Microtubule-associated proteins for icafolin tolerance

Abstract

The present disclosure relates to the field of biotechnology and provides novel recombinant DNA molecules for conferring tolerance to icafolin herbicides. The present disclosure also provides herbicide tolerant transgenic plants, seeds, cells, and plant parts comprising the recombinant DNA molecules, and methods of using the same.

Description

TITLE OF THE INVENTIONMICROTUBULE-ASSOCIATED PROTEINS FOR ICAFOLIN TOLERANCE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Application Serial No. 63 / 739,942, filed December 30, 2024, the entire disclosure of which is incorporated herein by reference.INCORPORATION OF SEQUENCE LISTING

[0002] The sequence listing contained in the file named “MONS579WO_ST26.xml”, which is 27.7 kilobytes (measured in MS-Windows) and was created on December 10. 2025, is filed herewith by electronic submission, and is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of biotechnology. More specifically, the disclosure relates to recombinant DNA molecules encoding proteins that provide tolerance to icafolin herbicides and methods of identifying proteins that provide tolerance to icafolin herbicides.BACKGROUND

[0004] Agricultural crop production often utilizes transgenic traits created using the methods of biotechnology. A heterologous gene, also known as a transgene, can be introduced into a plant to produce a transgenic trait. Expression of the transgene in the plant confers a trait, such as herbicide tolerance, to the plant. Non-limiting examples of transgenic herbicide tolerance traits include glyphosate tolerance, glufosinate tolerance, dicamba tolerance, and PPO herbicide tolerance. With the increase of weed species resistant to the commonly used herbicides, new herbicide tolerance traits are needed in the field. Herbicides of particular interest include icafolin herbicides. Icafolin herbicides provide control of a spectrum of herbicide-resistant weeds, thus making a trait conferring tolerance to these herbicides particularly useful in a cropping system. In particular embodiments, icafolin tolerance traits may be combined with one or more other herbicidetolerance trait(s).1US_ACTIVE\131878835W-1SUMMARY

[0005] In one aspect, the present disclosure provides a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide sequence that encodes a polypeptide conferring tolerance to an icafolin herbicide, wherein the polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO: 17, and wherein the heterologous promoter is functional in a plant cell. In another embodiment, the polynucleotide sequence comprises a nucleic acid sequence having at least about 90% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18. In yet another embodiment, the encoded polypeptide comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16. or SEQ ID NO: 17. In further embodiments, the encoded polypeptide comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO: 17. In still yet another embodiment, the nucleic acid sequence is SEQ ID NO:2 or SEQ ID NO: 18. The polynucleotide sequence, in one embodiment, further comprises a nucleic acid sequence that encodes a targeting sequence that functions to localize the encoded polypeptide within a cell. The icafolin herbicide, in another embodiment, is selected from the group consisting of: methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate; methyl(2R,4R)-4-[[(5S)-3-(3.5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]-amino] tetrahydrofuran-2-carboxylate; methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate; (2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; (2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5 -carbonyl] amino] tetrahydrofuran-2-carboxylic acid; (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; and combinations of any thereof. In yet another embodiment, the present disclosure provides a DNA construct comprising a recombinant DNA molecule as described herein. In one embodiment, the DNA construct may comprise a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 10,2US_ACTIVE\131878835W-1SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 wherein the heterologous promoter is functional in a plant cell. In another embodiment, a recombinant DNA molecule of the present disclosure is integrated into the genome of a transgenic plant, seed, plant part, or cell.

[0006] In another aspect, the present disclosure provides a transgenic plant, seed, cell, or plant part comprising a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide sequence that encodes a polypeptide conferring tolerance to an icafolin herbicide, wherein the polypeptide comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In one embodiment, the transgenic plant, seed, cell, or plant part comprises tolerance to an icafolin herbicide. In another embodiment, the transgenic plant, seed, cell, or plant part is a soybean, a maize, a cotton, a barley, a sorghum, a rice, a wheat, a sugar cane, a sugar beet, an alfalfa, or a Brassica plant, seed, cell, or plant part. In yet another embodiment, the icafolin herbicide is selected from the group consisting of: methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate; methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]-amino] tetrahydrofuran-2-carboxylate; methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate; (2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl] amino] tetrahydrofuran-2-carboxylic acid; (2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino] tetrahydrofuran-2-carboxylic acid; (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; and combinations of any thereof. In still yet another embodiment, a transgenic plant, seed, cell, or plant part of the present disclosure may comprise tolerance to at least one additional herbicide.

[0007] In yet another aspect, the present disclosure provides a method of conferring herbicide tolerance to a plant, seed, cell, or plant part, the method comprising expressing a recombinant DNA molecule of the present disclosure in the plant, seed, cell, or plant part. In one embodiment, the recombinant DNA molecule comprises a heterologous promoter operably linked to a polynucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 173US_ACTIVE\131878835W-1wherein the heterologous promoter is functional in a plant cell. In another embodiment, the plant, seed, cell, or plant part comprises tolerance to an icafolin herbicide. In yet another embodiment, the icafolin herbicide is selected from the group consisting of: methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino] tetrahydrofuran-2-carboxylate; methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]-amino]tetrahydrofuran-2-carboxylate; methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate; (2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino] tetrahydrofuran-2-carboxylic acid; (2R,4R)-4-[[(5S)-3-(3,5-difhiorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl] amino] tetrahydrofuran-2-carboxy lie acid; (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; and combinations of any thereof.

[0008] In still yet another aspect, the present disclosure provides a method of producing a transgenic plant or part thereof, the method comprising: a) introducing a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 into a plant cell; and b) regenerating the transgenic plant or part thereof from the cell or a descendant cell thereof that comprises the recombinant DNA molecule; wherein the regenerated transgenic plant or part thereof comprises tolerance to an icafolin herbicide. In another embodiment, the icafolin herbicide is selected from the group consisting of: methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5 -carbonyl] amino] tetrahydrofuran-2-carboxylate; methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]-amino]tetrahydrofuran-2-carboxylate; methyl (25.45)-4-[[(5S)-3-(3.5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate; (2R*,4R*)-4-[[(5S)-3-(3,5-difhiorophenyl)-5-vinyl-4H-isoxazole-5 -carbonyl] amino] tetrahydrofuran-2-carboxylic acid; (2R,4R)-4-[[(5S)-3-(3,5-difhiorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; (25.45)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran -2-carboxylic acid: and combinations of any thereof. In yet another embodiment, the method may further comprise crossing the regenerated transgenic plant with itself or with a second plant to4US_ACTIVE\131878835W-1produce seed. In still yet another embodiment, the encoded polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14. SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In one embodiment, the polynucleotide sequence comprises a nucleic acid sequence having at least about 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18. In another embodiment, the method may further comprise crossing the regenerated transgenic plant, or a descendant plant thereof that comprises the recombinant DNA molecule, with itself or a second plant to produce seed; wherein the seed comprises the recombinant DNA molecule. In yet another embodiment, the present disclosure provides a seed produced by the methods described herein. The present disclosure further provides, in still yet another embodiment, a transgenic plant or part thereof produced by the methods described herein, wherein the transgenic plant or part thereof comprises the recombinant DNA molecule.

[0009] In one aspect, the present disclosure provides a method for controlling weeds in a plant growth area that comprises a transgenic plant or seed of the present disclosure, the method comprising contacting the plant growth area with an icafolin herbicide, wherein the transgenic plant or seed is tolerant to the icafolin herbicide, and wherein weeds are controlled in the plant growth area. In one embodiment, the icafolin herbicide is selected from the group consisting of: methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino] tetrahydrofuran-2-carboxylate; methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]-amino]tetrahydrofuran-2-carboxylate; methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate;(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino] tetrahydrofuran-2-carboxylic acid; (2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid: (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; and combinations of any thereof. In another embodiment, the transgenic plant or seed is a soybean, a maize, a cotton, a barley, a sorghum, a rice, a wheat, a sugar cane, a sugar beet, an alfalfa, or a Brassica plant or seed.

[0010] In another aspect, the present disclosure provides a method of identifying a plant having tolerance to an icafolin herbicide and to at least one additional herbicide, the method comprising: a) obtaining a transgenic plant of the present disclosure comprising tolerance to an icafolin5US_ACTIVE\131878835W-1herbicide and to at least one additional herbicide; b) applying the at least one additional herbicide to the plant or a part thereof; and c) identifying the plant as exhibiting tolerance to the at least one additional herbicide.

[0011] In another aspect, the present disclosure provides a method for reducing the development of herbicide tolerant weeds in a plant growth area comprising a transgenic plant or seed of the present disclosure comprising tolerance to an icafolin herbicide and to at least one additional herbicide, the method comprising contacting the plant growth area with the icafolin herbicide and at least one additional herbicide, wherein the transgenic plant or seed is tolerant to the icafolin herbicide and the at least one additional herbicide. In one embodiment, the icafolin herbicide is selected from the group consisting of: methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate; methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]-amino]tetrahydrofuran-2-carboxylate; methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino] tetrahydrofuran-2-carboxylate; (2R*,4R*)-4-[[(5S)-3-(3,5-difhiorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; (2R,4R)-4-[[(5S)-3-(3.5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; and combinations of any thereof. In another embodiment, the at least one additional herbicide is selected from the group consisting of: an ACCase inhibitor, an ALS inhibitor, an EPSPS inhibitor, a synthetic auxin, a photosynthesis inhibitor, a glutamine synthesis inhibitor, a HPPD inhibitor, a PPO inhibitor, a PDS inhibitor, and a long-chain fatty acid inhibitor.

[0012] In yet another aspect, the present disclosure provides a method identifying candidate herbicide tolerance genes, the method comprising obtaining at least one protoplast derived from a plant of interest; transiently expressing one or more candidate herbicide tolerance genes in said at least one protoplast; exposing the at least one protoplast to a herbicidal compound; and measuring a cellular phenotype in said at least one protoplast following exposure to the herbicidal compound as compared to at least one control protoplast. In some embodiments, the method further comprises identifying at least one protoplast, wherein the cellular phenotype of said at least one protoplast following exposure to the herbicidal compound is similar to the cellular phenotype of at least one control protoplast. In another embodiment, the method further comprises identifying one or more candidate herbicide tolerance genes expressed in said at least one protoplast. In yet 6US_ACTIVE\131878835W-1another embodiment, the method further comprises transiently expressing one or more screenable marker genes in said at least one protoplast. In further embodiments, the one or more screenable marker genes comprises a fluorescent reporter gene. In still further embodiments, measuring the cellular phenotype further comprises filtering for fluorescent expression or detecting the fluorescent reporter gene. In some embodiments, the herbicidal compound is further defined as a cytoskeleton-targeting herbicide, a microtubule-targeting herbicide, or a cytoskeletal-associated protein-targeting herbicide. In other embodiments, the herbicidal compound comprises icafolin methyl. In specific embodiments, the icafolin herbicide is selected from the group consisting of: methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difhiorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate; methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]-amino]tetrahydrofuran-2-carboxylate; methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate; (2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl] amino] tetrahydrofuran-2-carboxy lie acid; (2R,4R)-4-[[(5S)-3-(3.5-difluorophenyl)-5-vinyl-4H-isoxazole-5 -carbonyl] amino]tetrahydrofuran-2-carboxylic acid ; (2S ,4S)-4-[[(5 S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5 carbonyl]amino]tetrahydrofuran-2-carboxylic acid; and combinations of any thereof. In still yet another embodiment, the cellular phenotype comprises a plastid phenotype. In another embodiment, the method further comprises identifying at least one protoplast, wherein the plastid phenotype of said at least one protoplast following exposure to the herbicidal compound is similar to the plastid phenotype of at least one control protoplast not exposed with the herbicidal compound. In other embodiments, measuring the cellular phenotype comprises automated analysis or manual analysis. In yet another embodiment, the one or more candidate herbicide tolerance genes are heterologous to the plant of interest. In further embodiments, measuring the cellular phenotype in said at least one protoplast is carrier out at one or more time points; or one or more concentrations of herbicidal compound. In still further embodiments, the at least one control protoplast lacks expression of the one or more candidate herbicide tolerance genes. In specific embodiments, the at least one control protoplast comprises tolerance to the herbicidal compound or the at least one control protoplast comprises susceptibility to the herbicidal compound.

[0013] Any aspect or embodiment of the present disclosure may be used in combination with any other aspect or embodiment described herein.7US_ACTIVE\131878835W-1BRIEF DESCRIPTION OF THE SEQUENCES

[0014] SEQ ID NO:1 is an amino acid sequence of Glycine max cv. Williams 82 MAP65-1 protein (GLYMA_02G295100).

[0015] SEQ ID NO:2 is the Glycine max nucleotide sequence encoding SEQ ID NO:1.

[0016] SEQ ID NO:3 is an amino acid sequence of Endoplasmic Reticulum-targeted mCherry protein.

[0017] SEQ ID NO:4 is an amino acid sequence of Thosea asigna virus 2A protein.

[0018] SEQ ID NO:5 is a nucleotide sequence of the expression element comprising the promoter and leader sequence of 35S Cauliflower mosiac virus.

[0019] SEQ ID NO:6 is a nucleotide sequence of the terminator sequence of Agrobacterium tumefaciens NOS gene.

[0020] SEQ ID NO:7 is an amino acid sequence of Beta-glucuronidase (GUS) protein.

[0021] SEQ ID NO:8 is an amino acid sequence of nuclear-targeted Cyan Fluorescent Protein (nucCFP).

[0022] SEQ ID NO:9 is an amino acid sequence of chloroplast (plastid) -targeted Green Fluorescent Protein (plGFP).

[0023] SEQ ID NO: 10 is an amino acid sequence variant of Arabidopsis thaliana MAP65-l(At5g55230.3) protein.

[0024] SEQ ID NO: 11 is an amino acid sequence variant of Arabidopsis thaliana MAP65-l(At5g55230.2) protein.

[0025] SEQ ID NO: 12 is an amino acid sequence variant of Arabidopsis thaliana MAP65-1 protein.

[0026] SEQ ID NO: 13 is an amino acid sequence variant of Arabidopsis lyrata MAP65-1 protein.

[0027] SEQ ID NO: 14 is an amino acid sequence variant of Arabidopsis lyrata MAP65-1 protein.

[0028] SEQ ID NO: 15 is an amino acid sequence of Glycine max cv. Williams 82 MAP65-1 protein homolog (GLYMA_14G018300).8US_ACTIVE\131878835W-1

[0029] SEQ TD NO: 16 is an amino acid sequence of Glycine max cv. A3555 MAP65-1 protein homolog (GEYMA_14G041935).

[0030] SEQ ID NO: 17 is an amino acid sequence variant of Glycine max cv. Williams 82 MAP65-1 protein (GLYMA_02G295100) with an alanine inserted at position two.

[0031] SEQ ID NO: 18 is a nucleotide sequence encoding SEQ ID NO: 17.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1: Representative images of tobacco BY-2 protoplast cells 24 hours after transformation with a plasmid encoding a GFP molecule that localizes to the stroma of plastids. Distinctively different phenotypes were observed for cells incubated for 18 hours in 0.2 pM ICM (right) compared to no ICM treatment (left). Scale bar represents 100 pm. Insets: Scale bar represents 10 pm. Cell membrane indicated by dashed circle “m”. Nucleus indicated by “N”. Plastids indicated by “pl”.

[0033] FIG. 2: Representative plastid GFP (Green Fluorescent Protein) assay ICM dose response curve in BY-2 protoplasts. Dose response curve was established by testing ICM concentrations ranging from 500 pM to 100 nM (and more broadly, 10 pM to 200 nM across experiments not included in the figure). These tests identified an effect transition between 1 and 100 nM. Graph summarizes data generated via the combined bright field (BF) and GFP image analysis protocol (“BF+GFP”). Error bars represent standard deviation in response value for three technical replicates.

[0034] FIG. 3: Power analysis results for plastid GFP response assay in BY-2 protoplasts. Power analysis model assumed tolerance of 10% false positives and calculated the estimated power for a varied number of technical replicates of each assay treatment based on untreated and 100 nM ICM treatment control samples. The four curves plotted represent power for different values of delta, “A”, given by the difference between the mean of the response value for a treatment sample and that for the 100 nM ICM control; for example, a treatment response yielding A = 20 produces an estimated power of roughly 98% for only three (3) technical replicates of that treatment.9US_ACTIVE\131878835W-1

[0035] FIG.4: Soy protoplast plastid GFP ICM dose response curve. Graph summarizes data from three technical replicates, with error bars representing standard deviation in response value. BF indicates Bright Field.

[0036] FIG. 5: ICM dose-dependent plastid GFP response value in soy protoplasts transiently transfected with MAP65-1 protein compared with GUS as a negative control using the assay protocol described in Example 1. ICM doses ranged from 0 to 30 nM. Cells expressing MAP65-1 (black circles) showed a higher frequency of “tolerant”-like phenotype — interpreted as tolerance to ICM — at every non-zero ICM concentration tested compared to cells expressing GUS (grey triangles), including the plateaus in response value for tested ICM concentrations > 3 nM. Unlike Figures 2 and 4, the data presented herein were generated using the improved organelle labeling and image analysis approach based on nuclear-directed CFP (Cyan Fluorescent Protein), endoplasmic reticulum-labeled mCherry, and plastid stroma-localized GFP fluorescent protein markers (“3FP”).DETAILED DESCRIPTION

[0037] The following is a detailed description provided to aid those skilled in the art in practicing the embodiments of the present disclosure. Modifications and variations to the embodiments described herein can be made without departing from the spirit or scope of the present disclosure.

[0038] Microtubule-associated proteins play a critical role in the organization, stability, and dynamics of microtubules, which are essential components of the cytoskeleton. Microtubules provide structural support, facilitate intracellular transport, and guide cell division and growth. In particular, MAPs regulate the assembly and disassembly of microtubules, mediate interactions between microtubules and other cellular structures, and respond to environmental and developmental signals to adjust the microtubule network. This regulation is vital for specific processes such as plant cell wall deposition, organelle positioning, and directional growth, which ultimately shape plant morphology and adaptation to environmental stresses. (Gardiner, The evolution and diversification of plant microtubule- associated proteins, The Plant Journal, 75, 219-229, 2013).

[0039] MAP65 proteins in plants act preferentially as crosslinkers of antiparallel microtubules to stabilize the cellular microtubule matrix. As such, MAP65 proteins maintain the integrity of both the midzone microtubules within the central spindle assembly and the phragmoplast, which is 10US_ACTIVE\131878835W-1essential for successful completion of cytokinesis. MAP65-1 is a key member of the MAP65 family and is known for its role in bundling and stabilizing microtubules. Beyond its role in cell division, MAP65-1 is involved in maintaining microtubule organization under stress conditions, such as cold or osmotic stress (Mazars, C. et al. Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts. Cell Calcium 22:413-420, 1997; and Wang, C. et al. Salt tolerance requires cortical microtubule reorganization in Arabidopsis. Plant Cell Physiol. 48:1534-1547, 2007). By regulating microtubule dynamics, MAP65-1 plays a pivotal role in plant growth, development, and survival.

[0040] MAP65 proteins have been shown to play a role in responses to challenges by various plant- associated microorganisms, including phytopathogenic and symbiotic microbes, e.g. AtMAP65-l is targeted by P. syringae effector HopEl to facilitate pathogenesis, suggesting it is an important component of plant immunity in bacterial pathogenesis. Furthermore, enhanced expression of MAP65-1 in transgenic plants increases resistance to both P. syringae pv. glycinea LN 10 and Phytophthora sojae. but not to the pathogenic nematode Heterodera glycines (Kim et al., Characterization of Soybean Events with Enhanced Expression of the Micro tubule- Associated Protein 65-1 (MAP65-1). MPM137:62-71, 2024). Enhanced MAP65-1 expression has also been shown to increase tolerance to the herbicide oryzalin, which is a member of the dinitroaniline class of herbicides. Regrettably, as observed with numerous existing herbicides, resistance to oryzalin has developed in various weed species. For example, some populations of green foxtail can tolerate six times more oryzalin than normal plants, and these populations are resistant to all herbicides in the dinitroaniline chemical family, as well as two other herbicides, dithiopyr and DCPA (Beckie, H.J. et al. Effect of ethalfluralin and other herbicides on trifluralinresistant green foxtail (Setaria viridis). Weed Technol. 7:6-14, 1993). Similarly, goosegrass in cotton fields has also developed resistance to oryzalin and other dinitroaniline herbicides (Vaughn, K.C., et al. A biotype of goosegrass (Eleusine indica) with an intermediate level of dinitroaniline resistance. WeedTechnol. 4:157-162, 1990).

[0041] In contrast to the dinitroaniline class of herbicides, icafolin is a relatively new herbicide belonging to the isoxazolin carboxamide chemical class that acts through plant- specific inhibition of tubulin polymerization. Icafolin has been shown to be highly effective on a broad spectrum of weeds with strong post-emergence activity. Moreover, its post-emergent efficacy is unique for this mode of action. Icafolin herbicides are effective against a range of herbicide-resistant weeds,11US_ACTIVE\131878835W-1making the identification and development of traits that confer tolerance to these herbicides a priority. However, identifying traits conferring tolerance to herbicides is a major challenge, especially herbicides having new and unique modes of action such as icafolin herbicides.

[0042] The present disclosure represents a significant advance in the art by providing proteins confering tolerance to icafolin herbicides. In particular, the present disclosure provides novel, recombinant DNA molecules that encode microtubule-associated proteins (MAPs), e.g. microtubule-associated protein 65-1 (MAP65-1), that confer tolerance to icafolin herbicides. As described herein, these MAP65-1 proteins were identified based on a novel assay, which used a plastid phenotype as an indicator of icafolin herbicide treatment along with a fluorescent protein reporter-based assay to screen traits for tolerance to icafolin.

[0043] The present disclosure therefore provides recombinant DNA molecules encoding a MAP65-1 -related protein, for example any one of SEQ ID NO:1, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, operably linked to a heterologous promoter. The present disclosure further provides vectors and expression cassettes encoding MAP65-1 proteins for expression in cells and plants. Methods for producing cells and plants tolerant to icafolin herbicides are also provided. The disclosure further provides methods and compositions for the use of protein engineering techniques and bioinformatic tools to obtain and improve proteins that confer icafolin tolerance.

[0044] The present disclosure also provides methods for identifying candidate herbicide tolerance genes. For example, the present disclosure demonstrates that protoplasts are amenable to scaling for high-throughput phenotypic screens to assess metabolic and / or morphological characteristics, such as in response to chemical and / or genetic perturbations, for the purpose of identifying and optimizing candidate herbicide tolerance genes including but not limited to genes conferring resistance to cytoskeleton-targeting herbicides, microtubule-targeting herbicides, cytoskeletal-associated protein-targeting herbicides, or icafolin herbicidesA. Icafolin Herbicides

[0045] Icafolin is a novel post-emergence isoxazole herbicide that acts on dicot (broadleaf) and monocot (narrow leaf / grass) weeds. Icafolin has the following structure:12US_ACTIVE\131878835W-1(2S, 4S)-isomer (acid form)

[0046] In certain embodiments, icafolin can be applied as a mixture of two isomers (2R, 4R or 2S, 4S), or can be applied as individual isomers. In some embodiments, icafolin can be applied in the acid form (shown above) or as a methyl ester (shown below).13US_ACTIVE\131878835W-1(2S, 4S)-isomer (methyl ester)

[0047] Non-limiting examples of an icafolin herbicide that may be used according to the embodiments of the present disclosure include methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate; methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]-amino]tetrahydrofuran-2-carboxylate; methyl(2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino] tetrahydrofuran-2-carboxylate; (2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; (2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid; (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5carbonyl]amino]tetrahydrofuran-2-carboxylic acid, and combinations of any thereof.14US_ACTIVE\131878835W-1

[0048] Icafolin and related compounds are described in U.S. Patent Application Publication No.2021 / 292312, the disclosure of which is incorporated herein by reference in its entirety.B. Recombinant Polynucleotide Molecules and Encoded Proteins

[0049] In specific aspects, the present disclosure provides recombinant DNA molecules and proteins. As used herein, the term “recombinant” refers to a non-naturally occurring DNA, protein, cell, seed, or organism that is the result of genetic engineering and as such would not normally be found in nature. As used herein, a “recombinant DNA molecule” is a DNA molecule comprising a DNA sequence that does not naturally occur in nature and as such is the result of human intervention, such as a DNA molecule comprised of at least two DNA molecules heterologous to each other. In one embodiment, a recombinant DNA molecule is a DNA molecule provided herein encoding a protein conferring tolerance to an icafolin herbicide operably linked to a heterologous regulatory or other element, such as a heterologous promoter. As used herein, a “recombinant protein” is a protein comprising an amino acid sequence that does not naturally occur and as such is the result of human intervention. In one embodiment, a recombinant protein may be an engineered protein or a chimeric protein. In another embodiment, a recombinant cell, seed, or organism may be a cell, seed, or organism comprising transgenic DNA. A recombinant cell, seed, or organisms may be, for example, a transgenic cell, seed, plant, or plant part produced as a result of plant transformation and comprising a recombinant DNA molecule.

[0050] As used herein the term “heterologous” refers to a polynucleotide molecule or protein that is not naturally present, or is not naturally present in the same form or structure, in the cell being genetically modified, without human intervention. For example, a heterologous polynucleotide molecule may not naturally occur in the plant species being transformed or modified, or may be expressed in a manner or genomic context that differs from the natural expression pattern or genomic context found in the species being transformed or modified. For example, in some embodiments the heterologous polynucleotide molecule may be overexpressed. In particular embodiments, the heterologous polynucleotide molecule may be the combination of two or more polynucleotide molecules, wherein such a combination is not normally found in nature. The two polynucleotide molecules may, in certain embodiments, be derived from different species or may be derived from different genes, such as, different genes from the same species or the same genes from different species. In some embodiments, a heterologous polynucleotide molecule may15US_ACTIVE\131878835W-1comprise two polynucleotide sequences that are not found juxtaposed or operably linked in any naturally occurring polynucleotide molecule. The heterologous polynucleotide molecule, in further embodiments, may comprise a promoter or other regulatory sequence operably linked to a transcribable polynucleotide sequence, wherein the promoter or other regulatory sequence and the transcribable polynucleotide sequence are not operably linked in any naturally occurring polynucleotide molecule.

[0051] As used herein, the phrase “not normally found in nature” means not found in nature without human intervention. A recombinant polynucleotide or protein molecule may comprise, in certain embodiments, polynucleotide or protein sequences that are (i) separated from other polynucleotide or protein sequence(s) that exist in proximity to each other in nature, or (ii) adjacent to (or contiguous with) other polynucleotide or protein sequences that are not naturally in proximity to each other. Recombinant polynucleotide molecules or proteins may also refer, in some embodiments, to a polynucleotide or protein molecule or sequence that has been genetically engineered or constructed outside of a cell. For example, a recombinant polynucleotide molecule may comprise any engineered or man-made plasmid, vector, or construct, and may include a linear or circular polynucleotide molecule. Such plasmids, vectors, or constructs, may contain, in certain embodiments, various maintenance elements including, for example, a prokaryotic origin of replication or selectable marker gene, as well as one or more transgenes or expression cassettes.

[0052] As used herein, the term “isolated” refers to at least partially separating a molecule from other molecules typically associated with it in its natural state. In one embodiment, the term isolated refers to a DNA molecule that is separated from the nucleic acids that normally flank the DNA molecule in its natural state. For example, a DNA molecule encoding a protein that is naturally present in a particular plant species would be an isolated DNA molecule if it was not within the DNA of the plant species from which the DNA molecule encoding the protein is naturally found. Thus, a DNA molecule fused to or operably linked to one or more other DNA molecule(s) with which it would not be associated in nature, for example as the result of recombinant DNA or plant transformation techniques, is considered isolated herein. Such molecules are considered isolated even when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules.16US_ACTIVE\131878835W-1

[0053] As used herein, the term “protein-coding DNA molecule” refers to a DNA molecule comprising a nucleotide sequence that encodes a protein. As used herein, a “protein-coding sequence” refers to a DNA sequence that encodes a protein. As used herein a “sequence” refers to a sequential arrangement of nucleotides or amino acids. In certain embodiments, the boundaries of a protein-coding sequence may be determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. In some embodiments, a protein-coding molecule may comprise a DNA sequence encoding a protein sequence. As used herein, “transgene expression”, “expressing a transgene”, “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 polypeptide chains, which are ultimately folded into proteins. A protein-coding DNA molecule may be, in certain embodiments, operably linked to a heterologous promoter in a DNA construct for expressing the protein in a cell transformed with the recombinant DNA molecule. As used herein, “operably linked” refers to two DNA molecules linked in manner so that one may affect the function of the other. Operably linked DNA molecules may be part of a single contiguous molecule and may or may not be adjacent. For example, a promoter is operably linked with a protein-coding DNA molecule in a DNA construct where the two DNA molecules are so arranged that the promoter may affect the expression of the transgene.

[0054] As used herein, a “DNA construct” refers to a recombinant DNA molecule comprising two or more heterologous DNA sequences. DNA constructs are useful for transgene expression and may be comprised within vectors or plasmids. DNA constructs may be used in vectors for the purpose of transformation, which is the introduction of heterologous DNA into a host cell, to produce transgenic plants and cells, and as such may also be contained in the plastid DNA or genomic DNA of a transgenic plant, seed, cell, or plant part. As used herein, a “vector” refers to any recombinant DNA molecule that may be used for the purpose of bacterial or plant transformation. Recombinant DNA molecules provided by the present disclosure may, for example, be inserted into a vector as part of a construct having the recombinant DNA molecule operably linked to a gene expression element that functions in a plant to affect expression of the engineered protein encoded by the recombinant DNA molecule. General methods useful for manipulating DNA molecules for making and using recombinant DNA constructs and plant transformation vectors are well known in the art and described in detail in, for example, handbooks and laboratory manuals including MR Green and J Sambrook, “Molecular Cloning: A Laboratory17US_ACTIVE\131878835W-1Manual” (Fourth Edition) ISBN:978-1-936113-42-2, Cold Spring Harbor Laboratory Press, NY (2012). The components for a DNA construct or a vector comprising a DNA construct, in certain embodiments, may include one or more gene expression elements operably linked to a transcribable DNA sequence, such as the following: a promoter for the expression of an operably linked DNA, an operably linked protein-coding DNA molecule, and an operably linked 3’ untranslated region (UTR). Gene expression elements useful in practicing the embodiments of the present disclosure include, but are not limited to, one or more of the following types of elements: promoter, 5’ UTR, enhancer, leader, cis-acting element, intron, targeting sequence, 3’ UTR, and one or more selectable marker transgenes.

[0055] The DNA constructs of the present disclosure may include a promoter operably linked to a protein-coding DNA molecule provided by the present disclosure, whereby the promoter drives expression of the recombinant protein molecule. Promoters useful in practicing the embodiments of the present disclosure include those that function in a cell for expression of an operably linked polynucleotide, such as a bacterial promoter, a viral promoter, or a plant promoter. Plant promoters are varied and well known in the art and include, for instance, those that are inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and / or spatio-temporally regulated.

[0056] In one embodiment of the present disclosure, a DNA construct provided herein includes a targeting sequence that is operably linked to a heterologous nucleic acid molecule encoding a polypeptide molecule that confers tolerance to an icafolin herbicide, whereby the targeting sequence facilitates localizing the polypeptide molecule within the cell. Targeting sequences are known in the art as signal sequences, targeting peptides, localization sequences, and transit peptides. Non-limiting examples of targeting sequences include a chloroplast transit peptide (CTP), a mitochondrial targeting sequence (MTS), and a dual chloroplast and mitochondrial targeting peptide. By facilitating protein localization within the cell, the targeting sequence may increase the accumulation of a recombinant protein, protect the protein from proteolytic degradation, and / or enhance the level of herbicide tolerance, thereby reducing levels of injury in the transgenic cell, seed, or organism following herbicide application.

[0057] CTPs and other targeting molecules that may be used in connection with the present disclosure are known in the art and include, but are not limited to, the Arabidopsis thaliana EPSPS18US_ACTIVE\131878835W-1CTP (Klee et al.. Mol Gen Genet. 210:437-442, 1987), the Petunia hybrida EPSPS CTP (della-Cioppa et al., PNAS 83:6873-6877, 1986), the maize cab-m7 signal sequence (Becker et al., Plant Mol Biol. 20:49-60, 1992; PCT WO 97 / 41228), a mitochondrial pre-sequence (e.g., Silva Filho et al., Plant Mol Biol 30:769-780, 1996), and the pea glutathione reductase signal sequence (Creissen et al., Plant J. 8:167-175, 1995; PCT WO 97 / 41228).

[0058] Recombinant DNA molecules of the present disclosure may be synthesized and modified by methods known in the art, either completely or in part, where it is desirable to provide sequences useful for DNA manipulation (such as restriction enzyme recognition sites or recombination-based cloning sites), plant-preferred sequences (such as plant-codon usage or Kozak consensus sequences), or sequences useful for DNA construct design (such as spacer or linker sequences). In particular embodiments, a recombinant DNA molecule or protein of the present disclosure may comprise or encode a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to any one of SEQ ID NOs:l-18 or fragments thereof, including any range derivable there between. In some embodiments, the recombinant DNA molecules or proteins of the present disclosure may confer icafolin herbicide tolerance when expressed in a plant cell. In certain embodiments, a sequence derived from any of SEQ ID NOs: 1-18 may have the activity of the base sequence from which it was derived, for example herbicide tolerance activity. As used herein, the term “percent sequence identity” or “% sequence identity” refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference (“query”) sequence (or its complementary strand) as compared to a test (“subject”) sequence (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide or amino acid insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the Sequence Analysis software package of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA), MEGAlign (DNAStar Inc., 1228 S. Park St., Madison, WI 53715), and MUSCLE (version 3.6) (Edgar, “MUSCLE: multiple sequence19US_ACTIVE\131878835W-1alignment with high accuracy and high throughput” Nucleic Acids Research 32(5): 1792-7 (2004)) for instance with default parameters. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in the portion of the reference sequence segment being aligned, that is, the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more sequences may be to a full-length sequence or a portion thereof, or to a longer sequence.

[0059] Recombinant DNA molecules or proteins of the present disclosure may comprise or encode a fragment of any of SEQ TD NOs:l-18. For example, the present disclosure provides DNA molecules comprising at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650. at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700 or more contiguous nucleotides of SEQ ID NO:2 or SEQ ID NO: 18. Such fragments of SEQ ID NO:2 or SEQ ID NO: 18 may have the activity of the base sequence from which they are derived, for example herbicide tolerance activity. In some embodiments, proteins of the present disclosure provide fragments comprising at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500, at least 525, at least 550, at least 560, at least 570, at least 580, or more contiguous amino acids of any of SEQ ID NO:1 or 10-17. Such fragments of SEQ ID NO: 1 or 10-17 may have the activity of the base sequence from which they are derived, for example herbicide tolerance activity.

[0060] Engineered proteins may be produced by changing (that is. modifying) a wild-type protein to produce a new protein with modified characteristic(s) including, but not limited to, a particular cellular localization pattern, such as targeted to the chloroplast or mitochondria, or a novel combination of useful protein characteristics, inhibitor / herbicide specificity, small molecule interactions, the ability to interact with other components in the cell such as partner proteins or membranes, and protein stability, among others. In certain embodiments, modifications may be made at specific amino acid positions in a protein. In one embodiment, a substitution of the amino acid found at a particular amino acid position in nature (that is, in the wild-type protein) with a 20US_ACTIVE\131878835W-1different amino acid may be made. Engineered proteins provided by the present disclosure thus provide a new protein with one or more altered protein characteristics relative to a similar protein found in nature. In one embodiment of the present disclosure, an engineered protein has altered protein characteristics, such as those that result in decreased sensitivity to one or more herbicides as compared to a similar wild-type protein, or an improved ability to confer herbicide tolerance to a transgenic plant expressing the engineered protein. In another embodiment, the present disclosure provides an engineered protein, and the recombinant DNA molecule encoding it, comprising at least one amino acid substitution selected from Table 1 and having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any of the engineered protein sequences provided herein, including but not limited to those protein sequences comprising or encoded by any one of SEQ ID NOs:l-18, including all ranges derivable therebetween. Amino acid substitutions may be made as a single amino acid substitution in the protein or in combination with one or more other mutation(s), such as one or more other amino acid substitution(s), deletions, or additions. Mutations may be made by any method known to those of skill in the art.Table 1: Amino Acid Substitutions.21US_ACTIVE\131878835W-1

[0061] As used herein, “wild-type” means a naturally occurring similar, but not identical, version of a protein. A “wild-type DNA molecule” or “wild-type protein” is a naturally occurring version of a DNA molecule or protein, that is, a version of a DNA molecule or protein pre-existing in nature.C. Genetically Modified Plants Produced by Genetic Engineering

[0062] Various genetic engineering technologies have been developed and may be used by those of skill in the art to introduce transgenic or edited traits into plants. The methods generally involve the delivery of a polynucleotide sequence into a plant cell, which may typically be a heterologous or recombinant polynucleotide molecule. A heterologous or recombinant polynucleotide molecule may comprise, for example, at least one transgene, expression cassette, or RNA molecule, such as a guide RNA (gRNA). In specific embodiments, the heterologous or recombinant polynucleotide molecule may be part of a ribonucleoprotein (RNP) or a fragment thereof or a guide RNA. Expression of the heterologous or recombinant polynucleotide molecule may, for example, produce a gRNA I site-specific nuclease complex for genome editing. In certain embodiments, traits are introduced into plants by altering or introducing a single genetic locus or transgene into the genome of a plant. Methods of genetic engineering to modify, delete, or insert transgenes, edits, mutations, and polynucleotide sequences into the genomic DNA of plants are known in the art. Molecular methods of editing a plant cell genome or endogenous plant gene using a genome editing technique are known in the art. According to present embodiments, a polynucleotide or DNA molecule comprising or encoding genome editing tools or machinery, such as a guide RNA, site-specific nuclease, and / or template DNA molecule, may be introduced into a plant cell using the methods described herein.

[0063] As used herein, a “genetically modified” plant, plant part, plant tissue, or plant cell comprises a genetic modification. As used herein, a “genetic modification” refers to one or more transgenes, mutations, or edits introduced into the genome of a plant, plant part or plant cell using a transformation, mutagenesis, or genome editing technique. Apart from a genome editing technique, a mutagenesis technique may include any chemical, physical, radiological, or biological (e.g., transposon-mediated) mutagenesis technique or mutagen.

[0064] As used herein, a “transgenic” plant, plant part, plant tissue, or plant cell has an exogenous nucleic acid sequence, genome edit, or transgene integrated into the genome of the plant, plant22US_ACTIVE\131878835W-1part, plant tissue, or plant cell. As used herein, a “transgenic trait” refers to a characteristic or phenotype conveyed or conferred by the presence of a transgene, nucleic acid sequence, genome edit, or transgene incorporated into the plant genome. Because of such a genomic alteration, the transgenic plant is something distinctly different from the related wild-type plant and the transgenic trait is a trait not naturally found in the wild-type plant. In certain embodiments, transgenic plants of the present disclosure comprise the recombinant DNA molecules and engineered proteins provided by the herein.

[0065] Genome editing can be used to make one or more edits or mutations at a desired target site in the genome of a plant, such as to change expression or activity of one or more genes, or to integrate an insertion sequence or transgene at a desired location in a plant genome. Any site or locus within the genome of a plant may potentially be chosen for making a gene edit or site-directed integration of a transgene, construct, or transcribable DNA sequence. As used herein, a “target site” for genome editing or site-directed integration refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by a site-specific nuclease to introduce a double- stranded break (DSB) or single-stranded nick into the nucleic acid backbone of the polynucleotide sequence and / or its complementary DNA strand within the plant genome. A target site may comprise, for example, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 consecutive nucleotides. A “target site” for an RNA-guided nuclease may comprise the sequence of either complementary strand of a double-stranded nucleic acid (DNA) molecule or chromosome at the target site. A site-specific nuclease may bind to a target site, such as via a non-coding guide RNA (e.g., without being limiting, a CRISPR RNA (crRNA) or a single-guide RNA (sgRNA) as described further herein). A non-coding guide RNA provided herein may be complementary to a target site (e.g., complementary to either strand of a double-stranded nucleic acid molecule or chromosome at the target site). It will be appreciated that perfect identity or complementarity may not be required for a non-coding guide RNA to bind or hybridize to a target site. For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 mismatches (or more) between a target site and a non-coding RNA may be tolerated. A “target site” also refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by any other site-specific nuclease that may not be guided by a non-coding RNA molecule, such as a zinc23US_ACTIVE\131878835W-1finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, etc., to introduce a DSB or single- stranded nick into the polynucleotide sequence and / or its complementary DNA strand. As used herein, a “site- specific nuclease” includes any zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), ribonucleoprotein, meganuclease, recombinase, transposase, or other nuclease that can introduce a double-stranded break (DSB) or single-stranded nick into a polynucleotide sequence at or near a target site, such as a target site within the genome of a plant cell. As used herein, a “target region” or a “targeted region” refers to a polynucleotide sequence or region that is flanked by two or more target sites. Without being limiting, in some embodiments, a target region may be subjected to a mutation, deletion, insertion, or inversion. As used herein, “flanked” when used to describe a target region of a polynucleotide sequence or molecule, refers to two or more target sites of the polynucleotide sequence or molecule surrounding the target region, with one target site on each side of the target region.

[0066] As used herein, a “targeted genome editing technique” refers to any method, protocol, or technique that allows the precise and / or targeted editing of a specific location in a genome of a plant (i.e., the editing is largely or completely non-random) using a site-specific nuclease, such as a meganuclease, a zinc-finger nuclease (ZFN), an RNA-guided endonuclease (e.g., the CRISPR / Cas9 system or CRISPR / Casl2a system), a TALE (transcription activator-like effector)-endonuclease (TALEN), a recombinase, or a transposase. As used herein, “editing” or “genome editing” refers to generating a targeted mutation, deletion, insertion, inversion, or substitution 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 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000. at least 10,000, or at least 25,000 nucleotides of an endogenous plant genome nucleic acid sequence. As used herein, “editing” or “genome editing” may also encompass the targeted insertion or site-directed integration 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 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75. at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 10,000, or at least 25,000 nucleotides into the endogenous genome of a plant. An “edit” or “genomic edit” in the singular refers to one such targeted mutation, deletion, inversion, substitution, or insertion,24US_ACTIVE\131878835W-1whereas “edits” or “genomic edits” refers to two or more targeted mutation(s), deletion(s), inversion(s), substitution(s) and / or insertion(s), with each “edit” being introduced via a targeted genome editing technique.

[0067] According to some embodiments, a site-specific nuclease may be co-delivered with a donor template molecule to serve as a template for making a desired edit, mutation, or insertion into the genome at the desired target site through repair of the double strand break (DSB) or nick created by the site-specific nuclease. According to some embodiments, a site-specific nuclease may be co-delivered with a DNA molecule comprising a selectable or screenable marker gene.

[0068] A site- specific nuclease provided herein may be selected from the group consisting of a zinc-finger nuclease (ZFN), a TALE-endonuclease (TALEN), a meganuclease, an RNA-guided endonuclease, a recombinase, a transposase, or any combination thereof. See, e.g., Khandagale et al. (Plant Biotechnol Rep 10:327-343, 2016); and Gaj et al. (Trends Biotechnol. 31(7):397-405, 2013. Zinc finger nucleases (ZFN) are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to a cleavage domain (or a cleavage half-domain), which may be derived from a restriction endonuclease (e.g., FokF). The DNA binding domain may be canonical (C2H2) or non-canonical e.g., C3H or C4). The DNA-binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers) depending on the target site but may typically be composed of 3-4 (or more) zinc-fingers. Multiple zinc fingers in a DNA-binding domain may be separated by linker sequence(s). ZFNs can be designed to cleave almost any stretch of double-stranded DNA by modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of a non-specific DNA cleavage domain (e.g., derived from the Fold nuclease) fused to a DNA-binding domain comprising a zinc finger array engineered to bind a target site DNA sequence. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger a-helix, which contribute to site-specific binding to the target site, can be changed and customized to fit specific target sequences. The other amino acids may form a consensus backbone to generate ZFNs with different sequence specificities.

[0069] Methods and rules for designing ZFNs for targeting and binding to specific target sequences are known in the art. See, e.g., U.S. Patent App. Pub. Nos. 2005 / 0064474, 2009 / 0117617, and 2012 / 0142062. The FokI nuclease domain may require dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are needed to bind opposite DNA25US_ACTIVE\131878835W-1strands of the cleavage site (separated by 5-7 bp). The ZFN monomer can cut the target site if the two-ZF-binding sites are palindromic. A ZFN, as used herein, is broad and includes a monomeric ZFN that can cleave double stranded DNA without assistance from another ZFN. The term ZFN may also be used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site. Because the DNA-binding specificities of zinc finger domains can be re-engineered using one of various methods, customized ZFNs can theoretically be constructed to target nearly any target sequence (e.g., at or near a gene in a plant genome). Publicly available methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly.

[0070] Transcription activator-like effectors (TALEs) can be engineered to bind practically any DNA sequence, such as at or near the genomic locus of a gene in a plant. TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat-variable diresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.

[0071] TALENs are artificial restriction enzymes generated by fusing the TALE DNA binding domain to a nuclease domain. In some aspects, the nuclease is selected from a group consisting of Pvull, MutH, TevI, FokI, Alwl, Mlyl, Sbfl, Sdal, StsI, CleDORF, Clo051, and Pept071. When each member of a TALEN pair binds to the DNA sites flanking a target site, the FokI monomers dimerize and cause a double-stranded DNA break at the target site. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN also refers to one or both members of a pair of TALENs that work together to cleave DNA at the same site.

[0072] Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites26US_ACTIVE\131878835W-1in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity. PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and FokI variants for use with TALEs. PvuII functions as a highly specific cleavage domain when coupled to a TALE (See Yank et al., PLoS One 8:e82539, 2013). MutH is capable of introducing strandspecific nicks in DNA (See Gabsalilow et al., Nucleic Acids Research. 41:e83, 2013). TevI introduces double-stranded breaks in DNA at targeted sites (See Beurdeley et al., Nature Communications 4:1762, 2013).

[0073] The relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. Software programs such as DNAWorks can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill in the art. See Doyle et al. (Nucleic Acids Research 4O:W117-122, 2012); Cermak et al. (Nucleic Acids Research 39:e82, 2011); and tale-nt.cac.cornell.edu / about. In another aspect, a TALEN provided herein is capable of generating a targeted DSB.

[0074] A site-specific nuclease may be a meganuclease. Meganucleases, which are commonly identified in microbes, such as the LAGLIDADG family of homing endonucleases, are unique enzymes with high activity and long recognition sequences (> 14 bp) resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 bp). The engineering of meganucleases can be more challenging than ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity.

[0075] A site-specific nuclease may be an RNA-guided nuclease. According to some embodiments, an RNA-guided endonuclease may be selected from the group consisting of Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3. Csf4, Casl2a (also known as Cpfl), CasX, CasY, and homologs or1US_ACTIVE\131878835W-1modified versions of any thereof, as well as Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo). and homologs or modified versions of any thereof). According to some embodiments, an RNA-guided endonuclease is a Cas9 or Casl2a enzyme. The RNA-guided nuclease may be delivered as a protein with or without a guide RNA, or the guide RNA may be complexed with the RNA-guided nuclease enzyme and delivered as a ribonucleoprotein (RNP).

[0076] For RNA-guided endonucleases, a guide RNA molecule may be further provided to direct the endonuclease to a target site in the genome of the plant via base-pairing or hybridization to cause a DSB or nick at or near the target site. The guide RNA may be transformed or introduced into a plant cell or tissue as a gRNA molecule, or as a recombinant DNA molecule, construct or vector comprising a transcribable DNA sequence encoding the guide RNA operably linked to a promoter. As understood in the art, a guide RNA may comprise, for example, a CRISPR RNA (crRNA), a tracrRNA, a single-chain guide RNA (sgRNA), or any other RNA molecule that may guide or direct an endonuclease to a specific target site in the genome. A prototypical CRISPR-associated protein, Cas9 from S. pyogenes, naturally binds two RNAs, a CRISPR RNA (crRNA) guide and a trans-acting CRISPR RNA (tracrRNA), to assemble a CRISPR ribonucleoprotein (crRNP). A “single-chain guide RNA” (or “sgRNA”) is an RNA molecule comprising a crRNA covalently linked a tracrRNA by a linker sequence, which may be expressed as a single RNA transcript or molecule. The guide RNA comprises a guide or targeting sequence (also referred to herein as a “spacer sequence”) that is identical or complementary to a target site within the plant genome, such as at or near a gene. The guide RNA is typically a non-coding RNA molecule that does not encode a protein. The guide sequence of the guide RNA may be at least 10 nucleotides in length, such as 12-40 nucleotides, 12-30 nucleotides, 12-20 nucleotides, 12-35 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, or 17-25 nucleotides in length, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length. The guide sequence may be at least 95%, at least 96%, at least 97%, at least 99% or 100% identical or complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15. at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a DNA sequence at the genomic target site.28US_ACTIVE\131878835W-1

[0077] As used herein, with respective to a given sequence, a “complement”, a “complementary sequence” and a “reverse complement” are used interchangeably. All three terms refer to the inversely complementary sequence of a nucleotide sequence, i.e., to a sequence complementary to a given sequence in reverse order of the nucleotides.

[0078] As used herein, the term “antisense” refers to DNA or RNA sequences that are complementary to a specific DNA or RNA sequence. Antisense RNA molecules are singlestranded nucleic acids which can combine with a sense RNA strand or sequence or mRNA to form duplexes due to complementarity of the sequences. The term “antisense strand” refers to a nucleic acid strand that is complementary to the “sense” strand. The “sense strand” of a gene or locus is the strand of DNA or RNA that has the same sequence as an RNA molecule transcribed from the gene or locus (with the exception of uracil in RNA and thymine in DNA).

[0079] A protospacer- adjacent motif (PAM) may be present in the genome immediately adjacent and upstream to the 5’ end of the genomic target site sequence complementary to the targeting sequence of the guide RNA - i.e., immediately downstream (3’) to the sense (+) strand of the genomic target site (relative to the targeting sequence of the guide RNA) as known in the art. See, e.g., Wu etal. (Quant Biol. 2(2):59-70, 2014). The genomic PAM sequence on the sense (+) strand adjacent to the target site (relative to the targeting sequence of the guide RNA) may comprise 5’-NGG-3’. However, the corresponding sequence of the guide RNA i.e., immediately downstream (3’) to the targeting sequence of the guide RNA) may generally not be complementary to the genomic PAM sequence.

[0080] In some embodiments, a site-specific nuclease is a recombinase. Non-limiting examples of recombinases that may be used include a serine recombinase attached to a DNA recognition motif, a tyrosine recombinase attached to a DNA recognition motif, or any recombinase enzyme known in the art attached to a DNA recognition motif. In certain embodiments, the site-specific nuclease is a recombinase or transposase, which may be a DNA transposase or recombinase attached or fused to a DNA binding domain. Non-limiting examples of recombinases include a tyrosine recombinase selected from the group consisting of a Cre recombinase, a Gin recombinase, a Flp recombinase, and a Tnpl recombinase attached to a DNA recognition motif provided herein. In one aspect of the present disclosure, a Cre recombinase or a Gin recombinase provided herein is tethered to a zinc-finger DNA-binding domain, a TALE DNA-binding domain, or a Cas929US_ACTIVE\131878835W-1nuclease. Tn another aspect, a serine recombinase selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase may be attached to a DNA recognition motif provided herein. In yet another aspect, a DNA transposase selected from the group consisting of a TALE-piggyBac and TALE-Mutator may be attached to a DNA binding domain provided herein.

[0081] Several site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, are not RNA-guided and instead rely on their protein structure to determine their target site for causing the DSB or nick, or they are fused, tethered, or attached to a DNA-binding protein domain or motif. The protein structure of the site-specific nuclease (or the fused / attached / tethered DNA binding domain) may target the site-specific nuclease to the target site. According to many of these embodiments, non-RNA-guided site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, may be designed, engineered, and constructed according to known methods to target and bind to a target site at or near the genomic locus of an endogenous gene of a plant to create a DSB or nick at such a genomic locus. The DSB or nick created by the non-RNA-guided site-specific nuclease may lead to knockdown of gene expression via repair of the DSB or nick, which may result in a mutation or insertion of a sequence at the site of the DSB or nick through cellular repair mechanisms. Such cellular repair mechanism may be guided by a donor template molecule.

[0082] As used herein, a “donor molecule”, “donor template”, or “donor template molecule” (collectively a “donor template”), which may be a recombinant polynucleotide, DNA or RNA donor template or sequence, is defined as a nucleic acid molecule having a homologous nucleic acid template or sequence (e.g., homology sequence) and / or an insertion sequence for site-directed, targeted insertion or recombination into the genome of a plant cell via repair of a nick or DSB in the genome of a plant cell. A donor template may be a separate DNA molecule comprising one or more homologous sequence(s) and / or an insertion sequence for targeted integration, or a donor template may be a sequence portion (i.e., a donor template region) of a DNA molecule further comprising one or more other expression cassettes, genes / transgenes, and / or transcribable DNA sequences. A donor template may be an Agrobacterium Transfer-DNA (T-DNA) molecule comprising one or more other expression cassettes, genes / transgenes, and / or transcribable DNA sequences. For example, a “donor template” may be used for site-directed integration of a transgene or construct, or as a template to introduce a mutation, such as an insertion, deletion, substitution, etc., into a target site within the genome of a plant. A targeted genome editing 30US_ACTIVE\131878835W-1technique provided herein may comprise the use of one or more, two or more, three or more, four or more, or five or more donor molecules or templates. A donor template provided herein may 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 gene(s) or transgene(s) and / or transcribable DNA sequence(s). Alternatively, a donor template may comprise no genes, transgenes, or transcribable DNA sequences.

[0083] Without being limiting, a gene / transgene or transcribable DNA sequence of a donor template may include, for example, an insecticidal resistance gene, an herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a yield enhancing gene, a nutritional quality gene, a DNA binding gene, a selectable marker gene, an RNAi or suppression construct, a site-specific genome modification enzyme gene, a single guide RNA of a CRISPR / Cas9 system, a guide RNA of a CRISPR / Casl2a system, a geminivirus-based expression cassette, or a plant viral expression vector system. According to other embodiments, an insertion sequence of a donor template may comprise a protein encoding sequence or a transcribable DNA sequence that encodes a non-coding RNA molecule, which may target an endogenous gene for suppression. A donor template may comprise a promoter operably linked to a coding sequence, gene, or transcribable DNA sequence, such as a constitutive promoter, a tissue- specific or tissue-preferred promoter, a developmental stage promoter, or an inducible promoter. A donor template may comprise a leader, enhancer, promoter, transcriptional start site, 5’-UTR, one or more exon(s), one or more intron(s), transcriptional termination site, region, or sequence, 3’-UTR, and / or poly adenylation signal, which may each be operably linked to a coding sequence, gene (or transgene) or transcribable DNA sequence encoding a non-coding RNA, a guide RNA, an mRNA and / or protein. A donor template may be a single- stranded or double- stranded DNA or RNA molecule or plasmid.

[0084] An “insertion sequence” of a donor template is a sequence designed for targeted insertion into the genome of a plant cell, which may be of any suitable length. For example, the insertion sequence of a donor template may be between 2 and 50,000, between 2 and 10,000, between 2 and 5000, between 2 and 1000, between 2 and 500, between 2 and 250, between 2 and 100, between 2 and 50, between 2 and 30, between 15 and 50, between 15 and 100, between 15 and 500, between 15 and 1000. between 15 and 5000, between 18 and 30, between 18 and 26. between 20 and 26, between 20 and 50, between 20 and 100, between 20 and 250, between 20 and 500, between 20 and 1000, between 20 and 5000, between 20 and 10,000, between 50 and 250, between 50 and 31US_ACTIVE\131878835W-1500, between 50 and 1000, between 50 and 5000, between 50 and 10,000, between 100 and 250, between 100 and 500, between 100 and 1000, between 100 and 5000, between 100 and 10,000, between 250 and 500, between 250 and 1000, between 250 and 5000. or between 250 and 10,000 nucleotides or base pairs in length. A donor template may also have at least one homology sequence or homology arm, such as two homology arms, to direct the integration of a mutation or insertion sequence into a target site within the genome of a plant via homologous recombination, wherein the homology sequence or homology arm(s) are identical or complementary, or have a percent identity or percent complementarity, to a sequence at or near the target site within the genome of the plant. When a donor template comprises homology arm(s) and an insertion sequence, the homology arm(s) will flank or surround the insertion sequence of the donor template. Each homology arm may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99% or 100% identical or complementary to at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 2500, or at least 5000 consecutive nucleotides of a target DNA sequence within the genome of a plant.

[0085] Any method known in the art for site-directed integration may be used with the present disclosure. In the presence of a donor template molecule with an insertion sequence, the DSB or nick can be repaired by homologous recombination between homology arm(s) of the donor template and the plant genome, or by non-homologous end joining (NHEJ), resulting in site-directed integration of the insertion sequence into the plant genome to create the targeted insertion event at the site of the DSB or nick. Thus, site-specific insertion or integration of a transgene, transcribable DNA sequence, construct, or sequence may be achieved if the transgene, transcribable DNA sequence, construct or sequence is located in the insertion sequence of the donor template.

[0086] The introduction of a DSB or nick may also be used to introduce targeted mutations in the genome of a plant. According to this approach, mutations, such as deletions, insertions, inversions, and / or substitutions may be introduced at a target site via imperfect repair of the DSB or nick to produce a knock-out or knock-down of a gene. Such mutations may be generated by imperfect repair of the targeted locus even without the use of a donor template molecule. A “knock-out” of a gene may be achieved by inducing a DSB or nick at or near the endogenous locus of the gene 32US_ACTIVE\131878835W-1that results in non-expression of the protein or expression of a non-functional protein, whereas a “knock-down” of a gene may be achieved in a similar manner by inducing a DSB or nick at or near the endogenous locus of the gene that is repaired imperfectly at a site that does not affect the coding sequence of the gene in a manner that would eliminate the function of the encoded protein. For example, the site of the DSB or nick within the endogenous locus may be in the upstream or 5’ region of the gene (e.g., a promoter and / or enhancer sequence) to affect or reduce its level of expression.

[0087] Similarly, such targeted knock-out or knock-down mutations of a gene may be generated with a donor template molecule to direct a particular or desired mutation at or near the target site via repair of the DSB or nick. The donor template molecule may comprise a homologous sequence with or without an insertion sequence and comprising one or more mutations, such as one or more deletions, insertions, inversions and / or substitutions, relative to the targeted genomic sequence at or near the site of the DSB or nick. For example, targeted knock-out or knock-down mutations of a gene may be achieved by substituting, inserting, deleting, or inverting at least a portion of the gene, such as by introducing a frame shift or premature stop codon into the coding sequence of the gene or disrupting a promoter sequence or the sequence of another non-coding regulatory element of the gene. A deletion of a portion of a gene may also be introduced by generating DSBs or nicks at two target sites and causing a deletion of the intervening target region flanked by the target sites.

[0088] In further embodiments, genetic modification of a plant may comprise transformation of a plant, plant part, plant tissue or plant cell to insert a polynucleotide or DNA sequence or transgene into the genome of the plant, plant part, plant tissue or plant cell. Methods for transformation of plants that are known in the art and applicable to many crop species include, but are not limited to, electroporation, microprojectile or particle bombardment, microinjection, PEG-mediated transformation. Agrobacterium-mediated transformation, and other modes of direct DNA uptake. Bacteria known to mediate plant cell transformation include a number of species of bacterial genera, species, and strains that may be assigned to the order Rhizobiales other than Agrobacterium, including but not limited to, bacterial species and strains from the taxonomic families Rhizobiaceae (e.g. Rhizobium spp., Sinorhizobium spp.), Phyllobacteriaceae (e.g. Mesorhizobium spp., Phyllobacterium spp.), Brucellaceae (e.g. Ochrobactrum spp.), Bradyrhizobiaceae (e.g. Brady rhizobium spp.), and Xanthobacteraceae (e.g. Azorhizobium spp.), among others. According to some embodiments, Agrobacterium-mediated transformation is 33US_ACTIVE\131878835W-1mediated by Agrobacterium tumefaciens. Targets for such transformation have often been undifferentiated callus tissues, although differentiated tissues have also been used for transient and stable plant transformation. As is well known in the art, other methods for plant transformation may be utilized, for instance as described by Miki et al., (1993, “Procedures for Introducing Foreign DNA into Plants,” in Methods in Plant Molecular Biology and Biotechnology, Glick B. R. and Thompson, J. E. Eds., CRC Press, Inc., Boca Raton, pages 67-88).

[0089] In specific embodiments, microprojectile bombardment may be employed to deliver a polynucleotide or DNA molecule, vector, sequence, segment, or RNP to a cell. In this method, particles are coated with a polynucleotide or polynucleotide / protein complex and delivered into cells by a propelling force. Exemplary particles may include those comprised of tungsten, platinum, or gold. For bombardment, target cells may be arranged on a solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the projectile stopping plate. A polynucleotide may be delivered into plant cells by acceleration using a biolistics particle delivery system, which may propel particles coated with a DNA or polynucleotide molecule through a screen, such as a stainless steel or Nytex screen, and toward the cells positioned on a surface. The screen may disperse the particles so that they are not delivered to the recipient cells in large aggregates. Microprojectile bombardment techniques are widely applicable and may be used to transform a variety of plant species.

[0090] Agrobacterium-mediated or Rhizobiales-mediated transformation of cells is another widely applicable system for introducing heterologous and / or recombinant DNA molecules into plant cells. Modem Agrobacterium-mediated transformation vectors are capable of replication in E. coli as well as in Agrobacterium, allowing for convenient manipulations (See, e.g., Klee et al., Nat. Biotechnol., 3(7):637-642, 1985). Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes. Additionally, Agrobacterium containing both armed and disarmed Ti plasmids can be used for transformation. Agrobacterium-mediated transformation is often the method of choice for many plant species. The use of Agrobacterium- mediated plant integrating vectors to introduce DNA34US_ACTIVE\131878835W-1into plant cells is known in the art (See, e.g., Fraley et al., Nat. Biotechnok, 3:629-635, 1985; U.S. Patent No. 5,563,055).

[0091] A number of promoters and expression elements have utility for plant gene expression of any selectable marker, scoreable marker, transgene, or any other gene of agronomic interest. Promoters may include any constitutive promoter, tissue-specific promoter, organ-specific promoter, tissue-preferred promoter, organ-preferred promoter, inducible promoter, reproductive tissue promoter, developmental stage promoter, viral promoter, or the like. Examples of various types of promoters and expression elements are known in art. Expression elements that may be useful for plant gene expression may include, for example, various promoters, enhancers, leaders, 5’ and 3’ untranslated regions, introns, terminators, and the like, as known in the art. A selectable or screenable marker or gene of interest may also be fused to a transit peptide or other targeting sequence. Transport of proteins produced by transgenes to a subcellular compartment such as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, nucleus, or mitochondrion or for secretion into the apoplast, may be accomplished by means of operably linking the nucleotide sequence encoding a signal or targeting sequence to the 5' and / or 3' region of a gene encoding the protein of interest. Targeting sequences at the 5' and / or 3' end of the structural gene may determine, during protein synthesis and processing, where the encoded protein is ultimately compartmentalized. The presence of a signal sequence directs a polypeptide to either an intracellular organelle or subcellular compartment or for secretion to the apoplast. Many signal sequences are known in the art. See, for example Becker et al. (Plant Mol. Biol., 20:49, 1992); Knox et al. (Plant Mol. Biol., 9:3-17, 1987); Lerner et al. (Plant Physiol., 91:124-129, 1989); Fontes et al. (Plant Cell, 3:483-496, 1991); Matsuoka et al. (Proc. Natl. Acad. Sci. USA, 88:834, 1991); Gould et al. (J. Cell. Biol., 108:1657. 1989); Creissen et al. (Plant J., 2:129. 1991); Kalderon et al. (Cell, 39:499-509, 1984); Steifel et al. (Plant Cell, 2:785-793, 1990).

[0092] Examples of constitutive promoters may include, for example, the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (See, e.g., Odel et al., Nature, 313:810, 1985), including monocots (See, e.g., Dekeyser et al., Plant Cell, 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990), a tandemly duplicated version of the CaMV 35S promoter, the enhanced 35S promoter (e35S). the nopaline synthase promoter (An et al., Plant Physiol., 88:547, 1988), the octopine synthase promoter (Fromm et al., Plant Cell, 1:977, 1989), and the fig wort mosaic virus (FMV) promoter as described 35US_ACTIVE\131878835W-1in U.S. Patent No. 5,378,619 and an enhanced version of the FMV promoter (eFMV) where the promoter sequence of FMV is duplicated in tandem, the cauliflower mosaic virus 19S promoter, a sugarcane bacilliform virus promoter, a commelina yellow mottle virus promoter, and other plant DNA virus promoters known to express in plant cells. Constitutive promoters may also include promoters derived from plant genomes such as the maize ubiquitin promoter (Christensen et al., Plant Mol. BioL, 18: 675, 689. 1992), maize actin promoter (McElroy, et al., Plant Cell, 2:163-171, 1990), or Arabidopsis S-Adenosylmethionine synthetase promoter (See U.S. Patent No.8,809,628).

[0093] With an inducible promoter, the rate of transcription increases in response to an inducing agent or signal. Any inducible promoter can be used according to the embodiments of the present disclosure. A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and / or developmental signals can be used for expression of an operably linked gene in plant cells, including promoters regulated by (1) heat (e.g., Callis et al., Plant Physiol., 88:965, 1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al., Plant Cell, 1:471, 1989; maize rbcS promoter, Schaffner and Sheen, Plant Cell, 3:997, 1991; or chlorophyll a / b-binding protein promoter, Simpson et al., EMBO J., 4:2723, 1985), (3) hormones, such as abscisic acid (Marcotte et al., Plant Cell, 1:969, 1989), (4) wounding (e.g., Siebertz et al., Plant Cell, 1:961, 1989), or (5) chemicals such as methyl jasmonate, salicylic acid, or Safener. It may also be advantageous to employ organ-specific or tissue specific promoters known in the art (e.g., Roshal et al., EMBO J., 6:1155, 1987; Schemthaner et al., EMBO J., 7:1249, 1988: Bustos et al., Plant Cell, 1:839, 1989).

[0094] Exemplary polynucleotide or DNA molecules which may be introduced include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. However, the term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps not present in the form, structure, or location. A polynucleotide may include a DNA molecule or sequence which is already present in the plant cell, is from another plant, is from a different organism, is exogenous or generated externally. A transgene or expression cassette may encode a mRNA and protein or an RNA molecule for suppression, such as a miRNA, siRNA, dsRNA, antisense RNA, inverted repeat RNA, or the like. A polynucleotide 36US_ACTIVE\131878835W-1may be an exogenous, heterologous and / or recombinant polynucleotide or DNA molecule or sequence.

[0095] Many different transgenes or genetic modifications could potentially be introduced according to the present disclosure, which may provide a beneficial agronomic trait of a crop plant having the transgene or modification. In particular embodiments, the transgene or genetic modification introduced into the genome of a plant cell may comprise or encode a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 2, 10, 11, 12, 13, 14, 15, 16, and 17, or fragments thereof, including any range derivable therebetween. In some embodiments, the transgene or genetic modification introduced into the genome of a plant cell may confer icafolin herbicide tolerance.

[0096] Additional non-limiting examples of particular transgenes or genetic modifications and corresponding phenotypes one may choose to introduce into a cell include one or more genes for insect resistance, such as a Bacillus thuringiensis (B.t.) gene, pest tolerance, such as genes for fungal disease control, herbicide tolerance, such as genes conferring glyphosate tolerance, and genes for quality improvements, such as yield, nutritional enhancements, environmental or stress tolerances, or any desirable changes in plant physiology, growth, development, morphology or plant product(s). For example, structural genes would include any gene that confers insect resistance, including, but not limited to, a Bacillus insect control protein gene as described in WO 99 / 31248, herein incorporated by reference in its entirety, U.S. Patent No. 5,689,052, herein incorporated by reference in its entirety, U.S. Patent Nos. 5,500.365 and 5,880,275. herein incorporated by reference in their entirety. In some embodiments, the structural gene can confer tolerance to the herbicide glyphosate as conferred by genes including, but not limited to, Agrobacterium strain CP4 glyphosate resistant EPSPS gene (rzroA:CP4) as described in U.S. Patent No. 5,633,435, herein incorporated by reference in its entirety, or glyphosate oxidoreductase gene (GOX) as described in U.S. Patent No. 5,463,175, herein incorporated by reference in its entirety.

[0097] A variety of assays are known in the art and may be used to confirm the presence of a genetic modification, exogenous DNA sequence, genetic marker, expression cassette or transgene in transformed, edited, or genetically modified cells, plants, or plant parts. Methods and techniques37US_ACTIVE\131878835W-1are provided for screening for, and / or identifying, cells or plants, etc., for the presence of targeted edits or transgenes, and selecting cells or plants comprising targeted edits or transgenes, which may be based on one or more phenotypes or traits, or on the presence or absence of a molecular marker or polynucleotide or protein sequence in the cells or plants. As used herein, a “molecular technique” refers to any method known in the fields of molecular biology, biochemistry, genetics, plant biology, or biophysics that involves the use, manipulation, or analysis of a nucleic acid, a protein, or a lipid. Without being limiting, molecular techniques useful for detecting the presence of a modified sequence in a genome include phenotypic screening; molecular marker technologies such as SNP analysis by TaqMan® or Illumina / Infinium technology; Southern blot hybridization; PCR; enzyme-linked immunosorbent assay (ELISA); and sequencing (e.g., Sanger, Illumina®, 454, Pac-Bio, Ion Torrent™). In one aspect, a method of detection provided herein comprises phenotypic screening. In another aspect, a method of detection provided herein comprises SNP analysis. In a further aspect, a method of detection provided herein comprises a Southern blot hybridization. In a further aspect, a method of detection provided herein comprises PCR. In an aspect, a method of detection provided herein comprises ELISA. In a further aspect, a method of detection provided herein comprises determining the sequence of a nucleic acid or a protein. Without being limiting, nucleic acids can be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0098] Nucleic acid or polynucleotide molecules can be isolated using techniques routine in the art. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology and / or PCR. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.

[0099] Detection (e.g., of an amplification product, of a hybridization complex, of a polypeptide) can be accomplished using detectable labels that may be attached or associated with a hybridization probe or antibody. The term “label” is intended to encompass the use of direct labels as well as indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials,38US_ACTIVE\131878835W-1luminescent materials, bioluminescent materials, and radioactive materials. The screening and selection of modified (e.g., edited) plants or plant cells can be through any methodologies known to those skilled in the art of molecular biology. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for detection of a polynucleotide, Northern blot hybridization, RNase protection, primer-extension, RT-PCR amplification for detecting RNA transcripts. Sanger sequencing, Next Generation sequencing technologies (e.g., Illumina®, PacBio®, Ion Torrent™, etc.) enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blot analysis, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immuno staining also can be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing all of the referenced techniques are known in the art.

[0100] As used herein, the term “polypeptide” refers to a chain of at least two covalently linked amino acids. Polypeptides can be encoded by polynucleotides provided herein. An example of a polypeptide is a protein. Proteins provided herein can be encoded by nucleic acid molecules provided herein. Polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A polypeptide also can be purified, for example, by expressing a nucleic acid in an expression vector. In addition, a purified polypeptide can be obtained by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0101] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. An antibody provided herein can be a polyclonal antibody or a monoclonal antibody. An antibody having specific binding affinity for a polypeptide provided herein can be generated using methods well known in the art. An antibody provided herein can be attached to a solid support such as a microtiter plate using methods known in the art.D. Genetically Modified Plants and Herbicide Tolerance

[0102] One aspect of the present disclosure includes genetically modified plant cells, plant tissues, plants, and seeds that comprise the recombinant DNA molecules or engineered proteins provided39US_ACTIVE\131878835W-1by the present disclosure. These cells, tissues, plants, and seeds, in certain embodiments, exhibit herbicide tolerance to at least one icafolin herbicide. In one embodiment, these cells, tissues, plants, and seeds may optionally exhibit tolerance to at least one additional herbicide.

[0103] Icafolin is a novel post-emergence isoxazole herbicide that acts on dicot (broadleaf) and monocot (narrow leaf / grass) weeds. Non-limiting examples of icafolin herbicides include methyl (2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino] tetrahydrofuran-2-carboxylate; methyl (2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]-amino]tetrahydrofuran-2-carboxylate; methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate;(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino] tetrahydrofuran-2-carboxylic acid; (2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid: (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5carbonyl]amino]tetrahydrofuran-2-carboxylic acid, and combinations of any thereof. In certain embodiments, cells, seeds, plants, and plant parts provided by the present disclosure exhibit herbicide tolerance to at least one icafolin herbicide.

[0104] Icafolin may be applied to a plant growth area comprising the plants and seeds provided by the present disclosure as a method for controlling weeds. Alternatively, or in addition, icafolin may be applied to a plant growth area prior to planting of the seeds provided by the present disclosure. In many embodiments, plants and seeds provided by the present disclosure comprise an herbicide tolerance trait and as such are tolerant to the application of at least one icafolin herbicide. The herbicide application rate of an icafolin herbicide may vary with external conditions, including, but not limited to temperature, humidity and the particular icafolin herbicide used. In a particular embodiment, the icafolin herbicide application may be the recommended commercial rate (IX) or any fraction or multiple thereof, such as half of the recommended commercial field rate (0.5X) or twice the recommended commercial rate (2X). Herbicide rates may be expressed as acid equivalent per pound per acre (lb ae / acre) or acid equivalent per gram per hectare (g ae / ha) or as pounds active ingredient per acre (lb ai / acre) or grams active ingredient per hectare (g ai / ha), depending on the herbicide and the formulation. In some embodiments, the application rate of an icafolin herbicide may be about 0.001 kg / ha to about 1.0 kg / ha or more of active substance, about 0.005 kg / ha to about 750 kg / ha of active substance, or about 5 g / ha to about 250 g / ha of active substance. The IX label rate for icafolin herbicides is about 5 g / ha to about 250 g / ha of active 40US_ACTIVE\131878835W-1substance. When a mixture of the (2R, 4R)-isomer and the (2S, 4S)-isomer is used, the isomers can be present in the mixture in a ratio of about 1.2:0.8 to about 0.8: 1.2, preferably in a ratio of about 1.1:0.9 to about 0.9: 1:1 (e.g. , the isomers can be present in a about a 1:1 ratio). The plant growth area may or may not comprise weed plants at the time of herbicide application. A herbicidally effective dose of an icafolin herbicide for use in an area for controlling weeds may be about 0.1X to about 30X label rate over a growing season. One (1) acre is equivalent to 2.47105 hectares and one (1) pound is equivalent to 453.592 grams. Herbicide application rates can be converted between English and metric as: (lb ai / ac) multiplied by 1.12 = (kg ai / ha) and (kg ai / ha) multiplied by 0.89 = (lb ai / ac).

[0105] Application of one or more herbicides may be performed sequentially or tank mixed with a combination of one or more herbicides. Multiple applications of one herbicide or of two or more herbicides, in combination or alone, may be used over a growing season to areas comprising transgenic plants of the present disclosure for the control of a broad spectrum of dicot weeds, monocot weeds, or both. In some embodiments, two applications of herbicide (such as a preplanting application and a post-emergence application or a pre-emergence application and a postemergence application) or three applications (such as a pre-planting application, a pre-emergence application, and a post-emergence application or a pre-emergence application and two postemergence applications) may be applied.

[0106] As used herein, “tolerance” or “herbicide tolerance” or “herbicide tolerance activity” refers to a plant, seed, or cell’s ability to resist the toxic effects of an herbicide when applied. Herbicide tolerant crops can continue to grow and are unaffected or minimally affected by the presence of the applied chemical. As used herein, an “herbicide tolerance trait” refers to a transgenic trait imparting improved herbicide tolerance to a plant, plant part, seed, or cell as compared to a wildtype plant. Non-limiting examples of plants which may be produced with an herbicide tolerance trait of the present disclosure include soybean plants, maize plants, cotton plants, barley plants, sorghum plants, rice plants, wheat plants, sugar cane plants, sugar beet plants, alfalfa plants, Brassica plants, and vegetable plants, and fruit plants (e.g., onion, tomato, pepper, curcurbit, pea, broccoli, cabbage, carrot, artichoke, lettuce, radish, spinach, cauliflower, zucchini, leek, potato, Brussels sprouts, tomatillo, beans, okra, apple, pear, cherry, peach, apricot, plum, banana, plantain, grape, citrus, avocado, mango, berry, etc.).41US_ACTIVE\131878835W-1

[0107] In certain aspects, the genetically modified plants, progeny, seeds, plant cells, and plant parts of the present disclosure may also contain one or more additional traits. Additional traits may be introduced by crossing a plant containing a recombinant DNA molecule provided by the disclosure with another plant containing one or more additional trait(s). As used herein, “crossing” refers to breeding two individual plants to produce a progeny plant. Two plants may thus be crossed to produce progeny that contain desirable traits from each parent. As used herein “progeny” refers the offspring of any generation of a parent plant, and transgenic progeny comprise a polynucleotide molecule provided by the present disclosure, inherited from at least one parent plant. Additional trait(s) also may be introduced by co-transforming a polynucleotide molecule for the additional transgenic trait(s) with a polynucleotide molecule comprising a recombinant polynucleotide molecule provided by the present disclosure, or by inserting the additional trait(s) into a transgenic plant comprising a polynucleotide molecule of the present disclosure or vice versa (for example, by using any of the methods of plant transformation or genome editing). Such additional traits include, but are not limited to. increased insect resistance, increased disease resistance (e.g., to Asian soybean rust), increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, and herbicide tolerance, in which the trait is measured with respect to a wild-type plant. Exemplary additional herbicide tolerance traits may include transgenic or non-transgenic tolerance to one or more herbicides such as ACCase inhibitors (for example aryloxyphenoxy propionates and cyclohexanediones), ALS inhibitors (for example sulfonylureas, imidazolinones, triazoloyrimidines, and triazolinones) EPSPS inhibitors (for example glyphosate), synthetic auxins (for example phenoxys, benzoic acids, carboxylic acids, semicarbazones), photosynthesis inhibitors (for example triazines, triazinones, nitriles, benzo thiadiazoles, and ureas), glutamine synthesis inhibitors (for example glufosinate), HPPD inhibitors (for example isoxazoles. pyrazolones, and triketones), PPO inhibitors (for example diphenylethers. N-phenylphthalimide, aryl triazinones, and pyrimidinediones), PDS inhibitors (for example, amides, pyridiazinones, and pyridines), and long-chain fatty acid inhibitors (for example chloroacetamindes, oxyacetamides, and pyrazoles), among others. Exemplary insect resistance traits may include resistance to one or more insect members within one or more of the orders of Lepidoptera, Coleoptera, Hemiptera, and Homoptera, among others. Such additional traits are well known to one of skill in the art; for example, and a list of such transgenic traits is provided42US_ACTIVE\131878835W-1by the United States Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS).

[0108] A cell transformed with a polynucleotide molecule of the present disclosure may, in certain embodiments, be selected for the presence of the polynucleotide molecule or its encoded polypeptide before or after regenerating such a cell into a transgenic plant. In some embodiments, transgenic plants comprising such a polynucleotide may thus be selected by identifying a transgenic plant that comprises the polynucleotide molecule or the encoded polypeptide, and / or displays an altered trait relative to an otherwise isogenic control plant. Such a trait may be, for example, tolerance to an icafolin herbicide.

[0109] Transgenic plants and progeny that contain a transgenic trait provided by the disclosure may be used according to any breeding method known in the art. In plant lines comprising two or more transgenic traits, the transgenic traits may be independently segregating, linked, or a combination of both in plant lines comprising three or more transgenic traits. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated by the present disclosure, as is vegetative propagation. Descriptions of breeding methods that are used for different traits and crops are well known to those of skill in the art. To confirm the presence of the transgene(s) in a particular plant or seed, a variety of assays may be performed. Such assays include, for example, molecular biology assays, such as Southern and northern blotting, PCR, and DNA sequencing; biochemical assays, such as detecting the presence of a protein product, for example, by immunological means (ELISAs and western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole plant.

[0110] Introgression of a transgenic trait into a plant genotype is achieved as the result of the process of backcross conversion. A plant genotype into which a transgenic trait has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid. Similarly, a plant genotype lacking the desired transgenic trait may be referred to as an unconverted genotype, line, inbred, or hybrid.E. Cell-Based Screening Methods for Identifying and Optimizing Herbicide Tolerance Genes

[0111] Crop yield loss due to weed pressure represents a major challenge in agriculture. Herbicides are an effective tool to control weeds. Crops engineered to tolerate herbicide applications afford 43US_ACTIVE\131878835W-1better control of weeds throughout the growing season. Herbicide tolerant (HT) trait discovery relying on in planta testing is hindered by a limited number of possible growth - and therefore test-and-leam - cycles per year as well as resource demand and controlled environment footprint.

[0112] Plant protoplasts are a versatile test system for cell-based assays, such as expression, localization, and signaling. The present invention demonstrates protoplasts are also amenable toward high-throughput phenotypic screens to assess metabolic and / or morphological characteristics, such as in response to chemical and / or genetic perturbations, for the purpose of identifying and optimizing HT leads. The examples provided herein disclose an approach to utilize dose-response to quantitatively describe a range of phenotypes corresponding to herbicide activity. Furthermore, screens of putative HT leads utilizing a phenotype recovery strategy are demonstrated. This approach assesses the protoplast phenotype for at least one dose within the established range (commonly IC50 and / or IC90) where the protective effect of a putative HT lead is evaluated by measuring at least a partial recovery of the untreated phenotype when cells are challenged with an herbicide. Hits can be ranked based on their magnitude and statistical significance of protective effect. The methods and techniques described herein encompass the use of cell painting, metabolic analysis, and co-expression of cellular markers to assess cellular phenotypes. In some embodiments, measuring the cellular phenotype comprises manual analysis. For example, cell images can be visually inspected and analyzed directly or with the assistance of imaging software tools. In other embodiments, measuring the cellular phenotype comprises automated analysis. For example, cell images can be inspected using software or algorithms without direct human input. In certain embodiments, measuring the cellular phenotype comprises manual analysis and automated analysis.

[0113] As described herein, fluorescent reporter proteins may be used to highlight or visualize components of a cell in response to chemical and / or genetic perturbations. For example, fluorescent organelle reporter gene-expressing vectors may be used to label the cytoplasm, cytoskeleton, endoplasmic reticulum, vacuoles, nucleus, nucleolus, mitochondria, or plastids of a cell. Furthermore, cells may be transformed with one or more plasmids encoding fluorescent protein molecule(s) (e.g., GFP) that localize to subcellular compartments of a cell, e.g. the stroma of plastids.44US_ACTIVE\131878835W-1

[0114] Provided herein are methods for identifying candidate herbicide tolerance genes, wherein the method comprises obtaining at least one protoplast derived from a plant of interest; transiently expressing one or more candidate herbicide tolerance genes in said at least one protoplast; exposing the at least one protoplast to a herbicidal compound; and measuring a cellular phenotype in said at least one protoplast following exposure to the herbicidal compound as compared to at least one control protoplast. In some embodiments, the method further comprises transiently expressing one or more screenable marker genes in said at least one protoplast. Furthermore, in certain aspects, measuring a cellular phenotype in at least one protoplast following exposure to the herbicidal compound as compared to at least one control protoplast comprises filtering for fluorescent expression or detecting a fluorescent signal associated with one or more reporter gene(s). Fluorescent proteins and their applications in imaging living cells and tissues are known in the art (e.g. Chudakov DM, et al. Physiol Rev. 2010 Jul;90(3): 1103-63). Fluorescent reporter proteins useful in practicing the embodiments of the present disclosure include, but are not limited to a nuclear-targeted Cyan Fluorescent Protein (nucCFP), a plastid-targeted Green Fluorescent Protein reporter, and an Endoplasmic Reticulum (ER)-targeted mCherry fluorophore protein. In some embodiments provided herein, measuring a cellular phenotype comprises detecting the localization of one or more fluorescent signals to a specific organelle or cellular structure following exposure to the herbicidal compound as compared to at least one control protoplast. In other embodiments, measuring a cellular phenotype comprises detecting the dispersal of a fluorescent signal in a cell following exposure to the herbicidal compound as compared to at least one control protoplast. In still other embodiments, measuring a cellular phenotype comprises detecting the movement of a fluorescent signal in a cell from one organelle or cellular structure to a distinct organelle or cellular structure following exposure to the herbicidal compound as compared to at least one control protoplast. As described herein, expression of multiple fluorescent reporter genes enables the ability to identify and selectively quantify a cellular phenotype in cells expressing a candidate herbicide tolerance gene. The identification and measurement of such cellular phenotypes may be facilitated by automated imaging and analysis software.

[0115] Turnaround time and throughput are two key factors in assay development. Provided herein are methods for identifying candidate herbicide tolerance genes within an approximately 24-hour period. Also provided herein are methods for identifying candidate herbicide tolerance genes within an approximately 48-hour or 56-hour period. According to some embodiments, the45US_ACTIVE\131878835W-1methods described herein comprise transiently expressing one or more candidate herbicide tolerance genes in at least one protoplast, and exposing said protoplast to a herbicidal compound about 2, about 3, about 4, about 5, about 6. about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33. about 34, about 35, about 36. about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, or about 56 hours post-transformation. Measuring a cellular phenotype in said at least one protoplast following exposure to the herbicidal compound as compared to at least one control protoplast may be carried out about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19. about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49. about 50, about 51, about 52, about 53, about 54, about 55, about 56 hours post-transformation. In some embodiments, a cellular phenotype may be measured in at least about 10, at least about 20, at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, or at least about 500 protoplasts.

[0116] Exposing the at least one protoplast to an herbicidal compound may be carried out at one or more concentrations of herbicidal compound. For example, the concentrations of herbicidal compound may comprise about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.5 nM, about 1 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 50 nM, about 100 nM, about 150 nM, about 200 nM, or about 300 nM, or about 500 nM.

[0117] The term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word “comprising” or other open language in the claims, the words “a” and “an” denote “one or more,” unless specifically noted otherwise. The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,”46US_ACTIVE\131878835W-1“includes,” and “including,” are also open-ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any system or method that “comprises,” “has,” or “includes” one or more components is not limited to possessing only those components and covers other unlisted components. As used herein, the term “consists essentially of’, when used in reference to a nucleotide or amino acid sequence of the present disclosure, means that the nucleotide sequence or amino acid sequence, may contain additional nucleotides or amino acids so long as the additional nucleotides or amino acids do not materially alter the function of the recited sequences. The term “materially alter,” as applied to a nucleotide sequence or amino acid sequence of the present disclosure, refers to a decrease in herbicide tolerance of the encoded polypeptide of at least 25%. For example, additional nucleotides or amino acids added to a nucleotide or an amino acid sequence of the present disclosure may be deemed to “materially alter” the encoded polypeptide or polypeptide sequence, if such additions decrease the conferred herbicide tolerance by at least 25%.

[0118] Other objects, features, and advantages of the present disclosure are apparent from the detailed description provided herein. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. Any embodiment of the present disclosure may be used in combination with any other embodiment described herein.

[0119] All references herein are incorporated herein by reference in their entirety.EXAMPLESExample 1: Development of a Cell-Based Assay to Screen for Icafolin (ICA) Herbicide Tolerance

[0120] This example describes the identification of a cell phenotype as an indicator of icafolin herbicide treatment followed by the development and optimization of a fluorescent protein reporter-based assay to screen traits for tolerance to icafolin, specifically icafolin-methyl (ICM).

[0121] Identification of Plastid Phenotype as Indicator of ICM Effect: The primary objective at the onset of assay development was to identify a characteristic phenotypic change in protoplasts 47US_ACTIVE\131878835W-1treated with ICM that could be used as a quantitative assay readout, in order to enable ranking of candidate genes of interest (GOIs) in the event that many exhibited signs of conferred tolerance to ICM. The initial assay development used protoplasts isolated from tobacco BY-2 cells and fluorescent cell reporters, and subsequently the assay was transferred to soy cotyledon protoplasts; in both cases, protoplast isolation and PEG-mediated transformation followed protocols similar to literature with minor modifications. For the desired assay turnaround time and throughput, the fluorescent reporter required rapid inducibility and easy traceability within an approximately 24-hour period. Various fluorescent organelle reporter gene-expressing vectors (plasmids) were transiently transformed into BY-2 protoplasts and observed over a 54-hour time frame. A comprehensive range of organelles was examined to identify significant phenotypic differences post-ICM treatment. Among them, reporters labeling the cytoplasm, endoplasmic reticulum, vacuoles, nucleus, nucleolus, and mitochondria did not produce a significant and traceable phenotypic change within the desired time frame. However, cells transformed with a plasmid encoding a green fluorescent protein (GFP) molecule that localized to the stroma of plastids exhibited a distinctively different phenotype when treated with ICM compared to no treatment. Cells not incubated with ICM produced a “speckled” phenotype beginning 12-18 hours after polyethylene glycol (PEG)-mediated transformation, arising from GFP directed to the stroma of plastids distributed throughout the cytoplasm of healthy cells. Notably, ICM treatment of GFP vector-transformed protoplasts significantly reduced or eliminated the speckled phenotype; that is, the GFP signal became concentrated around the nucleus, minimizing the presence of GFP-positive plastids in the cytoplasm. Outside the periphery of the nucleus, very few GFP-positive plastids were observed in each cell (See FIG. 1). This observed phenotype is consistent with organelle movement reported in literature as a response to environmental and physiological conditions via a variety of mechanisms, such as changes in organelle localization and signaling that lead to macroscale effects in planta (see Xin et.al., Stress Biology 2, 50 (2022); Sato et.al., J Cell Sci 114 (2): 269-279 (2001). Automated imaging and analysis software facilitated the identification of this phenotypic change. Brightfield (BF) imaging enabled cell identification and segmentation, while GFP imaging and advanced analysis techniques allowed for the identification and quantification of the spatial distribution of GFP-positive plastids within each cell (“BF+GFP” analysis). With these parameters, an ICM phenotype could be readily quantified within 18 hours post-ICM treatment (24 hours post-transformation).48US_ACTIVE\131878835W-1

[0122] ICM dosing was determined experimentally. ICM concentrations ranging from 0.01 pM to 0.2 pM significantly affected GFP-positive plastid expression in BY-2 protoplasts. Higher doses did not yield a more substantial effect.

[0123] The relative timing of ICM treatment and imaging significantly impacted the plastid phenotype. For example, delaying ICM treatment to 18 hours post-transformation did not result in a significant reduction in the “speckled” phenotype when cells were imaged 6 hours later at 24 hours post-transformation. Ultimately, ICM treatment at 6 hours post-transformation ICM produced a clear and reproducibly quantifiable phenotypic change 24 hours post-transformation.

[0124] Functional Assay Development and Statistical Power Analysis: For the establishment of a functional assay to be used to screen potential genes for tolerance to ICM, a reproducible ICM dose response curve was established by testing ICM concentrations ranging from 10 pM to 200 nM, which identified an effect transition between 1 and 100 nM (See FIG. 2).

[0125] Power analysis was performed to determine the number of technical replicates needed in a given assay run to achieve high power, with greater tolerance for false positives (“alpha”, a) than false negatives (1 - power) given the intent for this assay to serve as an initial, high-throughput screen after which promising candidates would be tested further for validation. Since the effect size (“delta”, A) between the untreated (0 nM ICM) and maximum-dose (100 nM ICA) controls reproducibly exceeded 20, power analysis modeled values for A of 5, 10, 15, and 20 to simulate ICM-tolerance levels ranging from weak to strong, respectively, for false positive rates of 5% and 10%. In both cases, three technical replicates were found to be sufficient to achieve greater than 90% power for an effect size of 20 (or greater); for a 10% false positive rate, three technical replicates additionally achieved nearly 90% power for a reduced effect size of 15 (See FIG. 3). In the interest of maximizing screening capacity, the standard assay protocol adopted the use of three technical replicates with the acknowledgement that retesting would be required to support tolerance for genes of interest (GOIs) producing statistically significant effect sizes less than 15. Additionally, Z-scores were calculated according to the expression:Zi,j = 1 3*(O(+Gy) / lp;+pyl, where

[0126] a, and G, refer to the standard deviation of response values for treatment i and j, respectively, and p, and p7refer to the mean response values for treatment z and. / , respectively. Typically, the 100 nM ICM treatment would be taken as a reference treatment, j. With the untreated control 49US_ACTIVE\131878835W-1taken as the second treatment, i, the Z-score represents a measure of the assay quality complementary to the power analysis, where a value greater than zero could be considered acceptable and a value greater than 0.5 is desirable. The Z-score for the untreated and 100 nM ICM controls using the “BF+GFP” analysis depicted in FIG. 2 was 0.14. Alternatively, with a test treatment taken as treatment i - that is, cells transfected with a candidate ICM-tolerance gene and challenged with an ICM concentration greater than 1 nM - the Z-score represents a measure of the statistical significance of a difference in the response for the test treatment from that for the 100 nM ICM control.

[0127] Protocol Transfer to Soy Protoplasts and Further Optimization via Dual-Reporter Strategy: To screen GOIs for tolerance in a relevant crop species, the assay described above was tested in soy cotyledon protoplasts, which were found to produce a plastid phenotype and dose response similar to BY-2 protoplasts with and without ICM treatment See FIG. 4). The assay protocol - both laboratory procedures and automated “BF+GFP” image analysis - for this experiment was the same as for the BY-2 protoplast data in FIG. 2. While the dose response in soy protoplasts was qualitatively similar, greater variability and smaller difference between untreated and maximum dose {i.e., 50 nM ICM) treatments indicated further optimization was warranted for use with soy protoplasts, as indicated by a Z-score of -0.27 for these treatments.

[0128] Two key limitations of the assay protocol were recognized. First, the use of bright field (BF) images for cell segmentation was complicated, meticulous, and potentially prone to errors due to low contrast of the cells of interest compared to unhealthy cells, debris, and cells outside the image focal plane. Second, the characteristic plastid phenotype could only be evaluated for cells expressing the GFP reporter. Since the GFP reporter and GOI were co-transfected in different plasmids, any cells that expressed the GFP reporter but not the GOI would contribute to a high background of the “susceptible” phenotype, reducing assay sensitivity. Measuring the cotransfection rate would be possible with downstream assays, such as sequencing, but this analysis would be incredibly challenging to couple with the image analysis for individual cells.

[0129] Two new co-transfection vectors were designed to improve the assay and address these limitations. The first vector included both a nuclear-targeted Cyan Fluorescent Protein (GFP) and the plastid GFP reporter in the same cassette separated by T2A sequence, thus allowing both proteins to be produced separately from a single vector and driven by the same promoter.50US_ACTIVE\131878835W-1Specifically, transformation vector pM514 comprised an expression cassette for a plant codon-optimized, nuclear-targeted Cyan Fluorescent Protein (nucCFP) (SEQ ID NO: 8) linked to a plant codon-optimized, plastid-targeted Green Fluorescent Protein (plGFP) (SEQ ID NO: 9) via a plant codon-optimized T2A self-cleaving peptide sequence (SEQ ID NO: 4). T2A is a 21 amino acid oligopeptide sequence that induces ribosomal skipping during translation of an mRNA thereby leading to the generation of polyproteins from a single Open Reading Frame. T2A thus mediates “cleavage” of polypeptides during translation thus ensuring that both gene products in the cassette can be expressed individually from a single promoter (See Liu, Z., Chen, O., Wall, LB ,J. et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193, 2017). The ORF comprising nucCFP and plGFP was placed under the control of the 35S promoter and leader sequence (SEQ ID NO: 5) and a 3’ terminator sequence from the Agrobacterium Nopaline synthase (NOS) gene (SEQ ID NO: 6).

[0130] The second vector similarly utilized T2A to allow the synthesis of both a GOI and Endoplasmic Reticulum (ER)-targeted mCherry fluorophore protein (ER-mCherry) (SEQ ID NO: 3) from a same ORF enabling the marking and analysis of cells expressing the GOI by filtering for mCherry expression. A polynucleotide sequence encoding for a selected GOI could be operably linked to a sequence encoding an Endoplasmic Reticulum (ER)-targeted mCherry fluorophore protein (ER-mCherry) (SEQ ID NO: 3) via a nucleotide sequence encoding a T2A self-cleaving peptide sequence (SEQ ID NO: 4). The resulting sequence was placed under the control of the 35S promoter and leader sequence (SEQ ID NO: 5) and a 3’ terminator sequence from the Agrobacterium Nopaline synthase (NOS) gene (SEQ ID NO: 6).

[0131] Co-transformation of the two constructs offered the ability to identify and selectively quantify the plastid GFP phenotype in cells expressing the GOI, improving sensitivity as well as providing a visual cue for GOI expression that can act as a quality control element to differentiate GOIs that are not expressed well from those that have no effect on the ICM plastid phenotype. In addition, the ER-mCherry labels the cell outline by visualizing the network of membranes that make up the ER and extend throughout the cell. Taken together with the nuclear CFP marker, these two plasmids express three proteins (“3FP”-3 Fluorescent Protein), that fluorescently label components necessary to facilitate critical aspects of the image analysis - namely, cell segmentation and profiling plastid spatial distribution within individual cells.51US_ACTIVE\131878835W-1

[0132] In summary, the use of fluorescent labeling contributed to the simplification of the analysis in several ways. First, by strategically labeling multiple organelles with fluorescent proteins, it allowed for easier and more precise visualization and identification of these cellular structures, which resulted in streamlining the process of locating and analyzing plastids within the cell. Second, the ability to introduce a gene of interest (GOI) linked to a fluorescent protein marker enabled identification and analysis of cells expressing the GOI by filtering based on the expression of mCherry, thereby streamlining the process of studying the impact of the GOI on plastid phenotype as well as improving assay sensitivity. Furthermore, the simultaneous production of multiple proteins from a single vector reduced the number of necessary vectors, in turn reducing the complexity of the experimental setup and allowing for more efficient analysis. Overall, the use of multiple fluorescent labels simplified the experimental workflow and facilitated selective cellular analysis, ultimately improving sensitivity of the plastid phenotype readout. Multiplexed fluorescent labeling also holds the potential to simplify automated analysis of the plastid phenotype, saving considerable time and resources required for large screens.Example 2: Icafolin Herbicide Tolerance Evaluation of Dicot Protoplasts Expressing MAP65-1

[0133] Morphological profiling at the single-cell level is commonly used in chemical and functional genomic screening. We have identified a plastid-based phenotype in dicot plant protoplasts that captures cellular responses to perturbations by the herbicide icafolin-methyl (ICM). We have developed a dicot plant protoplast assay leveraging this phenotype to demonstrate native tolerance or susceptibility to ICM. Exogeneous transgenes can be transiently expressed to prevent the ICM perturbation of the plastid phenotype. Such a transgene that preserves the untreated plastid phenotype when cells are challenged with ICM is considered to confer tolerance to ICM. This tolerance at the cellular level is expected to be retained in whole plants, making the transgene a candidate for a herbicide-tolerant trait. This assay was employed to test Glycine max Microtubule Associated Protein 65-1 (GmMAP65-l) for ICM tolerance in plant cells.

[0134] Briefly, plant protoplasts were isolated from a cell culture or plant tissue, such as tobacco BY-2 cells or soy cotyledon, following methods described in the literature with slight modification. Protoplasts were subjected to standard PEG-mediated transfection to transiently express both a green fluorescent protein (GFP) reporter directed to the plastid (chloroplast) and a putative52US_ACTIVE\131878835W-1tolerance gene of interest (GOT), incubated with icafolin methyl (TCM), and imaged using a fluorescence microscope. The resulting images were analyzed to quantify the fraction of cells expressing GFP that exhibit a plastid phenotype similar to that observed for untreated cells (i.e., transfected but not incubated with ICM). Any GOT that increases this fraction compared to negative controls would be interpreted as a potential tolerance gene and considered for advancement to the next testing phase.

[0135] Three plant transformation vectors were created. Vector pM505 comprised a functional cassette for the expression of G / nMAP65-l. The polynucleotide sequence (SEQ ID NO: 2) encoding for GmMAP65- 1 protein (SEQ ID NO: 1) was operably linked to a plant codon-optimized sequence encoding an Endoplasmic Reticulum (ER) targeted mCherry fluorophore protein (ER-mCherry) (SEQ ID NO:3) via a nucleotide sequence encoding a plant codon-optimized T2A selfcleaving peptide sequence (SEQ ID NO:4). An additional codon for alanine was introduced immediately after the methionine at the N-terminus of the protein as part of the standard Kozak sequence (SEQ ID NO: 17). The resulting sequence was placed under the control of the 35S promoter and leader sequence (SEQ ID NO:5) and a 3’ terminator sequence from the Agrobacterium Nopaline synthase (NOS) gene (SEQ ID NO:6).

[0136] Vector pM506 was a control plasmid identical to pM505 except that GmMAP65 was replaced with a plant codon-optimized sequence encoding the Beta-glucuronidase (GUS) protein (SEQ ID NOG). Transformation vector pM514 was generated to facilitate nucleus and plastid visualization for image analysis. It comprised an expression cassette for a plant codon-optimized, nuclear-targeted Cyan Fluorescent Protein (nucCFP) (SEQ ID NO:8) linked to a plant codon-optimized, plastid-targeted Green Fluorescent Protein (plGFP) (SEQ ID NO:9) via a plant codon-optimized T2A self-cleaving peptide sequence (SEQ ID NO: 4). The resulting sequence was placed under the control of the 35S promoter and leader sequence (SEQ ID NO:5) and a 3’ terminator sequence from the Agrobacterium Nopaline synthase (NOS) gene (SEQ ID NO:6).

[0137] Freshly isolated soy cotyledon protoplasts were transiently transformed using standard methods with pM514 (nucCFP:2A:plGFP) and pM504 (GmMAP65-l:2A:ERmCherry) plant transformation vectors. As a control, soy protoplasts were transformed with pM514 (nucCFP:2A:plGFP) and pM505 (GUS:2A:ERmCherry) plant transformation vectors. Beginning six hours after transformation, the protoplasts were incubated in the presence of the ICM herbicide at concentrations ranging from 0.3 nM to 30 nM or vehicle control treatment. Protoplasts were 53US_ACTIVE\131878835W-1then assayed for ICM herbicide tolerance approximately 24 hrs post-transformation by analyzing the number and spatial distribution of GFP -positive (GFP+) plastids. Individual protoplasts were classified as exhibiting either a “tolerant”-like phenotype if they contained > 1 GFP+ plastid in the area between the nucleus and ER labeled cell outline or a “susceptible” phenotype if all plastids were observed to be in very close proximity to the nucleus. Analysis was limited to only mCherry-positive cells so as to confirm GUS or MAP65-1 protein expression. Assays were done by analyzing > 50 cells in each of three replicate wells. Response values - that is, the percentage of analyzed cells exhibiting the “toleranf’-like phenotype - were averaged for GUS or MAP65-1 transformed cells in the vehicle control as well as at each ICM concentration, and standard deviation was calculated. The vehicle control showed a similar baseline response value (roughly 80%) for both GUS and MAP65-1 expressing cells. GUS expressing cells showed a decrease in the response value with increasing ICM concentration, plateauing around 24+3.0% for tested concentrations > 3 nM ICM. MAP65-1 expressing cells showed a higher response value at every ICM concentration tested as well as a higher plateau near 47+1.5% for ICM concentrations > 3 nM (See FIG. 5). These data indicate that GmMAP65-l provides tolerance to ICM, as supported by Z-scores of:a) 0.72 for the untreated and 3 nM ICM control and reference treatments in cells with GUS overexpression, demonstrating the high quality of the assay protocol; andb) 0.18 for the G / ;?MAP65- 1 and GUS overexpression treatments challenged with 3 nM ICM, indicating a meaningful difference in the response value for the candidate ICM-tolerant gene compared to a susceptible control.54US_ACTIVE\131878835W-1

Claims

CLAIMS1. A recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide sequence that encodes a polypeptide conferring tolerance to an icafolin herbicide, wherein the polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and wherein the heterologous promoter is functional in a plant cell.

2. The recombinant DNA molecule of claim 1, wherein the polynucleotide sequence comprises a nucleic acid sequence having at least about 90% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18.

3. The recombinant DNA molecule of claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

4. The recombinant DNA molecule of claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:

16. or SEQ ID NO: 17.

5. The recombinant DNA molecule of claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

6. The recombinant DNA molecule of claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.55US_ACTIVE\131878835W-17. The recombinant DNA molecule of claim 1 or 2, wherein the polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:

14. SEQ ID NO:

15. SEQ ID NO:

16. or SEQ ID NO: 17.

8. The recombinant DNA molecule of claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

9. The recombinant DNA molecule of any one of claims 2-8, wherein the polynucleotide sequence comprises a nucleic acid sequence having at least about 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18.

10. The recombinant DNA molecule of any one of claims 2-8, wherein the polynucleotide sequence comprises a nucleic acid sequence having at least about 96% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18.

11. The recombinant DNA molecule of any one of claims 2-8, wherein the polynucleotide sequence comprises a nucleic acid sequence having at least about 97% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18.

12. The recombinant DNA molecule of any one of claims 2-8, wherein the polynucleotide sequence comprises a nucleic acid sequence having at least about 98% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18.

13. The recombinant DNA molecule of any one of claims 2-8, wherein the nucleic acid sequence is SEQ ID NO:2 or SEQ ID NO:18.

14. The recombinant DNA molecule of any one of claims 1-13, wherein the polynucleotide sequence further comprises a nucleic acid sequence that encodes a targeting sequence that functions to localize the encoded polypeptide within a cell.

15. The recombinant DNA molecule of any one of claims 1-14, wherein said icafolin herbicide is selected from the group consisting of:56US_ACTIVE\131878835W-1methyl (2R*, 4R*)-4-[[(5S)-3-(3, 5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]- amino]tetrahydrofuran-2-carboxylate;methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxy lie acid;(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxylic acid;(2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxylic acid; andcombinations of any thereof.

16. A DNA construct comprising the recombinant DNA molecule of any one of claims 1-15.

17. The recombinant DNA molecule of any one of claims 1-16, wherein the recombinant DNA molecule is integrated into the genome of a transgenic plant, seed, plant part, or cell.

18. A transgenic plant, seed, cell, or plant part comprising a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide sequence that encodes a polypeptide conferring tolerance to an icafolin herbicide, wherein the polypeptide comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

19. The transgenic plant, seed, cell, or plant part of claim 18, wherein the transgenic plant, seed, cell, or plant part is a soybean, a maize, a cotton, a barley, a sorghum, a rice, a wheat, a sugar cane, a sugar beet, an alfalfa, or a Brassica plant, seed, cell, or plant part.

20. The transgenic plant, seed, cell, or plant part of claim 18 or 19, wherein the icafolin herbicide is selected from the group consisting of:57US_ACTIVE\131878835W-1methyl (2R*, 4R*)-4-[[(5S)-3-(3, 5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]- amino]tetrahydrofuran-2-carboxylate;methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxy lie acid;(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxylic acid;(2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxylic acid; andcombinations of any thereof.

21. The transgenic plant, seed, cell, or plant part of any one of claims 18-20, wherein the transgenic plant, seed, cell, or plant part comprises tolerance to at least one additional herbicide.

22. A method of conferring herbicide tolerance to a plant, seed, cell, or plant part, the method comprising expressing the recombinant DNA molecule of any one of claims 1-17 in said plant, seed, cell, or plant part, wherein said plant, seed, cell, or plant part comprises tolerance to an icafolin herbicide.

23. The method of claim 22, wherein the icafolin herbicide is selected from the group consisting of:methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]- amino]tetrahydrofuran-2-carboxylate;methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxy lie acid;58US_ACTIVE\131878835W-1(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxy lie acid;(25.45)-4-[[(5S)-3-(3.5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylic acid; andcombinations of any thereof.

24. A method of producing a transgenic plant or part thereof, the method comprising:a) introducing a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:

12. SEQ ID NO: 13, SEQ ID NO:

14. SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 into a plant cell; andb) regenerating the transgenic plant or part thereof from said cell or a descendant cell thereof that comprises the recombinant DNA molecule;wherein the regenerated transgenic plant or part thereof comprises tolerance to an icafolin herbicide.

25. The method of claim 24, wherein the icafolin herbicide is selected from the group consisting of:methyl (2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]- amino]tetrahydrofuran-2-carboxylate;methyl (2S,4S)-4-[[(5S)-3-(3.5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxy lie acid;(2R,4R)-4-[[(5S)-3-(3,5-difhiorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxylic acid;(25.45)-4-[[(5S)-3-(3,5-difhiorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxylic acid; andcombinations of any thereof.59US_ACTIVE\131878835W-126. The method of claim 24 or 25, further comprising crossing the regenerated transgenic plant with itself or with a second plant to produce seed.

27. The method of any one of claims 24-26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

28. The method of any one of claims 24-27, wherein the polynucleotide sequence comprises a nucleic acid sequence having at least about 95% sequence identity to SEQ ID NO:2 or SEQ ID NO:18.

29. The method of any one of claims 24-28, further comprising crossing the regenerated transgenic plant, or a descendant plant thereof that comprises the recombinant DNA molecule, with itself or a second plant to produce seed; wherein the seed comprises the recombinant DNA molecule.

30. A transgenic plant or part thereof produced by the method of any one of claims 24-29, wherein said transgenic plant or part thereof comprises the recombinant DNA molecule.

31. A seed produced by the method of claim 29.

32. A method for controlling weeds in a plant growth area that comprises the transgenic plant or seed of any one of claims 18-21, the method comprising contacting the plant growth area with an icafolin herbicide, wherein the transgenic plant or seed is tolerant to the icafolin herbicide, and wherein weeds are controlled in the plant growth area.

33. The method of claim 32, wherein the icafolin herbicide is selected from the group consisting of:methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]- amino]tetrahydrofuran-2-carboxylate;methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;60US_ACTIVE\131878835W-1(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxy lie acid;(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylic acid;(2S,4S)-4-[[(5S)-3-(3,5-difhrorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxylic acid; andcombinations of any thereof.

34. The method of claim 32 or 33, wherein the transgenic plant or seed is a soybean, a maize, a cotton, a barley, a sorghum, a rice, a wheat, a sugar cane, a sugar beet, an alfalfa, or a Brassica plant or seed.

35. A method of identifying a plant having tolerance to an icafolin herbicide and to at least one additional herbicide, the method comprising:a) obtaining a plant according to any one of claims 18-21;b) applying the at least one additional herbicide to said plant or a part thereof; and c) identifying said plant as exhibiting tolerance to the at least one additional herbicide.

36. A method for reducing the development of herbicide tolerant weeds in a plant growth area comprising the transgenic plant or seed according to any one of claims 18-21, the method comprising contacting the plant growth area with an icafolin herbicide and at least one additional herbicide, wherein the transgenic plant or seed is tolerant to the icafolin herbicide and the at least one additional herbicide.

37. The method of claim 36, wherein the icafolin herbicide is selected from the group consisting of:methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;methyl(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]- amino]tetrahydrofuran-2-carboxylate;methyl (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylate;61US_ACTIVE\131878835W-1(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxy lie acid;(2R,4R)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl]amino]tetrahydrofuran-2-carboxylic acid;(2S,4S)-4-[[(5S)-3-(3,5-difhrorophenyl)-5-vinyl-4H-isoxazole-5- carbonyl] amino] tetrahydrofuran-2-carboxylic acid; andcombinations of any thereof.

38. The method of claim 36 or 37, wherein the at least one additional herbicide is selected from the group consisting of: an ACCase inhibitor, an ALS inhibitor, an EPSPS inhibitor, a synthetic auxin, a photosynthesis inhibitor, a glutamine synthesis inhibitor, a HPPD inhibitor, a PPO inhibitor, a PDS inhibitor, and a long-chain fatty acid inhibitor.

39. A method for identifying candidate herbicide tolerance genes, the method comprising:a) obtaining at least one protoplast derived from a plant of interest;b) transiently expressing one or more candidate herbicide tolerance genes in said at least one protoplast;c) exposing the at least one protoplast to a herbicidal compound; andd) measuring a cellular phenotype in said at least one protoplast following exposure to the herbicidal compound as compared to at least one control protoplast.

40. The method of claim 39. wherein the method further comprises identifying at least one protoplast, wherein the cellular phenotype of said at least one protoplast following exposure to the herbicidal compound is similar to the cellular phenotype of at least one control protoplast.

41. The method of claim 40, wherein the method further comprises identifying one or more candidate herbicide tolerance genes expressed in said at least one protoplast.

42. The method of claim 39, wherein the method further comprises transiently expressing one or more screenable marker genes in said at least one protoplast.

43. The method of claim 42, wherein the one or more screenable marker genes comprises a fluorescent reporter gene.62US_ACTIVE\131878835W-144. The method of claim 43, wherein measuring the cellular phenotype further comprises filtering for fluorescent expression or detecting the fluorescent reporter gene.

45. The methods of claim 39, wherein the herbicidal compound is further defined as a cytoskeleton-targeting herbicide, a microtubule-targeting herbicide, or a cytoskeletal-associated protein-targeting herbicide.

46. The methods of claim 39, wherein the herbicidal compound comprises icafolin methyl.

47. The method of claim 39, wherein the cellular phenotype comprises a plastid phenotype.

48. The method of claim 47, wherein the method further comprises identifying at least one protoplast, wherein the plastid phenotype of said at least one protoplast following exposure to the herbicidal compound is similar to the plastid phenotype of at least one control protoplast not exposed with the herbicidal compound.

49. The method of claim 39, wherein measuring the cellular phenotype comprises automated analysis.

50. The method of claim 39, wherein the one or more candidate herbicide tolerance genes are heterologous to the plant of interest.

51. The method of claim 39, wherein measuring the cellular phenotype in said at least one protoplast is carried out at:a) one or more time points; orb) one or more concentrations of herbicidal compound.

52. The method of claim 39, wherein the at least one control protoplast lacks expression of the one or more candidate herbicide tolerance genes.63US_ACTIVE\131878835W-1

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