Methods and compositions for resistance to Icaforin herbicides

Abstract

This disclosure provides a novel recombinant DNA molecule for conferring resistance to the herbicide icaforin in the field of biotechnology. This disclosure also provides herbicide-resistant transgenic plants, seeds, cells, and plant parts containing the recombinant DNA molecule, as well as methods for using the above.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 508,094, filed on June 14, 2023, and U.S. Provisional Application No. 63 / 606,492, filed on December 5, 2023, the entire disclosures of each of which are incorporated herein by reference.

[0002] Incorporation of Sequence Listing The sequence listing contained in a file named "MONS562WO_ST26.xml", which is 77.1 kilobytes (measured in MS - Windows), created on May 23, 2024, and submitted electronically with this specification, is incorporated herein by reference in its entirety.

[0003] This disclosure relates to the field of biotechnology. More specifically, this disclosure relates to recombinant DNA molecules encoding enzymes that provide resistance to the ikafolin herbicide.

Background Art

[0004] Agricultural crop production often utilizes transgenic traits created using biotechnology methods. A heterologous gene, also known as a transgene, can be introduced into a plant to create a transgenic trait. Expression of a transgene in a plant confers traits such as herbicide resistance to the plant. Non - limiting examples of transgenic traits that are herbicide - resistant include glyphosate resistance, glufosinate resistance, dicamba resistance, and PPO herbicide resistance. With the increasing number of weed species resistant to commonly used herbicides, new herbicide - resistant traits are needed in the art. Particularly targeted herbicides include the ikafolin herbicide. The ikafolin herbicide provides control of a wide variety of herbicide - resistant weeds, and thus, in cropping systems, traits that confer resistance to these herbicides are particularly useful. In certain embodiments, the ikafolin - resistant trait can be combined with one or more other herbicide - resistant traits (plural possible). [Overview of the project]

[0005] In one embodiment, the disclosure provides a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide sequence encoding a polypeptide having at least about 94% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, which functions in plant cells. In one embodiment, the encoded polypeptide confers resistance to the herbicide Icaforin. In another embodiment, the polynucleotide sequence comprises a nucleic acid sequence having at least about 95% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, or a nucleic acid sequence having at least about 85% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36. In yet another embodiment, the encoded polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In yet another embodiment, the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36. In one embodiment, the polynucleotide sequence further comprises a nucleic acid sequence that encodes a targeting sequence that functions to localize the encoded polypeptide into a cell.In another embodiment, the herbicide is 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-carboxy Silates; (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 any combination thereof: selected from the group. In further embodiments, the present invention provides DNA constructs comprising the recombinant DNA molecules described herein. In one embodiment, the DNA construct comprises a heterologous promoter functionally linked to a polynucleotide sequence encoding a polypeptide having at least about 94% sequence identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, wherein the heterologous promoter may comprise a recombinant DNA molecule that is functional in plant cells. In another embodiment, the recombinant DNA molecule of the present disclosure is incorporated into the genome of a transgenic plant, seed, plant part, or cell.

[0006] In another embodiment, a transgenic plant, seed, cell, or plant part comprising a recombinant DNA molecule comprising a heterologous promoter functionally linked to a polynucleotide sequence encoding a polypeptide having at least about 85% sequence identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In one embodiment, the transgenic plant, seed, cell, or plant part is resistant to the herbicide icaforin. In another embodiment, the transgenic plant, seed, cell, or plant part is a soybean, corn, cotton, barley, sorghum, rice, wheat, sugarcane, sugar beet, alfalfa, or a plant, seed, cell, or plant part of the Brassicaceae family. In yet another embodiment, the herbicide is methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate;(2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate;(2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran- 2-methyl 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 any combination thereof. In yet another embodiment, the transgenic plant, seed, cell, or plant part of the present disclosure may include resistance to at least one additional herbicide.

[0007] In yet another embodiment, the Disclosure provides a method for conferring herbicide resistance to a plant, seed, cell, or plant part, the method comprising expressing a recombinant DNA molecule of the Disclosure in the plant, seed, cell, or plant part. In one embodiment, the recombinant DNA molecule comprises a heteropromoter functionally linked to a polynucleotide sequence encoding a polypeptide having at least about 94% sequence identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, wherein the heteropromoter is functional in a plant cell. In another embodiment, the plant, seed, cell, or plant part is resistant to the herbicide Icaforin. In yet another embodiment, the herbicide is methyl(2R*,4R*)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate;(2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate;(2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran- 2-methyl 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 any combination thereof.

[0008] In yet another embodiment, the Disclosure provides a method for producing a transgenic plant or a portion thereof, the method comprising: a) a recombinant DNA molecule comprising a heterologous promoter functionally linked to a polynucleotide sequence encoding a polypeptide having at least about 85% sequence identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28 in a plant cell; and b) regenerating the transgenic plant or a portion thereof from the cell or its progeny cells containing the recombinant DNA molecule. In one embodiment, the method may further include selecting the regenerated transgenic plant having resistance to the herbicide Icaforin. In another embodiment, the herbicide is 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; (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2 -methyl 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 any combination thereof. In yet another embodiment, the method may further include crossing the regenerated transgenic plant with itself or with a second plant to produce seeds.In yet another embodiment, the encoded polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In one embodiment, the polynucleotide sequence comprises a nucleic acid sequence having at least about 95% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, or a nucleic acid sequence having at least about 85% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36. In yet another embodiment, the method may further comprise crossing the regenerated transgenic plant, or its offspring plant containing a recombinant DNA molecule, with itself or a second plant to produce seeds, the seeds containing the recombinant DNA molecule. In yet another embodiment, the present disclosure provides seeds produced by the method described herein. In yet another embodiment of this disclosure, a transgenic plant or a portion thereof produced by the method described herein, the transgenic plant or a portion thereof comprising the recombinant DNA molecule.

[0009] In one embodiment, the Disclosure provides a method for controlling weeds in a plant growing area including a transgenic plant or seed of the Disclosure, the method comprising contacting the plant growing area with the herbicide icaforin, wherein the transgenic plant or seed is resistant to the herbicide icaforin, and weeds are controlled in the plant growing area. In one embodiment, the herbicide is 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; (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate Chil; (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 any combination thereof are selected from the group. In another embodiment, the transgenic plant or seed is soybean, maize, cotton, barley, sorghum, rice, wheat, sugarcane, sugar beet, alfalfa, or a plant of the Brassicaceae family.

[0010] In another aspect, the Disclosure provides a method for identifying plants resistant to the herbicide icaforin and at least one additional herbicide, the method comprising: a) obtaining a transgenic plant of the Disclosure having resistance to the herbicide icaforin and at least one additional herbicide; b) applying the at least one additional herbicide to the plant or a portion thereof; and c) identifying the plant as exhibiting resistance to the at least one additional herbicide.

[0011] In yet another aspect, the Disclosure provides a method for suppressing the growth of herbicide-resistant weeds in a plant growing area comprising a transgenic plant or seed of the Disclosure that is resistant to the herbicide ichafolin and at least one additional herbicide, the method comprising bringing the plant growing area into contact with the herbicide ichafolin and at least one additional herbicide, wherein the transgenic plant or seed is resistant to the herbicide ichafolin and at least one additional herbicide. In one embodiment, the herbicide is 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; (2S,4S)-4-[[(5S)-3-(3,5-difluorophenyl)-5-vinyl-4H-isoxazole-5-carbonyl]amino]tetrahydrofuran-2-carboxylate Chill; (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 any combination thereof. In another embodiment, at least one additional herbicide is selected from the group consisting of ACCase inhibitors, ALS inhibitors, EPSPS inhibitors, synthetic auxins, photosynthesis inhibitors, glutamine synthesis inhibitors, HPPD inhibitors, PPO inhibitors, PDS inhibitors, and long-chain fatty acid inhibitors.

[0012] Any aspect or embodiment of this disclosure may be used in combination with any other aspect or embodiment described herein. A brief explanation of arrays

[0013] Sequence ID 1 is the amino acid sequence of the Amaranthus palmeri CYP12 protein.

[0014] Sequence ID 2 is the amino acid sequence of the Amaranthus palmeri CYP13 protein.

[0015] Sequence ID 3 is the amino acid sequence of the Amaranthus palmeri CYP14 protein.

[0016] Sequence ID 4 is the amino acid sequence of the Amaranthus palmeri CYP15 protein.

[0017] Sequence ID 5 is the amino acid sequence of a variant of the CYP12 protein that contains a serine amino acid inserted at position 2.

[0018] Sequence ID 6 is the Amaranthus palmeri nucleotide sequence that encodes Sequence ID 1.

[0019] Sequence ID 7 is the Amaranthus palmeri nucleotide sequence that encodes Sequence ID 2.

[0020] Sequence ID 8 is the Amaranthus palmeri nucleotide sequence that encodes Sequence ID 3.

[0021] Sequence ID 9 is the Amaranthus palmeri nucleotide sequence that encodes Sequence ID 4.

[0022] Sequence ID 10 is a nucleotide sequence encoding Sequence ID 5, codon-optimized for expression in dicotyledons.

[0023] Sequence ID 11 is a nucleotide sequence encoding Sequence ID 2, codon-optimized for expression in dicotyledons.

[0024] SEQ ID NO: 12 is a nucleotide sequence encoding SEQ ID NO: 3, which is codon-optimized for dicotyledonous expression.

[0025] SEQ ID NO: 13 is a nucleotide sequence encoding SEQ ID NO: 4, which is codon-optimized for dicotyledonous expression.

[0026] SEQ ID NO: 14 is a nucleotide sequence of an expression element containing the promoter, leader, and intron of ubiquitin 3 of Arabidopsis thaliana.

[0027] SEQ ID NO: 15 is a nucleotide sequence of the terminator sequence of Medicago truncatula photosystem II.

[0028] SEQ ID NO: 16 is the amino acid sequence of the Amaranthus palmeri CYP21 protein.

[0029] SEQ ID NO: 17 is the amino acid sequence of the Amaranthus palmeri CYP52 protein.

[0030] SEQ ID NO: 18 is the Amaranthus palmeri nucleotide sequence encoding SEQ ID NO: 16.

[0031] SEQ ID NO: 19 is the Amaranthus palmeri nucleotide sequence encoding SEQ ID NO: 17.

[0032] SEQ ID NO: 20 is a nucleotide sequence encoding SEQ ID NO: 16, which is codon-optimized for dicotyledonous expression.

[0033] SEQ ID NO: 21 is a nucleotide sequence encoding SEQ ID NO: 17, which is codon-optimized for dicotyledonous expression.

[0034] SEQ ID NO: 22 is a nucleotide sequence encoding SEQ ID NO: 17, which is codon-optimized for monocotyledonous expression.

[0035] Sequence ID 23 is the amino acid sequence of the Amaranthus tuberculatus CYP28 protein.

[0036] Sequence ID 24 is the Amaranthus tuberculatus nucleotide sequence that encodes Sequence ID 23.

[0037] Sequence ID 25 is a nucleotide sequence encoding Sequence ID 23, codon-optimized for expression in dicotyledons.

[0038] Sequence ID 26 is the amino acid sequence of the Amaranthus cruentus CYP34 protein.

[0039] Sequence ID 27 is the amino acid sequence of the Amaranthus palmeri CYP55 protein.

[0040] Sequence ID 28 is the amino acid sequence of the Amaranthus palmeri CYP56 protein.

[0041] Sequence ID 29 is the Amaranthus cruentus nucleotide sequence that encodes Sequence ID 26.

[0042] Sequence ID 30 is the Amaranthus palmeri nucleotide sequence that encodes Sequence ID 27.

[0043] Sequence ID 31 is the Amaranthus palmeri nucleotide sequence that encodes Sequence ID 28.

[0044] Sequence ID 32 is a nucleotide sequence encoding Sequence ID 26, codon-optimized for expression in dicotyledons.

[0045] Sequence ID 33 is a nucleotide sequence encoding Sequence ID 27, codon-optimized for expression in dicotyledons.

[0046] Sequence ID 34 is a nucleotide sequence encoding Sequence ID 27, codon-optimized for expression in monocots.

[0047] Sequence ID 35 is a nucleotide sequence encoding Sequence ID 28, codon-optimized for expression in dicotyledons.

[0048] Sequence ID 36 is a nucleotide sequence encoding Sequence ID 28, codon-optimized for expression in monocots. [Brief explanation of the drawing]

[0049] [Figure 1] Representative soybean plants treated with various icaforin regimens are shown. Photographs were taken three weeks after treatment. [Modes for carrying out the invention]

[0050] The following is a detailed description provided to assist those skilled in the art in carrying out embodiments of the disclosure. Modifications and alterations to embodiments described herein can be made without departing from the spirit or scope of the disclosure.

[0051] This disclosure provides a novel recombinant DNA molecule encoding a cytochrome P450 enzyme (CYP) that confers resistance to the herbicide icaforin. CYP enzymes are members of a superfamily of enzymes reported to catalyze numerous reactions on different substrates and are found in both prokaryotes and eukaryotes. The cytochrome P450 superfamily is the largest family of enzyme proteins in plants, comprising hundreds of members, and is involved in many metabolic pathways with unique and complex functions (Xu et. al., The cytochrome P450 superfamily: Key players in plant development and defense, J. of Integrative Agric., 14:9, 1673-1686, 2015). Some CYP enzymes are involved in herbicide resistance observed in certain herbicide species (e.g., Siminszky, B. Plant cytochrome P450-mediated herbicide metabolism. Phytochem Rev 5, 445-458, 2006). Plant P450 enzymes have been named and classified into the CYP71-CYP99 gene family and the CYP701-CYP999 gene family (Nelson DR. Cytochrome P450 diversity in the tree of life. Biochim Biophys Acta Proteins Proteom. 1866(1):141-154. 2018).

[0052] CYP14 and CYP15 belong to the CYP81E subfamily of cytochrome P450 enzymes. The CYP81E8 gene has been shown to be differentially expressed in populations of Amaranthus tuberculatus resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) (see PCT Publication WO2022 / 051340 and Giacomini, et al., Coexpression Clusters and Allele-Specific Expression in Metabolism-Based Herbicide Resistance. Genome Biol Evol.;12:2267-2278, 2020). Similarly, increased expression of the CYP81E8 gene was observed in Amaranthus palmeri populations resistant to the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide tembotrione (see Kuepper, A., Molecular genetics of herbicide resistance in palmer amaranth (Amaranthus palmeri): Metabolic tembotrione resistance and geographic origin of glyphosate resistance PhD Thesis., Colorado State University, Fort Collins, CO, 2018 and Aarthy et.al., Front. Agron., Vol 4, Rapid metabolism, and increased expression of CYP81E8 gene confer high level of resistance to tembotrione in a multiple-resistant Palmer amaranth, 2022 https: / / doi.org / 10.3389 / fagro.2022.1010292).

[0053] In certain embodiments, the Disclosure further provides vectors and expression cassettes encoding CYP proteins for expression in cells and plants. Methods for producing cells and plants tolerant to the herbicide Icaforin are also provided. The Disclosure further provides methods and compositions for using protein engineering techniques and bioinformatics tools to obtain and improve enzymes that confer Icaforin resistance.

[0054] A. Icaforin herbicide Ikaforin is a novel post-emergence isoxazole herbicide that acts on dicotyledonous (broadleaf) and monocotyledonous (narrowleaf / grass) herbicides. Ikaforin has the following structure: [ka]

[0055] In certain embodiments, icaforin may be applied as a mixture of two isomers (2R, 4R or 2S, 4S) or as individual isomers. In some embodiments, icaforin may be applied in the form of an acid (shown above) or as a methyl ester (shown below). [ka]

[0056] Non-limiting examples of icaforin herbicides that may be used in accordance with embodiments of this 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-5-carbonyl]amino]tetrahydrofuran-2-carboxylic acid, and any combination thereof.

[0057] Icaforin 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.

[0058] B. Recombinant polynucleotide molecules and encoded proteins In certain embodiments, this disclosure provides recombinant DNA molecules and proteins. As used herein, the term “recombinant” means non-natural DNA, proteins, cells, seeds, or organisms that are the result of genetic engineering and therefore do not normally exist in nature. As used herein, “recombinant DNA molecule” means a DNA molecule containing a DNA sequence that does not exist in nature and is therefore the result of human intervention, such as a DNA molecule containing at least two DNA molecules heterogeneous to each other. In one embodiment, the recombinant DNA molecule is a DNA molecule provided herein that encodes a protein that confers resistance to the herbicide icaforin, operably linked to heterogeneous controls or other elements such as heterogeneous promoters. As used herein, “recombinant protein” means a protein containing an amino acid sequence that does not exist in nature and is therefore the result of human intervention. In one embodiment, the 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 containing transgenic DNA. Recombinant cells, seeds, or organisms may be transgenic cells, seeds, plants, or plant parts that contain recombinant DNA molecules, produced, for example, as a result of plant transformation.

[0059] As used herein, the term “heterogeneous” means a polynucleotide molecule or protein that does not exist naturally, or that does not exist naturally in genetically modified cells in the same form or structure without artificial intervention. For example, a heterogeneous polynucleotide molecule may not exist naturally in the transformed or modified plant species, or may be expressed in a manner or genomic context different from the natural expression pattern or genomic context found in the transformed or modified species. For example, in some embodiments, heterogeneous polynucleotide molecules may be overexpressed. In certain embodiments, a heterogeneous polynucleotide molecule may be a combination of two or more polynucleotide molecules, such a combination not typically found in nature. In certain embodiments, the two polynucleotide molecules may originate from different species, or from different genes, for example, different genes from the same species or the same gene from different species. In some embodiments, a heterogeneous polynucleotide molecule may comprise two polynucleotide sequences that are not juxtaposed or operably linked in any naturally occurring polynucleotide molecule. In further embodiments, heterogeneous polynucleotide molecules may include a promoter or other regulatory sequence operably ligated to a transcribable polynucleotide sequence, where the promoter or other regulatory sequence and the transcribable polynucleotide sequence are not operably ligated in any naturally occurring polynucleotide molecule.

[0060] As used herein, the phrase “not typically found in nature” means not found in nature without artificial intervention. In some embodiments, recombinant polynucleotide or protein molecules may include polynucleotide or protein sequences that (i) are isolated from other polynucleotide or protein sequences that are in close proximity to each other in nature, or (ii) are in close proximity to (or contiguous with) other polynucleotide or protein sequences that are not in close proximity to each other in nature. In some embodiments, recombinant polynucleotide molecules or proteins may also refer to polynucleotide or protein molecules or sequences that are produced by genetic engineering or constructed extracellularly. For example, recombinant polynucleotide molecules may include any engineered or artificial plasmid, vector, or construct, and may include linear or cyclic polynucleotide molecules. In some embodiments, such plasmids, vectors, or constructs may contain various maintenance elements, such as prokaryotic origin of replication or selection marker genes, and one or more transgenes or expression cassettes.

[0061] As used herein, the term “isolated” means separating a molecule at least partially from other molecules that are typically associated with it in its natural state. In one embodiment, the term “isolated” means a DNA molecule that is separated in its natural state from nucleic acids that are normally adjacent to it. For example, a DNA molecule that encodes a protein naturally present in a particular plant species would be an isolated DNA molecule if the DNA molecule encoding that protein were not present in the DNA of the plant species in which it is naturally found. Thus, a DNA molecule that is fused or operably ligated to one or more other DNA molecules that would not be associated in nature, for example, as a result of recombinant DNA or plant transformation techniques, is considered isolated herein. Such a molecule is considered isolated even if it is incorporated into the chromosome of a host cell or present in nucleic acid solution together with other DNA molecules.

[0062] As used herein, the term “protein-coding DNA molecule” refers to a DNA molecule containing a nucleotide sequence that encodes a protein. As used herein, “protein-coding sequence” refers to a DNA sequence that encodes a protein. As used herein, “sequence” refers to a sequential arrangement of nucleotides or amino acids. In some embodiments, the boundaries of a protein-coding sequence may be determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. In some embodiments, a protein-coding molecule may include a DNA sequence that encodes a protein sequence. As used herein, “transgene expression,” “expressing a transgene,” “protein expression,” and “expressing a protein” mean the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA), translating the mRNA into a polypeptide chain, and folding this polypeptide chain into a protein. In some embodiments, a protein-coding DNA molecule may be operably linked in a DNA construct to a heterologous promoter for expressing the protein in cells transformed with a recombinant DNA molecule. As used herein, “operably linked” means two DNA molecules linked in such a manner that one can influence the function of the other. Operatively linked DNA molecules may be part of a single contiguous molecule, and may be adjacent or not. For example, a promoter is operationally linked to a protein-coding DNA molecule in a DNA construct if the two DNA molecules are positioned such that the promoter can influence the expression of the transgene.

[0063] As used herein, “DNA construct” means a recombinant DNA molecule containing two or more heterologous DNA sequences. DNA constructs are useful for the expression of transgenes and may be contained in a vector or plasmid. DNA constructs may be used in vectors for transformation, i.e., for the introduction of heterologous DNA into host cells, in order to produce transgenic plants and cells. They may also be contained in plasmid DNA or genomic DNA of transgenic plants, seeds, cells, or plant parts. As used herein, “vector” means any recombinant DNA molecule that can be used for the purpose of bacterial or plant transformation. Recombinant DNA molecules provided by the present invention can be inserted into a vector, for example, as part of a construct having the recombinant DNA molecule operably linked to a gene expression element that functions in a plant and affects the expression of an engineered protein encoded by the recombinant DNA molecule in the plant. General methods useful for manipulating DNA molecules for the preparation and use of DNA constructs and plant transformation vectors are well known in the art and are detailed in manuals and laboratory guides, including, for example, MR Green and J Sambrook, “Molecular Cloning: A Laboratory Manual” (Fourth Edition), ISBN: 978-1-936113-42-2, Cold Spring Harbor Laboratory Press, NY (2012). In some embodiments, the components for a DNA construct or a vector containing a DNA construct include one or more gene expression elements operably ligated to a transcriptionable DNA sequence, and thus include, as follows: a promoter for expressing the operably ligated DNA, an operably ligated protein-coding DNA molecule, and an operably ligated 3' untranslated region (UTR).Gene expression elements useful in implementing this disclosure include, but are not limited to, one or more of the following types of elements: promoters, 5'UTRs, enhancers, leaders, cis-acting elements, introns, targeting sequences, 3'UTRs, and one or more selectable marker transgenes.

[0064] The DNA constructs of this disclosure may include promoters operably ligated to the protein-coding DNA molecules provided herein, thereby driving the expression of the recombinant protein molecules. Promoters useful in embodiments of this disclosure include promoters that function in cells to express operably ligated polynucleotides, such as bacterial promoters, viral promoters, or plant promoters. Plant promoters are diverse and well known in the art, and include, for example, inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and / or spatio-temporally regulated promoters.

[0065] In one embodiment of the present disclosure, the DNA construct provided herein comprises a target sequence operably linked to a heterologous nucleic acid molecule encoding a polypeptide molecule that confers resistance to the herbicide icaforin, thereby promoting the intracellular localization of the polypeptide molecule. Targeting sequences are known in the art as signal sequences, targeting peptides, localization sequences, and transit peptides. Non-limiting examples of targeting sequences include chloroplast transport peptides (CTPs), mitochondrial targeting sequences (MTSs), or chloroplast-mitochondrial dual targeting peptides. By facilitating the intracellular localization of proteins, targeting sequences may increase the accumulation of recombinant proteins, protect proteins from proteolysis, and / or improve the level of herbicide resistance, thereby reducing the level of damage to cells, seeds, or organisms after herbicide application.

[0066] CTPs and other targeting molecules that may be used in connection with this disclosure are known in the art, including the Arabidopsis thaliana EPSPS CTP (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. (Listed in, but not limited to, 8:167-175, 1995; PCT WO 97 / 41228).

[0067] Where it is desirable to provide sequences useful for DNA manipulation (such as restriction enzyme recognition sites or recombination-based cloning sites), sequences preferred in plants (such as plant codon usage frequencies or Kozak consensus sequences), or sequences useful for designing DNA constructs (such as spacer or linker sequences), the recombinant DNA molecules of the present invention may be synthesized and modified, either entirely or partially, by methods known in the art. In certain embodiments, the recombinant DNA molecules or proteins of the present disclosure may include or encode any one of SEQ ID NOs: 1 to 36 and a sequence having a fragment thereof, including 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, or any range in between. In some embodiments, the recombinant DNA molecules or proteins of the present disclosure may confer resistance to the herbicide Icaforin when expressed in plant cells. In certain embodiments, sequences derived from any of Sequence IDs 1 to 36 may possess the activity of the nucleotide sequence from which they are derived, for example, herbicide resistance activity. As used herein, the terms “sequence identity percent” or “sequence identity %” refer to the percentage of identical nucleotides or amino acids in the linear polynucleotide or polypeptide sequences of a reference sequence (or its complementary chain) compared to a test sequence ("Target") sequence (or its complementary chain), when the two sequences are optimally aligned (using appropriate nucleotide or amino acid insertions, deletions, or gaps that result in a total of less than 20 percent of the reference sequence across the comparison window).Optimal sequence alignment for aligning comparison windows is well known to those skilled in the art and may be performed, for example, using default parameters, by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, and the similarity search method of Pearson and Lipman, as well as by computer implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, available as part of the sequence analysis software package of 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 sequence alignment with high accuracy and high throughput” Nucleic Acids Research 32(5):1792-7(2004)). The "identity fraction" for an aligned segment of a test sequence and a reference sequence is calculated by dividing the number of identical components shared by the two aligned sequences in a portion of the aligned reference sequence segment, i.e., the entire reference sequence or a smaller specified portion thereof, by the total number of components. The sequence identity percentage is expressed as the identity fraction multiplied by 100. The comparison of one or more sequences may be of the full sequence, a portion thereof, or a longer sequence.

[0068] The recombinant DNA molecules or proteins of this disclosure may contain or encode any fragment of SEQ ID NOs. 1 to 36. For example, the DNA molecules provided in this disclosure contain 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, at least 1800, at least 1900, and at least 2000 consecutive nucleotides of SEQ ID NOs. 6 to 15, 18 to 22, 24 to 25, or 29 to 36. The fragments of sequence numbers 6-15, 18-22, 24-25, or 29-36 may possess the activity of the nucleotide sequence from which they are derived, such as herbicide resistance activity. In some embodiments, the 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 575, at least 600, at least 625, at least 650, at least 675, at least 700, at least 725, at least 750, at least 775, at least 800, at least 825, at least 850, at least 875, at least 900, at least 925, at least 950, at least 975, and at least 1000 consecutive amino acids of any of the S.E. codes 1-5, 16, 17, 23 or 26-28. The fragments of sequence numbers 1-5, 16, 17, 23, or 26-28 may possess the activity of the nucleotide sequence from which they are derived, such as herbicide resistance activity.

[0069] The manipulated proteins may exhibit specific cellular localization patterns targeting chloroplasts or mitochondria, or novel combinations of useful protein properties, particularly modified V max , K m , K i ,I C 50 , k catThe modified proteins may be produced by altering (i.e., modifying) a wild-type protein to produce a novel protein having modified properties such as substrate specificity, inhibitor / herbicide specificity, substrate selectivity, ability to interact with other intracellular components such as partner proteins or membranes, and protein stability. In certain embodiments, the modification may be carried out at specific amino acid positions in the protein. In one embodiment, the modification may be carried out by substituting different amino acids for amino acids found at specific amino acid positions in nature (i.e., the wild-type protein). Thus, the engineered proteins provided by this disclosure provide a novel protein having one or more modified protein characteristics compared to a similar protein found in nature. In some embodiments of this disclosure, the engineered protein has modified protein properties such as a property that results in reduced sensitivity to one or more herbicides compared to the same wild-type protein, or a property that results in an improved ability to confer herbicide resistance to transgenic plants expressing the engineered protein. In another embodiment, the Disclosure provides recombinant DNA molecules encoding an engineered protein and at least one amino acid substitution selected from Table 1, 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, and including, but not limited to, protein sequences composed of or encoded by any one of SEQ ID NOs: 1 to 36, and including all derivable ranges between them. The amino acid substitution may be made as a single amino acid substitution in the protein, or in combination with one or more other mutations, such as one or more other amino acid substitutions, deletions, or additions. Mutations may be made by any method known to those skilled in the art. [Table 1]

[0070] As used herein, “wild-type” means a naturally occurring, similar but not identical version of a protein. “Wild-type DNA molecule” or “wild-type protein” means the naturally occurring version of a DNA molecule or protein, i.e., the version of a DNA molecule or protein that exists in nature.

[0071] C. Genetically modified plants through genetic engineering Various genetic engineering techniques have been developed to introduce transgenic or edited traits into plants and can be used by those skilled in the art. The methods generally involve delivering a polynucleotide sequence to plant cells, which is typically a heterogeneous and / or recombinant polynucleotide molecule. A heterogeneous or recombinant polynucleotide molecule may include, for example, at least one transgene, an expression cassette, or an RNA molecule such as guide RNA (gRNA). In specific embodiments, the heterogeneous or recombinant polynucleotide molecule may be a ribonucleoprotein (RNP) or a fragment thereof or part of a guide RNA. Expression of a heterogeneous or recombinant polynucleotide molecule can result in, for example, a gRNA / site-specific nuclease complex for genome editing. In certain embodiments, the trait is introduced into the plant by modifying or introducing a single locus or transgene into the plant genome. Genetic engineering methods for modifying, deleting, or introducing transgenes, editing, mutations, and polynucleotide sequences into plant genomic DNA are known in the art. Molecular methods for editing plant cell genomes or endogenous plant genes using genome editing techniques are known in the art. According to this embodiment, genome editing tools or mechanisms, such as guide RNA, site-specific nucleases, and / or template DNA molecules, or polynucleotides or DNA molecules encoding them, can be introduced into plant cells using the methods described herein.

[0072] As used herein, “genetically modified” plant, plant part, plant tissue, or plant cell includes genetic modification. As used herein, “genetic modification” means one or more introduced genes, mutations, or edits into the genome of a plant, plant part, or plant cell using transformation, mutagenesis, or genome editing techniques. Apart from genome editing techniques, mutagenesis techniques may include any chemical, physical, radiological, or biological (e.g., transposon-mediated) mutagenesis techniques or mutagens.

[0073] As used herein, “transgenic” plant, plant part, plant tissue, or plant cell has an exogenous nucleic acid sequence, genome edit, or transgene incorporated into the genome of a plant, plant part, plant tissue, or plant cell. As used herein, “transgenic trait” refers to a characteristic or phenotype transmitted or conferred by the presence of a transgene, nucleic acid sequence, genome edit, or transgene incorporated into the plant genome. As a result of such genome modification, the transgenic plant is distinctly different from the associated wild-type plant, and the transgenic trait is a trait not naturally found in the wild-type plant. In some embodiments, the transgenic plant described herein comprises recombinant DNA molecules and engineered proteins provided herein.

[0074] Genome editing can be used to modify, for example, the expression or activity of one or more genes, or to insert an insertion sequence or transgene at a desired location in the plant genome by applying one or more edits or mutations to a desired target site in the plant genome. Any site or locus in the plant genome may potentially be selected for genome editing or site-directed insertion of a transgene, construct, or transcriptionable DNA sequence. As used herein, “target site” for genome editing or site-directed insertion refers to a location in a polynucleotide sequence in the plant genome that is bound and cleaved by a site-specific nuclease to introduce a double-strand break (DSB) or single-strand nick into the nucleic acid backbone and / or complementary DNA strand of the polynucleotide sequence in the plant genome. The target site may, for example, comprise 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. The “target site” for RNA-inducible nucleases may comprise the sequence of the complementary strand of either a double-stranded nucleic acid (DNA) molecule or a chromosome at the target site. Site-specific nucleases may bind to the target site via non-coding guide RNA (e.g., CRISPR RNA (crRNA) or single guide RNA (sgRNA) as further described herein, but not limited to these). The non-coding guide RNAs provided herein may be complementary to the target site (e.g., complementary to either strand of a double-stranded nucleic acid molecule or a chromosome at the target site). It should be understood that complete identity or complementarity is not required for the non-coding guide RNA to bind to or hybridize with the target site. For example, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight mismatches (or more) between the target site and the non-coding RNA may be acceptable.The “target site” also refers to a location in the plant genome of a polynucleotide sequence that is bound and cleaved by any other site-specific nuclease, which does not necessarily have to be induced by a non-coding RNA molecule such as a zinc finger nuclease (ZFN), activator-like effector nuclease (TALEN), or meganuclease, to introduce double-strand breaks (DSBs) or single-strand nicks into the polynucleotide sequence and / or its complementary DNA strand. As used herein, “site-specific nuclease” includes any zinc finger nuclease (ZFN), activator-like effector nuclease (TALEN), ribonucleoprotein, meganuclease, recombinase, transposase, or other nuclease that can introduce double-strand breaks (DSBs) or single-strand nicks into a polynucleotide sequence at or near a target site, such as a target site in the genome of a plant cell. As used herein, “target region” or “targeted region” refers to a polynucleotide sequence or region where two or more target sites are in close proximity. In some embodiments, the target region may undergo mutation, deletion, insertion, or inversion. As used herein, “proximity” refers to two or more target sites of a polynucleotide sequence or molecule surrounding the target region, along with one target site on each side of the target region, when used to describe a target region of a polynucleotide sequence or molecule.

[0075] As used herein, “targeted genome editing technology” means any method, protocol, or technique that enables precise and / or targeted editing of specific locations within the plant genome (i.e., editing is largely or completely non-random) using site-specific nucleases such as meganucleases, zinc finger nucleases (ZFNs), RNA-induced endonucleases (e.g., CRISPR / Cas9 or CRISPR / Cas12a), TALE (transmission activator-like effector) endonucleases (TALENs), recombinases, or transposases. As used herein, “editing” or “genome editing” means generating targeted mutations, deletions, inversions, or substitutions of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 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 in an endogenous plant genome nucleic acid sequence. As used herein, “editing” or “genome editing” may also include targeted insertions or site-directed incorporations 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.The singular “edit” or “genome editing” refers to one targeted mutation, deletion, inversion, substitution, or insertion; “edit” or “genome editing” refers to two or more targeted mutations, deletions, inversions, substitutions, and / or insertions, each “edit” being introduced via a targeted genome editing technique.

[0076] According to some embodiments, site-specific nucleases may be co-delivered with a donor template molecule that functions as a template for performing desired edits, mutations, or insertions into the genome at desired target sites via repair of double-strand breaks (DSBs) or nicks created by the site-specific nuclease. According to some embodiments, site-specific nucleases may be co-delivered with a DNA molecule containing a selectable or screenable marker gene.

[0077] The site-specific nucleases provided herein may be selected from the group consisting of zinc finger nucleases (ZFNs), TALE endonucleases (TALENs), meganucleases, RNA-induced endonucleases, recombinases, transposases, or any combination thereof. For example, Khandagale et al. (Plant Biotechnol Rep 10:327-343, 2016) and Gaj et al. (Trends Biotechnol. See 31(7):397-405, 2013. Zinc finger nucleases are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to a cleavage domain (or half of a cleavage domain) that may be derived from a restriction enzyme (e.g., FokI). The DNA-binding domain may be standard (C2H2) or non-standard (e.g., C3H or C4). The DNA-binding domain may contain one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more) depending on the target site, but is usually composed of 3-4 (or more) zinc fingers. Multiple zinc fingers in the DNA-binding domain are separated by a 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 consisting of a nonspecific DNA cleavage domain (e.g., derived from FokI nuclease) fused to a DNA-binding domain containing a zinc finger array engineered to bind to a target site DNA sequence. Amino acids at positions -1, +2, +3, and +6 (which contribute to site-specific binding to the target site) relative to the starting point of the zinc finger α-helix can be altered and customized to fit a specific target sequence. Other amino acids can form a consensus skeleton to produce ZFNs with different sequence specificities.

[0078] Methods and rules for designing ZFNs to target and bind to specific target sequences are known in the art. See, for example, U.S. Patent Applications 2005 / 0064474, 2009 / 0117617, and 2012 / 0142062. The FokI nuclease domain may require dimerization to cleave DNA, and therefore two ZFNs with C-terminal regions are needed to bind to the opposite DNA strands of the cleavage site (separated at 5-7 bp). If the two ZF binding sites are palindromic, the ZFN monomer can cleave the target site. As used herein, ZFN broadly encompasses monomeric ZFNs 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 engineered to work together to cleave DNA at the same site. Since the DNA binding specificity of zinc finger domains can be remanufactured using one of several methods, customized ZFNs can theoretically be constructed to target almost any target sequence (e.g., genes in or near plants in the genome). Publicly available methods for manipulating zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly.

[0079] Transcription activator-like effectors (TALEs) can be manipulated to bind to virtually any DNA sequence, for example, at or near genomic loci of plant genes. TALEs have a central DNA-binding domain composed of 13–28 repeat monomers of 33–34 amino acids. The amino acids in each monomer are highly conserved, except for the hypervariable amino acid residues at positions 12 and 13. Two variable amino acids are called repeat variable duos (RVDs). The amino acid pairs NI, NG, HD, and NN of the RVD preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and can recognize a sequence of DNA bases through the modification of the RVDs. This simple relationship between amino acid sequences and DNA recognition has made it possible to manipulate specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs.

[0080] TALENs are artificial restriction enzymes produced by fusing a TALEN DNA-binding domain to a nuclease domain. In some embodiments, the nuclease is selected from the group consisting of PvuII, MutH, TevI, FokI, AlwI, MlyI, SbfI, SdaI, StsI, CleDORF, Clo051, and Pept071. When each member of a TALEN pair binds to a DNA site adjacent to the target site, the FokI monomer dimerizes, causing a break in double-stranded DNA at the target site. As used herein, the term TALEN broadly encompasses monomeric TALENs that can break double-stranded DNA without the assistance of another TALEN. The term TALEN also refers to one or both members of a TALEN pair that work together to break DNA at the same site.

[0081] Beyond the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and activity. The FokI domain functions as a dimer, requiring two constructs with specific DNA-binding domains for a site in the target genome, oriented and spaced appropriately. 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. The PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and its variants for use with TALE. PvuII functions as a highly specific cleavage domain when coupled to TALE (Yank et al., PLoS One 8:e82539, 2013). MutH can introduce specific nicks into strands in DNA (Gabsalilow et al., Nucleic Acids Research. 41:e83, 2013). TevI introduces double-strand breaks in DNA at targeted sites (Beurdeley et al., Nature Communications 4:1762, 2013).

[0082] The relationship between the amino acid sequence of the TALE-binding domain and DNA recognition enables the design of protein constructs. TALE constructs can be designed using software programs such as DNAWorks. Other methods for designing TALE constructs are known to those skilled in the art. See Doyle et al. (Nucleic Acids Research 40:W117-122, 2012), Cermak et al. (Nucleic Acids Research 39:e82, 2011) and tale-nt.cac.cornell.edu / about. In another embodiment, the TALENs provided herein are capable of generating targeted DSBs.

[0083] Site-specific nucleases can be meganucleases. Meganucleases commonly identified in microorganisms (such as the LAGLIDADG family of homing endonucleases) are unique enzymes that possess 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 (e.g., 14–40 bp). Because the DNA recognition and cleavage functions of meganucleases are intertwined within a single domain, manipulating meganucleases can be more challenging than manipulating ZFNs and TALENs. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and retain improved nuclease activity.

[0084] Site-specific nucleases can be RNA-inducible nucleases. According to some embodiments, RNA-inducible endonucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, The group may be selected from Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cas12a (also known as Cpf1), CasX, CasY, and any homologs or modified versions thereof, as well as Argonaut (non-limiting examples of Argonaut proteins include Thermus thermophilus Argonaut (TtAgo), Pyrococcus furiosus Argonaut (PfAgo), Natronobacterium gregoryi Argonaut (NgAgo), and any homologs or modified versions thereof). According to some embodiments, the RNA-inducible endonuclease is a Cas9 or Cas12a enzyme. RNA-induced nucleases may be delivered as proteins with or without guide RNA, or the guide RNA may be delivered as a ribonucleoprotein (RNP) by forming a complex with the RNA-induced nuclease enzyme.

[0085] For RNA-induced endonucleases, guide RNA molecules may be further provided to direct the endonuclease to a target site in the plant genome via base pairing or hybridization, and to generate a double-segment break (DSB) or nick at or near the target site. The guide RNA may be transformed into or introduced into plant cells or tissues as a gRNA molecule, or as a recombinant DNA molecule, construct, or vector containing a transcribable DNA sequence encoding a guide RNA operably linked to a promoter. As understood in the art, the guide RNA may include, for example, CRISPR RNA (crRNA), tracrRNA, single-stranded guide RNA (sgRNA), or any other RNA molecule that can induce or direct an endonuclease to a specific target site in the genome. Cas9, a prototype CRISPR-associated protein derived from Streptococcus pyogenes, spontaneously binds to two RNAs, CRISPR RNA (crRNA) guide and trans-acting CRISPR RNA (tracrRNA), to construct CRISPR ribonucleoprotein (crRNP). A “single-chain guide RNA” (or “sgRNA”) is an RNA molecule containing crRNA covalently bound to tracrRNA by a linker sequence, and can be expressed as a single RNA transcript or molecule. The guide RNA contains a guide or targeting sequence (also referred to herein as a “spacer sequence”) that is identical or complementary to a target site in the plant genome, such as in or near a gene. The guide RNA is usually a non-coding RNA molecule that does not encode a protein. The guide sequence of the guide RNA may be, for example, 12–40 nucleotides long, 12–30 nucleotides long, 12–20 nucleotides long, 12–35 nucleotides long, 12–30 nucleotides long, 15–30 nucleotides long, 17–30 nucleotides long, or 17–25 nucleotides long, or at least 10 nucleotides long, such as approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more nucleotides long.The guide sequence may be at least 95%, at least 96%, at least 97%, at least 99%, or 100% identical to, 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, or at least 25 consecutive nucleotides of the DNA sequence at the target site of the genome.

[0086] In this specification, the terms “complementary strand,” “complementary sequence,” and “reverse complementary strand” are used interchangeably with respect to a given sequence. All three terms refer to the reverse complementary sequence of a nucleotide sequence, that is, a sequence in which the nucleotides are complementary to a given sequence in reverse order.

[0087] As used herein, the term “antisense” refers to a DNA or RNA sequence that is complementary to a particular DNA or RNA sequence. An antisense RNA molecule is a single-stranded nucleic acid that, in combination with a sense RNA strand, sequence, or mRNA, can form a double helix by sequence complementarity. The term “antisense strand” refers to a nucleic acid strand complementary to the “sense” strand. The “sense strand” of a gene or locus is a strand of DNA or RNA (excluding uracil in RNA and thymine in DNA) that has the same sequence as the RNA molecule transcribed from the gene or locus.

[0088] Protospacer adjacency motifs (PAMs) can exist in the genome, as is known in the art, i.e., immediately downstream (3') of the sense (+) strand of the genomic target site (complementary to the target sequence of the guide RNA), and directly adjacent to and upstream of the genomic target site complementary 5' end of the guide RNA target site. See, for example, Wu et al. (Quant Biol. 2(2):59-70, 2014). Genomic PAM sequences on the sense (+) strand adjacent to the target site (relative to the targeting sequence of the guide RNA) may include 5'-NGG-3'. However, generally speaking, the corresponding sequence of the guide RNA (i.e., directly downstream (3') of the target sequence of the guide RNA) does not have to be complementary to the genomic PAM sequence.

[0089] In some embodiments, the site-specific nuclease is a recombinase. Non-limiting examples of recombinases that can be used include serine recombinases attached to a DNA recognition motif, tyrosine recombinases 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 to or fused to a DNA-binding domain. Non-limiting examples of recombinases include tyrosine recombinases selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase attached to a DNA recognition motif provided herein. In one aspect of this disclosure, the Cre recombinase or Gin recombinase provided herein is tethered to a zinc finger DNA-binding domain, a TALE DNA-binding domain, or a Cas9 nuclease. In another embodiment, a serine recombinase selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase may be attached to the DNA recognition motif provided herein. In yet another embodiment, a DNA transposase selected from the group consisting of TALE-piggyBac and TALE mutagenesis may be attached to the DNA binding domain provided herein.

[0090] Some site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, are not RNA-inducible; instead, they rely on their protein structure to determine their target site to induce a double-stroke spread (DSB) or nick, or they are fused, tethered, or attached to a DNA-binding protein domain or motif. The protein structure of a site-specific nuclease (or fused / attached or ligated DNA-binding domain) can target the site-specific nuclease to a target site. According to many of these embodiments, non-RNA-inducible site-specific nucleases such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs can be designed, manipulated, and constructed according to known methods to target and bind to a target site at or near a genomic locus of an endogenous gene in a plant, thereby creating a DSB or nick at such a genomic locus. DSBs or nicks created by non-RNA-inducible site-specific nucleases may cause knockdown of gene expression through DSB or nick repair, resulting in sequence mutations or insertions at the DSB or nick site via cellular repair mechanisms. These cellular repair mechanisms can be induced by donor template molecules.

[0091] As used herein, a “donor molecule,” “donor template,” or “donor template molecule” (collectively, “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., homologous sequence) and / or an insertion sequence for site-directed, targeted insertion or recombination into the genome of a plant cell via repair of nicks or DSBs in the genome of the plant cell. A donor template may be an isolated DNA molecule containing one or more homologous sequences and / or an insertion sequence for targeted integration, or a donor template may be a sequence portion of a DNA molecule (i.e., a donor template region) further containing one or more other expression cassettes, genes / transgenes, and / or transcriptionable DNA sequences. A donor template may be an Agrobacterium Transfer-DNA (T-DNA) molecule containing one or more other expression cassettes, genes / transgenes, and / or transcriptionable DNA sequences. For example, a “donor template” may be used for site-directed integration of a transgene or construct, or as a template for introducing mutations such as insertions, deletions, or substitutions into target sites within a plant genome. Targeted genome editing techniques provided herein may involve the use of one or more, two or more, three or more, four or more, or five or more donor molecules or templates. Donor templates provided herein may include 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 genes or transgenes and / or transcriptional DNA sequences. Alternatively, a donor template may not include genes, transgenes, or transcriptional DNA sequences.

[0092] Examples of donor template genes / transgenes or transcriptable DNA sequences may include, but are not limited to, insecticide resistance genes, herbicide resistance genes, nitrogen use efficiency genes, water use efficiency genes, yield improvement genes, nutritional quality genes, DNA binding genes, selectable marker genes, RNAi or repression constructs, site-directed genome modification enzyme genes, single guide RNAs for CRISPR / Cas9 systems, CRISPR / Cas12a system guide RNAs, geminivirus-based expression cassettes, or plant virus expression vector systems. In other embodiments, the donor template insertion sequence may include a protein-encoding sequence or a transcriptable DNA sequence encoding a non-coding RNA molecule that can target an endogenous gene for repression. The donor template may include a promoter (such as a constitutive promoter, tissue-specific or tissue-preferential promoter, developmental stage promoter, or inducible promoter) operably bound to the coding sequence, gene, or transcriptable DNA sequence. The donor template may include a leader, enhancer, promoter, transcription start site, 5'-UTR, one or more exons, one or more introns, transcription termination sites, regions, or sequences, 3'-UTR, and / or polyadenylation signals, each of which may be operably bound to a coding sequence, gene (or transgene), or transcriptionally viable DNA sequence encoding non-coding RNA, guide RNA, mRNA, and / or protein. The donor template may be a single-stranded or double-stranded DNA or RNA molecule or plasmid.

[0093] The "insertion sequence" of a donor template is a sequence designed for targeted insertion into the genome of a plant cell, and it can be of any suitable length. For example, the insertion sequence of a donor template can be between 2 and 50,000, between 2 and 10,000, between 2 and 5,000, between 2 and 1,000, 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 1,000, between 15 and 5,000, 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, and between 20 and 500. The length of a nucleotide or base pair can be between 1,000, between 20 and 5,000, between 20 and 10,000, between 50 and 250, between 50 and 500, between 50 and 1,000, between 50 and 5,000, between 50 and 10,000, between 100 and 250, between 100 and 500, between 100 and 1,000, between 100 and 5,000, between 250 and 500, between 250 and 1,000, between 250 and 5,000, or between 250 and 10,000. The donor template also has at least one homologous sequence or homologous arm(s) such that homologous recombination directs the insertion of a mutation or insertion sequence into a target site in the plant genome, and the homologous sequence or homologous arm(s) are identical or complementary, or have percent identity or percent complementarity to a sequence at or near the target site in the plant genome. If the donor template contains homologous arm(s) and an insertion sequence, the homologous arm(s) will be adjacent to or surround the insertion sequence in the donor template.Each homologous arm is 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 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 in the plant genome.

[0094] Any method known in the art for site-directed integration may be used in this disclosure. In the presence of a donor template molecule containing an insertion sequence, a DSB or nick may be repaired by homologous recombination between the donor template and a homologous arm(s) of the plant genome, or by non-homologous end joining (NHEJ), resulting in site-directed integration of the insertion sequence into the plant genome and generating a targeted insertion event at the site of the DSB or nick. Thus, site-directed insertion or integration of a transgene, transcriptable DNA sequence, construct, or sequence may be achieved if the transgene, transcriptable DNA sequence, construct, or sequence is located within the insertion sequence of the donor template.

[0095] Targeted mutations can also be introduced into the plant genome using DSBs or nicks. According to this approach, mutations (such as deletions, insertions, inversions, and / or substitutions) are introduced at a target site via incomplete repair of DSBs or nicks, potentially producing gene knockout or knockdown. Such mutations can sometimes be generated by incomplete repair of a targeted locus, without the use of a donor template molecule. Gene "knockout" can be achieved by inducing a DSB or nick at or near the gene's endogenous locus, resulting in non-expression of the protein or expression of a non-functional protein; gene "knockdown" can be achieved in a similar manner by inducing a DSB or nick at or near the gene's endogenous locus, which is incompletely repaired at a site that does not affect the gene's coding sequence, resulting in loss of function of the encoded protein. For example, the site of a DSB or nick within an endogenous locus may be located upstream or in the 5' region of the gene (e.g., promoter and / or enhancer sequences) and may affect or reduce the level of expression.

[0096] Similarly, such targeted knockout or knockdown mutations of a gene can be generated by a donor template molecule that directs a specific or desired mutation at or near the target site via DSB or nick repair. The donor template molecule may contain a homologous sequence with or without an insertion sequence that contains one or more mutations (one or more deletions, insertions, inversions, and / or substitutions, etc.) at or near the DSB or nick site compared to the targeted genomic sequence. For example, a targeted knockout or knockdown mutation of a gene can be achieved by substituting, inserting, deleting, or inverting at least a portion of the gene (for example, by introducing a frameshift or immature stop codon into the gene's coding sequence, or by disrupting the sequence of a promoter sequence or other non-coding regulatory elements of the gene). A partial deletion of a gene can also be introduced by generating a DSB or nick at two target sites, resulting in a deletion of a target region adjacent to and interposed by the target sites.

[0097] In further embodiments, plant genetic modification may include transformation of plants, plant parts, plant tissues, or plant cells to insert polynucleotides or DNA sequences or transgenes into the genome of a plant, plant part, plant tissue, or plant cell. Plant transformation methods 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 forms of direct DNA incorporation. Bacteria known to mediate plant cell transformation include, but are not limited to, numerous bacterial genera, species, and strains that may be classified in the order Rhizobiales other than Agrobacterium, as well as bacterial species and strains from taxonomic families such as Rhizobiaceae (e.g., Rhizobium, Sinorhizobium), Phyllobacteriaceae (e.g., Mesorhizobium, Phyllobacterium), Brucellaceae (e.g., Ochrobactrum), Bradyrhizobiaceae (e.g., Bradyrhizobium), and Xanthobacteraceae (e.g., Azorhizobium). According to some embodiments, Agrobacterium-mediated transformation is mediated by Agrobacterium tumefaciens. The target of such transformation is often undifferentiated callus tissue, but differentiated tissue has also been used for transient and stable plant transformation.Other methods of plant transformation, as are well known in the art, may also be used, such as those described in Miki et al., (1993, “Procedures for Introducing Foreign DNA into Plants,” in Methods in Plant Molecular Biology and Biotechnology, Glick BRand Thompson, JE Eds., CRC Press, Inc., Boca Raton, pages 67-88).

[0098] In specific embodiments, microprojectile bombardment can be used to deliver polynucleotides or DNA molecules, vectors, sequences, segments, or RNPs to cells. In this method, particles are coated with polynucleotides or polynucleotide / protein complexes and delivered to cells by propulsion. Exemplary particles may consist of tungsten, platinum, or gold. For bombardment, target cells may be placed on a solid culture medium. The cells to be bombarded are positioned at an appropriate distance below a projection stop plate. Polynucleotides can be delivered into plant cells by acceleration using a bioristic particle delivery system, which can propel particles coated with DNA or polynucleotide molecules through a screen such as a stainless steel or Nytex screen towards cells positioned on the surface. The screen can disperse the particles so that they are not delivered to recipient cells as large aggregates. Microprojectile bombardment technology is widely applicable and can be used for transformation in various plant species.

[0099] Agrobacterium-mediated or Rhizobiales-mediated transformation of explants is another widely applicable system for incorporating heterologous and / or recombinant DNA molecules into plant cells. Modern Agrobacterium-mediated transformation vectors are replicable not only in Agrobacterium but also in E. coli and are easy to handle (see, e.g., Klee et al., Nat. Biotechnol., 3(7):637-642, 1985). Furthermore, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites within the vector, making it easier to construct vectors capable of expressing genes encoding various polypeptides. The described vectors have a convenient multilinker region adjacent to the promoter and polyadenylation site for direct expression of the inserted polypeptide-coding gene. Additionally, Agrobacterium containing both armed and unarmed Ti plasmids can be used for transformation. Agrobacterium-mediated transformation is a popular method for many plant species. The use of Agrobacterium-mediated plant-integrated vectors for introducing DNA into plant cells is known in the art (see, for example, Fraley et al., Nat. Biotechnol., 3:629-635, 1985; U.S. Patent No. 5,563,055).

[0100] Many promoters and expression elements are useful for the expression of any selectable marker, scoreable marker, transgene, or any other gene of agricultural interest in plant genes. Promoters may include any constitutive promoter, tissue-specific promoter, organ-specific promoter, tissue-selective promoter, organ-selective promoter, inductive promoter, germ tissue promoter, developmental stage promoter, viral promoter, and the like. Examples of various types of promoters and expression elements are known in the art. Expression elements that may be useful for plant gene expression include, for example, various promoters, enhancers, leaders, 5' and 3' untranslated regions, introns, terminators, and the like, as known in the art. The selectable marker or screenable marker or target gene may also be fused to a signaling peptide or other target sequence. The transport of a protein produced by a transgene into intracellular compartments such as chloroplasts, vacuoles, peroxisomes, glyoxisomes, the cell wall, the nucleus, or mitochondria, or secretion into the apoplast, can be achieved by operably ligating a signal sequence or a nucleotide sequence encoding a target sequence to the 5' and / or 3' regions of the gene encoding the protein of interest. The target sequence at the 5' and / or 3' ends of the structural gene can determine where the encoded protein is ultimately compartmentalized during protein synthesis and processing. The presence of a signal sequence directs the polypeptide to either an intracellular organelle or intracellular compartment, or to secretion into the apoplast. Many signal sequences are known in the art.For example, see 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).

[0101] Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S promoter (see, e.g., Odel et al., Nature, 313:810, 1985), which gives constitutively high levels of expression in most plant tissues, including monocots (see, e.g., Dekeyser et al., Plant Cell, 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990), a tandem duplicate version of the CaMV 35S promoter, the enhanced 35S promoter (e35S), the nopalin synthase promoter (see, An et al., Plant Physiol., 88:547, 1988), and the octopin synthase promoter (see, Fromm et al., Plant Examples include the figwolt mosaic virus (FMV) promoter described in Cell, 1:977, 1989, and U.S. Patent No. 5,378,619, as well as enhanced versions of the FMV promoter (eFMV) in which the FMV promoter sequence is tandem-dubbed, the cauliflower mosaic virus 19S promoter, the sugarcane rod-type virus promoter, the Commelina yellow mottle virus promoter, and other plant DNA virus promoters known to be expressed in plant cells. Constitutive promoters include the maize ubiquitin promoter (Christensen et al., Plant Mol. Biol., 18:675, 689, 1992), the maize actin promoter (McElroy, et al., Plant Cell, 2:163-171, 1990), or the Arabidopsis S-adenosylmethionine synthetase promoter (see U.S. Patent Nos. 8,809, 628).

[0102] Using an inductive promoter increases the transcription rate in response to an inducer or inductive signal. Any inductive promoter can be used according to embodiments of this disclosure. To express operably linked genes in plant cells, various plant gene promoters controlled in response to environmental, hormonal, chemical, and / or developmental signals can be used, including (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, e.g., abscisic acid (Marcotte et al., Plant Cell, 1:969, 1989), (4) injury (e.g., Siebertz et al., Plant (5) Promoters controlled by chemical substances, such as methyl jasmonate, salicylic acid, or Safener. It may also be advantageous to utilize organ-specific or tissue-specific promoters known in the art (e.g., Roshal et al., EMBO J., 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988; Bustos et al., Plant Cell, 1:839, 1989).

[0103] Exemplary polynucleotides or DNA molecules that can be introduced include, for example, DNA sequences or genes from another species, or even genes or sequences that originate from or exist in the same species but are incorporated into recipient cells by genetic engineering methods rather than by classical reproductive or breeding techniques. However, the term “exogenous” is also intended to refer to genes that are not normally present in the transformed cells, or genes that are unlikely to be present in their form, structure, or location. Polynucleotides may include DNA molecules or sequences that are already present in plant cells, originate from another plant, originate from a different organism, are exogenous, or are produced externally. Transgenes or expression cassettes may encode mRNA and proteins or RNA molecules for repression, such as miRNA, siRNA, dsRNA, antisense RNA, reverse repeat RNA, etc. Polynucleotides may be exogenous, heterogeneous, and / or recombinant polynucleotides or DNA molecules or sequences.

[0104] Many different transgenes or genetic modifications can potentially be introduced in accordance with this disclosure, thereby providing beneficial agrochemical traits in crop plants having the transgene or modification. In certain embodiments, the genetic modification or genetic modification introduced into the genome of a plant cell may include 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 its fragments, including SEQ ID NOs: 1-36, or any range that can be derived between them. In some embodiments, the transgene or genetic modification introduced into the genome of a plant cell may confer resistance to the herbicide icaforin.

[0105] Additional non-limiting examples of specific transgenes or genetic modifications for introduction into cells and their corresponding phenotypes include one or more genes for insect resistance, e.g., the Bacillus thuringiensis (Bt) gene; pest resistance, e.g., genes for controlling fungal diseases; herbicide resistance, e.g., genes conferring glyphosate resistance; and quality improvement, e.g., genes for yield, vegetative enhancement, environmental tolerance or stress tolerance, or any desired change in plant physiological function, growth, development, morphology or plant products. For example, structural genes may include, but are not limited to, any genes conferring insect resistance, including Bacillus insect regulatory protein genes described in WO99 / 31248 (in whole, incorporated herein by reference), U.S. Patent No. 5,689,052 (in whole, incorporated herein by reference), U.S. Patent No. 5,500,365 and U.S. Patent No. 5,880,275 (in whole, incorporated herein by reference). In some embodiments, the structural gene can confer resistance to the herbicide glyphosate, such as that conferred by a gene including the Agrobacterium strain CP4 glyphosate-resistant EPSPS gene (aroA:CP4) described in U.S. Patent No. 5,633,435 (the entirety of which is incorporated herein by reference), or the glyphosate oxidoreductase gene (GOX) described in U.S. Patent No. 5,463,175 (the entirety of which is incorporated herein by reference).

[0106] Various assays are known and can be used in the art to confirm the presence of genetic modifications, exogenous DNA sequences, genetic markers, expression cassettes, or transgenes in transformed, edited, or genetically modified cells, plants, or plant parts. Methods and techniques are provided for screening and / or identifying cells or plants, etc., for the presence of targeted edits or transgenes, and for selecting cells or plants containing targeted edits or transgenes, which may be based on the presence or absence of one or more phenotypes or traits, or molecular markers or polynucleotide or protein sequences in the cells or plants. As used herein, “molecular techniques” means any method known in the fields of molecular biology, biochemistry, genetics, plant biology, or biophysics that involves the use, manipulation, or analysis of nucleic acids, proteins, or lipids. Molecular techniques useful for detecting the presence of modified sequences in a genome include, but are not limited to, phenotypic screening; molecular marker techniques, e.g., SNP analysis by TaqMan® or Illumina / Infinium techniques; Southern blot hybridization; PCR; enzyme-linked immunosorbent assay (ELISA); and sequencing (e.g., Sanger, Illumina®, 454, Pac-Bio, Ion Torrent®). In one embodiment, the detection method provided herein includes phenotypic screening. In another embodiment, the detection method provided herein includes SNP analysis. In a further embodiment, the detection method provided herein includes Southern blot hybridization. In a further embodiment, the detection method provided herein includes PCR. In one embodiment, the detection method provided herein includes ELISA. In a further embodiment, the detection method provided herein includes sequencing of nucleic acids or proteins. Nucleic acids can be detected using hybridization, but are not limited to these methods.Nucleic acid hybridization 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).

[0107] Nucleic acid or polynucleotide molecules can be isolated using techniques conventional in the art. For example, nucleic acids can be isolated using any method, including, but not limited to, recombinant nucleic acid techniques and / or PCR. Common 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, for example, include restriction enzyme digestion and ligation, which can be used to isolate nucleic acids. Isolated nucleic acids can also be chemically synthesized as either a single nucleic acid molecule or a series of oligonucleotides.

[0108] Detection (e.g., of amplification products, hybridization complexes, or polypeptides) can be achieved using detectable labels that can adhere to or associate with hybridization probes or antibodies. The term “label” is intended to encompass the use of direct and indirect labeling. Detectable labels include enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Screening and selection of modified (e.g., edited) plants or plant cells can be carried out by any methodology known to those skilled in the field of molecular biology. Examples of screening and selection methodologies include, but are not limited to, Southern spectroscopy, PCR amplification for polynucleotide detection, Northern blot hybridization, RNase protection, primer extension, RT-PCR amplification for RNA transcript detection, Sanger sequencing, next-generation sequencing technologies (e.g., Illumina®, PacBio®, Ion Torrent®, etc.), enzyme assays for detecting the enzymatic or ribozyme activity of polypeptides and polynucleotides, as well as protein gel electrophoresis, Western blot analysis, immunoprecipitation, and enzyme-linked immunoassays for detecting polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining may also be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing all of the referenced techniques are publicly known in the art.

[0109] As used herein, the term “polypeptide” refers to a chain of at least two covalently bonded 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., biological samples) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. Polypeptides can also be purified, for example, by expressing nucleic acids in an expression vector. Furthermore, purified polypeptides can be obtained by chemical synthesis. The degree of purity of polypeptides can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0110] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. The antibodies provided herein may be polyclonal or monoclonal antibodies. Antibodies having a specific binding affinity to the polypeptides provided herein can be prepared using methods well known in the art. The antibodies provided herein can be conjugated to a solid support, such as a microtiter plate, using methods known in the art.

[0111] D. Genetically modified plants and herbicide-resistant plants In one embodiment, the Disclosure includes genetically modified plant cells, plant tissues, plants, and seeds comprising recombinant DNA molecules or engineered proteins provided in the Disclosure. These cells, tissues, plants, and seeds exhibit herbicide resistance to at least one icaforin herbicide in certain embodiments. In one embodiment, these cells, tissues, plants, and seeds may optionally exhibit resistance to at least one additional herbicide.

[0112] Icaforin is a novel post-emergence isoxazole herbicide that acts on dicotyledonous (broadleaf) and monocotyledonous (narrowleaf / grass) herbicides. Non-limiting examples of Icaforin 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 Examples include carboxylates; (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 any combination thereof. In certain embodiments, the cells, seeds, plants, and plant parts provided by this disclosure exhibit herbicide resistance to at least one icaforin herbicide.

[0113] Icaforin may be sprayed onto plant growing areas containing the plants and seeds provided by this disclosure as a method for controlling weeds. Alternatively, or in addition, Icaforin may be applied to plant growing areas before sowing the seeds provided by this disclosure. In many embodiments, the plants and seeds provided by the present invention contain herbicide-resistant traits and are therefore resistant to the application of at least one Icaforin herbicide. The herbicide application rate of Icaforin herbicides may vary depending on external conditions, including but not limited to temperature, humidity, and the specific Icaforin herbicide used. In certain embodiments, the spraying of Icaforin herbicides may be at the recommended commercial rate (1×) or any fraction or multiple thereof, for example, half the recommended commercial rate (0.5X) or twice the recommended commercial rate (2×). The herbicide concentration may be expressed as pound-based acid equivalents per acre (lb ae / acre) or gram-based acid equivalents per hectare (g ae / ha), or as pound-based active ingredient per acre (lb ai / acre) or gram-based active ingredient per hectare (g ai / ha), depending on the herbicide and formulation. In some embodiments, the application concentration of the icaforin herbicide may include about 0.001 kg / ha to about 1.0 kg / ha or more of active ingredient, about 0.005 kg / ha to about 750 kg / ha of active ingredient, or about 5 g / ha to about 250 g / ha of active ingredient. A 1x label concentration for the icaforin herbicide corresponds to about 5 g / ha to about 250 g / ha of active ingredient. When using a mixture of (2R,4R)-isomers and (2S,4S)-isomers, the isomers may be present in the mixture in a ratio of about 1.2:0.8 to about 0.8:1.2, preferably about 1.1:0.9 to about 0.9:1:1 (for example, the isomers may be present in a ratio of about 1:1). The plant growing area may or may not contain weed plants at the time of herbicide application. The herbicidally effective dose of icaforin herbicide for use in a certain area to control weeds may range from about 0.1X to about 30X of the label rate over the growing season. One acre is equivalent to 2.47105 hectares, and one pound is equivalent to 453.592 grams.Herbicide rates can be converted between the British system and the metric system as follows: (lb ai / ac) × 1.12 = (kg ai / ha) and (kg ai / ha) × 0.89 = (lb ai / ac).

[0114] The application of one or more herbicides may be carried out sequentially, or a combination of one or more herbicides may be mixed in a tank. To control a wide area of ​​dicotyledonous, monocotyledonous, or both, one or more herbicides may be applied multiple times in combination or individually to an area containing the transgenic plants of this disclosure. In some embodiments, two herbicides (such as pre-planting and post-germination application, or pre-germination and post-germination application) or three applications (such as pre-planting, pre-germination, and post-germination application, or pre-germination and two post-germination applications) may be used.

[0115] As used herein, “resistance,” “herbicide resistance,” or “herbicide resistance activity” refers to the ability of a plant, seed, or cell to exhibit resistance to the toxic effects of a herbicide when applied. Herbicide-resistant crops can continue to grow and are unaffected, or only slightly affected, by the presence of the applied chemical. As used herein, “herbicide resistance trait” refers to a transgenic trait that confers improved herbicide resistance to a plant, plant part, seed, or cell compared to a wild-type plant. Non-limiting examples of plants that can be produced using the herbicide-resistant traits of this disclosure include soybean plants, maize plants, cotton plants, barley plants, sorghum plants, rice plants, wheat plants, sugarcane plants, sugar beet plants, alfalfa plants, rapeseed plants, and vegetable plants and fruit plants (e.g., onions, tomatoes, peppers, cucumbers, peas, broccoli, cabbage, carrots, artichokes, lettuce, radishes, spinach, cauliflower, zucchini, leeks, potatoes, Brussels sprouts, tomatillos, beans, okra, apples, pears, cherries, peaches, apricots, plums, bananas, plantains, grapes, oranges, avocados, mangoes, berries, etc.).

[0116] In certain embodiments, the genetically modified plants, offspring, seeds, plant cells, and plant parts of the Disclosure may also contain one or more additional traits. Additional traits may be introduced by crossing a plant containing the recombinant DNA molecules provided by the Disclosure with another plant containing one or more additional traits. As used herein, “crossing” means breeding two individual plants to produce offspring plants. The two plants may be crossed to produce progeny containing the desired traits derived from each parent. As used herein, “progeny” means offspring of any generation of the parent plants, and the transgenic progeny contains polynucleotide molecules provided by the Disclosure and inherited from at least one parent plant. Additional traits may also be introduced by co-transforming the polynucleotide molecules for the additional transgenic trait(s) with polynucleotide molecules containing the recombinant polynucleotide molecules provided by the Disclosure, or by introducing the additional trait(s) into a transgenic plant containing the polynucleotide molecules of the Disclosure, or vice versa (for example, by using either plant transformation or genome editing methods). These additional traits include, but are not limited to, increased insect resistance, increased disease resistance (e.g., 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, and the traits herein are measured in terms of their characteristics compared to wild-type plants.Examples of additional herbicide resistance traits include, in particular, ACCase inhibitors (e.g., aryloxyphenoxypropionate and cyclohexanedione), ALS inhibitors (e.g., sulfonylurea, imidazolinone, triazolopyrimidine, and triazolinone), EPSPS inhibitors (e.g., glyphosate), synthetic auxins (e.g., phenoxybenzoic acid, carboxylic acid, semicarbazone), photosynthesis inhibitors (e.g., triazine, triazinon, nitrile, benzothiadiazole, and urea), and glutamine synthesis inhibitors (e.g., This may include genetically modified or non-genetically modified resistance to one or more herbicides, such as glufosinate, HPPD inhibitors (e.g., isoxazole, pyrazolone, and triketone), PPO inhibitors (e.g., diphenyl ether, N-phenylphthalimide, aryltriazinon, and pyrimidinedione), PDS inhibitors (e.g., amide, pyridiazinon, and pyridine), and long-chain fatty acid inhibitors (e.g., chloroacetamindes, oxyacetamide, and pyrazole). Examples of insect resistance traits may include resistance to one or more insect members from the orders Lepidoptera, Coleoptera, Hemiptera, and Homoptera. Such additional traits are well known to those skilled in the art, and a list of such transgenic traits is provided by the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS).

[0117] Cells transformed with the polynucleotide molecules of this disclosure may, in certain embodiments, be selected for the presence of the polynucleotide molecules or their encoded polypeptides before or after regenerating the cells into transgenic plants. Therefore, in some embodiments, transgenic plants containing such polynucleotides may be selected by identifying transgenic plants that contain polynucleotide molecules or encoded polypeptides and / or exhibit altered traits compared to other homogeneous control plants. Such traits may, for example, be resistance to the herbicide icaforin.

[0118] Transgenic plants and progeny containing transgenic light traits provided by the disclosure may be used by any propagation method known in the art. In plant lines containing two or more transgenic traits, the transgenic traits may be independently isolated, linked, or in plant lines containing three or more transgenic traits, a combination of both. Backcrossing to parent plants and outcrossing with non-transgenic plants are also intended, as are vegetative propagation. Descriptions of propagation methods used for different traits and crops are well known to those skilled in the art. Various assays may be performed to confirm the presence of a transgene(s) in a particular plant or seed. Such assays include, for example, molecular biological assays such as Southern blotting and Northern blotting, PCR, and DNA sequencing; biochemical assays such as detection of the presence of protein products by immunological means (ELISA and Western blotting) or by enzymatic function; plant part assays such as leaf or root assays; and analytical assays of the phenotype of the whole plant.

[0119] The gene transfer of a transgenic trait into a plant genotype is achieved as a result of a backcross conversion process. A plant genotype into which a transgenic trait has been transferred may be referred to as a backcross conversion genotype, strain, inbred strain, or hybrid strain. Similarly, a plant genotype lacking the desired transgenic trait may be referred to as an unconverted genotype, strain, inbred strain, or hybrid strain.

[0120] The term “approximately” is used to indicate that the device or method employed to determine the value includes the standard deviation of the mean. The use of the term “or” in the claims is used to mean “and / or” unless it is explicitly indicated that only the options are being referred to, or that the options are mutually exclusive. When used in the claims with the word “comprising” or other open wording, the words “a” and “an” mean “one or more” unless otherwise specified. The terms “comprise,” “comprising,” and “include” are open-ended linking verbs. Any one or more forms or tenses of these verbs, such as “comprises,” “comprising,” “have,” “have,” “includes,” and “including,” are also open-ended. For example, any method that “comprises,” “have,” or “includes” one or more steps is not limited to having only those one or more steps, but also includes other unlisted steps. Similarly, any system or method that “comprises,” “has,” or “includes” one or more components is not limited to having only those components, but also includes other components not enumerated. Where used herein, the term “essentially from” means, when used in reference to a nucleotide or amino acid sequence of this disclosure, that the nucleotide or amino acid sequence may include additional nucleotides or amino acids, provided that the additional nucleotides or amino acids do not substantially alter the function of the enumerated sequence. Where used in reference to a nucleotide or amino acid sequence of this disclosure, the term “substantially altered” means a reduction of at least 25% in the herbicide resistance of the encoded polypeptide.For example, an additional nucleotide or amino acid added to the nucleotide or amino acid sequence of the present disclosure may be considered to "substantially alter" the encoded polypeptide or polypeptide sequence if the herbicide resistance conferred by such addition is reduced by at least 25%.

[0121] Other purposes, features, and advantages of this disclosure are apparent from the detailed description provided herein. However, various changes and modifications within the spirit and scope of this disclosure will be apparent to those skilled in the art from the detailed description, so it should be understood that this detailed description and any particular examples provided are given only as examples, while illustrating specific embodiments of this disclosure. Any embodiment of this disclosure may be used in combination with any other embodiment described herein.

[0122] All references cited herein are incorporated in their entirety by reference. [Examples]

[0123] Example 1: Evaluation of Icaforin herbicide resistance in soybean plants expressing CYP protein. This example describes testing the ability of different cytochrome P450 genes to confer resistance to the herbicide icaforin to transgenic plants.

[0124] Using a transgenic plant-based screening approach, we identified enzymes capable of inactivating icaforin. One of the gene families was the cytochrome P450 (CYP) superfamily. Forty-one CYP proteins, including two CYP72A family members, CYP12 (SEQ ID NO: 1) and CYP13 (SEQ ID NO: 2), and two CYP81E family members, CYP14 (SEQ ID NO: 3) and CYP15 (SEQ ID NO: 4) (previously identified from a population of Palmer pigweed (Amaranthus palmeri)), were screened for resistance to icaforin. Gene expression profiling experiments in a population of water hemp (Amaranthus tuberculatus) resistant to the herbicide 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor and 2,4-D amine have previously shown that the CYP81E gene is significantly upregulated after 2,4-D treatment. See PCT Publication WO2022 / 051340. CYP14 and CYP15 proteins share 98.98% sequence identity and differ by 5 amino acids.

[0125] Prior to plant transformation, the Palmer pigweed nucleotide sequence encoding the CYP enzyme was codon-optimized for expression in dicotyledons. A variant of the CYP12 protein, CYP12_var, containing a serine amino acid inserted at position 2, was also generated and screened for resistance to the herbicide icaforin. Table 2 provides the sequence identifiers (SEQ ID NOs) corresponding to the tested proteins and nucleotide sequences. [Table 2]

[0126] Codon-optimized polynucleotide sequences were placed in plant transformation cassettes, and each CYP coding sequence was operably linked to a constitutive expression element containing promoter, reader, and intron sequences, including Arabidopsis thaliana Ubiquitin 3 (SEQ ID NO: 14) and Medicago truncatula photosystem II terminator (SEQ ID NO: 15). Four plant transformation vectors conferring resistance to the antibiotics spectinomycin and streptomycin were created, each containing one CYP gene expression cassette and an aadA selectable marker expression cassette. Embryoexplants derived from soybean variety AG3555 seeds were transformed with the vectors by Agrobacterium-mediated transformation using methods known in the art, and transformed plantlets were selected based on growth in the presence of spectinomycin. Plantlets containing single-copy events were grown in a greenhouse to produce R1 seeds. Subsequently, R1 plants were selected as homozygous CYP-positive plants by either molecular analysis of young leaf tissue or by seed chipping before sowing. Next, these selected plants were screened for resistance to the herbicide icaforin.

[0127] Herbicide resistance screening was performed by selecting R1 plants at approximately the V2-V3 development stage and applying the herbicide icaforin using a track sprayer under standard greenhouse conditions. Two independent transgenic events were screened for each transgene. The herbicide was formulated with 1% v / v methylated seed oil (MSO) and 2.5% v / v ammonium sulfate (AMS) and sprayed at 15 gallons / hectare (GPA) using an XR nozzle to mimic field conditions. The icaforin used in this example and the experiments described in Examples 2-4 was a mixture of methyl ester forms of 2R, 4R, and 2S, 4S isomers. The icaforin formulation was sprayed at two treatment rates: 5 g (ai) / hectare (ha) and 25 g ai / ha of the active ingredient. Soybean plants treated with the herbicide were returned to the greenhouse after application and grown under standard conditions. Plants were evaluated for herbicide damage approximately 14 and 21 days (DAT) after treatment. Untreated transgenic plants and control (wild-type) plants were used for phenotypic comparison. Herbicide damage evaluation considered plant deformation, inhibition, chlorosis, necrosis, and plant death compared to untreated control plants. A damage rate of approximately 90% or more indicates near-complete plant death. A damage rate of less than 30% indicates partial acceptance, and a damage rate of less than 20% indicates good acceptance. Damage evaluations were collected and analyzed, and representative plant photographs were taken. Damage rates are summarized in Table 3. [Table 3]

[0128] Treated control (wild-type) soybeans suffered severe damage at a treatment rate of 5 g ai / ha and were almost completely killed at a treatment rate of 25 g ai / ha. CYP14-expressing and CYP15-expressing transgenic plants showed unexpected resistance to icaforin. Herbicide application was repeated twice with two additional transgenic events generated from CYP14 and CYP15 transformations, respectively. As shown in Table 4, resistance to icaforin was reproduced in these events. Figure 1 shows photographs of representative control and CYP15-expressing transgenic plants after icaforin treatment. [Table 4]

[0129] At low treatment rates of 5g ai / ha, most CYP14 and CYP15 transgenic events showed less than 30% damage, and often less than 20% damage, while treated control (wild-type) plants tended to show around 50% damage at a treatment rate of 5g ai / ha. At higher treatment rates (25g ai / ha), plant damage increased correspondingly and showed greater variability, with some events showing a damage rate of approximately 30% and others showing a damage rate close to that of treated controls (approximately 90%). Transgenic plants expressing CYP12_var or CYP13 did not show significant resistance to icaforin, as the damage rates at both low and high treatment rates of 5g ai / ha and 25g ai / ha were similar to those observed in treated control plants (see Table 3). When combined with data from 37 other CYPs tested in a transgenic plant screening assay, these results suggest that only CYP14 and CYP15 can provide significant resistance to the herbicide icaforin.

[0130] Example 2: Evaluation of herbicide resistance in CYP15-expressing transgenic plants This example describes the results of evaluating the resistance of CYP15-expressing transgenic soybean plants to four different classes of herbicides.

[0131] A screening assay was designed to determine whether transgenic soybean plants expressing CYP15 exhibit increased resistance to herbicides other than icaforin. Transgenic soybean plants expressing CYP15 and control (wild-type) plants were treated with eight common herbicides representing four different herbicide families, as shown in Table 5. [Table 5]

[0132] CYP15-expressing plants were grown in a greenhouse alongside control plants and tested with the herbicides listed in Table 5 at growth stages V2-V3 using the same method as the Icaforin spray application described in Example 1. For complete and partial resistance assays, the herbicides were applied at full-field (1X) and half-field (0.5X) rates, respectively. Soybean plants treated with the herbicides were returned to the greenhouse after spray application and grown under standard conditions. Plants were evaluated for herbicide damage at approximately 14DAT and 21DAT after spray application. Untreated transgenic plants and control plants were used for phenotypic comparison. Herbicide damage evaluation considered plant deformation, inhibition, chlorosis, necrosis, and plant death compared to untreated control plants. A damage rate of approximately 90% or more indicates near-complete plant death. A damage rate of less than 30% indicates partial acceptance, and a damage rate of less than 20% indicates good acceptance. Damage assessments were collected and analyzed, and representative plant photographs were taken. Damage rates are summarized in Table 6. [Table 6]

[0133] The results indicate that herbicide damage ranged from approximately 30% to over 90% at 14DAT. No significant differences in damage assessment were observed in CYP15-expressing plants compared to control plants at 14DAT. At 21DAT, some variability was observed in plant regrowth between treatments, but both control and CYP15-expressing plants showed over 40% damage across all treatments, suggesting a lack of resistance. Taken together, these data suggest that CYP15 expression in soybean plants did not confer resistance to nicosulfuron, formusulfuron, tenbotrione, mesotrione, saflufenacil, sulfenthrazone, 2,4-D, or dicamba.

[0134] Example 3: Evaluation of Icaforin herbicide resistance in individuals expressing additional CYP proteins. This example describes testing the ability of additional cytochrome P450 genes to confer resistance to the herbicide icaforin to plants.

[0135] The presented plant genome database and internal plant genome databases (including the Amaranthus database) were screened to identify additional cytochrome P450 genes for testing for icaforin resistance. Twenty-one additional cytochrome P450 genes, including nine Amaranthus CYP genes, were selected and screened for icaforin resistance. Among these were CYP21 (SEQ ID NO: 16) supplied from Amaranthus palmeri and CYP28 (SEQ ID NO: 23) supplied from Amaranthus tuberculatus. At the amino acid level, CYP21 and CYP28 share 98.8% and 95.1% identity with CYP15, respectively.

[0136] The selected CYP enzymes were screened for icaforin resistance using the transgenic plant-based screening approach described in Example 1. For plant transformation, the protein sequences encoding the CYP enzymes were codon-optimized for dicotyledonous expression. Table 7 provides sequence identifiers (SEQ ID NOs) corresponding to the protein and nucleotide sequences. [Table 7]

[0137] Codon-optimized genes were placed in plant transformation cassettes and operably linked at each CYP coding sequence to constitutive expression elements including promoters, leaders, and introns of Arabidopsis thaliana Ubiquitin 3 (SEQ ID NO: 14) and Medicago truncatula photosystem II terminator (SEQ ID NO: 15). Two plant transformation vectors conferring resistance to the antibiotics spectinomycin and streptomycin were created, each containing one CYP gene cassette and an aadA selectable marker expression cassette. Embryoexplants derived from soybean variety AG3555 seeds were transformed with the vectors by Agrobacterium-mediated transformation using methods known in the art, and positively transformed plantlets were selected based on growth in the presence of spectinomycin. Plantlets containing single-copy events were grown in a greenhouse to produce R1 seeds. Subsequently, R1 plants were selected as homozygous CYP-positive plants by either molecular analysis of young leaf tissue or by seed chipping before sowing. Next, these selected plants were screened for resistance to the herbicide icaforin.

[0138] Herbicide resistance screening was performed by selecting R1 plants at approximately the V2-V3 development stage and applying the herbicide icaforin using a track sprayer under standard greenhouse conditions. Two independent transgenic events were selected for screening for each transgene. The herbicide was formulated with 1% v / v methylated seed oil (MSO) and 2.5% v / v ammonium sulfate (AMS) and sprayed at 15 gallons / hectare (GPA) using an XR nozzle to mimic field conditions. The icaforin formulation was sprayed at two treatment rates: 5 g (ai) / hectare (ha) and 25 g ai / ha of the active ingredient. Soybean plants treated with the herbicide were returned to the greenhouse after spray application and grown under standard conditions. Plants were evaluated for herbicide damage approximately 14 and 21 days (DAT) post-treatment. Untreated transgenic plants and control (wild-type) plants were used for phenotypic comparison. Herbicide damage assessment considers plant deformation, inhibition, chlorosis, necrosis, and plant death compared to untreated control plants. A damage rate of approximately 90% or more indicates near-complete plant death. A damage rate of less than 30% indicates partial acceptance, and a damage rate of less than 20% indicates good acceptance. Damage assessments were collected and analyzed, and representative plant photographs were taken. Damage rates are summarized in Table 8. [Table 8]

[0139] In 14 DATs, at lower treatment rates of 5g ai / ha, CYP21 transgenic events showed less than 10% damage, and CYP28 transgenic events showed less than 30% damage. Treated control (wild-type) plants tended to suffer approximately 45% damage at a treatment rate of 5g ai / ha. As the treatment rate (25g ai / ha) increased, plant damage increased correspondingly, and variability increased. Control plants showed approximately 78% damage. One CYP21 event showed approximately 50% damage, while another event showed a damage rate close to that of the treated control, with approximately 72% damage. CYP28 events tended to show a damage rate of around 67.5%. The same effect was evident in 21 DAT evaluations. Overall, the data indicate that CYP21 provides good resistance to icaforin, and CYP28 provides partial resistance to icaforin at lower application rates. None of the other 19 CYP proteins tested showed resistance to Icaforin.

[0140] Example 4: Evaluation of Icaforin herbicide resistance in individuals expressing additional CYP proteins. This example describes testing the ability of the cytochrome P450 gene CYP34 to confer resistance to the herbicide icaforin to plants.

[0141] CYP34 (SEQ ID NO: 26) was supplied from Amaranthus cruentus. At the amino acid level, CYP34 shares 96.1% identity with CYP15.

[0142] CYP34 was screened for icaforin resistance using the transgenic plant-based screening approach described in Example 1. For plant transformation, the protein sequence encoding the CYP34 enzyme was codon-optimized for dicotyledonous expression. Table 9 provides sequence identifiers (SEQ ID NOs) corresponding to the protein and nucleotide sequences. [Table 9]

[0143] Codon-optimized genes were placed in plant transformation cassettes, and the CYP34 coding sequence was operably linked to constitutive expression elements including promoters, leaders, and introns of Arabidopsis thaliana Ubiquitin 3 (SEQ ID NO: 14) and Medicago truncatula photosystem II terminator (SEQ ID NO: 15). Plant transformation vectors conferring resistance to the antibiotics spectinomycin and streptomycin were created, each containing a CYP34 gene cassette and an aadA selectable marker expression cassette. Embryoexplants derived from soybean variety AG3555 seeds were transformed with the vectors by Agrobacterium-mediated transformation using methods known in the art, and positively transformed plantlets were selected based on growth in the presence of spectinomycin. Plantlets containing single-copy events were grown in a greenhouse to produce R1 seeds. Subsequently, R1 plants were selected as homozygous CYP-positive plants by either molecular analysis of young leaf tissue or by seed chipping before sowing. Next, these selected plants were screened for resistance to the herbicide icaforin.

[0144] Herbicide resistance screening was performed by selecting R1 plants at approximately the V2-V3 development stage and applying the herbicide icaforin using a track sprayer under standard greenhouse conditions. Two independent transgenic events were selected for screening for each transgene. The herbicide was formulated with 1% v / v methylated seed oil (MSO) and 2.5% v / v ammonium sulfate (AMS) and sprayed at 15 gallons / hectare (GPA) using an XR nozzle to mimic field conditions. The icaforin formulation was sprayed at two treatment rates: 5 g (ai) / hectare (ha) and 25 g ai / ha of the active ingredient. Soybean plants treated with the herbicide were returned to the greenhouse after spray application and grown under standard conditions. Plants were evaluated for herbicide damage approximately 13 and 21 days (DAT) post-treatment. Untreated transgenic plants and control (wild-type) plants were used for phenotypic comparison. Herbicide damage assessment considers plant deformation, inhibition, chlorosis, necrosis, and plant death compared to untreated control plants. A damage rate of approximately 90% or more indicates near-complete plant death. A damage rate of less than 30% indicates partial acceptance, and a damage rate of less than 20% indicates good acceptance. Damage assessments were collected and analyzed, and representative plant photographs were taken. Damage rates are summarized in Table 10. [Table 10]

[0145] In 13 DATs, at lower treatment rates of 5g ai / ha, the CYP34 transgenic event showed less than 15% damage. Treated control (wild-type) plants tended to suffer approximately 42% damage at a treatment rate of 5g ai / ha. At higher treatment rates (25g ai / ha), control plants showed approximately 70% damage, while the CYP34 event showed less than 50% damage. Similar trends were observed in 21 DAT assessments, with the CYP34 transgenic event showing less damage compared to control plants. Overall, the data indicate that CYP34 provides good resistance to icaforin.

[0146] Example 5: Field trials of selected soybean plants expressing CYP14 and CYP15. Soybean transgenic plants expressing CYP14 or CYP15 were assayed for resistance to icaforin spray in small field trials at nine facilities in the United States. Plants were grown in the field and sprayed at the early vegetative stage, approximately V3. The icaforin formulation was applied in two treatment repeats per site at either a concentration of 5 g ai / ha or 25 g ai / ha. Plants were evaluated for herbicide damage approximately 7, 14, 21, and 28 days (DAT) post-treatment. A total of 12 events were tested for CYP14 and 11 events for CYP15. Events in non-herbicide-resistant constructs served as negative controls to provide a comparison of damage.

[0147] Damage data from 21DAT are presented in Table 11. Similar to what was observed in the greenhouse experiment, both CYP14 and CYP15 provided visual tolerance to the application of icaforin at a treatment rate of 5 g ai / ha, with damage assessments of less than 15%. At higher application rates of 25 g ai / ha, plants showed less damage than the control, but suffered significant damage typical of icaforin damage, characterized by inhibition, abnormal leaf formation, and cessation of development. This indicates that expression of both CYP14 and CYP15 using the expression cassette described in Example 1 provides soybeans with tolerance to low application rates of icaforin under both greenhouse and field growing conditions. [Table 11]

Claims

1. A recombinant DNA molecule comprising a heterologous promoter functionally linked to a polynucleotide sequence encoding a polypeptide having at least about 94% sequence identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, wherein the heterologous promoter is functional in plant cells.

2. The recombinant DNA molecule according to claim 1, wherein the polypeptide confers resistance to the herbicide icaforin.

3. The recombinant DNA molecule according to claim 1 or 2, wherein the polynucleotide sequence comprises a nucleic acid sequence having at least about 95% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, or a nucleic acid sequence having at least about 85% sequence identity with SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO:

36.

4. The recombinant DNA molecule according to any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:

28.

5. The recombinant DNA molecule according to any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:

28.

6. The recombinant DNA molecule according to any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:

28.

7. The recombinant DNA molecule according to any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:

28.

8. The recombinant DNA molecule according to any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:

28.

9. The recombinant DNA molecule according to any one of claims 1 to 3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:

28.

10. The recombinant DNA molecule according to any one of claims 3 to 9, wherein the polynucleotide sequence includes a nucleic acid sequence having at least about 96% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:

31.

11. The recombinant DNA molecule according to any one of claims 3 to 9, wherein the polynucleotide sequence includes a nucleic acid sequence having at least about 97% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:

31.

12. The recombinant DNA molecule according to any one of claims 3 to 9, wherein the polynucleotide sequence includes a nucleic acid sequence having at least about 98% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:

31.

13. The recombinant DNA molecule according to any one of claims 3 to 9, wherein the polynucleotide sequence includes a nucleic acid sequence having at least about 99% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:

31.

14. A recombinant DNA molecule according to any one of claims 3 to 9, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO:

36.

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

16. The aforementioned icaforin herbicide is a recombinant DNA molecule selected from the group consisting of the following, according to any one of claims 2 to 15: 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 any combination thereof.

17. A DNA construct comprising a recombinant DNA molecule according to any one of claims 1 to 16.

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

19. A transgenic plant, seed, cell, or plant part comprising a recombinant DNA molecule, the recombinant DNA molecule comprising a heterologous promoter functionally linked to a polynucleotide sequence encoding a polypeptide having at least about 85% sequence identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:

28.

20. The transgenic plant, seed, cell, or plant part according to claim 19, wherein the transgenic plant, seed, cell, or plant part is resistant to the icaforin herbicide.

21. The transgenic plant, seed, cell, or plant part according to claim 19 or 20, wherein the transgenic plant, seed, cell, or plant part is a soybean, corn, cotton, barley, sorghum, rice, wheat, sugarcane, sugar beet, alfalfa, or a plant, seed, cell, or plant part of a Brassicaceae family.

22. The transgenic plant, seed, cell, or plant part according to claim 20 or 21, wherein the icaforin herbicide is selected from the group consisting of the following: 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 any combination thereof.

23. The transgenic plant, seed, cell, or plant part according to any one of claims 20 to 22, wherein the transgenic plant, seed, cell, or plant part is resistant to at least one additional herbicide.

24. The method described above is a method for conferring herbicide resistance to a plant, seed, cell, or plant part, comprising expressing a recombinant DNA molecule according to any one of claims 1 to 18 in the plant, seed, cell, or plant part.

25. The method according to claim 24, wherein the plant, seed, cell, or plant part is resistant to the icaforin herbicide.

26. The method according to claim 25, wherein the icaforin herbicide is selected from the group consisting of the following. 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 any combination thereof.

27. A method for producing transgenic plants or parts of those plants, a) The recombinant DNA molecule is a recombinant DNA molecule that is introduced into plant cells, the recombinant DNA molecule comprising a heterologous promoter functionally linked to a polynucleotide sequence encoding a polypeptide having at least about 85% sequence identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, and b) The method comprising regenerating the transgenic plant or a portion thereof from the cell or its progeny cell containing the recombinant DNA molecule.

28. The method according to claim 27, further comprising selectivity for regenerated transgenic plants having resistance to the icaforin herbicide.

29. A method according to claim 28, comprising a icaforin herbicide selected from the group consisting of the following: 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 any combination thereof.

30. The method according to any one of claims 27 to 29, further comprising crossing the regenerated transgenic plant with itself or with a second plant to produce seeds.

31. The method according to any one of claims 27 to 30, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:

28.

32. The method according to any one of claims 27 to 31, wherein the polynucleotide sequence includes a nucleic acid sequence having at least about 95% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, or a nucleic acid sequence having at least about 85% sequence identity with SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO:

36.

33. The method according to any one of claims 27 to 32, comprising crossing the regenerated transgenic plant, or its offspring plant containing the recombinant DNA molecule, with itself or a second plant to produce seeds; wherein the seeds contain the recombinant DNA molecule.

34. A transgenic plant or a part thereof produced by the method according to any one of claims 27 to 33, wherein the transgenic plant or part thereof contains the recombinant DNA molecule.

35. Seeds produced by the method of claim 33.

36. The method comprises bringing the plant growing area into contact with the ikaforin herbicide, wherein the transgenic plant or seed is resistant to the ikaforin herbicide, and weeds are controlled in the plant growing area, as described in any one of claims 19 to 23.

37. The method according to claim 36, wherein the icaforin herbicide is selected from the group consisting of the following: 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 any combination thereof.

38. The method according to claim 36 or 37, wherein the transgenic plant or seed is soybean, corn, cotton, barley, sorghum, rice, wheat, sugarcane, sugar beet, alfalfa, or a plant or seed of the Brassicaceae family.

39. A method for identifying plants resistant to the herbicide icaforin and at least one additional herbicide, a) To obtain the plant described in any one of claims 20 to 23; b) Applying the at least one additional herbicide to the plant or a portion thereof; and c) The method comprising identifying the plant as being resistant to the at least one additional herbicide.

40. A method for suppressing the emergence of herbicide-resistant weeds in a plant growing area comprising a transgenic plant or seed according to any one of claims 20 to 23, wherein the method comprises contacting the plant growing area with the ikaforin herbicide and at least one additional herbicide, and the transgenic plant or seed is resistant to the ikaforin herbicide and at least one additional herbicide.

41. The method according to claim 40, wherein the icaforin herbicide is selected from the group consisting of the following: 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 any combination thereof.

42. The method according to claim 40 or 41, wherein the at least one additional herbicide is selected from the group consisting of ACCase inhibitors, ALS inhibitors, EPSPS inhibitors, synthetic auxins, photosynthesis inhibitors, glutamine synthesis inhibitors, HPPD inhibitors, PPO inhibitors, PDS inhibitors, and long-chain fatty acid inhibitors.

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