing large quantities of seeds or vegetative propagation sites, or both, which contaminate agricultural products and allow these weed species to persist in agricultural land; and 4) generating large amounts of vegetation on both agricultural and non-agricultural land that requires treatment.

[0007] Chemical herbicides are frequently used to control the growth and spread of weeds. Chemical herbicides are active at one or more target sites in plants, where they inhibit normal plant functions. Auxins are important regulators of plant growth and development, involved in many developmental and response pathways, including cell division and elongation, plant patterning, root growth, vascular tissue development, leaf shape, phototropism, and gravitropism. Because this plant hormone is key to many important plant pathways, disturbances in auxin levels or responses often lead to plant death. This has led to the identification and commercialization of several compounds that mimic the function of the naturally occurring active auxin indole-3-acetic acid (IAA), making them effective herbicides. These auxin herbicides include compounds such as 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 2,4-dichlorophenoxyacetic acid (2,4-D). Crop plants resistant to auxin herbicides are required. Summary of the Invention

[0008] In one aspect, a modified plant or seed is provided, wherein the modified plant or seed is sensitive to at least one endogenous auxin, and wherein the modified plant or seed exhibits tolerance to at least one synthetic auxin herbicide. The endogenous auxin may be selected from the group consisting of IAA and NAA, and the synthetic auxin herbicide may be selected from the group consisting of dicamba, 2,4-D, picloram, triclid, quinclorac, and fluroxypyr. In some embodiments, the modified plant or seed includes at least one modification in an endogenous gene encoding an AUX / IAA protein, wherein said at least one modification confers tolerance to the auxin herbicide compared to a control plant without said at least one modification. In a further embodiment, the modification produces an AUX / IAA polypeptide comprising a modified degron domain containing motif X1WPPX2, wherein X1 is modified relative to the wild-type AUX / IAA polypeptide to be asparagine (N), isoleucine (I), or leucine (L), and wherein X2 is isoleucine (I), valine (V), leucine (L), or methionine (M). In a further embodiment, the modification produces an AUX / IAA polypeptide comprising an amino acid sequence having at least 90% identity with SEQ ID NO:219, wherein the modified plant or seed is not Kochia scoparia. The modification can produce an AUX / IAA polypeptide comprising the amino acid sequence SEQ ID NO:219, wherein the modified plant or seed is not Kochia scoparia. In some embodiments, the modified plant or seed is a crop plant or crop seed, such as a monocotyledonous crop selected from the group consisting of: corn, sorghum, wheat, rapeseed, soybean, cotton, grape, tomato, potato, lettuce, broccoli, cucumber, peanut, melon, leek, onion, rice, and barley, or a dicotyledonous crop selected from the group consisting of: soybean, alfalfa, sunflower, cotton, rapeseed, and sugar beet. The modification may result in increased tolerance to auxin herbicides compared to a control plant or seed without the modification.

[0009] In another aspect, a method for producing modified plants is provided, the method comprising the steps of: (a.) introducing a modification into the AUX / IAA gene of a plant cell; (b.) identifying and selecting one or more plant cells from step (a.) that contain the modification in the degradation determinant domain of AUX / IAA; and (c.) regenerating at least one plant from the at least one or more cells selected in step (b).

[0010] On the other hand, a modified DNA molecule is provided encoding a modified auxin / indole-3-acetic acid (AUX / IAA) polypeptide, wherein the AUX / IAA polypeptide comprises a modified degradation determinant domain containing motif X1WPPX2, wherein X1 is modified relative to the wild-type AUX / IAA polypeptide to asparagine (N), isoleucine (I), or leucine (L), and wherein X2 is isoleucine (I), valine (V), leucine (L), or methionine (M). In some embodiments, X1 has been modified from glycine to asparagine. In a further embodiment, the modified AUX / IAA polypeptide comprises a polypeptide sequence selected from the group consisting of: (a.) a sequence having at least 90% sequence identity with SEQ ID NO:2n-1, wherein n = 1-110, or any of SEQ ID NO:247-284; and (b.) a fragment of the sequence in (a.) having auxin / indole-3-acetic acid binding activity. In some embodiments, the modified AUX / IAA polypeptide comprises a polynucleotide sequence having at least 95% sequence identity with SEQ ID NO:220 or contains the sequence SEQ ID NO:220.

[0011] On the other hand, a DNA construct is provided comprising a modified DNA molecule, as described herein, operatively linked to a heterologous promoter. The promoter may be an inducible promoter or a promoter of a natural auxin / indole-3-acetic acid gene in a plant genome.

[0012] On the other hand, a recombinant plant, plant part, cell, or seed is provided, comprising the modified DNA molecule provided herein. A plant genome is also provided, comprising the modified DNA molecule provided herein.

[0013] On the other hand, a method for conferring tolerance to auxin herbicides to plants is provided, comprising expressing the modified DNA molecule described herein in the plant. The method may include transforming the plant or its progenitor cells with the modified DNA molecule. In some embodiments, the auxin herbicide is selected from the group consisting of: dicamba, 2,4-D, chlorpyrifos, chlorpyrifos, cypermethrin, and fluroxypyr.

[0014] In another embodiment, a method for producing auxin-resistant herbicide-tolerant plants is provided, the method comprising: modifying an AUX / IAA polypeptide of the plant to alter residue X1 in a degradation determinant domain comprising motif X1WPPX2 relative to SEQ ID NO:217, wherein X2 is isoleucine, valine, leucine, or methionine. In some embodiments, X1 is modified from glycine to asparagine, isoleucine, or leucine. In further embodiments, the modification comprises site-specific mutagenesis. In some embodiments, the degradation determinant domain is modified by a base-editing enzyme.

[0015] Plants produced by the methods described herein are also provided.

[0016] On the other hand, a method is provided for detecting plants exhibiting sensitivity to at least one endogenous auxin and tolerance to at least one auxin herbicide, comprising the steps of: a. obtaining a genomic DNA sample from said plant; b. amplifying said DNA sample using a first primer and a second primer to generate an amplicon; c. detecting in said amplicon a polynucleotide sequence encoding motif X1WPPX2 within SEQ ID NO:219. In some embodiments, the first primer has the sequence SEQ ID NO:221 and the second primer has the sequence SEQ ID NO:222. Detection may include restriction fragment length polymorphism (RFLP) analysis. In some embodiments, the first primer has the sequence SEQ ID NO:223 and the second primer has the sequence SEQ ID NO:224. Detection may further include using a probe selected from the group consisting of SEQ ID NO:225, SEQ ID NO:226, or a combination thereof. Attached Figure Description

[0017] Figure 1 The gene structure of KsIAA16 is shown, highlighting the nucleotide and amino acid differences between the sensitive (S) and resistant (R) alleles.

[0018] Figure 2This diagram shows a phylogenetic tree containing Arabidopsis AUX / IAA proteins and putative orthologous Kochia scoparia AUX / IAA proteins identified in transcriptomic sequences. The members of this phylogenetic tree are: AtIAA1, (SEQ ID NO:247); AtIAA2, (SEQ ID NO:248); AtIAA3, (SEQ ID NO:249); AtIAA4, (SEQ ID NO:250); AtIAA5, (SEQ ID NO:251); AtIAA6, (SEQ ID NO:252); AtIAA7, (SEQ ID NO:253); AtIAA8, (SEQ ID NO:254); AtIAA9, (SEQ ID NO:255); AtIAA10, (SEQ ID NO:256); AtIAA11, (SEQ ID NO:257); AtIAA12, (SEQ ID NO:258); AtIAA13, (SEQ ID NO:259); AtIAA14, (SEQ ID NO:260); AtIAA15, (SEQ ID NO:261); AtIAA16, (SEQ ID NO:247); AtIAA2, (SEQ ID NO:248); AtIAA3, (SEQ ID NO:249); AtIAA4, (SEQ ID NO:250); AtIAA5, (SEQ ID NO:251); AtIAA16, (SEQ ID NO:252); AtIAA7, (SEQ ID NO:253); AtIAA8, (SEQ ID NO:254); AtIAA9, (SEQ ID NO:255); AtIAA10, (SEQ ID NO:256); AtIAA11, (SEQ ID NO:257); AtIAA12, (SEQ ID NO:258); AtIAA13, (SEQ ID NO:259); AtIAA14, (SEQ ID NO:260); AtIAA15, (SEQ ID NO:261); AtIAA16 ...2, ID NO:262); AtIAA17, (SEQ ID NO:263); AtIAA18, (SEQ ID NO:264); AtIAA19, (SEQ ID NO:265); AtIAA20, (SEQ ID NO:266); AtIAA26, (SEQ ID NO:267); AtIAA27, (SEQ ID NO:268); AtIAA28, (SEQ ID NO:269); AtIAA29, (SEQ ID NO:270); AtIAA30, (SEQ ID NO:271); AtIAA31, (SEQ ID NO:272); AtIAA32, (SEQ ID NO:273); AtIAA33, (SEQ ID NO:274); AtIAA34, (SEQ ID NO:275); KsIAA1, (SEQ ID NO:276); KsIAA3, (SEQ ID NO:277); KsIAA6, (SEQ ID NO:278); KsIAA7, (SEQ ID NO:279); KsIAA8, (SEQ ID NO:280); KsIAA9, (SEQ ID NO:281); KsIAA13, (SEQ ID NO:282); KsIAA18, (SEQ ID NO:283); KsIAA31, (SEQ ID NO:284).

[0019] Figure 3 The following is a phylogenetic tree showing the amino acid sequences of orthologs of the KsIAA16 clade from the following plants: European rapeseed (Brassica napus), cultivated soybean (Glycine max), common wheat (Triticumaestivum), upland cotton (Gossypium hirsutum), bicolor sorghum (Sorghum bicolor), maize (Zeamays), Eurasian grape (Vitis vinifera), thick-skinned melon (Cucumis melo), potato (Solanum tuberosum), tomato (Solanum lycopersicum), lettuce (Lactuca sativa), cucumber (Cucumissativus), alfalfa (Medicago truncatula), and sunflower (Helianthus annuus).

[0020] Figure 4 It shows Figure 2 Multiple sequence alignment of representative members of the phylogenetic tree, where the boxed amino acids represent degradation determinant motifs.

[0021] Figure 5 The root elongation response of resistant (R) and sensitive (S) Kochia scoparia biotype seedlings was shown in the presence and absence of natural and synthetic auxins.

[0022] Figure 6: Shows the fresh weight (percentage of untreated control) of dicamba (Figure A), 2,4-D (Figure B) and fluroxypyr (Figure C). Detailed Implementation

[0023] Unless otherwise defined herein, terms should be understood according to their conventional usage by those skilled in the art. Examples of resources describing many of the molecular biology-related terms used herein can be found in Alberts et al., *Molecular Biology of the Cell*, 5th ed., Garland Science Publishing, Inc., New York, 2007; Rieger et al., *Glossary of Genetics: Classical and Molecular*, 5th ed., Springer-Verlag: New York, 1991; King et al., *A Dictionary of Genetics*, 6th ed., Oxford University Press: New York, 2002; and Lewin, *Genes IX*, Oxford University Press: New York, 2007. DNA base nomenclature as stated in Section 1.822 of 37 C.FR was used.

[0024] Plant hormones, specifically auxins, are important regulators of genes involved in many plant growth, development, 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 many compounds that act as effective herbicides.

[0025] Maize and other monocotyledonous crops are naturally tolerant to low levels of synthetic auxin herbicides, while dicotyledonous crops such as soybeans and cotton are highly susceptible. Efforts to develop auxin-tolerant varieties have focused on the heterologous expression of enzymes that inactivate auxin herbicides, thereby enabling herbicide tolerance in otherwise susceptible plants. For example, dicamba-tolerant soybean plants have been produced containing a gene from *Stenotrophomonas maltophilia* encoding dicamba monooxygenase (DMO), an enzyme that inactivates dicamba by converting it into the herbicide-inactive 3,6-dichlorosalicylic acid.

[0026] While this approach has been successful, recent advances in genome editing technology have sparked considerable interest in developing crops with modified traits. Regarding the trait of auxin herbicide tolerance, genome editing can be used to generate specific mutations that alter the auxin-sensing mechanism in plants, thereby making the plant resistant to the effects of synthetic auxins, including auxin herbicides, while remaining sensitive to endogenous auxins. This alteration in auxin sensing may not lead to any significant fitness effects. Without being bound by any particular theory, plants tolerant due to altered auxin sensing will not need to expend resources to highly express any metabolic proteins used for herbicide detoxification, which in turn will free up more resources for other growth traits.

[0027] To alter auxin sensing in tolerant plants using genome editing, it is essential to identify specific mutations that confer the desired auxin sensing phenotype. Extensive work has been done to identify and characterize mutations that alter auxin sensing in the model plant Arabidopsis thaliana; however, none of the known mutations have produced resistance to synthetic auxins while maintaining sensitivity to endogenous IAAs. Consequently, it has been found that most known mutations result in plant growth defects in Arabidopsis thaliana.

[0028] Auxin-resistant field isolates potentially provide a unique set of examples in which plants exhibit altered auxin perception yet retain sufficient fitness to reproduce under conditions more severe than typically provided by controlled environments. Previously, efforts to identify the genetic basis of auxin resistance in weed species from any field source have been hampered by the low frequency of resistance alleles and the slow spread of auxin-resistant weeds.

[0029] One particularly important agricultural weed is Kochia scoparia L. Schrad, a resilient dicotyledonous weed species. Although dicamba-resistant Kochia scoparia establishes and spreads extremely rapidly, the genetic basis of this resistance has not been reported. This disclosure provides the first evidence of the genetic basis for the dicamba-resistant phenotype exhibited by a Kochia scoparia biotype from Western Nebraska. Surprisingly, the inventors have demonstrated that dicamba resistance in this Kochia scoparia biotype is due to a two-nucleotide base change that results in a glycine-to-asparagine amino acid shift within the highly conserved "GWPPV / I" motif of the auxin / indole-3-acetic acid (AUX / IAA) protein, which is most similar to IAA16 in Arabidopsis thaliana. Furthermore, it has been demonstrated that the mutant IAA16 protein from the resistant Kochia scoparia biotype appears to possess altered auxin sensing.

[0030] As used herein, the singular and singular forms of the terms “a,” “an,” and “the” include, for example, plural objects unless the context clearly specifies otherwise. Thus, for example, references to “plant,” “the plant,” or “a plant” also include multiple plants; similarly, depending on the context, the use of the term “plant” may also include offspring that are genetically similar to or identical to said plant; indeed, the use of the term “nucleic acid” optionally includes many copies of said nucleic acid molecule; similarly, the term “probe” optionally (and generally) covers many similar or identical probe molecules.

[0031] As used herein, "plant" means the whole plant, any part thereof, or a cell or tissue culture derived from a plant, including any of the following: the whole plant, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and / or their progeny. Progeny plants can be derived from any filiform generation, such as F1, F2, F3, F4, F5, F6, F7, etc. Plant cells are the biological cells of a plant, obtained from the plant or by culturing cells derived from the plant.

[0032] As used herein, “modification” in plants, seeds, plant components, plant cells, and plant genomes refers to a state containing changes or variations from their natural or original state. For example, the “natural transcript” of a gene refers to the RNA transcript produced by an unmodified gene. Typically, natural transcripts are sense transcripts. Modified plants or seeds contain molecular changes in their genetic material, including genetic or epigenetic modifications. Typically, modified plants or seeds, or their parental or progenitor cell lines, have undergone mutagenesis, genome editing (e.g., but not limited to methods using site-specific nucleases), genetic transformation (e.g., but not limited to methods using Agrobacterium-mediated transformation or particle bombardment), or a combination thereof. On one hand, the modified plants described herein do not contain non-plant genetic material or sequences. On the other hand, the modified plants described herein do not contain interspecific genetic material or sequences.

[0033] As used herein, an "endogenous gene" or a "natural copy" of a gene refers to a gene that originates from a given organism, cell, tissue, genome, or chromosome. An "endogenous gene" or a "natural copy" of a gene is a gene that has not been previously modified by humans. Similarly, an "endogenous protein" refers to a protein encoded by an endogenous gene.

[0034] As used herein, “synthetic auxin herbicides” or “auxin herbicides” means any herbicide that produces herbicidal activity by mimicking endogenous plant auxins or inhibiting the removal of auxin compounds from cells. Examples of synthetic auxin herbicides include benzoic acid, phenoxycarboxylic acid, pyridinecarboxylic acid, quinolinecarboxylic acid, hemicarbazone, diflufenzopyr, 2,4-D, 2,4-DB, MCPA, MCPB, mecoprop, dicamba, clopyralid, fluroxypyr, chlorpyrifos, aminopyralid, aminocyclopyrachlor, and cypermethrin.

[0035] In one aspect, this disclosure provides non-transgenic plants containing synthetic mutations in the degradation determinant domain of the AUX / IAA protein. In another aspect, this disclosure provides non-transgenic plants containing synthetic mutations in the degradation determinant domain of the IAA16 protein. As used herein, the term "synthetic mutation" refers to a non-spontaneous mutation that occurs due to genomic modification, for example, by site-specific genomic modifying enzymes.

[0036] Several embodiments relate to a modified auxin / indole-3-acetic acid (AUX / IAA) polypeptide comprising a modified degradation determinant domain containing a motif X1WPPX2, wherein X1 has been modified from glycine to asparagine, isoleucine, or leucine, and wherein X2 is isoleucine, valine, leucine, or methionine. Several embodiments also relate to nucleic acid sequences encoding a modified auxin / indole-3-acetic acid (AUX / IAA) polypeptide comprising a modified degradation determinant domain containing a motif X1WPPX2, wherein X1 has been modified from glycine to asparagine, isoleucine, or leucine, and wherein X2 is isoleucine, valine, leucine, or methionine. Several embodiments relate to plants, plant parts, plant cells, or plant seeds that contain a nucleic acid sequence encoding a modified auxin / indole-3-acetic acid (AUX / IAA) polypeptide and / or a modified auxin / indole-3-acetic acid (AUX / IAA) polypeptide, said AUX / IAA polypeptide comprising a modified degradation determinant domain containing a motif X1WPPX2, wherein X1 has been modified from glycine to asparagine, isoleucine, or leucine, and wherein X2 is isoleucine, valine, leucine, or methionine. In some embodiments, a plant, plant part, plant cell, or plant seed comprises a modified auxin / indole-3-acetic acid (AUX / IAA) polypeptide containing a modified degradation determinant domain comprising a motif X1WPPX2, wherein X1 has been modified from glycine to asparagine, isoleucine, or leucine, and wherein X2 is isoleucine, valine, leucine, or methionine. The plant, plant part, plant cell, or plant seed is resistant to the effects of synthetic plant auxins, including auxin herbicides, but remains sensitive to endogenous auxins.

[0037] On the one hand, modifications to nucleic acid molecules encoding proteins result in at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 missense mutations compared to the unmodified sequence. On the other hand, modifications to nucleic acid molecules encoding AUX / IAA proteins result in at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 missense mutations compared to the unmodified sequence. As used herein, a “missense mutation” refers to a single nucleotide change that results in a codon encoding a different amino acid. For example, the codon “CGU” encodes the amino acid arginine. If a missense mutation changes G to U, producing the "CUU" codon, then that codon now encodes the leucine amino acid. Missense mutations can be caused by insertions, deletions, substitutions, or inversions.

[0038] In further embodiments, methods are provided for designing and generating crop plants that exhibit tolerance to auxin herbicides. In some embodiments, methods are provided for designing plants that exhibit tolerance to auxin herbicides by modifying the genome of plant cells to alter genes involved in auxin herbicide tolerance, as described herein. Genome modification can be accomplished through targeted mutagenesis as described herein.

[0039] Several embodiments involve modified auxin / indole-3-acetic acid (AUX / IAA) peptides capable of providing auxin resistance to plants. Several embodiments involve nucleic acid sequences encoding the modified auxin / indole-3-acetic acid (AUX / IAA) peptides, and compositions and methods using the modified peptides. In some embodiments, the nucleic acid sequence encoding the modified auxin / indole-3-acetic acid (AUX / IAA) peptide provides tolerance to auxin herbicides in plants containing recombinant DNA molecules.

[0040] Several embodiments involve nucleic acid molecules, polynucleotides, peptides, proteins, cells, seeds, or plants produced through genetic engineering, and therefore they are not typically found in nature and are produced through artificial intervention. Several embodiments involve plant genomes comprising DNA sequences that are not naturally present in plant genomes and are produced through artificial intervention. In some embodiments, the DNA sequence provided herein encodes a modified AUX / IAA peptide comprising a modified degradation determinant domain. In some embodiments, a modified AUX / IAA peptide is provided that includes a change relative to the peptide encoded by KsIAA16S (SEQ ID NO: 218) in the degradation determinant domain comprising motif X1WPPX2. In some embodiments, a recombinant peptide comprising a modified degradation determinant domain containing motif X1WPPX2 is provided. ， X1 has been modified from glycine to a different amino acid, for example, a hydrophilic amino acid such as arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, or threonine, and X2 is a hydrophobic amino acid such as leucine or isoleucine. In some embodiments, a recombinant polypeptide comprising a modified degradation determinant domain containing motif X1WPPX2 is provided, wherein X1 has been modified from glycine to asparagine, isoleucine, or leucine, and X2 is isoleucine, valine, leucine, or methionine. In some embodiments, a polypeptide comprising any of the following polypeptide sequences: any one of SEQ ID NO:2n-1, where n = 1-110, or any one of SEQ ID NO:247-284, has been modified to have a modified degradation determinant domain comprising motif X1WPPX2, wherein X1 has been modified from glycine to asparagine, isoleucine, or leucine, and X2 is isoleucine, valine, leucine, or methionine.

[0041] Several embodiments involve polynucleotides encoding polypeptide sequences capable of conferring altered auxin sensing in plants, such as any of the following polypeptides: SEQ ID NO:2n-1, where n = 1-110, or any of SEQ ID NO:247-284, which have been modified to have a modified degradation determinant domain comprising the motif X1WPPX2, wherein X1 has been modified from glycine to asparagine, isoleucine, or leucine, and X2 is isoleucine, valine, leucine, or methionine. In some embodiments, the polynucleotide comprises the polynucleotide sequence SEQ ID NO:220 (KsIAA16R). In some embodiments, plants, plant parts, plant cells, or seeds comprising the polynucleotide or polypeptide sequences described herein are provided.

[0042] In some embodiments, the nucleic acid molecule may be synthesized and modified, either wholly or partially, by methods known in the art, wherein it is desirable to provide sequences that can be used for DNA manipulation (such as restriction enzyme recognition sites or recombination-based cloning sites), plant-preferred sequences (such as plant codon-used or Kozak concordant sequences), or sequences that can be used for DNA construct design (such as spacer regions or adapter sequences). In some embodiments, a nucleic acid molecule is provided that has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with the polynucleotide sequence SEQ ID NO:2n-1, where n = 1-110, and is capable of conferring altered auxin sensing in plants, thereby conferring tolerance to auxin herbicides.

[0043] Several embodiments involve polypeptide sequences that have at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with a polynucleotide sequence SEQ ID NO:2n-1, where n = 1-110, and are capable of conferring altered auxin sensing to plants, thereby conferring tolerance to auxin herbicides.

[0044] In some embodiments, fragments of polynucleotide or polypeptide sequences capable of conferring altered auxin sensing in plants, thereby conferring tolerance to auxin herbicides, are provided. In a particular embodiment, a fragment of any of SEQ ID NO:2n-1, wherein n = 1-110, is provided, comprising at least about 50, 95, 150, 250, 500, 750, or at least about 1000 consecutive nucleotides or longer of any of SEQ ID NO:2n-1, wherein n = 1-110, and capable of conferring altered auxin sensing in plants, thereby conferring tolerance to auxin herbicides.

[0045] The AUX / IAA protein family comprises four conserved domains, with domain II comprising a 17-amino acid sequence as a degradation determinant domain that confers auxin-dependent degradation. Specifically, this degradation determinant domain contains the motif "GWPPV / I," which is strictly conserved across all AUX / IAA proteins. In some embodiments, a polynucleotide encoding an AUX / IAA polypeptide is provided, the AUX / IAA polypeptide comprising a fragment or variant of the amino acid sequence provided herein and comprising a modified degradation determinant domain containing the motif X1WPPX2, wherein the modification of the degradation determinant domain provides a polypeptide capable of conferring altered auxin sensing in plants, thereby conferring tolerance to synthetic auxin herbicides. In some embodiments, the polypeptide encoded by the polynucleotide comprises a fragment or variant of the amino acid sequence provided herein and comprises a modified degradation determinant domain containing the motif X1WPPX2, wherein X1 has been modified from glycine to asparagine, isoleucine, or leucine, and X2 is isoleucine, valine, leucine, or methionine.

[0046] As used herein, the term “DNA” or “DNA molecule” refers to a double-stranded DNA molecule of genomic or synthetic origin, that is, a polymer or polynucleotide molecule of deoxyribonucleotide bases 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. The nomenclature used herein corresponds to Title 37 of Section 1.822 of the Code of Federal Regulations and is stated in the tables of WIPO Standard ST.25 (1998), Annex 2, Tables 1 and 3.

[0047] As used herein, the term "isolated DNA molecule" refers to a DNA molecule that is at least partially separated from other molecules that are normally associated with it in their original or native state. In one embodiment, the term "isolated" refers to a DNA molecule that is at least partially separated from some nucleic acids that are normally side-attached to DNA molecules in their original or native state. Thus, DNA molecules fused, for example, to regulatory or coding sequences that are normally unrelated, are considered isolated herein. Such molecules are also considered isolated when integrated into the chromosome of a host cell or when present in a nucleic acid solution with other DNA molecules, because they are not in their native state.

[0048] As disclosed herein, numerous methods known to those skilled in the art can be used to isolate and manipulate polynucleotides or fragments thereof. For example, polymerase chain reaction (PCR) technology can be used to amplify specific starting polynucleotide molecules and / or produce variants of the original molecule. Polynucleotide molecules or fragments thereof can also be obtained by other techniques, such as direct synthesis of fragments by chemical methods, as commonly practiced using automated oligonucleotide synthesizers. Polynucleotides can be single-stranded (ss) or double-stranded (ds). “Double-stranded” refers to base pairing that typically occurs between sufficiently complementary antiparallel nucleic acid strands under physiologically relevant conditions to form a double-stranded nucleic acid structure. Embodiments of the methods include embodiments in which the polynucleotide is at least one selected from the group consisting of: positive single-stranded DNA (ssDNA), positive single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), double-stranded DNA / RNA hybrids, antisense ssDNA, or antisense ssRNA; mixtures of any of these types of polynucleotides can be used.

[0049] As used herein, the terms "sequence identity percentage" or "sequence identity %" refer to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference ("query") sequence (or its complement) compared to the test ("target") sequence (or its complement) when two sequences are optimally aligned (provided that the total number of appropriate nucleotide or amino acid insertions, deletions, or gaps on the comparison window is less than 20% of the reference sequence). Optimal alignment of sequences for the comparison window is well known to those skilled in the art and can be achieved using tools such as Smith and Waterman's local homology algorithm, Needleman and Wunsch's homology alignment algorithm, Pearson and Lipman's similarity search method, and computerized implementations of these algorithms, such as those using default parameters. Wisconsin The sequence analysis software packages available as part of Accelrys (San Diego, CA), MEGAlign (DNAStar, Madison, WI), and MUSCLE (version 3.6) (Edgar, “MUSCLE: multiple sequence alignment with high accuracy and high throughput” Nucleic Acids Research 32(5):1792-7(2004)) are GAP, BESTFIT, FASTA, and TFASTA. The “identity score” of the aligned fragments of the test and reference sequences is calculated by dividing the number of identical components shared by the two aligned sequences by the total number of components in the portion of the reference sequence fragment being compared (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). The sequence identity percentage is expressed as the identity score multiplied by 100. Comparisons of one or more sequences can be performed on the full-length sequence or a portion thereof, or on a longer sequence.

[0050] As used herein, the term "protein-coding polynucleotide molecule" refers to a polynucleotide molecule containing a nucleotide sequence that encodes a protein. "Protein-coding sequence" means a polynucleotide sequence that encodes a protein. Such polynucleotides are at least one selected from the group consisting of: positive single-stranded DNA (ssDNA), positive single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), double-stranded DNA / RNA hybrids, antisense ssDNA, or antisense ssRNA; or any mixture of these types of polynucleotides.

[0051] “Sequence” refers to the sequential arrangement of nucleotides or amino acids. The boundaries of a protein-coding sequence can be determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. In some embodiments, a protein-coding molecule may contain a DNA sequence encoding a protein sequence. In some embodiments, a protein-coding molecule may contain an RNA sequence encoding a protein sequence. As used herein, “transgenic expression,” “expressing a transgenic,” “protein expression,” and “expressing a protein” refer to the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into a polypeptide chain (which ultimately folds into a protein).

[0052] Due to the degeneracy of the genetic code, a variety of different polynucleotide sequences can encode polypeptides, such as the modified polypeptides disclosed herein. A table is described in Lewin (1985) showing all possible triplet codons (where U also substitutes for T) and the amino acid encoded by each codon. Furthermore, alternative polynucleotide sequences that produce polypeptides encoding the same or substantially the same modifications described herein are entirely within the capabilities of those skilled in the art. These variants or alternative polynucleotide sequences are within the scope of the embodiments described herein. As used herein, the reference to “substantially the same” means a sequence with amino acid substitutions, deletions, additions, or insertions that do not substantially alter the functional activity of the polypeptide encoded by the polynucleotide of the embodiments described herein. Allelic variants of the nucleotide sequences encoding wild-type or modified polypeptides are also covered within the scope of the embodiments described herein. Substitutions of amino acids other than those specifically exemplified or naturally occurring in wild-type or modified AUX / IAA polypeptides are also considered within the scope of the embodiments described herein, provided that the polypeptide with said substitutions retains substantially the same functional activity (e.g., selective auxin sensing).

[0053] I. Genome Editing

[0054] Targeted modifications of the plant genome using genome editing methods can be used to produce improved plant lines by modifying plant genomic DNA. Additionally, genome editing methods can allow one or more target nucleic acids to be inserted into the plant genome. Examples of methods for introducing donor polynucleotides into the plant genome or modifying the plant's genomic DNA include the use of sequence-specific nucleases, such as zinc finger nucleases, engineered or naturally occurring broad-spectrum nucleases, TALE endonucleases, or RNA-guided endonucleases (e.g., the regularly spaced clustered short palindromic repeats (CRISPR) / Cas9 system, CRISPR / Cpf1 system, CRISPR / CasX system, CRISPR / CasY system, CRISPR / Cascade system). Several embodiments relate to genome editing methods that introduce precise base-pair modifications into the plant genome using single-stranded oligonucleotides, as described by Sauer et al. (Plant Physiol. 2016 Apr; 170(4):1917–1928). Genome editing methods for modifying, deleting, or inserting nucleic acid sequences into genomic DNA are known in the art.

[0055] Several embodiments involve a CRISPR / Cas9 system for modifying or replacing existing coding sequences in a plant genome, such as sequences encoding AUX / IAA peptides. Several embodiments involve a CRISPR / Cpf1 system for modifying or replacing existing coding sequences in a plant genome, such as sequences encoding AUX / IAA peptides. Several embodiments involve Cas9-cytidine deaminase for modifying existing coding sequences in a plant genome, such as sequences encoding AUX / IAA peptides. Several embodiments involve Cpf1-cytidine deaminase for modifying existing coding sequences in a plant genome, such as sequences encoding AUX / IAA peptides. In a further embodiment, a transcription activator-like effector (TALE) is used to modify or replace existing coding sequences in a plant genome, such as sequences encoding AUX / IAA peptides. In some embodiments, existing AUX / IAA peptide coding sequences in a plant genome are modified using non-template genome editing with sequence-specific nucleases. In some embodiments, existing AUX / IAA peptide coding sequences in a plant genome are modified using template genome editing with sequence-specific nucleases. In some embodiments, a sequence-specific nuclease and a donor nucleic acid encoding a modified degradation determinant domain are introduced into plant cells. In some embodiments, the modified degradation determinant domain encodes an X1WPPX2 motif that confers tolerance to auxin herbicides to the plant. In some embodiments, X1 has been modified from glycine to asparagine, isoleucine, or leucine. In some embodiments, X2 is isoleucine, valine, leucine, or methionine. Modification or substitution of the endogenous AUX / IAA coding sequence according to the methods provided herein results in the expression of a polypeptide containing a modified degradation determinant domain comprising the motif X1WPPX2, for example, wherein one or more of X1 and X2 have been altered. Several embodiments therefore involve providing a sequence-specific nuclease capable of recognizing specific target nucleotide sequences (such as AUX / IAA sequences) in the plant genome to achieve alteration of the AUX / IAA sequence through non-template editing or integration of the donor nucleic acid at said site.

[0056] Modification of the AUX / IAA encoding sequence, such as one of SEQ ID NO:2n-1 provided herein, where n = 1-110, can produce a sequence encoding a polypeptide comprising a modified degradation determinant domain containing the motif X1WPPX2 as described herein, which confers altered auxin sensing in plants, thereby conferring tolerance to auxin herbicides. In some embodiments, X1 has been modified from glycine to asparagine, isoleucine, or leucine. In some embodiments, X2 is isoleucine, valine, leucine, or methionine.

[0057] On the one hand, “modification” includes the insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. On the other hand, "modification" includes the deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. On the other hand, "modification" includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotide inversions. On the other hand, "modification" includes substitutions of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. In some embodiments, "modification" includes replacing "C," "G," or "T" with "A" in the nucleic acid sequence. In some embodiments, "modification" includes replacing "A," "G," or "T" with "C" in the nucleic acid sequence. In some embodiments, "modification" includes replacing "A," "C," or "T" with "G" in the nucleic acid sequence. In some implementations, "modification" includes replacing "A", "C", or "G" with "T" in the nucleic acid sequence. In some implementations, "modification" includes replacing "U" with "C" in the nucleic acid sequence. In some implementations, "modification" includes replacing "A" with "G" in the nucleic acid sequence. In some implementations, "modification" includes replacing "G" with "A" in the nucleic acid sequence.In some implementations, "modification" includes replacing "C" with "T" in the nucleic acid sequence.

[0058] In some embodiments, a recombinant DNA construct is provided, such as a transgene or expression cassette containing a nucleic acid sequence encoding an AUX / IAA protein. In some embodiments, the AUX / IAA protein is a homolog of IAA16. In some embodiments, the nucleic acid sequence encodes an AUX / IAA protein containing a modified degradation determinant domain. In some embodiments, the modified degradation determinant domain contains an X1WPPX2 motif capable of conferring plant tolerance to auxin herbicides. In some embodiments, X1 has been modified from glycine to asparagine, isoleucine, or leucine. In some embodiments, X2 is isoleucine, valine, leucine, or methionine. In some embodiments, the transgene or expression cassette contains a promoter derived from an endogenous promoter operatively linked to an IAA gene encoding the AUX / IAA coding sequence. In some embodiments, the transgene or expression cassette can be inserted into or integrated into a locus within the genome of a plant or plant cell. In some embodiments, the transgene or expression cassette can be inserted into or integrated into a specific site or locus within the genome of a plant or plant cell via site-directed integration. In some embodiments, the recombinant DNA construct comprises a nucleotide sequence encoding a polypeptide of SEQ ID NO:2n-1, where n = 1-110, which has been modified to have a modified degradation determinant domain comprising motif X1WPPX2; or a sequence encoding a fragment of such polypeptide. In some embodiments, the recombinant DNA construct may further comprise one or two homologous arms flanking an insert sequence (i.e., a sequence, transgene, cassette, etc., to be inserted into the plant genome).

[0059] Several implementations involve recombinant DNA constructs containing expression cassettes encoding site-specific nucleases and / or any associated proteins for genomic modification. These nuclease expression cassettes may be present in the same molecule or vector as the donor template used for template editing, or in expression cassettes containing nucleic acid sequences encoding AUX / IAA proteins as described herein (cis), or in separate molecules or vectors (trans). Several methods for site-directed integration are known in the art, involving the cleavage of genomic DNA to generate double-strand breaks (DSBs) or gaps at desired genomic sites or loci, using different sequence-specific nucleases (or complexes of proteins and / or guide RNA). As understood in the art, during the repair of DSBs or gaps introduced by nucleases, donor template DNA, transgenes, or expression cassettes may be integrated into the genome at the site of the DSB or gap. Although insertion events can occur via non-homologous end joining (NHEJ), the presence of homologous arms in the DNA to be integrated can facilitate the adoption and targeting of the inserted sequence in the plant genome during the repair process through homologous recombination. Examples of site-specific nucleases that can be used include zinc finger nucleases, engineered or naturally occurring broad-spectrum nucleases, TALE endonucleases, and RNA-guided endonucleases (e.g., Cas9, CasX, CasY, or Cpf1). For methods using RNA-guided site-specific nucleases (e.g., Cas9, CasX, CasY, or Cpf1), the recombinant DNA construct may also contain sequences encoding one or more guide RNAs to guide the nuclease to the desired site within the plant genome. In some embodiments, one or more guide RNAs (cis-) may be provided on a separate molecule or vector.

[0060] II. Site-specific genome-modifying enzymes

[0061] As used herein, the term "double-strand break inducer" refers to any agent that can induce double-strand breaks (DSBs) in DNA molecules. In some embodiments, the double-strand break inducer is a site-specific genome-modifying enzyme.

[0062] As used herein, the term "site-specific genome-modifying enzyme" refers to any enzyme capable of modifying a nucleotide sequence in a sequence-specific manner. In some embodiments, a site-specific genome-modifying enzyme modifies the genome by inducing single-strand breaks. In some embodiments, a site-specific genome-modifying enzyme modifies the genome by inducing double-strand breaks. In some embodiments, a site-specific genome-modifying enzyme comprises cytidine deaminase. In some embodiments, a site-specific genome-modifying enzyme comprises adenine deaminase. In this disclosure, site-specific genome-modifying enzymes include endonucleases, recombinases, transposases, deaminases, helicases, and any combination thereof. In some embodiments, a site-specific genome-modifying enzyme is a sequence-specific nuclease.

[0063] Several embodiments involve promoting recombination by providing plant cells with site-specific genome-modifying enzymes, such as sequence-specific nucleases. In some embodiments, recombination is promoted by providing a single-strand break inducer. In some embodiments, recombination is promoted by providing a double-strand break inducer. In some embodiments, recombination is promoted by providing a strand separation inducer. In one aspect, the site-specific genome-modifying enzyme is selected from endonucleases, recombinases, transposases, deaminases, helicases, or any combination thereof. In some embodiments, recombination occurs between chromosome B. In some embodiments, recombination occurs between chromosome B and chromosome A.

[0064] Several embodiments involve facilitating the integration of one or more target DNAs by providing a site-specific genome-modifying enzyme. In some embodiments, the integration of one or more target DNAs is facilitated by providing a single-strand break inducer. In some embodiments, the integration of one or more target DNAs is facilitated by providing a double-strand break inducer. In some embodiments, the integration of one or more target DNAs is facilitated by providing a strand separation inducer. In one aspect, the site-specific genome-modifying enzyme is selected from endonucleases, recombinases, transposases, helicases, or any combination thereof.

[0065] On the one hand, the endonuclease is selected from a wide range of nucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), Argonaute proteins (non-restricted examples of Argonaute proteins include: Argonaute protein (TtAgo) from *Thermus thermophilus*, Argonaute protein (PfAgo) from *Pyrococcus furiosus*, Argonaute protein (NgAgo) from *Natronobacterium gregoryi*), and RNA-guided nucleases such as CRISPR-associated nucleases (non-restricted examples of CRISPR-associated nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, and Csc1). Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, Mad7, their homologs or their modified forms).

[0066] In some embodiments, the site-specific genome-modifying enzyme is a dCas9-Fok1 fusion protein. In another embodiment, the site-specific genome-modifying enzyme is a dCas9-recombinase fusion protein. As used herein, “dCas9” refers to a Cas9 endonuclease protein having one or more amino acid mutations that result in the Cas9 protein lacking endonuclease activity but retaining RNA-guided site-specific DNA binding. As used herein, “dCas9-recombinase fusion protein” is dCas9 fused to a protein in a manner in which the recombinase catalyzes DNA.

[0067] In some embodiments, the site-specific genome-modifying enzyme is a dCas9-cytosine deaminase fusion protein. In another embodiment, the site-specific genome-modifying enzyme is a dCas9-adenine deaminase fusion protein. In some embodiments, one or more of the dCas9-cytosine deaminase and dCas9-adenine deaminase fusion proteins are used to modify the nucleic acid sequence encoding the degradation determinant domain of the AUX / IAA protein. In some embodiments, the nucleic acid sequence encoding the degradation determinant domain is modified to encode a degradation determinant domain containing the motif X1WPPX2, wherein X1 has been modified from glycine to asparagine, isoleucine, or leucine, and X2 is isoleucine, valine, leucine, or methionine. In some embodiments, the AUX / IAA protein is an ortholog of KsIAA16.

[0068] In some embodiments, the site-specific genome-modifying enzyme is a dCpf1-cytosine deaminase fusion protein. In another embodiment, the site-specific genome-modifying enzyme is a dCpf1-adenine deaminase fusion protein. In some embodiments, one or more of the dCpf1-cytosine deaminase and dCpf1-adenine deaminase fusion proteins are used to modify the nucleic acid sequence encoding the degradation determinant domain of the AUX / IAA protein. In some embodiments, the nucleic acid sequence encoding the degradation determinant domain is modified to encode a degradation determinant domain containing the motif X1WPPX2, wherein X1 has been modified from glycine to asparagine, isoleucine, or leucine, and X2 is isoleucine, valine, leucine, or methionine. In some embodiments, the AUX / IAA protein is an ortholog of KsIAA16.

[0069] In some embodiments, the site-specific genome-modifying enzyme is a recombinase. Non-limiting examples of recombinases include tyrosine recombinases linked to the DNA recognition motifs provided herein, selected from the group consisting of Cre recombinases, Gin recombinases, Flp recombinases, and Tnp1 recombinases. In one aspect, the Cre or Gin recombinases provided herein are linked to a zinc finger DNA-binding domain, a TALE DNA-binding domain, or a Cas9 nuclease. In another aspect, serine recombinases linked to the DNA recognition motifs provided herein are selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. In yet another aspect, DNA transposases linked to the DNA-binding domains provided herein are selected from the group consisting of TALE-piggyBac and TALE-Mutator.

[0070] Site-specific genome-modifying enzymes, such as broad-spectrum nucleases, ZFN, TALEN, Argonaute proteins (non-restrictive examples of Argonaute proteins include Argonaute proteins from *Thermophila thermophila* (TtAgo), Argonaute proteins from *Extremophila* (PfAgo), Argonaute proteins from *Haloxybacterium gargearii* (NgAgo), their homologs or modified forms thereof), RNA-guided nucleases (non-restrictive examples of RNA-guided nucleases include CRISPR-related nucleases such as Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas... 10. Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, Mad7, their homologs or modified forms thereof) and engineered RNA-guided nucleases (RGN) induce genome modifications, such as double-stranded DNA breaks (DSBs) or single-stranded DNA breaks at genome sequence target sites. In some implementations, breaks or gaps in the target DNA sequence are repaired through natural processes such as homologous recombination (HR) or non-homologous end joining (NHEJ). In some implementations, sequence modifications occur at or near the site of the cleavage or gap, and may include deletions or insertions that result in nucleic acid sequence modifications, or integration of exogenous nucleic acids through homologous recombination or NHEJ.

[0071] By introducing target DNA and the provided site-specific genome-modifying enzymes, any target DNA described herein can be integrated into the target site of a chromosomal sequence. Any method described herein can utilize any site-specific genome-modifying enzyme provided herein.

[0072] Several embodiments relate to the methods and / or compositions provided herein that comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific genome-modifying enzymes. In another aspect, the methods and / or compositions provided herein comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten polynucleotides encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific genome-modifying enzymes.

[0073] In one aspect, targeted genome modification as described herein includes the use of recombinases. In another aspect, the tyrosine recombinase linked to the DNA recognition motif provided herein is selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase. In yet another aspect, the Cre or Gin recombinase provided herein is linked to a zinc finger DNA-binding domain. The Flp-FRT site-directed recombination system is derived from a 2μ plasmid in *Saccharomyces cerevisiae*. In this system, the Flp recombinase (a reversing enzyme) recombines the sequence between target (FRT) sites recognized by the reversing enzyme. The FRT site contains 34 nucleotides. Flp binds to the "arm" of the FRT site (one arm in the opposite direction) and cleaves the FRT site at either end of the intervening nucleic acid sequence. After cleavage, Flp recombines the nucleic acid sequence between the two FRT sites. Cre-lox is a site-directed recombination system derived from bacteriophage P1, which is similar to the Flp-FRT recombination system. Cre-lox can be used to reverse, delete, or translocate nucleic acid sequences. In this system, Cre recombinase recombines a pair of lox nucleic acid sequences. The lox site contains 34 nucleotides, with the first and last 13 nucleotides (arms) forming a palindromic structure. During recombination, the Cre recombinase protein binds to two lox sites on different nucleic acids and cleaves them at the lox sites. The cleaved nucleic acids are spliced ​​together (reciprocal translocation) and recombination is completed. Alternatively, the lox sites provided herein are loxP, lox 2272, loxN, lox 511, lox5171, lox71, lox66, M2, M3, M7, or M11.

[0074] On the other hand, the serine recombinases ligated to the DNA recognition motifs provided herein are selected from the group consisting of: PhiC31 integrase, R4 integrase, and TP-901 integrase. On the other hand, the DNA transposases ligated to the DNA-binding domains provided herein are selected from the group consisting of: TALE-piggyBac and TALE-Mutator.

[0075] In some implementations, targeted genome modification, as described herein, includes the use of zinc finger nucleases (ZFNs). ZFNs are synthetic proteins composed of engineered zinc finger DNA-binding domains fused with a cleavage domain of a FokI restriction nuclease. ZFNs can be engineered to cleave virtually any long double-stranded DNA for modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers consisting of non-specific DNA-cleaving domains of a FokI nuclease fused with an array of zinc fingers engineered to bind to a target DNA sequence. The DNA-binding domain of a ZFN typically consists of an array of 3-4 zinc fingers. Amino acids located at positions -1, +2, +3, and +6 relative to the zinc finger ∞-helix origin, which contribute to site-specific binding to the target DNA, can be modified and customized to fit a specific target sequence. Other amino acids form a common backbone to produce ZFNs with different sequence specificities. The rules for selecting the target sequence of a ZFN are known in the art. The FokI nuclease domain requires dimerization to cleave DNA, and therefore requires two ZFNs with C-terminal regions to bind to the opposite DNA strands (separated by 5-7 nt) at the cleavage site. If the two ZF binding sites are palindromic, the ZFN monomer can cleave the target site. As used herein, the term ZFN is broad and includes monomeric ZFNs capable of cleaving double-stranded DNA without the assistance of another ZFN. The term ZFN is also used to refer to one or both members of a pair of ZFNs engineered to work together to cleave DNA at the same site.

[0076] Unrestricted by any scientific theory, because in principle, the DNA binding specificity of zinc finger domains can be reengineered using one of several methods, it is theoretically possible to construct custom ZFNs targeting almost any gene sequence. Publicly available methods for engineering zinc finger domains include context-dependent assembly (CoDA), oligomer library engineering (OPEN), and modular assembly.

[0077] Several embodiments relate to the methods and / or compositions provided herein that comprise one or more, two or more, three or more, four or more, or five or more ZFNs targeting a target sequence in a gene encoding the AUX / IAA protein. In another aspect, the ZFNs provided herein are capable of generating targeted DSBs. In yet another aspect, a vector comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more ZFNs is provided to cells via methods known in the art (e.g., but not limited to viral transfection, particle bombardment, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation).

[0078] Several implementation schemes involve the use of macronucleases to generate modifications as described herein. Macronucleases are commonly identified in microorganisms and are unique enzymes with high activity and long recognition sequences (>14 nt), resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring macronucleases typically have extended DNA recognition sequences (e.g., 14 to 40 nt). Engineering macronucleases can be more challenging than ZFNs and TALENs because their DNA recognition and cleavage functions are intertwined within a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to generate novel macronuclease variants that recognize unique sequences and possess improved nuclease activity.

[0079] In one aspect, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more broad-spectrum nucleases targeting a target sequence in a gene encoding the AUX / IAA protein. In some embodiments, the broad-spectrum nucleases provided herein are capable of generating targeted DSBs. In some embodiments, a vector comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more broad-spectrum nucleases is provided to cells via methods known in the art (e.g., but not limited to viral transfection, particle bombardment, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation).

[0080] In some implementations, targeted genome modification as described herein includes the use of transcription activator-like effector nucleases (TALENs). TALENs are artificial restriction enzymes created by fusing a transcription activator-like effector (TALE) DNA-binding domain with a nuclease domain. In one aspect, nucleases are selected from the group consisting of: PvuII, MutH, TevI and FokI, AlwI, MlyI, SbfI, SdaI, StsI, CleDORF, Clo051, and Pept071. As used herein, the term TALEN is broad and includes monomeric TALENs capable of cleaving double-stranded DNA without the assistance of another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs engineered to act together to cleave DNA at the same site. Transcription activator-like effectors (TALEs) can be engineered to bind to target sequences in nucleic acids encoding virtually any DNA sequence, such as those encoding AUX / IAA proteins. TALE proteins are DNA-binding domains derived from various plant bacterial pathogens of the genus Xanthomonas. During infection, pathogen X secretes TALE into the host plant cell. The TALE migrates to the nucleus, where it recognizes and binds to specific DNA sequences within the promoter regions of specific genes in the host genome. The TALE has a central DNA-binding domain consisting of 13–28 repeating monomers of 33–34 amino acids. Except for the highly variable amino acid residues at positions 12 and 13, the amino acids in each monomer are highly conserved. These two variable amino acids are called repeating variable diresidues (RVDs). The amino acids in RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine for NI, NG, HD, and NN, respectively, and regulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition allows for the engineering of specific DNA-binding domains by selecting combinations of repeating fragments containing appropriate RVDs.

[0081] In one aspect, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more TALENs. In another aspect, the TALENs provided herein are capable of generating targeted DSBs in the target sequence of a nucleic acid encoding the AUX / IAA protein. In another aspect, a vector comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more TALENs is provided to cells via methods known in the art (e.g., but not limited to viral transfection, particle bombardment, PEG-mediated protoplast transfection, or Agrobacterium-mediated transformation).

[0082] In some embodiments, targeted genome modification as described herein includes the use of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-directed nucleases. In some embodiments, the CRISPR / Cas9 system, CRISPR / Cpf1 system, CRISPR / CasX system, or CRISPR / CasY system may be used to generate modifications to nucleic acids encoding AUX / IAA proteins. CRISPR systems are RNA-directed engineered nucleases that use complementary base pairing to recognize DNA sequences at target sites. CRISPR (Regularly Spaced Clustered Short Palindromic Repeats) / Cas (CRISPR-associated) systems are alternatives to synthetic proteins whose DNA-binding domains enable them to modify specific sequences of genomic DNA (e.g., ZFN and TALEN). CRISPR / Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them from invading nucleic acids (such as viruses) by cleaving foreign DNA in a sequence-dependent manner. Immunity is acquired by integrating a short fragment of invading DNA, called a spacer, between two adjacent repetitive sequences proximal to the CRISPR locus. The CRISPR array, including the spacer, is transcribed upon subsequent contact with the invading DNA and processed into a small interfering CRISPR RNA (crRNA) approximately 40 nt in length, which binds to a trans-activating CRISPR RNA (tracrRNA) to activate and guide the Cas9 nuclease. This cleaves a homologous double-stranded DNA sequence in the invading DNA called the protospacer. A prerequisite for cleavage is the presence of a conserved protospacer neighbor motif (PAM) downstream of the target DNA, typically with the sequence 5'-NGG-3', but less frequently NAG. Specificity is provided by a so-called "seed sequence" approximately 12 bases upstream of the PAM, which must match between the RNA and the target DNA. Cpf1 behaves similarly to Cas9, but Cpf1 does not require tracrRNA. The specificity of the CRISPR / Cas system is based on RNA guidance that uses complementary base pairing to recognize the target DNA sequence. In some implementations, the site-specific genome-modifying enzyme is a CRISPR / Cas system.On the one hand, the site-specific genome-modifying enzymes provided herein may include any RNA-guided Cas nuclease (non-restricted examples of RNA-guided nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, ... Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, Mad7, their homologs or modified forms thereof); and optionally, guide RNA necessary for targeting the corresponding nuclease.

[0083] Several implementations involve plant cells, plant tissues, plant seeds, and plants produced by the methods disclosed herein. The plants may be monocotyledonous or dicotyledonous and may include, for example, rice, wheat, barley, oats, rye, sorghum, corn, grapes, tomatoes, potatoes, lettuce, broccoli, cucumbers, peanuts, melons, leeks, onions, soybeans, alfalfa, sunflowers, cotton, rapeseed, and sugar beets.

[0084] III. Expression Constructs

[0085] The polynucleotides described herein can be provided in expression constructs. Expression constructs typically include regulatory elements that function in the intended host cell to which the expression construct is to be expressed. Therefore, those skilled in the art can choose regulatory elements for use in bacterial host cells, yeast host cells, plant host cells, insect host cells, mammalian host cells, and human host cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. As used herein, the term "expression construct" refers to a combination of transcribed nucleic acid sequences that provide operably linked nucleic acid sequences. As used herein, "operably linked" means that two DNA molecules are linked in such a way that one DNA molecule can influence the function of the other. The operably linked DNA molecules can be part of a single adjacent molecule and may be adjacent or not adjacent. For example, a promoter is operably linked to a polypeptide-coding DNA molecule in a DNA construct, wherein the two DNA molecules are arranged such that the promoter can influence the expression of the DNA molecule.

[0086] As used herein, the term "heterogeneous" refers to a relationship between two or more substances that originate from different sources and are therefore not typically related in nature. For example, a protein-coding recombinant DNA molecule is heterogeneous relative to an operatively linked promoter if this combination is not typically found in nature. Furthermore, a particular recombinant DNA molecule may be heterogeneous relative to the specific cell, seed, or organism in which it is inserted, when it occurs unnaturally in that particular cell, seed, or organism.

[0087] The expression construct may contain a promoter sequence operatively linked to a polynucleotide sequence encoding the modified polypeptide described herein. The promoter can be bound to the polynucleotide using standard techniques known in the art. Multiple copies of the promoter or multiple promoters may be used in the expression construct described herein. In some embodiments, the promoter may be located at approximately the same distance from the transcription start site of the expression construct as it is in its native genetic environment. Variations in this distance are permitted without a substantial reduction in promoter activity. The transcription start site is typically included in the expression construct.

[0088] Several embodiments involve recombinant DNA molecules encoding modified AUX / IAA peptides comprising a modified degradation determinant domain containing the motif X1WPPX2, wherein X1 has been modified from glycine to asparagine, and wherein X2 is isoleucine or valine, wherein the recombinant DNA molecule is further defined to be operatively linked to a heterologous regulatory element. In a particular embodiment, the heterologous regulatory element is a promoter functioning in a plant cell. In a further embodiment, the promoter is an inducible promoter.

[0089] If the expression construct is to be provided or introduced into plant cells, plant virus promoters such as, for example, cauliflower mosaic virus (CaMV) 35S (including the enhanced CaMV 35S promoter (see, for example, U.S. Patent No. 5,106,739)) or CaMV 19S promoter or cassavavein mosaic virus can be used. Other promoters that can be used for expression constructs in plants include, for example, the zein promoter (including the maize zein promoter), the prolifera promoter, the Ap3 promoter, the heat shock promoter, the T-DNA 1'- or 2'- promoter of Agrobacterium tumefaciens, the polygalacturonase promoter, the petunia chalcone synthase A (CHS-A) promoter, the tobacco PR-1a promoter, the ubiquitin promoter, the actin promoter, the alcA gene promoter, the pin2 promoter (Xu et al., 1993), the maize WipI promoter, the maize trpA gene promoter (US Patent No. 5,625,136), the maize CDPK gene promoter, and the RUBISCO SSU promoter (US Patent No. 5,034,322). Constitutive promoters (such as CaMV, ubiquitin, actin, or NOS promoters), developmental regulatory promoters, and inducible promoters (such as those induced by heat, light, hormones, or chemicals) are also considered for use in the polynucleotide expression constructs described herein.

[0090] Expression constructs may optionally include transcription termination sequences, translation termination sequences, sequences encoding signal peptides, and / or enhancer elements. Transcription termination regions are typically derived from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences may be located downstream of the coding sequence to provide efficient termination. Signal peptide sequences are short amino acid sequences typically present at the amino terminus of proteins, responsible for retargeting operatively linked mature polypeptides to a wide range of post-translational cellular destinations, from specific organelle compartments to protein action sites and the extracellular environment. Targeting gene products to intended cellular and / or extracellular destinations using operatively linked signal peptide sequences is intended for use with the polypeptides described herein. Classical enhancers are cis-acting elements that increase gene transcription and may also be included in expression constructs. Classical enhancer elements are known in the art and include, but are not limited to, the CaMV 35S enhancer element, the 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 transcriptional region and be orientation-dependent. Examples include the corn shrunken-1 enhancer element (Clancy and Hannah, 2002).

[0091] IV. Conversion Methods

[0092] Several embodiments involve plant cells, plant tissues, plants, and seeds containing the recombinant DNA described herein. In some embodiments, the recombinant DNA encodes a sequence-specific enzyme. In some embodiments, the cells, tissues, plants, and seeds containing the recombinant DNA molecule exhibit tolerance to auxin herbicides. In some embodiments, the recombinant DNA encoding a sequence-specific enzyme is selected from plants exhibiting tolerance to auxin herbicides.

[0093] Methods suitable for transforming host plant cells include virtually any method capable of introducing DNA or RNA into cells (e.g., methods for stably integrating recombinant DNA constructs into plant chromosomes, or transiently providing recombinant DNA constructs or RNA to plant cells), and such methods are well known in the art. Two effective cell transformation methods are Agrobacterium-mediated transformation and particle bombardment-mediated transformation. The particle bombardment method is described, for example, in U.S. Patent Nos. 5,550,318; 5,538,880; 6,160,208; and 6,399,861. The Agrobacterium-mediated transformation method is described, for example, in U.S. Patent No. 5,591,616, which is incorporated herein by reference in its entirety. The transformation of plant material is practiced in tissue culture on a nutrient medium (e.g., a mixture of nutrients that allows cells to grow in vitro). Recipient cell targets include, but are not limited to, meristematic cells, shoot tips, hypocotyls, callus, immature or mature embryos, and gametophyte cells such as microspores and pollen. Callus can originate from tissues including, but not limited to, immature or mature embryos, hypocotyls, seedling apical meristems, and microspores. Cells containing transgenic nuclei grow into transgenic plants.

[0094] During transformation, DNA is typically introduced into only a small percentage of target plant cells in any transformation experiment. Marker genes are used to provide an effective system for identifying those cells that have been stably transformed by accepting recombinant DNA molecules and integrating them into their genome. Preferred marker genes provide selective markers that confer resistance to selectors such as antibiotics or herbicides. Any herbicide to which the plant of this disclosure may be resistant is a reagent used for selective marking. Potentially transformed cells are exposed to the selector. The surviving cell population consists of those cells in which the gene that typically confers resistance is integrated and expressed at a level sufficient to allow cell survival. Cells may be further tested to confirm stable integration of the exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin (nptII), hygromycin B (aph IV), spectinomycin (aadA), and gentamicin (aac3 and aacC4) or to herbicides such as glufosinate (bar or pat), dicamba (DMO), and glyphosate (aroA or EPSPS). Examples of such selective markers are described in U.S. Patent Nos. 5,550,318; 5,633,435; 5,780,708; and 6,118,047. Markers that provide the ability to visually screen transformants may also be used, such as genes expressing chromogenic or fluorescent proteins (e.g., luciferase or green fluorescent protein (GFP)) or genes expressing β-glucuronidase or the uidA gene (GUS), known for its various chromogenic substrates.

[0095] Several implementation schemes involve methods and constructs for regenerating plants using cells with modified genomic DNA derived from genome editing. The regenerated plants can then be used to propagate other plants.

[0096] Several embodiments relate to a method for introducing an allelic variant of a gene encoding an AUX / IAA protein, comprising: (a) introducing at least one modification of an endogenous AUX / IAA gene into at least one plant cell to generate the allelic variant; (b) identifying and selecting at least one plant cell from step (a) containing the allelic variant exhibiting tolerance to auxin herbicides; and (c) regenerating a plant from the at least one plant cell selected in step (b). Several embodiments relate to a method for introducing an allelic variant of a gene encoding an AUX / IAA protein, comprising: (a) introducing at least one modification of an endogenous AUX / IAA gene into at least one plant cell to generate the allelic variant; (b) identifying and selecting at least one plant cell from step (a) containing the allelic variant exhibiting tolerance to auxin herbicides; (c) identifying and selecting at least one plant cell from step (b) exhibiting sensitivity to endogenous auxin IAA; and (d) regenerating a plant from the at least one plant cell selected in step (c). In one aspect, the method includes introducing a modification into the sequence encoding the degradation determinant domain of the AUX / IAA protein. In some embodiments, the modified degradation determinant domain encodes an X1WPPX2 motif that confers plant tolerance to auxin herbicides. In some embodiments, X1 has been modified from glycine to asparagine, isoleucine, or leucine. In some embodiments, X2 is isoleucine, valine, leucine, or methionine. In another aspect, the introduction method includes introducing a recombinant DNA vector containing a polynucleotide sequence-specific enzyme targeting a target sequence in the nucleic acid encoding the AUX / IAA protein.

[0097] V. Auxin-based herbicides and plants tolerant to auxin-based herbicides

[0098] Several embodiments relate to plant cells, plant tissues, plants, and seeds comprising an AUX / IAA protein containing a modified degradation determinant domain. In some embodiments, the modified degradation determinant domain comprises an X1WPPX2 motif capable of conferring tolerance to synthetic auxin herbicides to plant cells, plant tissues, plants, and seeds. In some embodiments, X1 has been modified from glycine to asparagine, isoleucine, or leucine. In some embodiments, X2 is isoleucine, valine, leucine, or methionine. The plant may be a monocot or dicot and may include, for example, rice, wheat, barley, oats, rye, sorghum, corn, grapes, tomatoes, potatoes, lettuce, broccoli, cucumbers, peanuts, melons, peppers, carrots, squash, onions, soybeans, alfalfa, sunflowers, cotton, rapeseed, and sugar beets.

[0099] Based on their mode of action, synthetic auxin herbicides are also known as auxin-based growth regulator herbicides, or Group O or Group 4 herbicides. Synthetic auxin herbicides work by appearing to affect cell wall plasticity and nucleic acid metabolism, potentially leading to uncontrolled cell division and growth. The group of synthetic auxin herbicides comprises four chemical families: phenoxy, carboxylic acids (or pyridines), benzoic acid, and the newest quinoline carboxylic acid family.

[0100] Phenoxy herbicides are the most common and have been used as herbicides since the discovery of (2,4-dichlorophenoxy)acetic acid (2,4-D) in the 1940s. Other examples include 4-(2,4-dichlorophenylmethyl)butyric acid (2,4-DB), 2-(2,4-dichlorophenylmethyl)propionic acid (2,4-DP), (2,4,5-trichlorophenylmethyl)acetic acid (2,4,5-T), 2-(2,4,5-trichlorophenylmethyl)propionic acid (2,4,5-TP), 2-(2,4-dichloro-3-methylphenoxy)-N-phenylpropionamide (clomeprop), (4-chloro-2-methylphenoxy)acetic acid (MCPA), 4-(4-chloro-o-tolyloxy)butyric acid (MCPB), and 2-(4-chloro-2-methylphenoxy)propionic acid (MCPP).

[0101] The second largest family of chemicals is carboxylic acid herbicides, also known as pyridine herbicides. Examples include 3,6-dichloro-2-pyridinecarboxylic acid (chlorpyrifos), 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (toxamethonium), (2,4,5-trichlorophenoxy)acetic acid (chlorpyrifos), and 4-amino-3,5-dichloro-6-fluoro-2-pyridineoxyacetic acid (flumethoxam). The third family of chemicals is benzoic acid, with examples including 3,6-dichloro-o-anisolic acid (dicamba) and 3-amino-2,5-dichlorobenzoic acid (choramben). The fourth and newest family of auxin herbicides is the quinoline carboxylic acid family, which includes 7-chloro-3-methyl-8-quinoline carboxylic acid (quinmerac) and 3,7-dichloro-8-quinoline carboxylic acid (quickgrass). The latter is unique in that it also controls some grassy weeds, unlike other auxin-like herbicides, which primarily control broadleaf or dicotyledonous plants.

[0102] Synthetic auxin herbicides can be applied to plant growing areas containing plants and seeds provided by the compositions and methods described herein as a method of weed control. The plants and seeds provided by the compositions and methods described herein contain synthetic auxin herbicide tolerance traits and are therefore tolerant to the application of one or more auxin herbicides. Herbicide application can be at the recommended commercial ratio (1X) or any fraction or multiple thereof, such as twice the recommended commercial ratio (2X). Depending on the herbicide and formulation, the auxin herbicide ratio can be expressed as acid equivalents / pound / acre (lb ae / acre) or acid equivalents / gram / ha (g ae / ha) or as pounds of active ingredient / acre (lb ai / acre) or grams of active ingredient / ha (g ai / ha). The plant growing area may or may not contain weed plants at the time of herbicide application.

[0103] The herbicide can be applied sequentially or in a container with one, two, or a combination of several auxin herbicides or any other compatible herbicide. Multiple applications of one herbicide or in combination or alone of two or more herbicides can be used during the growing season in areas containing plants expressing the modified AUX / IAA protein as described herein to control a wide range of dicotyledonous weeds, monocotyledonous weeds, or both, for example, two applications (such as pre-planting and post-emergence or pre-emergence and post-emergence) or three applications (such as pre-planting, pre-emergence and post-emergence or pre-emergence and two post-emergence applications).

[0104] As used herein, “tolerance” or “herbicide tolerance” refers to the ability of a plant, seed, or cell to resist the toxic effects of a herbicide upon application. Herbicide tolerance in plants, seeds, plant tissues, plant parts, or cells can be measured by comparing the plant, seed, plant tissue, plant part, or cell to a suitable control. For example, herbicide tolerance can be measured or assessed by applying a herbicide to a plant (test plant) containing a recombinant DNA molecule encoding a modified polypeptide that confers herbicide tolerance and to a plant (control plant) not containing such a molecule, and then comparing the plant damage to the two types of plants. A lower damage rate compared to the control plant indicates herbicide tolerance in the test plant. Herbicide-tolerant plants, seeds, plant tissues, plant parts, or cells exhibit a reduced response to the toxic effects of herbicide compared to control plants, seeds, plant tissues, plant parts, or cells. As used in this article, "herbicide tolerance trait" is a transgenic trait that confers improved herbicide tolerance on a plant compared to the wild type.

[0105] As used herein, the term "including" means "including but not limited to".

[0106] Several embodiments have been described in detail, and it will be apparent that modifications, variations, and equivalent embodiments may exist. Furthermore, it should be understood that the embodiments in this disclosure are provided as non-limiting examples.

[0107] Example

[0108] Example 1: Identification of genetic differences between dicamba-resistant and dicamba-sensitive Kochia scoparia biotypes

[0109] A dicamba-resistant Kochia scoparia biotype exhibiting a nearly 30-fold increase in dicamba tolerance was obtained, believed to be conferred by a single dominant or semi-dominant locus. Transcriptome sequencing of cDNA isolated from the aerial tissues of vegetative plants of dicamba-resistant (R) and dicamba-sensitive (S) Kochia scoparia biotypes allowed the identification of putative Kochia scoparia orthologs known to be involved in auxin synthesis, transport, and regulatory responses in Arabidopsis thaliana. Comparatives included one AUX1 / LAX transporter, one ABP homolog, several multidrug resistance-like proteins (MRPs), four TIR / AFB co-receptors, and ten AUX / IAA proteins.

[0110] Leaf tissues were harvested from greenhouse-grown S and R Kochia scoparia biotypes and frozen in liquid nitrogen. Total RNA was isolated from three bioreplicas of each tissue using TRIzol (Invitrogen) according to the manufacturer's specifications. RNA quantity was determined using a Nanodrop 8000 spectrophotometer, and integrity was assessed using BioAnalyzer analysis with RIN > 6. 2 mg of total RNA was used for sequencing library preparation using an Illumina TruSeq RNA Sample Prep Kit V2, according to the manufacturer's specifications. qPCR (SYBR PCR Master Mix, Applied Biosystems) was used for quantitative sequencing of the library. Sequencing was performed using a HiSeq2000 sequencer with 50-base-pair reads, as previously described (Rice et al., 2014). Kochia scoparia transcriptome assemblies were constructed using paired end reads of R (82, 123, 945 pairs) and S (68, 928, 684 pairs) biotypes. First, the reads were quality-trimmed using the FASTX toolkit (version 0.0.13, at least 50% of the bases with a quality fraction >= 30), and then assembled using Trinity (version r2013_08_14, default settings, minimum transcription length = 300) (Grabherr et al., Nat Biotechnol. 2011 29(7):644-52.).

[0111] Significant differences were observed between R-type and S-type kochia transcripts. Compared to transcript sequences from the S-type, the R-type showed a GG-to-AA nucleotide change. This GG-to-AA nucleotide change resulted in an amino acid substitution of glycine (G) for asparagine (N). Figure 1 The protein containing this amino acid substitution (SEQ ID NO:219) (KsIAA16; SEQ ID NO:217) is most closely related to the Arabidopsis AUX / IAA protein At.IAA16 (SEQ ID NO:262), which belongs to the same clade as At.IAA7 (SEQ ID NO:253), At.IAA14 (SEQ ID NO:260), and At.IAA17 (SEQ ID NO:263). Figure 2 The observed amino acid changes occurred within a highly conserved degradation determinant region (GWPPV / I). Figure 1 Changes occurring within this five-amino acid degradation determinant region have been reported to enhance the stability of the AUX / IAA protein and lead to dominant auxin resistance in many Arabidopsis mutants, including axr5(IAA1) (Yang et al., 2004), shy2(IAA3) (Tian and Reed, 1999), axr2(IAA7, (SEQ ID NO:247)) (Nagpal et al., 2000), slr(IAA14, (SEQ ID NO:260)) (Fukaki et al., 2002), axr3(IAA17, (SEQ ID NO:263)) (Rouse et al., 1998), crane(IAA18, (SEQ ID NO:264)) (Uehara et al., 2008), bodenlos(IAA12, (SEQ ID NO:258)) (Hamann et al., 2002), and msg2(IAA19, (SEQ ID NO:264)) as shown in Table 1. KsIAA16 (SEQ ID NO:265) (Tatematsu et al., 2004), iaa16 (SEQ ID NO:262) (Rinaldi et al., 2012), and iaa28 (SEQ ID NO:269) (Rogg et al., 2001). These findings suggest that the observed GG to AA nucleotide change leading to the glycine (G) to asparagine (N) amino acid substitution in KsIAA16 is likely the basis for the dicamba resistance observed in this Kochia scoparia biotype.

[0112] Table 1. Amino acid sequences within the degradation determinant domain of Arabidopsis thaliana gain-of-function AUX / IAA mutants.

[0113]

[0114]

[0115] While mutations in the highly conserved GWPPV degradation determinant sequence have previously been reported to confer resistance to the endogenous auxin compound indole-3-acetic acid (IAA), it has not been observed that these mutations confer resistance to herbicidal auxin compounds (e.g., reduced binding affinity) while maintaining sensitivity to endogenous auxin IAA (e.g., still being able to bind to it). Degradation determinant sequence mutants identified in Arabidopsis exhibit significantly altered abilities to sense endogenous IAA, resulting in detrimental phenotypes and undesirable inferior forms.

[0116] Example 2: Sequence-based identification of TIR / AFB homologs of Kochia scoparia

[0117] Numerous auxin-sensing pathways have been identified in plants. One of the most thoroughly investigated pathways involves the degradation of the AUX / IAA transcriptional repressor catalyzed by SCF ubiquitin ligases, mediated by the TIR1 / AFB family of F-box proteins. In the presence of auxin, the TIR1 subunit of the SCF complex acts as an auxin receptor. Once TIR1 binds to auxin, it binds with high affinity to members of the AUX / IAA protein family, forming a complex between the two proteins and promoting ubiquitination of the AUX / IAA protein, thus tagging it for degradation. When the AUX / IAA protein binds to the auxin-binding TIR1 protein, the "GWPPV / I" motif in the degradation determinant region of the AUX / IAA protein is thought to cover the entire auxin molecule. Mutations in the degradation determinant motif have been observed to lead to stabilization of the AUX / IAA protein, which in turn results in reduced auxin response and various growth defects in plant model systems.

[0118] Since AUX / IAA and TIR / AFB proteins co-act as auxin co-receptors, a putative homolog of the TIR / AFB protein from Kochia scoparia was identified to determine whether the KsIAA16 protein interacts with the TIR / AFB protein in the presence of auxin. Such an interaction would indicate that KsIAA16 is an auxin co-receptor.

[0119] BLAST analysis of the Kochia transcriptome dataset was performed using known Arabidopsis TIR / AFB proteins as query sequences. Using this method, four TIR / AFB orthologs were identified in Kochia. The proteins were compared with orthologs TIR1, AFB1, AFB2, AFB3, AFB4, and AFB5 in Arabidopsis, and AFB6 in date palm (Phoenix dactylifera L., datepalm) and slash pine (Pinus taeda L., loblolly pine) (Parry et al., 2009). Alignment was performed using the Clustal W method in MegAlign software (DNASTAR, Madison, WI). The results indicate that Kochia contains one ortholog from each known clade of the Arabidopsis TIR / AFB family.

[0120] Example 3: Analysis of the interaction between Kochia scoparia auxin response factors and auxin

[0121] Yeast two-hybrid (Y2H) research uses Golden yeast two-hybrid system: vector, transformation protocol and yeast strain (Clonetech Laboratories, Inc.). PCR amplification was performed using the primer pairs specified in Table 2 that encode the sequences of KsIAA16R (SEQ ID NO: 220), KsIAA16S (SEQ ID NO: 218), KsTIR1 (SEQ ID NO: 285), KsAFB6 (SEQ ID NO: 287), KsAFB2 (SEQ ID NO: 289), and KsAFB5 (SEQ ID NO: 291).

[0122] Table 2: Primer sequences used in the construction of the Y2H vector

[0123]

[0124]

[0125] The PCR products were purified by gel electrophoresis and then... The cloning system (Clonetech Laboratories, Inc.) recombined the pGADT7 or pGBKT7 vectors digested with NdeI / NotI. The pGBKT7 vector contains a GAL4 DNA-binding domain, making the fusion a "bait" while the "prey" is a fusion containing the pGADT7 vector, which contains a GAL4 activation domain. All vectors were first sequence-verified, and then the listed vector combinations were co-transformed into the yeast strain Y2HGold. The vector combinations were as follows: pGADT7-T+pGBKT7 empty (negative control), pGADT7-T+pGBKT7-Lam (weak interaction control), pGADT7-T+pGBKT7-53 (strong interaction control). The pGBKT7 empty vector was co-transformed with the pGADT&KsIAA16 construct as a negative control, and pGADT7-T was co-transformed with each pGDKT7TIR / AFB construct as a negative control. Transformant plates were inoculated onto SC-Trp-Leu plates to select transformants containing both plasmids. Four healthy colonies were randomly selected and resuspended in 300 μL of 0.1% agar solution. 5 μL of this dilution was replicated onto each plate. The plates were incubated at 30°C for 3 days before evaluating colony growth.

[0126] This method enables the study of the binding / interaction of the R- and S-type Kochia scoparia alleles of KsIAA16 with each Kochia scoparia TIR / AFB homolog (KsTIR1 (SEQ ID NO:285) and KsAFB6 (SEQ ID NO:287)) in the absence or presence of exogenous IAA, NAA, dicamba and 2,4-D, atrazine, chlorpyrifos, cypermethrin, flufenoxuron, or PAA. All combinations of bait and prey vectors showed good growth without selection, indicating that transcript expression has no inherent negative impact on yeast growth. The results obtained from Y2H analysis are shown in Tables 3 and 4.

[0127] Table 3: Y2H analysis results of the binding of KsIAA16S and KsIAA16R to KsTIR1

[0128]

[0129] Table 4: Y2H analysis results of the binding of KsIAA16S and KsIAA16R to KsAFB6

[0130]

[0131] In the absence of auxin, growth of both the R and S alleles of KsIAA16 with orthologs of KsTIR1 and KsAFB6 was barely observed, indicating that proteins do not interact in the absence of auxin. KsIAA16S showed interaction with KsTIR1 in the presence of 100M and 500M IAA, 2-(1-naphthyl)acetic acid (NAA), dicamba, 2,4-D, and 500M chlormequat chloride, but KsIAA16R only showed interaction with KsTIR1 in the presence of 100M and 500M IAA and NAA. In the presence of dicamba and 2,4-D, KsIAA16R showed a reduced ability to interact with KsTIR1. Without being bound by any particular theory, the reduced interaction between KsIAA16R and KsTIR1 in the presence of dicamba and 2,4-D, leading to increased stability of KsIAA16R in the presence of dicamba and 2,4-D, could explain the resistance to these compounds observed in dicamba-resistant Kochia scoparia biotypes. Similarly, interactions between KsIAA16S and KsAFB6 were observed in the presence of 100 μM and 500 μM IAA, NAA, and chlorpyrifos, and 500 μM 2,4-D, cypermethrin, and cypermethrin, but the interaction between KsIAA16R and KsAFB6 was only observed in the presence of the highest concentrations of IAA and NAA (500 M) tested. This indicates that the ability of KsIAA16R to interact with KsAFB6 is reduced in the presence of IAA and NAA, as only higher concentrations promote their binding, and no interaction between KsIAA16R and KsAFB6 was observed at the tested concentrations of chlorpyrifos, 2,4-D, cypermethrin, or chlorpyrifos. No interaction was observed with any of the tested protein combinations in the presence of PAA or fluopyram. Without being bound by any particular theory, the lack of Y2H interaction observed in the presence of fluopyram may be due to the instability of the interaction, which prevents detection in yeast systems; or it may be due to the absence of the metabolism of fluopyram methyl heptyl ester, which is reportedly lysed and released by plants and bacteria as free acid. These findings suggest that KsIAA16 synergistically acts as a co-receptor for auxin with KsTIR1 and / or KsAFB6, and that the combination of KsIAA16 with KsTIR1 and / or KsAFB6 is at least partially responsible for the sensing of each of these auxin herbicides. Furthermore, this is the first reported characterization of an AFB6 homolog, as this clade has disappeared from several studied species, including Arabidopsis thaliana, rice, sorghum, and maize (Hayashi, 2012; Mironova et al., 2010; Parry et al., 2009).Without being bound by any particular theory, these findings suggest that KsAFB6 can act as a co-receptor for auxin and works synergistically with KsIAA16. KsAFB6 can interact with several tested auxin herbicides in the presence of these herbicides.

[0132] Example 4: Characterization of auxin-mediated root elongation response in dicamba-resistant and dicamba-sensitive Kochia scoparia

[0133] Root elongation responses from seedlings of R-type and S-type Kochia scoparia biotypes were tested to determine whether the R-type response was consistent with the Y2H results. For root elongation studies, a plant nutrient medium containing 0.5% sucrose was used. Seeds were surface-sterilized in a 30% bleach solution containing 0.1% Triton-X100 for 10–30 minutes, rinsed twice in sterile water, and then resuspended in 0.1% agar for plate inoculation. Root lengths were measured to the nearest millimeter, and the mean root length of 12 individuals was calculated unless otherwise specified. Root elongation data are as follows: Figure 5 As shown, the R-type Kochia biotype remains sensitive to the root elongation inhibition of IAA and NAA, but is resistant to the inhibition of dicamba and 2,4-D. Additionally, a short-root phenotype was observed in the absence of auxin. These findings are consistent with the observed Y2H results, which showed that at the tested concentrations, IAA and NAA could promote some interactions between KsIAA16R and KsTIR1 and / or KsAFB6, but not with dicamba, 2,4-D, chlorpyrifos, chlorpyrifos, cypermethrin, and fluroxypyr. The natural auxin PAA also appears to have an effect on root elongation. In addition to the Y2H results, the observations summarized here provide further support for the hypothesis that the auxin resistance observed in this Kochia biotype is due to a mutation in KsIAA16R.

[0134] Example 5: Development of KsIAA16R allele molecular marker

[0135] Based on base pair substitutions identified in KsIAA16 between S-type and R-type Kochia scoparia biotypes, restriction fragment length polymorphism (RFLP) analysis was developed for genotyping purposes. analyze.

[0136] For RFLP analysis, forward primers (SEQ ID NO: 221) and reverse primers (SEQ ID NO: 222) were designed to amplify 146 bp products from genomic DNA of resistant and susceptible plants. The mutation identified in the R biotype was found to produce additional MluCI restriction sites in the KsIAA16R allele. This allowed for differentiation between the R and S alleles when the digested products were electrophoresed on a 4% agarose gel, as the S allele product had only two MluCI restriction endonuclease cleavage sites and produced fragments of 120, 19, and 7 base pairs after digestion, while the R allele had three cleavage sites and produced fragments of 90, 30, 19, and 7 base pairs.

[0137] for Analysis revealed that forward primers (SEQ ID NO: 223) and reverse primers (SEQ ID NO: 224) were designed to amplify the genomic DNA of the two KsIAA16 alleles. A VIC-labeled probe (SEQ ID NO: 225) was designed to detect the R allele, while a FAM-labeled probe (SEQ ID NO: 226) was designed to detect the S allele. The mixture was analyzed using TaqMan. Universal Master Mix II was used for each PCR reaction in a final volume of 20 μL in 96-well plates. Analysis was performed on the ViiA7 system (Applied Biosystems) using the following standard Taqman genotyping conditions, according to the manufacturer's specifications: 95°C for 15 seconds, 60°C for 60 seconds, with 40 cycles per plate. Allele calls were specified using the ViiA7 software, and each experiment included known homozygous Kochia scoparia R and S genomic DNA, as well as a DNA-free control. These markers can be used to detect R alleles in individuals of field populations and to determine plant genotypes resulting from hybridization events, thereby identifying segregation patterns and conducting fitness studies.

[0138] Example 6: Separation pattern of dicamba resistance trait in Kochia scoparia

[0139] To determine whether the KsIAA16R allele cosegregates with the dicamba resistance phenotype, a spray experiment was conducted on a Kochia scoparia population containing both S-type and R-type plants. Crosses were performed between S-type and R-type individuals, using the S-type as female and the R-type as male, to identify individuals produced by cross-pollination. Due to the anatomical structure of Kochia scoparia's reproductive organs, it is difficult to prevent some self-pollination, but... Genotyping analysis distinguished between KsIAA16 (R / S) heterozygous offspring and simple-pollinated (S / S) offspring. Several validated heterozygous F1 plants were selected for F2 seed production. Additionally, dicamba was sprayed onto subsets of S / S and R / S individuals from the F1 generation to ensure the markers effectively identified the R allele. The best correlation was observed between plants identified as heterozygous for the R allele and those showing resistance to dicamba in the F1 spray test. This result suggests that dicamba resistance is a dominant or semi-dominant trait.

[0140] To determine whether the KsIAA16R allele co-segregated with the dicamba resistance phenotype, 720 F2 progeny plants from validated heterozygous F1 parents were sampled and genotyped, and tolerance to dicamba spray concentrations at twice the typical field application rate (1120 g / ha) was determined. Plant identity was maintained throughout the experiment, and genotypes and the percentage of visible dicamba damage for each F2 progeny were determined. The mean damage for each genotype was approximately 80% for S / S, approximately 40% for R / S, and approximately 20% for R / R. Based on Taqman genotyping analysis, 234 homozygous susceptible plants (S / S), 371 heterozygous plants (R / S), and 105 homozygous resistant plants (R / R) were identified. Fourteen plants reported indeterminate genotypes, thus representing a 2% PCR failure rate. The genotype ratios (1:1.6:0.4) differed significantly from the expected Mendelian 1:2:1 segregation pattern (P<0.05), suggesting that resistance alleles may influence germination and / or seedling formation. A strong correlation was found between genotype results and observed dicamba damage, with a mean damage rate of 82.3% for S / S individuals, 35.9% for R / S individuals, and 24.6% for R / R individuals. Statistical analysis of these data using ANOVA showed a high certainty that the dicamba resistance phenotype was associated with the presence of the KsIAA16R allele (p<0.0001), and Cramér-von Mises statistical analysis indicated that the trait was semi-dominant.

[0141] Example 7: Expression and detection of KsIAA16 in Arabidopsis thaliana

[0142] To determine whether the presence of the KsIAA16R allele alone is necessary and sufficient to confer dicamba resistance, several transgenic expression constructs were developed for studying KsIAA16 in Arabidopsis thaliana. Binary vectors were introduced into the plants using Agrobacterium tumefaciens via flower dip (Clough and Bent, 1998). The vectors contained an ethanol-inducible alcA promoter element (Salter et al., 1998), a constitutive CaMV35S promoter, or the native Arabidopsis thaliana IAA16 promoter (AtIAA16P) driving KsIAA16R (SEQ ID NO: 220) or KsIAA16S (SEQ ID NO: 218). The GUS reporter gene construct contained a 2.5 kb genomic DNA fragment directly upstream of KsIAA16, expected to contain most of the promoter elements as well as the 5' UTR driving uidA. All constructs included either the spectinomycin resistance gene (SPEC) or the glufosinate resistance gene (BAR) in the T-DNA region as a selection marker. For all constructs, T1 seeds were harvested, surface-sterilized, and then plated on suitable selective media to identify independent T1 transformants. Individual seed decentization and segregation on selective media were used to estimate the number of transgene insertions and identify homozygous lines. To induce the P-Alc promoter, ethanol was added to a final concentration of 0.01% just before pouring the plates, and the plates were used immediately. All plates were grown in a Percival growth chamber (Percival Scientific, Inc.) at 22°C under a 16-hour / 8-hour dark-light cycle at a light intensity of 200 μmol / min / m². 2 The relative humidity was maintained at 50%. GUS analysis was performed according to the standard protocol. In short, a 20 mg / mL stock solution of X-Gluc (GoldBio, St. Louis, MO) in dimethylformamide was diluted to a final concentration of 1 mg / mL in 50 mM phosphate buffer at pH 7.0. This solution was added to the plant tissue, and the sample was placed under vacuum for 5 minutes to ensure the solution penetrated the tissue. The sample was covered with foil to prevent light from entering and incubated at 37°C for 24 hours to allow for color development. The staining solution was removed, and chlorophyll was removed by incubating the sample in 50% ethanol for several hours. For seedling stagnation counts, seedlings exhibiting obvious protrusions but whose root length never exceeded 2–3 mm by day 8 after plate inoculation were considered stagnated.

[0143] In lines carrying both the 35S:KsIAA16 and P-Alc:KsIAA16 constructs, a significant proportion of seedlings appeared to arrest shortly after germination, likely due to high levels of constitutive / basal expression (Table 5). Induction of P-Alc promoter expression with 0.01% ethanol slightly increased the arrest rate, but not significantly. There was no difference in the 35S:KsIAA16 lines in the presence or absence of ethanol.

[0144] Table 5: Data on seedling stagnation in transgenic Arabidopsis thaliana seedlings

[0145]

[0146] Lines carrying the AtIAA16P:KsIAA16 construct did not appear to have significant seedling retardation and were therefore used for further investigation. As shown in Table 6, four independent transformant lines carrying AtIAA16P:KsIAA16R showed resistance to dicamba in root elongation inhibition analysis, while only one of the four lines carrying AtIAA16P:KsIAA16S showed very slight resistance. Several lines from both constructs also showed slight resistance to the root inhibition of IAA. In summary, these data support the hypothesis that the function of the KsIAA16R allele in Arabidopsis is similar to that in Kochia scoparia. Furthermore, putative overexpression of the KsIAA16S allele appears to sometimes induce resistance to exogenous auxin, albeit to a lesser degree. Unwilling to be bound by any particular theory, this may be due to the increased total amount of AUX / IAA protein present compared to wild-type plants, as a slight increase in IAA resistance in root elongation analysis has previously been reported to be due to heterologous expression of the wild-type AUX / IAA gene PtrIAA14 (Liu et al., 2015). Overall, these results suggest that the expression mode / level of the KsIAA16 transgene affects auxin sensing, as only those plants with an endogenous promoter most closely resembling the Arabidopsis homolog (AtAA16P) produced plants with the fewest inferior morphologies.

[0147] Table 6: Root length (mm) of T2 transgenic plants with phytonutrients plus IAA or wheatgrass bran.

[0148]

[0149] To understand the expression pattern of KsIAA16 and how its spatiotemporal expression compares to the observed Kochia scoparia resistance phenotype, a northern analysis was performed on Kochia scoparia tissues. Additionally, a KsIAA16 promoter (P):β-glucuronidase (GUS) reporter gene construct was generated and transformed in Arabidopsis thaliana to visualize KsIAA16 expression in a heterologous system. Visualization of the expression pattern of the KsIAA16P:GUS construct in Arabidopsis thaliana by X-glucose staining showed that the reporter gene was not expressed in dry or imbibition seeds, but was strongly expressed at germination or immediately after germination. Expression was widespread in most tissues, with the highest expression in vascular tissues and lateral root primordia. Strong expression was maintained throughout development, with diffuse staining observed in all leaf and inflorescence tissues, and the strongest expression in vascular tissues. Significant staining was also observed in pollen and carpels of flowers, septa of young siliques, and abscission zones of mature siliques. Northern blot analysis of Kochia scoparia tissues from susceptible and resistant lines showed that the endogenous allele of the KsIAA16 transcript was transcribed in all analyzed tissues. This widespread expression pattern of KsIAA16 could explain the strong resistance to foliar dicamba spraying.

[0150] Example 8: Modification of AUX / IAA genes in crop plants

[0151] Based on the base pair substitutions identified within the degradation determinant domain of the KsIAA16 protein that cause a change in amino acid composition from glycine to asparagine, corresponding base pair substitutions can be performed in IAA16 orthologs of crops such as maize, sorghum, wheat, rapeseed, soybean, cotton, grape, tomato, potato, lettuce, broccoli, cucumber, peanut, melon, leek, onion, rice, barley, or other orthologs identified herein. These sequences can be modified using targeted genome editing techniques (e.g., single-stranded oligonucleotide-mediated genome editing, broad-spectrum nucleases, zinc finger nucleases, TALENs, or RNA-guided nucleases such as CRISPR).

[0152] Table 7: Orthologs of the KsIAA16 clade

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] Precise genome editing can be performed as described (Sauer et al. (2016)). Single-stranded oligonucleotides were used to engineer the GG to AA nucleotide changes in the degradation determinant motif of the AUX / IAA 16 orthologs of the desired plant species. Selection of introduced mutations was performed through molecular analysis, genomic sequence confirmation of the nucleotide changes, and auxin sensitivity analysis.

[0160] Alternatively, one or more site-specific nucleases, such as zinc finger nucleases, TALENs, or RNA-guided nucleases (e.g., CRISPR-Cas9, CRISPR-Cpf1, CRISPR-CasX, etc.), are introduced into plant cells along with a template containing an exon encoding a mutated degradation determinant motif (a GG to AA nucleotide change). The site-specific nuclease induces a double-strand break (DSB) on the chromosome around an exon encoding an AUX / IAA16 ortholog of the degradation determinant motif. Through homologous recombination, the template replaces the endogenous exon encoding the wild-type degradation determinant motif. Plants carrying the mutated degradation determinant motif are selected using standard molecular analysis and auxin sensitivity analysis.

[0161] Alternatively, one or more site-specific nucleases, such as zinc finger nucleases, TALENs, or RNA-guided nucleases (e.g., CRISPR), are introduced into plant cells along with a template containing an exon encoding a mutated degradation determinant motif (a GG to AA nucleotide change). The site-specific nuclease induces two double-strand breaks (DSBs) on the chromosome: one DSB 5'- and one DSB 3'- of an exon encoding an AUX / IAA16 ortholog of the degradation determinant motif. By joining non-homologous ends, the template replaces the endogenous exon encoding the wild-type degradation determinant motif. Plants carrying the mutated degradation determinant motif are selected using standard molecular analyses and auxin sensitivity assays.

[0162] Alternatively, base-editing enzymes (e.g., Cas9-cytidine deaminase, Cpf1-cytidine deaminase, etc.) can be introduced into plant cells. These enzymes catalyze a C-to-T base conversion in the exons of the AUX / IAA16 ortholog encoding the degradation determinant motif, resulting in a glycine (G) to asparagine (N) amino acid substitution in the motif. Plants carrying the mutated degradation determinant motif can be selected using standard molecular analysis and auxin sensitivity analysis.

[0163] Example 9. Cross-resistance of R-type Kochia scoparia biotype to 2,4-D and fluroxypyr.

[0164] To clarify whether the R-type Kochia scoparia biotype exhibits cross-resistance to 2,4-D or fluopyram, Kochia scoparia plants grown in greenhouses at a height of 10-15 cm were sprayed with 0, 140, 280, 560, 2240, 4480, 6720, 8960, or 17920 g of aeha using a tracked sprayer. -1 Dicamba; or 0.70, 140, 280, 560, 1120, 2240, 4480, 6720, 8960g aeha -1 2,4-D, or 0, 17.5, 35, 70, 140, 280, 420, 560, 1120, and 2240 g aeha-1 of fluopyram were administered. Fresh weight was determined at 21 DAT and used to calculate dose-response curves using the "R" software. Curves for R-type Kochia scoparia (solid circles) and S-type Kochia scoparia (hollow circles) are shown in Figure 6, with dicamba in Figure A, 2,4-D in Figure B, and fluopyram in Figure C. Visible damage (not shown) was observed in both R and S biotypes at 14 days (DAT) after treatment for all treatments.

Claims

1. A method for producing modified plants, the method comprising the steps of: a. Introducing a modification into the auxin / indole-3-acetic acid (AUX / IAA) gene in plant cells, wherein the modification produces an AUX / IAA polypeptide containing a modified degradation determinant domain comprising motif X1WPPX2, wherein X1 is modified to asparagine (N) relative to the wild-type AUX / IAA polypeptide, and wherein X2 is valine (V). b. Identify and select one or more plant cells from step (a) whose degradation determinant domain of AUX / IAA contains the modification described above; and c. Regenerate at least one plant from at least one or more cells selected from step (b); The modified AUX / IAA polypeptide is encoded by the polynucleotide sequence SEQ ID NO:

220.

2. A modified DNA molecule encoding a modified AUX / IAA polypeptide, wherein the AUX / IAA polypeptide comprises a modified degradation determinant domain containing a motif X1WPPX2, wherein X1 is modified to be asparagine (N) relative to the wild-type AUX / IAA polypeptide, and wherein X2 is valine (V), wherein the modified AUX / IAA polypeptide is encoded by a polynucleotide sequence SEQ ID NO:

220.

3. A DNA construct comprising a modified DNA molecule as described in claim 2, operably linked to a heterologous promoter.

4. The DNA construct of claim 3, wherein the promoter is an inducible promoter.

5. The DNA construct of claim 3, further defined as a promoter of a natural auxin / indole-3-acetic acid gene operatively linked to the plant genome.

6. A method for conferring plant tolerance to auxin herbicides, comprising expressing in the plant a modified DNA molecule as claimed in claim 2, wherein the method is further defined as comprising transforming the plant or its progenitor cells with the modified DNA molecule as claimed in claim 2.

7. A method for producing auxin-tolerant plants, the method comprising: The polynucleotide encoding the AUX / IAA polypeptide of the plant is modified to encode a modified AUX / IAA polypeptide comprising a modified degradation determinant domain containing motif X1WPPX2, wherein X1 is modified to be asparagine (N) relative to the wild-type AUX / IAA polypeptide, and wherein X2 is valine (V), wherein the modified AUX / IAA polypeptide is encoded by the polynucleotide sequence SEQ ID NO:

220.

8. The method of claim 7, wherein the modification comprises site-specific mutagenesis.

9. A method for detecting plants exhibiting sensitivity to at least one endogenous auxin and tolerance to at least one auxin herbicide, comprising the steps of: a. Obtaining genomic DNA samples from the plant; b. Amplify the DNA sample using the first and second primers to generate amplicon; c. Detect the polynucleotide sequence encoding motif X1WPPX2 within SEQ ID NO: 219 in the amplicon; wherein X1 is modified with asparagine (N) relative to the wild-type AUX / IAA polypeptide, and wherein X2 is valine (V).

10. The method of claim 9, wherein the first primer has the sequence SEQ ID NO: 221 and the second primer has the sequence SEQ ID NO:

222.

11. The method of claim 10, wherein the detection includes restriction fragment length polymorphism (RFLP) analysis.

12. The method of claim 9, wherein the first primer has the sequence SEQ ID NO: 223 and the second primer has the sequence SEQ ID NO:

224.

13. The method of claim 12, wherein the detection comprises using a probe selected from the group consisting of SEQ ID NO: 225, SEQ ID NO: 226, or a combination thereof.

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