WO2024020316A2 - Procédés et compositions pour la tolérance aux herbicides ppo - Google Patents

Procédés et compositions pour la tolérance aux herbicides ppo Download PDF

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WO2024020316A2
WO2024020316A2 PCT/US2023/070191 US2023070191W WO2024020316A2 WO 2024020316 A2 WO2024020316 A2 WO 2024020316A2 US 2023070191 W US2023070191 W US 2023070191W WO 2024020316 A2 WO2024020316 A2 WO 2024020316A2
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plant
methyl
protein
inhibitor
herbicide
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PCT/US2023/070191
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WO2024020316A3 (fr
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Udo Bickers
Jens Frackenpohl
Hendrick Helmke
Anna Maria REINGRUBER
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Monsanto Technology Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • the present disclosure relates to the fields of agriculture, plant biotechnology, and molecular biology. More specifically, the disclosure relates to the use of certain substituted phenyl uracils, such as those comprising a cyclopropylcarboxylic acid-based side chain, or an agrochemically acceptable salt thereof, for controlling or preventing weed growth in plant growth areas of transgenic crop plants that are tolerant to protoporphyrinogen oxidase (PPO) inhibiting herbicides.
  • PPO protoporphyrinogen oxidase
  • herbicides are often used to control the growth and spread of weeds or other plants that are unwanted in a particular environment. These chemicals are active at one or more target sites within a plant where they interrupt normal plant functions. Herbicides vary in their modes of action, in their effects on weeds and crop plants, and how they are used. While herbicides are very effective in controlling growth of undesirable vegetation, their use may also cause incidental damage to desired plants located in the same vicinity, such as crop plants. In order to minimize crop damage, extensive research has been directed toward the development of herbicide tolerant plants, especially through use of transgenic traits. Examples of transgenic herbicide tolerance traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance.
  • Herbicides of particular interest include herbicides that inhibit protoporphyrinogen oxidase (PPO, EC 1.3.3.4), referred to as PPO inhibitor herbicides.
  • PPO inhibitor herbicides provide control of a spectrum of herbicide-resistant weeds, thus making a trait conferring tolerance to these herbicides particularly useful in a cropping system combined with one or more other herbicide-tolerance trait(s).
  • a method for controlling or preventing weed growth in a plant growth area comprises the steps of: (a) providing in said plant growth area a plant or a seed that when grown produces said plant, wherein the plant comprises a recombinant DNA molecule comprising a DNA sequence encoding a heterologous HemG protein, wherein said protein confers tolerance in said plant to an herbicidally active compound corresponding to a compound selected from the group consisting of Al, A2, and A3, or an agrochemically acceptable salt thereof, wherein:
  • Al corresponds to:
  • A2 corresponds to:
  • A3 corresponds to:
  • the heterologous HemG protein has herbicide-insensitive protoporphyrinogen oxidase activity.
  • the heterologous HemG protein has at least 85% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NOs:l-20 and 65-193.
  • the DNA sequence encoding the heterologous HemG protein is selected from the group consisting of SEQ ID NOs:22-64 and 194-322.
  • the heterologous HemG protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:l-20 and 65-193.
  • the DNA sequence encoding a heterologous HemG protein is operably linked to a DNA sequence encoding a chloroplast transit peptide (CTP).
  • CTP comprises an amino acid sequence with at least 97% sequence identity to a sequence selected from the group consisting of SEQ ID NOs:323-328, 340, and 342-407.
  • the DNA sequence encoding the CTP comprises at least 97% identity to a sequence selected from the group consisting of SEQ ID NOs:329-339, 341, and 408-483.
  • said recombinant DNA molecule further comprises a heterologous promoter operably linked to the DNA sequence encoding said HemG protein.
  • the plant comprising the recombinant DNA molecule is a monocotyledonous plant. In other embodiments, the plant comprising the recombinant DNA molecule is a dicotyledonous plant.
  • the herbicidally active compound is applied to the area at a rate of about 0.02 g a.i./ha to about 750 g a.i./ha, about 0.05 g a.i./ha to about 400 g a.i./ha, about 0.25 g a.i./ha to about 300 g a.i./ha, about 0.3 g a.i./ha to about 250 g a.i./ha, about 0.4 g a.i./ha to about 150 g a.i./ha, or about 0.5 g a.i./ha to about 120 g a.i./ha.
  • the method is further defined as comprising applying said compound to said area at least twice.
  • the herbicidally active compound is applied in an amount that does not damage said plant comprising the recombinant DNA molecule.
  • said applying of the compound is carried out pre-emergence.
  • said applying of the compound is carried out post-emergence.
  • said applying of the compound comprises contacting said plant with the compound.
  • said applying of the compound comprises an over the top application of said compound.
  • said applying of the compound results in an increase in the growth or yield of said plant relative to a plant of the same genotype cultivated in a growth area in which said compound has not been applied.
  • the method further comprises applying to said area an effective amount of at least a second herbicide.
  • the second herbicide is selected from the group consisting of: an ACCase inhibitor, an ALS inhibitor, an EPSPS inhibitor, a synthetic auxin, a photosynthesis inhibitor, a glutamine synthesis inhibitor, a HPPD inhibitor, a PPO inhibitor, and a long-chain fatty acid inhibitor.
  • the ACCase inhibitor is an aryloxyphenoxy propionate or a cyclohexanedione;
  • the ALS inhibitor is a sulfonylurea, imidazolinone, triazoloyrimidine, or a triazolinone;
  • the EPSPS inhibitor is glyphosate;
  • the synthetic auxin is a phenoxy herbicide, a benzoic acid, a carboxylic acid, or a semicarbazone;
  • the photosynthesis inhibitor is a triazine, a triazinone, a nitrile, a benzothiadiazole, or a urea;
  • the glutamine synthesis inhibitor is glufosinate;
  • the HPPD inhibitor is an isoxazole, a pyrazolone, or a triketone;
  • the PPO inhibitor is a diphenylether, a N- phenylphthalimide, an aryl triazinone, or a
  • SEQ ID NO:1 is the amino acid sequence of the HemG PPO H_N90.
  • SEQ ID NO:2 is the amino acid sequence of the HemG PPO H_N20.
  • SEQ ID NO:3 is the amino acid sequence of the HemG PPO H_N60.
  • SEQ ID NO:4 is the amino acid sequence of H_N10, which is the E. coli wild-type
  • HemG protoporphyrinogen oxidase NCBI GenBank Accession No. WP_021498199.
  • SEQ ID NO:5 is the amino acid sequence of the HemG PPO H_N30.
  • SEQ ID NO:6 is the amino acid sequence of the HemG PPO H_N40.
  • SEQ ID NO:7 is the amino acid sequence of the HemG PPO H_N50.
  • SEQ ID NO:8 is the amino acid sequence of the HemG PPO H_N70.
  • SEQ ID NO:9 is the amino acid sequence of the HemG PPO H_N100.
  • SEQ ID NO: 10 is the amino acid sequence of the HemG PPO H_N 110.
  • SEQ ID NOs: 11-17 are amino acid sequences lacking the start methionine and corresponding to SEQ ID NOs:l, 2, 4, 5, 6, 7, and 9, respectively.
  • SEQ ID Nos: 18-19 are amino acid sequences of two variants of SEQ ID NO: 11.
  • SEQ ID NO:20 is the amino acid sequence of a variant of SEQ ID NO: 17.
  • SEQ ID NO:21 is the amino acid sequence of the wild-type PPO from Amaranthus tuberculatus (waterhemp).
  • SEQ ID NOs:22-31 are nucleotide sequences encoding SEQ ID NOs:l-10, respectively, codon optimized for E. coli expression.
  • SEQ ID NOs:32-41 are nucleotide sequences encoding SEQ ID NOs: 1-10, respectively, codon optimized for dicot expression.
  • SEQ ID NOs:42-48 are nucleotide sequences encoding SEQ ID NOs: 11-17, respectively, codon optimized for dicot expression.
  • SEQ ID NOs:49-50 are recombinant nucleotide sequences encoding SEQ ID NO: 11.
  • SEQ ID NO:51 is a recombinant nucleotide sequence encoding SEQ ID NO: 12.
  • SEQ ID NOs:52-54 are recombinant nucleotide sequences encoding SEQ ID NOs: 18-
  • SEQ ID NOs:55-64 are nucleotide sequences encoding SEQ ID NOs:l-10, respectively, codon optimized for monocot expression.
  • SEQ ID Nos:65-77 are amino acid sequences of HemG PPOs from different species with variations in the long chain insert loop.
  • SEQ ID NOs:78-193 are amino acid sequences of recombinant HemG PPG variants, each incorporating a mutation to the long chain insert loop.
  • SEQ ID NOs: 194-206 are nucleotide sequences encoding the HemG PPOs of SEQ ID NOs: 194-206.
  • SEQ ID NOs:207-322 are nucleotide sequences encoding the recombinant HemG PPO variants of SEQ ID NOs:78-193.
  • SEQ ID NO:323 is the amino acid sequence of the Arabidopsis thaliana albino and pale green (APG6) chloroplast transit peptide (CTP).
  • SEQ ID NO:324 is the amino acid sequence of an amino-terminal optimized variant of the APG6 CTP.
  • SEQ ID NO:325 is the amino acid sequence of the Arabidopsis thaliana 90 kDa heat shock protein (CR88) CTP.
  • SEQ ID NO:326 is the amino acid sequence of the petunia ShkG-EPSPS CTP.
  • SEQ ID NO:327 is the amino acid sequence of the pea rbcS-3C CTP.
  • SEQ ID NO:328 is the amino acid sequence of the rice Waxy CTP.
  • SEQ ID NOs:329-333 are the nucleotide sequences encoding APG6 CTP of SEQ ID NOs:329-333 are the nucleotide sequences encoding APG6 CTP of SEQ ID NOs:329-333.
  • SEQ ID NO:334 is the nucleotide sequence encoding APG6 CTP of SEQ ID NO:324.
  • SEQ ID NOs:335-336 are nucleotide sequences encoding AtCR88 CTP optimized for dicot and monocot expression, respectively.
  • SEQ ID NOs:337-339 are nucleotide sequences encoding SEQ ID NOs:326-328.
  • SEQ ID NO:340 is the amino acid sequence of the cotton 12G088600TP CTP.
  • SEQ ID NO:341 is the nucleotide sequence encoding the cotton 12G088600TP CTP, optimized for dicot expression.
  • SEQ ID NOs: 342-407 are amino acid sequences of transit peptides from different species.
  • SEQ ID NOs:408-483 are nucleotide sequences encoding SEQ ID NOs:342-407.
  • the present disclosure provides for the use of certain substituted phenyl uracils, specifically those comprising a cyclopropylcarboxylic acid-based side chain or an agrochemically acceptable salt thereof, in controlling or preventing weed growth in plant growth areas of transgenic crop plants that express herbicide insensitive PPOs (protoporphyrinogen oxidases), and that are therefore tolerant to PPO inhibiting herbicides.
  • PPO is an essential enzyme in plants that catalyzes the dehydrogenation of protoporphyrinogen IX to form protoporphyrin IX, which is the precursor to heme and chlorophyll.
  • PPO inhibition in plant cells causes accumulation of intermediate tetrapyrroles and the formation of reactive oxygen species, resulting in membrane disruption and ultimately cell death.
  • PPO inhibitors such as diphenyl ethers, aryl triazolinones, pyrimidinediones, and N- phenylphthalimides.
  • Certain substituted phenyl uracils specifically those comprising a cyclopropylcarboxylic acid-based side chain, have been identified as having herbicidal activity and can be used for controlling monocotyledonous and dicotyledonous weeds. These compounds are effective against a broad spectrum of harmful plants when applied both preemergence and postemergence, with the possibility of non-selective use for control of unwanted plant growth or selective use in plant crops.
  • the present application shows that certain substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, or agrochemically acceptable salts thereof, can be applied to transgenic crop plants that comprise one or more genes conferring tolerance to PPO inhibitor herbicides.
  • the herbicide tolerance trait described herein provides tolerance to one of more of the herbicidally active compounds described herein or agrochemically acceptable salts thereof.
  • phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain or agrochemically acceptable salts thereof for controlling or preventing weed growth in plant growth areas of transgenic crop plants that are tolerant to PPO inhibiting herbicides wherein the plants comprise a recombinant DNA molecule comprising a DNA sequence encoding a heterologous HemG protein that has herbicide-insensitive PPO activity.
  • the specific substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain are referenced herein as compounds Al, A2, and A3 and are further characterized as shown in Table 1 below.
  • a “herbicide” is any molecule that is used to control, prevent, or interfere with the growth of one or more plants.
  • Illustrative herbicides include acetyl-CoA carboxylase (ACCase) inhibitors (for example, aryloxyphenoxy propionates and cyclohexanediones); acetolactate synthase (ALS) inhibitors (for example, sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones); 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors (for example, glyphosate), synthetic auxins (for example, phenoxys, benzoic acids, carboxylic acids, and semicarbazones), photosynthesis (photosystem II) inhibitors (for example, triazines, triazinones,
  • ACCase acetyl-CoA carboxylase
  • ALS aceto
  • PPO inhibiting herbicides are known in the art and commercially available.
  • PPO inhibiting herbicides include, but are not limited to, diphenylethers (such as acifluorfen, its salts and esters, bifenox, its salts and esters, ethoxyfen, its salts and esters, fluoronitrofen, furyloxyfen, halosafen, chlomethoxyfen, fluoroglycofen, its salts and esters, lactofen, its salts and esters, oxyfluorfen, and fomesafen, its salts and esters); thiadiazoles (such as fluthiacet-methyl and thidiazimin); pyrimidinediones or phenyluracils (such as benzfendizone, butafenacil, ethyl [3-2-chloro-4-fluoro-5-( 1 -methyl-6-trifluoromethyl-2,4-di
  • herbicide-tolerant or “herbicide-tolerance” means the ability to be wholly or partially unaffected by the presence or application of one or more herbicide(s), for example to resist the toxic effects of an herbicide when applied.
  • a cell or organism is “herbicide- tolerant” if it is able to maintain at least some normal growth or phenotype in the presence of one or more herbicide(s).
  • a trait is an herbicide-tolerance trait if its presence can confer improved tolerance to an herbicide upon a cell, plant, or seed as compared to the wild-type or control cell, plant, or seed. Crops comprising a herbicide-tolerance trait can continue to grow and are minimally affected by the presence of the herbicide.
  • a target enzyme is “herbicide-tolerant” if it exhibits improved enzyme activity relative to a wild-type or control enzyme in the presence of the herbicide.
  • Herbicide-tolerance may be complete or partial insensitivity to a particular herbicide, and may be expressed as a percent (%) tolerance or insensitivity to a particular herbicide.
  • Contemplated plants which might be produced with an herbicide tolerance trait of the present disclosure could include, for instance, any plant susceptible to a PPO inhibitor herbicide, including crop plants such as soybean (Glycine max), maize (Zea mays), cotton (Gossypium sp.), Brassica plants, alfalfa, barley, beans, beet, broccoli, cabbage, carrot, canola, cauliflower, celery, Chinese cabbage, cucumber, eggplant, leek, lettuce, melon, oat, onion, pea, pepper, peanut, potato, pumpkin, radish, rice, sweet com, sorghum, spinach, squash, sugar beet, sugar cane, sunflower, tomato, watermelon, and wheat, among others.
  • crop plants such as soybean (Glycine max), maize (Zea mays), cotton (Gossypium sp.), Brassica plants, alfalfa, barley, beans, beet, broccoli, cabbage, carrot, canola, cauliflower, celery, Chinese cabbage, cucumber,
  • Herbicides may be applied to a plant growth area comprising the plants and seeds provided by the disclosure as a method for controlling weeds.
  • Plants and seeds provided by the disclosure comprise an herbicide tolerance trait and as such are tolerant to the application of one or more PPO inhibiting herbicides.
  • the herbicide application may be the recommended commercial rate (IX) or any fraction or multiple thereof, such as twice the recommended commercial rate (2X).
  • Herbicide rates may be expressed as acid equivalent per pound per acre (lb ae/acre) or acid equivalent per gram per hectare (g ae/ha) or as pounds active ingredient per acre (lb ai/acre) or grams active ingredient per hectare (g ai/ha), depending on the herbicide and the formulation.
  • the herbicide application comprises at least one PPO inhibiting herbicide.
  • the plant growth area may or may not comprise weed plants at the time of herbicide application.
  • a herbicidally-effective dose of PPO inhibiting herbicide(s) for use in an area for controlling weeds may consist of a range from about 0.1X to about 30X label rate(s) over a growing season.
  • One (1) hectare is equivalent to 2.47105 acres and one (1) pound is equivalent to 453.592 grams.
  • the desired application rate of the compounds Al, A2, and A3 and/or their salts is generally impacted to a certain extent by external conditions such as temperature, humidity, etc.
  • the application rate may therefore vary within wide limits, and can be determined empirically by one of skill in the art in view of the present disclosure.
  • the total amount of the compound Al, A2, or A3 and/or their salts is often desirably in the range from about 0.02 g a.i./ha to about 750 g a.i./ha, about 0.05 g a.i./ha to about 400 g a.i./ha, or about 0.25 g a.i./ha to about 300 g a.i./ha, but preferably from about 0.3 g a.i./ha to about 250 g a.i./ha, especially from about 0.4 g a.i./ha to about 150 g a.i./ha, and most preferably from about 0.5 g a.i./ha to about 120 g a.i./ha. This applies both to the pre-emergence or the post-emergence application.
  • Herbicide applications may be sequentially or tank mixed with one, two, or a combination of several PPO inhibiting herbicides or any other compatible herbicide. Multiple applications of one herbicide or of two or more herbicides, in combination or alone, may be used over a growing season to areas comprising transgenic plants of the disclosure for the control of a broad spectrum of dicot weeds, monocot weeds, or both, for example, two applications (such as a pre-planting application and a post-emergence application or a pre-emergence application and a post-emergence application) or three applications (such as a pre-planting application, a preemergence application, and a post-emergence application or a pre-emergence application and two post-emergence applications).
  • two applications such as a pre-planting application and a post-emergence application or a pre-emergence application and a post-emergence application
  • three applications such as a pre-planting application, a preemergence application, and a post-emergence application or a pre-emergence application and two post-emergence applications.
  • Certain substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, such as compounds Al, A2, or A3 as defined above, to be used according to the disclosure and its salts, have excellent herbicidal efficacy against a broad spectrum of economically important monocotyledonous and dicotyledonous annual harmful plants.
  • the present disclosure therefore provides methods for controlling weeds, in areas of transgenic crop plants being tolerant to PPO inhibitor herbicides wherein the plants comprises a recombinant DNA molecule comprising a DNA sequence encoding a heterologous HemG protein, wherein the protein confers tolerance to such herbicides, comprising the application of the compound of Al, A2, or A3 and/or salts as defined above, to the plants (for example harmful plants such as monocotyledonous or dicotyledonous weeds or undesired crop plants), to the seed (for example grains, seeds or vegetative propagules such as tubers or shoot parts with buds) or to the area on which the plants grow (for example the area under cultivation), including any possible combinations thereof.
  • the spectrum of action extends to species such as, for example, Abutilon, Amaranthus, Ambrosia, Anoda, Anthemis, Aphanes, Artemisia, Atriplex, Bellis, Bidens, Capsella, Carduus, Cassia, Centaurea, Chenopodium, Cirsium, Convolvulus, Datura, Desmodium, Emex, Erodium, Erysimum, Euphorbia, Galeopsis, Galinsoga, Galium, Geranium, Hibiscus, Ipomoea, Kochia, Lamium, Lepidium, Lindemia, Matricaria, Mentha, Mercurialis, Mullugo, Myosotis, Papaver, Pharbitis, Plantago, Polygonum, Portulaca, Ranunculus, Raphanus, Rorippa, Rotala, Rumex, Salsola, Senecio, Sesbania, Sida, Sinapis
  • substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain described herein display outstanding herbicidal activity against monocotyledonous and dicotyledonous weeds, many economically important crop plants, depending on the structure of the respective active ingredients and the application rate thereof, are damaged only insignificantly, if at all.
  • Economically important crops here are, for example, dicotyledonous crops from the genera of Arachis, Beta, Brassica, Cucumis, Cucurbita, Helianthus, Daucus, Glycine, Gossypium, Ipomoea, Lactuca, Linum, Lycopersicon, Nicotiana, Phaseolus, Pisum, Solanum, and Vicia, or monocotyledonous crops from the genera of Allium, Ananas, Asparagus, Avena, Hordeum, Oryza, Panicum, Saccharum, Secale, Sorghum, Triticale, Triticum, and Zea.
  • the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain and/or salts thereof can be formulated in various ways according to which biological and/or physicochemical parameters are required.
  • general formulation options are: wettable powders (WP), water-soluble powders (SP), emulsifiable concentrates (EC), water-soluble concentrates, aqueous solutions (SL), emulsions (EW), such as oil-in-water and water-in-oil emulsions, sprayable solutions or emulsions, dispersions based on oil or water, oil dispersions (OD), suspoemulsions (SE), suspension concentrates (SC), oil-miscible solutions, capsule suspensions (CS), dusting products (DP), dressings, granules for soil application or scattering, granules (GR) in the form of microgranules, spray granules, absorption and adsorption granules
  • Wettable powders are preparations which can be dispersed uniformly in water and, in addition to the active ingredient, apart from a diluent or inert substance, also comprise surfactants of the ionic and/or nonionic type (wetting agents, dispersants), for example polyoxyethylated alkylphenols, polyoxyethylated fatty alcohols, polyoxyethylated fatty amines, fatty alcohol polyglycol ether sulfates, alkanesulfonates, alkylbenzene sulfonates, sodium lignosulfonate, sodium 2,2'-dinaphthylmethane-6,6'-disulfonate, sodium dibutylnaphthalenesulfonate, or sodium oleoyl methyltaurate.
  • the active herbicidal ingredients are finely ground, for example, in customary apparatuses such as hammer mills, blower mill
  • Emulsifiable concentrates are produced by dissolving the active ingredient in an organic solvent, for example butanol, cyclohexanone, dimethylformamide, xylene, or else relatively high-boiling aromatics or hydrocarbons or mixtures of the organic solvents, with addition of one or more ionic and/or nonionic surfactants (emulsifiers).
  • organic solvent for example butanol, cyclohexanone, dimethylformamide, xylene, or else relatively high-boiling aromatics or hydrocarbons or mixtures of the organic solvents.
  • emulsifiers which may be used are: calcium alkyl aryl sulfonate salts, such as calcium dodecylbenzenesulfonate, or nonionic emulsifiers such as fatty acid polyglycol esters, alkyl aryl polyglycol ethers, fatty alcohol polyglycol ethers, propylene oxide-ethylene oxide condensation products, alkyl polyethers, sorbitan esters, for example sorbitan fatty acid esters, or for example polyoxyethylene sorbitan fatty acid esters.
  • calcium alkyl aryl sulfonate salts such as calcium dodecylbenzenesulfonate
  • nonionic emulsifiers such as fatty acid polyglycol esters, alkyl aryl polyglycol ethers, fatty alcohol polyglycol ethers, propylene oxide-ethylene oxide condensation products, alkyl polyethers, sorbitan esters, for example sorbit
  • Dusting products are obtained by grinding the active ingredient with finely distributed solids, for example talc, natural clays, such as kaolin, bentonite and pyrophyllite, or diatomaceous earth.
  • finely distributed solids for example talc, natural clays, such as kaolin, bentonite and pyrophyllite, or diatomaceous earth.
  • Suspension concentrates may be water- or oil-based. They may be produced, for example, by wet-grinding by means of commercial bead mills and optional addition of surfactants as already listed above, for example, for the other formulation types.
  • Emulsions for example oil-in-water emulsions (EW)
  • EW oil-in-water emulsions
  • Active compounds that can be employed in combination with substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain described herein in compositions described herein are, for example, known active compounds which are based on inhibition of, for example, acetolactate synthase, acetyl-CoA carboxylase, cellulose synthase, enolpyruvylshikimate-3-phosphate synthase, glutamine synthetase, p-hydroxyphenylpyruvate dioxygenase, phytoene desaturase, photosystem I, photosystem II, or protoporphyrinogen oxidase, as are described in, for example, “Glossary of Common Names and Abbreviations of Herbicides,” Weed Research 26:441-445, 1986 or MacBean, "The Pesticide Manual,” 19 th ed., British Crop Protection Council, Alton
  • Known herbicides or plant growth regulators which can be combined with the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain described herein are, for example, the following, where said active compounds are designated either with their "common name" in accordance with the International Organization for Standardization (ISO) or with the chemical name or with the code number. They always encompass all the use forms, for example acids, salts, esters and also all isomeric forms such as stereoisomers and optical isomers, even if they are not mentioned explicitly.
  • ISO International Organization for Standardization
  • herbicidal mixing partners include one or more of the following: acetochlor, acifhiorfen, acifhiorfen-methyl, acifhiorfen-sodium, aclonifen, alachlor, allidochlor, alloxydim, alloxydim-sodium, ametryn, amicarbazone, amidochlor, amidosulfuron, 4-amino-3- chloro-6-(4-chloro-2-fluoro-3-methylphenyl)-5-fluoropyridine-2-carboxylic acid, aminocyclo- pyrachlor, aminocyclopyrachlor-potassium, aminocyclopyrachlor-methyl, aminopyralid, aminopyralid-dimethylammonium, aminopyralid-tripromine, amitrole, ammonium sulfamate, anilofos, asularn, asulam-potassium, asularn sodium, atrazine, azafen
  • Examples of plant growth regulators as possible mixing partners are: abscisic acid and related analogs [e.g. (2Z,4E)-5-[6-Ethynyl-l-hydroxy-2,6-dimethyl-4-oxocyclohex-2-en-l-yl]-3- methylpenta-2,4-dienoic acid, methyl-(2Z,4E)-5- [6-ethynyl- 1 -hydroxy-2, 6-dimethyl-4-oxocyclo- hex-2-en-l-yl]-3-methylpenta-2,4-dienoate, (2Z,4E)-3-ethyl-5-(l-hydroxy-2,6,6-trimethyl-4-oxo- cyclohex-2-en- 1 -yl)penta-2,4-dienoic acid, (2E,4E)-5-( 1 -hydroxy-2, 6, 6-trimethyl-4-oxocyclo- hex-2-en-l-yl-y
  • COs sometimes referred to as N-acetylchitooligosaccharides, are also composed of GlcNAc residues but have side chain decorations that make them different from chitin molecules [(CgHigNOsjn, CAS No. 1398-61-4] and chitosan molecules [(CsHnNOzQn, CAS No.
  • chitinous compounds chlormequat chloride, cloprop, cyclanilide, 3-(Cycloprop- l-enyl)propionic acid, l-[2-(4-cyano-3,5-dicyclopropylphenyl)acetamido]cyclohexanecarboxylic acid, l-[2-(4-cyano-3-cyclopropylphenyl)acetamido]cyclohexanecarboxylic acid, daminozide, dazomet, dazomet-sodium, n-decanol, dikegulac, dikegulac-sodium, endothal, endothal- dipotassium, -disodium, and mono(N,N-dimethylalkylammonium), ethephon, flumetralin, flurenol, flurenol-butyl, flurenol-methyl, flurprimidol, forchlorfenur
  • LCO lipo-chitooligosaccharides
  • Nod symbiotic nodulation
  • Myc factors consist of an oligosaccharide backbone of P-l,4-linked A-acetyl-D-glucosamine (“GlcNAc”) residues with an N-linked fatty acyl chain condensed at the non-reducing end.
  • LCOs differ in the number of GlcNAc residues in the backbone, in the length and degree of saturation of the fatty acyl chain and in the substitutions of reducing and non-reducing sugar residues), linoleic acid or derivatives thereof, linolenic acid or derivatives thereof, maleic hydrazide, mepiquat chloride, mepiquat pentaborate, 1 -methylcyclopropene, 3-methylcyclopropene, 1 -ethylcyclopropene, 1-n-propylcyclopropene, 1- cyclopropenylmethanol, methoxyvinylglycin (MVG), 3’ -methyl abscisic acid, l-(4- methylphenyl)-N-(2-oxo-l-propyl-l,2,3,4-tetrahydroquinolin-6-yl)methanesulfon-amide and related substituted tetrahydroquinolin
  • Active compounds which can be employed in combination with the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain according to the present disclosure in compositions according to the present disclosure are, for example, the following safeners:
  • nA is an integer value in the range of 0 to 5, preferably 0 to 3;
  • RA 1 is halogen, (Ci-C4)-alkyl, (Ci-C4)-alkoxy, nitro or (Ci-C4)-haloalkyl;
  • WA is an unsubstituted or substituted divalent heterocyclic moiety selected from the group of partially unsaturated or aromatic five-membered heterocycles carrying 1 to 3 hetero ring atoms selected from the group of nitrogen (N) und oxygen (O), and carrying at least one N-atom and not more than one O-atom in the ring, preferably a five-membered heterocyclic moiety selected from the group (WA 1 ) to (WA 4 ),
  • RA 2 is ORA 3 , SRA 3 or NRA 3 RA 4 or a saturated or unsaturated 3- to 7-membered heterocycle containing at least one N-atom and up to 3 heteroatoms, preferably combined with other heteroatoms from the group of O (oxygen) and S (sulfur), and which is linked to the carbonyl group in (SI) via a nitrogen atom, and which is unsubstituted or substituted by moieties selected from the group of (Ci-C4)-alkyl, (Ci-C4)-alkoxy or possibly substituted phenyl, preferably ORA 3 , NHRA 4 or N(CHS)2, particularly ORA 3
  • RA 3 is hydrogen or an unsubstituted or substituted aliphatic hydrocarbon moiety, preferably containing 1 to 18 C-atoms;
  • RA 4 is hydrogen, (Ci-C6)-alkyl, (Ci-C6)-alkoxy or substituted or unsubstituted phenyl;
  • RA 5 is hydrogen, (Ci-C8)-alkyl, (Ci-C8)-haloalkyl, (Ci-C4)-alkoxy-(Ci-C8)-alkyl, cyano or COORA 9 , wherein RA 9 is hydrogen, (Ci-C8)-alkyl, (Ci-C8)-haloalkyl, (Ci-C4)-alkoxy-(Ci-C4)- alkyl, (Ci-C6)-hydroxyalkyl, (C3-Ci2)-cycloalkyl, or tris-(Ci-C4)-alkylsilyl;
  • RA 6 , RA 7 , RA 8 are independently hydrogen, (Ci-C8)-alkyl, (Ci-C8)-haloalkyl, (C3-Ci2)-cycloalkyl or substituted or unsubstituted phenyl;
  • RA 10 is hydrogen, (C3-Ci2)-cycloalkyl, substituted or unsubstituted phenyl, or substituted or unsubstituted heteroaryl;
  • Sl a Compounds of the dichlorophenylpyrazoline-3-carboxylic acid type (Sl a ), preferably compounds such as l-(2,4-dichlorophenyl)-5-(ethoxycarbonyl)-5-methyl-2-pyrazoline-3- carboxylic acid, ethyl l-(2,4-dichlorophenyl)-5-(ethoxycarbonyl)-5-methyl-2-pyrazoline- 3-carboxylate (S 1-1) ("mefenpyr-diethyl”), and related compounds, as described in WO- A-91/07874;
  • Sl b Derivatives of dichlorophenylpyrazolecarboxylic acid (Sl b ), preferably compounds such as ethyl l-(2,4-dichlorophenyl)-5-methylpyrazole-3-carboxylate (Sl-2), ethyl l-(2,4- dichlorophenyl)-5-isopropylpyrazole-3-carboxylate (Sl-3), ethyl l-(2,4-dichlorophenyl)- 5-(l,l-dimethylethyl)pyrazole-3-carboxylate (SI -4) and related compounds as described in EP-A-333131 and EP-A-269806;
  • Sl c Derivatives of l,5-diphenylpyrazole-3-carboxylic acid (Sl c ), preferably compounds such as ethyl l-(2,4-dichlorophenyl)-5-phenylpyrazole-3-carboxylate (SI -5), methyl l-(2- chlorophenyl)-5-phenylpyrazole-3-carboxylate (Sl-6) and related compounds as described, for example, in EP-A-268554;
  • Sl d Compounds of the triazolecarboxylic acid type (Sl d ), preferably compounds such as fenchlorazole(-ethyl ester), i.e. ethyl l-(2,4-dichlorophenyl)-5-trichloromethyl-lH-l,2,4- triazole-3-carboxylate (Sl-7), and related compounds, as described in EP-A- 174562 and EP-A-346620;
  • Sl f compounds of the triazolyloxy acetic acid type (Sl f ), preferably compounds such as methyl- ⁇ [l,5-bis(4-chloro-2-fluorophenyl)-lH-l,2,4-triazol-3-yl]oxy ⁇ acetate (Sl-14) or ⁇ [l,5-Bis(4-chloro-2-fluorophenyl)-lH-l,2,4-triazol-3-yl]oxy ⁇ acetic acid (Sl-15) or methyl- ⁇ [5-(4-chloro-2-fluorophenyl)- 1 -(2,4-difluorophenyl)-lH- 1 ,2,4-triazol-3- yl]oxy ⁇ acetate (Sl-16) or ⁇ [5-(4-chloro-2-fluorphenyl)-l-(2,4-difluorophenyl)-lH-l,2,4- triazol-3-yl
  • S2 a Compounds of the 8-quinolinoxyacetic acid type (S2 a ), preferably 1 -methylhexyl (5- chloro-8-quinolinoxy)acetate ("cloquintocet-mexyl") (S2-1), 1,3-dimethylbut-l-yl (5- chloro-8-quinolinoxy)acetate (S2-2), 4-allyloxybutyl (5-chloro-8-quinolinoxy)acetate (S2- 3), l-allyloxyprop-2-yl (5-chloro-8-quinolinoxy)acetate (S2-4), ethyl (5-chloro-8- quinolinoxy)acetate (S2-5), methyl (5-chloro-8-quinolinoxy)acetate (S2-6), allyl (5- chloro-8-quinolinoxy)acetate (S2-7), 2-(2-propylideneiminoxy)-l -ethyl (5-chloro-8- quinolinoxy)acetate (S2-8)
  • S2 b Compounds of the (5-chloro-8-quinolinoxy)malonic acid type (S2 b ), preferably compounds such as diethyl (5-chloro-8-quinolinoxy)malonate, diallyl (5-chloro-8- quinolinoxy)malonate, methyl ethyl (5-chloro-8-quinolinoxy)malonate and related compounds, as described in EP-A-0582 198.
  • S3 Active ingredients of the dichloroacetamide type (S3), which are frequently used as preemergence safeners (soil-acting safeners), for example "dichlormid” (N,N-diallyl-2,2-dichloroacetamide) (S3-1),
  • R-29148 (3-dichloroacetyl-2,2,5-trimethyl-l,3-oxazolidine) from Stauffer (S3-2)
  • R-28725" (3-dichloroacetyl-2,2-dimethyl-l,3-oxazolidine) from Stauffer (S3-3)
  • benoxacor (4-dichloroacetyl-3,4-dihydro-3-methyl-2H-l,4-benzoxazine) (S3-4)
  • PPG- 1292 N-allyl-N-[(l,3-dioxolan-2-yl)methyl]dichloroacetamide
  • AD-67 or "MON 4660” (3-dichloroacetyl-l-oxa-3-azaspiro[4.5]decane) from Nitrokemia or Monsanto (S3-7),
  • TI-35 (1-dichloroacetylazepane) from TRI-Chemical RT (S3-8), "diclonon” (dicyclonon) or "BAS 145138” or “LAB 145138” (S3-9) ((RS)- 1 -dichloroacetyl-3 , 3 , 8a-trimethylperhydropyrrolo [ 1 ,2-a]pyrimidin-6-one) from
  • RA 1 is (Ci-Ce)-alkyl, (C3-C6)-cycloalkyl, where the 2 latter radicals are substituted by VA substituents from the group of halogen, (Ci-C4)-alkoxy, (Ci-C6)-haloalkoxy and (Ci-C4)-alkylthio and, in the case of cyclic radicals, also by (Ci-C4)-alkyl and (Ci- C4)-haloalkyl;
  • RA 2 is halogen, (Ci-C4)-alkyl, (Ci-C4)-alkoxy, CF3; mA is 1 or 2;
  • VA is 0, 1, 2 or 3;
  • RB 1 , Ra 2 are independently hydrogen, (Ci-Ce)-alkyl, (C3-C6)-cycloalkyl, (C3-Ce)-alkenyl, (C3-Ce)-alkynyl;
  • RB 3 is halogen, (Ci-C4)-alkyl, (Ci-C4)-haloalkyl or (Ci-C4)-alkoxy; and ms is 1 or 2, e.g. those in which
  • RB 1 cyclopropyl
  • RB 2 hydrogen
  • (RB 3 ) 2-OMe
  • RB 1 cyclopropyl
  • RB 2 hydrogen
  • (RB 3 ) 5-Cl-2-OMe (S4-2)
  • RB 1 ethyl
  • RB 2 hydrogen
  • (RB 3 ) 2-OMe (S4-3)
  • RB 1 isopropyl
  • RB 2 hydrogen
  • (RB 3 ) 5-Cl-2-OMe (S4-4) and
  • RB 1 isopropyl
  • RB 2 hydrogen
  • (RB 3 ) 2-OMe (S4-5);
  • Rc 1 , Rc 2 are independently hydrogen, (Ci-C8)-alkyl, (C3-C8)-cycloalkyl, (C3-C6)- alkenyl, (C3-C6)-alkynyl,
  • Rc 3 is halogen, (Ci-C4)-alkyl, (Ci-C4)-alkoxy, CF3 and me is 1 or 2; for example l-[4-(N-2-methoxybenzoylsulfamoyl)phenyl]-3-methylurea, l-[4-(N-2-methoxybenzoylsulfamoyl)phenyl]-3,3-dimethylurea, l-[4-(N-4,5-dimethylbenzoylsulfamoyl)phenyl]-3-methylurea;
  • RD 4 is halogen, (Ci-C4)-alkyl, (Ci-C4)-alkoxy, CF3;
  • IDD is 1 or 2;
  • RD 5 is hydrogen, (Ci-Ce)-alkyl, (C3-C6)-cycloalkyl, (C2-Cg)-alkenyl, (C2-Ce)-alkynyl, (C5-Ct,)-cycloalkenyl.
  • Active ingredients from the class of the hydroxyaromatics and the aromatic-aliphatic carboxylic acid derivatives (S5) for example ethyl 3,4,5-triacetoxybenzoate, 3,5-dimethoxy-4-hydroxybenzoic acid, 3,5- dihydroxybenzoic acid, 4-hydroxysalicylic acid, 4-fluorosalicylic acid, 2-hydroxycinnamic acid, 2,4-dichlorocinnamic acid, as described in WO-A-2004/084631, WO-A- 2005/015994, WO-A-2005/016001.
  • Active ingredients from the class of the l,2-dihydroquinoxalin-2-ones (S6) for example
  • RD 1 is halogen, (Ci-C4)-alkyl, (Ci-C4)-haloalkyl, (Ci-C4)-alkoxy, (Ci-C4)-haloalkoxy, RD 2 is hydrogen or (Ci-C4)-alkyl,
  • RD 3 is hydrogen, (Ci-C8)-alkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl or aryl, where each of the aforementioned carbon-containing radicals is unsubstituted or substituted by one or more, preferably up to three, identical or different radicals from the group consisting of halogen and alkoxy; or salts thereof, no is an integer from 0 to 2.
  • Active ingredients from the class of the 3-(5-tetrazolylcarbonyl)-2-quinolones (S9) for example l,2-dihydro-4-hydroxy-l-ethyl-3-(5-tetrazolylcarbonyl)-2-quinolone (CAS Reg. No.: 219479- 18-2), 1 ,2-dihydro-4-hydroxy- 1 -methyl-3-(5-tetrazolylcarbonyl)-2-quinolone
  • RE 1 is halogen, (Ci-C4)-alkyl, methoxy, nitro, cyano, CF3, OCF3,
  • YE, ZE are independently O or S, ns is an integer from 0 to 4,
  • RE 2 is (Ci-Ci6)-alkyl, (C2-C6)-alkenyl, (C3-C6)-cycloalkyl, aryl; benzyl, halobenzyl, RE 3 is hydrogen or (Ci-Ce)-alkyl.
  • oxabetrinil ((Z)-l,3-dioxolan-2-ylmethoxyimino(phenyl)acetonitrile) (SI 1-1), which is known as a seed-dressing safener for millet/sorghum against metolachlor damage
  • fluorofenim 1 -(4-chlorophenyl)-2,2,2-trifluoro- 1 -ethanone O-( 1 ,3-dioxolan-2- ylmethyl)oxime
  • naphthalic anhydride (1,8-naphthalenedicarboxylic anhydride) (S 13-1), which is known as a seed-dressing safener for maize against thiocarbamate herbicide damage
  • fenclorim (4,6-dichloro-2-phenylpyrimidine) (SI 3-2), which is known as a safener for pretilachlor in sown rice
  • flurazole (benzyl 2-chloro-4-trifluoromethyl-l,3-thiazole-5-carboxylate) (S13-3), which is known as a seed-dressing safener for millet/sorghum against alachlor and metolachlor damage,
  • JC-940 3-(2-chlorophenylmethyl)-l-(l-methyl-l-phenylethyl)urea, see JP-A-60087254), which is known as a safener for rice against damage by some herbicides
  • methoxyphenone or "NK 049” (3,3'-dimethyl-4-methoxybenzophenone), which is known as a safener for rice against damage by some herbicides
  • RH 1 is a (Ci-Ce)-haloalkyl radical
  • RH 2 is hydrogen or halogen
  • RH 3 , RH 4 are independently hydrogen, (Ci-Ci6)-alkyl, (C2-Ci6)-alkenyl or (C2-C16)- alkynyl, where each of the 3 latter radicals is unsubstituted or substituted by one or more radicals from the group of halogen, hydroxyl, cyano, (Ci-C4)-alkoxy, (C1-C4)- haloalkoxy, (Ci-C4)-alkylthio, (Ci-C4)-alkylamino, di[(Ci-C4)-alkyl]amino, [(Ci- C4)-alkoxy]carbonyl, [(Ci-C4)-haloalkoxy]carbonyl, (C3-C6)-cycloalkyl which is unsubstituted or substituted, phenyl which is unsubstituted or substituted, and heterocyclyl which is unsubstituted or substituted, or
  • RH 4 is hydrogen or (Ci-C4)-alkyl or
  • RH 3 and RH 4 together with the directly attached nitrogen atom represent a four- to eightmembered heterocyclic ring which, as well as the nitrogen atom, may also contain further ring heteroatoms, preferably up to two further ring heteroatoms from the group of N, O, and S, and which is unsubstituted or substituted by one or more radicals from the group of halogen, cyano, nitro, (Ci-C4)-alkyl, (Ci-C4)-haloalkyl, (Ci-C4)-alkoxy, (C1-C4)- haloalkoxy and (Ci-C4)-alkylthio.
  • Safeners that may be used in combination with the herbicidal compounds described herein include, but are not limited to, cloquintocet-mexyl, cyprosulfamide, fenchlorazole ethyl ester, isoxadifen-ethyl, mefenpyr-diethyl, fenclorim, cumyluron, S4-1, and S4-5.
  • Preferred safeners include cloquintocet-mexyl, cyprosulfamide, isoxadifen-ethyl, and mefenpyr-diethyl.
  • the herbicide combinations described herein may comprise further components, for example plant growth regulators or compounds that prevent or eliminate unwanted species.
  • Such compounds include, but are not limited to herbicides, fungicides, insecticides, acaricides, nematicides, miticides, and related substances.
  • plant growth regulators examples include, but are not limited to, acibenzolar, acibenzolar-S-methyl, 5-aminolevulinic acid, ancymidol, 6-benzylaminopurine, brassinolide, catechol, chlormequat chloride, cloprop, cyclanilide, 3-(cycloprop-l-enyl)propionic acid, daminozide, dazomet, n-decanol, dikegulac, dikegulac-sodium, endothal, endothal- dipotassium, -disodium, and mono(N,N-dimethylalkylammonium), ethephon, flumetralin, flurenol, flurenol-butyl, flurprimidol, forchlorfenuron, gibberellic acid, inabenfide, indole-3-acetic acid (IAA), 4-indol-3-ylbutyric acid, is
  • Active compounds that may be used in combination with the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain described herein (in, for example, mixed formulations or a tank mix) are, for example, fungicidally active compounds.
  • the preferred fungicidally active compounds comprise at least one standard commercial active ingredient, and include, but are not limited to:
  • Ergosterol biosynthesis inhibitors for example (1.001) cyproconazole, (1.002) difenoconazole, (1.003) epoxiconazole, (1.004) fenhexamid, (1.005) fenpropidin, (1.006) fenpropimorph, (1.007) fenpyrazamine, (1.008) fluquinconazole, (1.009) flutriafol, (1.010) imazalil, (1.011) imazalil sulfate, (1.012) ipconazole, (1.013) metconazole, (1.014) myclobutanil, (1.015) paclobutrazole, (1.016) prochloraz, (1.017) propiconazole, (1.018) prothioconazole, (1.019) pyrisoxazole, (1.020) spiroxamine, (1.021) tebuconazole, (1.022) tetraconazole, (1.023) triadimen
  • Inhibitors of the respiratory chain in complex I or II for example (2.001) benzovindiflupyr, (2.002) bixafen, (2.003) boscalid, (2.004) carboxin, (2.005) fluopyram, (2.006) flutolanil, (2.007) fluxapyroxad, (2.008) furametpyr, (2.009) isofetamid, (2.010) isopyrazam (anti-epimeric enantiomer 1R,4S,9S), (2.011) isopyrazam (anti-epimeric enantiomer 1S,4R,9R), (2.012) isopyrazam (anti-epimeric racemate 1RS,4SR,9SR), (2.013) isopyrazam (mixture of the syn-epimeric racemate 1RS,4SR,9RS and the anti- epimeric racemate 1RS,4SR,9SR), (2.014) isopyrazam (syn-epimeric enantiomer 1R,4S,
  • Respiratory chain inhibitors acting on complex III for example (3.001) ametoctradin, (3.002) amisulbrom, (3.003) azoxystrobin, (3.004) coumethoxystrobin, (3.005) coumoxystrobin, (3.006) cyazofamid, (3.007) dimoxystrobin, (3.008) enoxastrobin, (3.009) famoxadon, (3.010) fenamidon, (3.011) flufenoxystrobin, (3.012) fluoxastrobin, (3.013) kresoxim-methyl, (3.014) metominostrobin, (3.015) orysastrobin, (3.016) picoxystrobin, (3.017) pyraclostrobin, (3.018) pyrametostrobin, (3.019) pyraoxystrobin, (3.020) trifloxystrobin, (3.021) (2E)-2- ⁇ 2-[( ⁇ [(lE)-l-(3- ⁇ [(E)
  • Amino acid and/or protein biosynthesis inhibitors for example (7.001) cyprodinil, (7.002) kasugamycin, (7.003) kasugamycin hydrochloride hydrate, (7.004) oxytetracycline, (7.005) pyrimethanil, (7.006) 3-(5-fluoro-3,3,4,4-tetramethyl-3,4- dihydroisoquinolin- 1 -yl)quinoline.
  • ATP production inhibitors for example (8.001) silthiofam.
  • Cell wall synthesis inhibitors for example (9.001) benthiavalicarb, (9.002) dimethomorph, (9.003) flumorph, (9.004) iprovalicarb, (9.005) mandipropamid, (9.006) pyrimorph, (9.007) valifenalate, (9.008) (2E)-3-(4-tert-butylphenyl)-3-(2-chloropyridin-4- yl)- 1 -(morpholin-4-yl)prop-2-en- 1 -one, (9.009) (2Z)-3-(4-tert-butylphenyl)-3-(2- chloropyridin-4-yl)- 1 -(morpholin-4-yl)prop-2-en- 1 -one.
  • Lipid and membrane synthesis inhibitors for example (10.001) propamocarb, (10.002) propamocarb hydrochloride, (10.003) tolclofos-methyl.
  • Nucleic acid synthesis inhibitors for example (12.001) benalaxyl, (12.002) benalaxyl-M (kiralaxyl), (12.003) metalaxyl, (12.004) metalaxyl-M (mefenoxam).
  • Signal transduction inhibitors for example (13.001) fludioxonil, (13.002) iprodione, (13.003) procymidone, (13.004) proquinazid, (13.005) quinoxyfen, (13.006) vinclozolin.
  • Preferred fungicides are selected from the group consisting of benalaxyl, bitertanol, bromuconazole, captafol, carbendazim, carpropamid, cyazofamid, cyproconazole, diethofencarb, edifenphos, fenpropimorph, fentin acetate, fluquinconazole, fosetyl, fluoroimide, folpet, iminoctadine, iprodione, iprovalicarb, kasugamycin, maneb, nabam, pencycuron, prochloraz, propamocarb, propineb, prothioconazole, pyrimethanil, spiroxamine, quintozene, tebuconazole, tolylfluanid, triadimefon, triadimenol, trifloxystrobin, and zineb.
  • Active compounds which can be employed in combination with the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain according to the present disclosure in compositions described herein (for example in mixed formulations or in the tank mix) are, for example, insecticidal, acaricidal, nematicidal, miticidal and related active ingredients are, for example (the compounds are, if possible, referred to by their common names):
  • Acetylcholinesterase (AChE) inhibitors preferably carbamates selected from alanycarb, aldicarb, bendiocarb, benfuracarb, butocarboxim, butoxycarboxim, carbaryl, carbofuran, carbosulfan, ethiofencarb, fenobucarb, formetanate, furathiocarb, isoprocarb, methiocarb, methomyl, metolcarb, oxamyl, pirimicarb, propoxur, thiodicarb, thiofanox, triazemate, trimethacarb, XMC, and xylylcarb; or organophosphates selected from acephate, azamethiphos, azinphos-ethyl, azinphos-methyl, cadusafos, chlorethoxyfos, chlorfenvinphos, chlormephos, chlorpyrifos-methyl,
  • GABA-gated chloride channel blockers preferably cyclodiene-organochlorines selected from chlordane and endosulfan, or phenylpyrazoles (fiproles) selected from ethiprole and fipronil.
  • Sodium channel modulators preferably pyrethroids selected from acrinathrin, allethrin, d-cis-trans allethrin, d-trans allethrin, bifenthrin, bioallethrin, bioallethrin S- cyclopentenyl isomer, bioresmethrin, cycloprothrin, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, gamma-cyhalothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, theta-cypermethrin, zeta-cypermethrin, cyphenothrin [(lR)-trans isomer], deltamethrin, empenthrin [(EZ)-(IR) isomer], esfenvalerate
  • Nicotinic acetylcholine receptor (nAChR) competitive modulators preferably neonicotinoids selected from acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam, or nicotine, or sulfoximines such as sulfoxaflor, or butenolides such as flupyradifurone.
  • nAChR Nicotinic acetylcholine receptor
  • Nicotinic acetylcholine receptor (nAChR) allosteric modulators preferably spinosyns selected from spinetoram and spinosad.
  • Glutamate-gated chloride channel (GluCl) allosteric modulators preferably avermectins/milbemycins selected from abamectin, emamectin benzoate, lepimectin, and milbemectin.
  • Juvenile hormone mimics preferably juvenile hormone analogues selected from hydroprene, kinoprene and methoprene, or fenoxycarb or pyriproxyfen.
  • Miscellaneous non-specific (multi-site) inhibitors preferably alkyl halides selected from methyl bromide and other alkyl halides; or chloropicrin or sulfuryl fluoride or borax or tartar emetic or methyl isocyanate generators selected from diazomet and metam.
  • Mite growth inhibitors selected from clofentezine, hexythiazox, diflovidazin, and etoxazole.
  • Microbial disruptors of insect midgut membranes selected from Bacillus thuringiensis subspecies israelensis, Bacillus sphaericus, Bacillus thuringiensis subspecies aizawai, Bacillus thuringiensis subspecies kurstaki, Bacillus thuringiensis subspecies tenebrionis, and B.t. plant proteins selected from CrylAb, CrylAc, CrylFa, CrylA.105, Cry2Ab, VIP3A, mCry3A, Cry3Ab, Cry3Bb, and Cry34Abl/35Abl.
  • Inhibitors of mitochondrial ATP synthase preferably ATP disruptors selected from diafenthiuron, or organotin compounds selected from azocyclotin, cyhexatin and fenbutatin oxide, or propargite or tetradifon.
  • Nicotinic acetylcholine receptor channel blockers selected from bensultap, cartap hydrochloride, thiocyclam, and thiosultap-sodium.
  • Inhibitors of chitin biosynthesis type 0, selected from bistrifluron, chlorfluazuron, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, noviflumuron, teflubenzuron, and triflumuron.
  • Molting disruptors (especially in the case of Diptera) such as cyromazine.
  • Ecdysone receptor agonists selected from chromafenozide, halofenozide, methoxyfenozide, and tebufenozide.
  • Octopamine receptor agonists such as amitraz.
  • Mitochondrial complex III electron transport inhibitors selected from hydramethylnon, acequinocyl, and fluacrypyrim.
  • Mitochondrial complex I electron transport inhibitors preferably METI acaricides selected from fenazaquin, fenpyroximate, pyrimidifen, pyridaben, tebufenpyrad and tolfenpyrad, or rotenone (Derris).
  • METI acaricides selected from fenazaquin, fenpyroximate, pyrimidifen, pyridaben, tebufenpyrad and tolfenpyrad, or rotenone (Derris).
  • Voltage-dependent sodium channel blockers selected from indoxacarb and metaflumizone.
  • Inhibitors of acetyl-CoA carboxylase preferably tetronic and tetramic acid derivatives selected from spirodiclofen, spiromesifen, and spirotetramat.
  • Mitochondrial complex IV electron transport inhibitors preferably phosphines selected from aluminum phosphide, calcium phosphide, phosphine, and zinc phosphide, or cyanides selected from calcium cyanide, potassium cyanide, and sodium cyanide.
  • Mitochondrial complex II electron transport inhibitors preferably beta-keto nitrile derivatives selected from cyenopyrafen and cyflumetofen, or carboxanilides such as pyflubumide.
  • Insecticides that can preferably be used together with the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain are, for example, as follows: acetamiprid, acrinathrin, aldicarb, amitraz, acinphos-methyl, cyfluthrin, carbaryl, cypermethrin, deltamethrin, endosulfan, ethoprophos, fenamiphos, fenthion, fipronil, imidacloprid, methamidophos, methiocarb, niclosamide, oxydemeton-methyl, prothiophos, silafluofen, thiacloprid, thiodicarb, tralomethrin, triazophos, trichlorfon, triflumuron, terbufos, fonofos, phorate, chlorpyriphos, carbo
  • the disclosure relates, in certain embodiments, to recombinant DNA molecules that encode herbicide-insensitive protoporphyrinogen oxidases (PPOs) and the proteins encoded thereby.
  • PPOs herbicide-insensitive protoporphyrinogen oxidases
  • engineered refers to a non-natural DNA, protein, cell, or organism that would not normally be found in nature and was created by human intervention.
  • an “engineered protein,” “engineered enzyme,” or “engineered PPO,” refers to a protein, enzyme, or PPO whose amino acid sequence was conceived of and created in the laboratory using one or more of the techniques of biotechnology, protein design, or protein engineering, such as molecular biology, protein biochemistry, bacterial transformation, plant transformation, site-directed mutagenesis, directed evolution using random mutagenesis, genome editing, gene editing, gene cloning, DNA ligation, DNA synthesis, protein synthesis, and DNA shuffling.
  • an engineered protein may have one or more deletions, insertions, or substitutions relative to the coding sequence of the wild-type protein and each deletion, insertion, or substitution may consist of one or more amino acids.
  • Genetic engineering can be used to create a DNA molecule encoding an engineered protein, such as an engineered PPO that is herbicide tolerant and comprises at least a first amino acid substitution relative to a wild-type PPO protein as described herein.
  • proteins provided herein have herbicide-tolerant protoporphyrinogen oxidase activity.
  • herbicide-tolerant protoporphyrinogen oxidase means the ability of a protoporphyrinogen oxidase to maintain at least some of its protoporphyrinogen oxidase activity in the presence of one or more PPO inhibiting herbicide(s).
  • protoporphyrinogen oxidase activity means the ability to catalyze the six-electron oxidation (removal of electrons) of protoporphyrinogen IX to form protoporphyrin IX, that is, to catalyze the dehydrogenation of protoporphyrinogen to form protoporphyrin.
  • Enzymatic activity of a protoporphyrinogen oxidase can be measured by any means known in the art, for example, by an enzymatic assay in which the production of the product of protoporphyrinogen oxidase or the consumption of the substrate of protoporphyrinogen oxidase in the presence of one or more PPO inhibiting herbicide(s) is measured via fluorescence, high performance liquid chromatography (HPLC), or mass spectrometry (MS).
  • HPLC high performance liquid chromatography
  • MS mass spectrometry
  • an assay for measuring enzymatic activity of a protoporphyrinogen oxidase is a bacterial assay, such as the assays described herein, whereby a recombinant protoporphyrinogen oxidase is expressed in a bacterial cell otherwise lacking PPO activity and the ability of the recombinant protoporphyrinogen oxidase to complement this knockout phenotype is measured.
  • a "hemG knockout strain” means an organism or cell of an organism, such as E.
  • a hemG knockout strain of, for instance, E. coli may be prepared in view of knowledge in the art, for instance in view of the E. coli HemG PPO sequence (Ecogene Accession No. EG11485 ; Sasarman et al., "Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of E. coli KI 2" Can. J. Microbiol. 39:1155-1161, 1993).
  • the term “recombinant” refers to a non-naturally occurring DNA, protein, cell, seed, or organism that is the result of genetic engineering and was created by human intervention.
  • a “recombinant DNA molecule” is a DNA molecule comprising a DNA sequence that does not naturally occur and as such is the result of human intervention, such as a DNA molecule comprising at least two DNA molecules heterologous to each other.
  • An example of a recombinant DNA molecule is a DNA molecule provided herein encoding an herbicide-tolerant protoporphyrinogen oxidase operably linked to a heterologous promoter.
  • a “recombinant protein” is a protein comprising an amino acid sequence that does not naturally occur and as such is the result of human intervention, such as an engineered protein.
  • a recombinant cell, seed, or organism is a cell, seed, or organism comprising transgenic or heterologous DNA or protein, for example a transgenic plant cell, seed, or plant comprising a DNA construct or engineered protein described herein.
  • wild-type means a naturally occurring.
  • a “wild-type DNA molecule,” “wild-type protein” is a naturally occurring version of a DNA molecule or protein, that is, a version of a DNA molecule or protein pre-existing in nature.
  • a wild-type version of a DNA molecule or protein may be useful for comparison with a recombinant or engineered DNA molecule or protein.
  • An example of a wild-type protein useful for comparison with the engineered proteins provided by the present disclosure is the PPO enzyme from E. cloacae (H_N90) provided as SEQ ID NO:l.
  • a “wild-type plant” is a naturally occurring plant. Such wild-type plants may also be useful for comparison with a plant comprising a recombinant or engineered DNA molecule or protein.
  • An example of a wild-type plant useful for comparison with plants comprising a recombinant or engineered DNA molecule or protein may be a plant of the same type as the plant comprising the engineered DNA molecule or protein, such as a protein conferring an herbicide tolerance trait, and as such is genetically distinct from the plant comprising the herbicide tolerance trait.
  • control plants means an experimental control designed for comparison purposes.
  • a control plant in a transgenic plant analysis is a plant of the same type as the experimental plant (that is, the plant to be tested) but does not contain the transgenic insert, recombinant DNA molecule, or DNA construct of the experimental plant.
  • Examples of control plants useful for comparison with transgenic plants include: for maize plants, non-transgenic LH244 maize (U.S. Patent No. 6,252,148) or non-transgenic 01DKD2 maize (U.S. Patent No.
  • DNA refers to a double-stranded DNA molecule of genomic or synthetic origin (that is, a polymer of deoxyribonucleotide bases or a polynucleotide molecule) read from the 5' (upstream) end to the 3' (downstream) end.
  • DNA sequence refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to that of by Title 37 of the United States Code of Federal Regulations ⁇ 1.822, and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.
  • protein-coding DNA molecule refers to a DNA molecule comprising a DNA sequence that encodes a protein.
  • protein refers to a chain of amino acids linked by peptide (amide) bonds and includes both polypeptide chains that are folded or arranged in a biologically functional way and polypeptide chains that are not.
  • a “protein-coding sequence” means a DNA sequence that encodes a protein.
  • a “sequence” means a sequential arrangement of nucleotides or amino acids.
  • a “DNA sequence” may refer to a sequence of nucleotides or to the DNA molecule comprising of a sequence of nucleotides; a “protein sequence” may refer to a sequence of amino acids or to the protein comprising a sequence of amino acids.
  • the boundaries of a protein-coding sequence are usually determined by a translation start codon at the 5 '-terminus and a translation stop codon at the 3'-terminus.
  • the term “isolated” refers to at least partially separating a molecule from other molecules typically associated with it in its natural state.
  • the term “isolated” refers to a DNA molecule that is separated from the nucleic acids that normally flank the DNA molecule in its natural state.
  • a DNA molecule encoding a protein that is naturally present in a bacterium would be an isolated DNA molecule if it was not within the DNA of the bacterium from which the DNA molecule encoding the protein is naturally found.
  • DNA molecule fused to or operably linked to one or more other DNA molecule(s) with which it would not be associated in nature is considered isolated herein.
  • Such molecules are considered isolated even when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules.
  • DNA molecules, or fragment thereof can also be obtained by other techniques, such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer.
  • DNA sequences can encode proteins, such as the altered or engineered proteins disclosed herein. It is well within the capability of one of skill in the art to create alternative DNA sequences encoding the same, or essentially the same, altered or engineered proteins as described herein. These variant or alternative DNA sequences are within the scope of the embodiments described herein. As used herein, references to “essentially the same” sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions that do not materially alter the functional activity of the protein encoded by the DNA molecule of the embodiments described herein.
  • nucleotide sequences encoding a wild-type or engineered protein are also encompassed within the scope of the embodiments described herein.
  • Substitution of amino acids other than those specifically exemplified or naturally present in a wild-type or engineered PPO enzyme are also contemplated within the scope of the embodiments described herein, so long as the PPO enzyme having the substitution still retains substantially the same functional activity described herein.
  • Recombinant DNA molecules of the present disclosure may be synthesized and modified by methods known in the art, either completely or in part, where it is desirable to provide sequences useful for DNA manipulation (such as restriction enzyme recognition sites or recombination-based cloning sites), plant-preferred sequences (such as plant-codon usage or Kozak consensus sequences), or sequences useful for DNA construct design (such as spacer or linker sequences).
  • sequences useful for DNA manipulation such as restriction enzyme recognition sites or recombination-based cloning sites
  • plant-preferred sequences such as plant-codon usage or Kozak consensus sequences
  • sequences useful for DNA construct design such as spacer or linker sequences.
  • the present disclosure includes recombinant DNA molecules and engineered proteins having at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, and at least 99% sequence identity to any of the recombinant DNA molecule or amino acid sequences provided herein, and having herbicide-tolerant protoporphyrinogen oxidase activity.
  • percent sequence identity refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or amino acid sequence of a reference (“query”) sequence (or its complementary strand) as compared to a test (“subject”) sequence (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide or amino acid insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison).
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the Sequence Analysis software package of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA), MEGAlign (DNAStar Inc., 1228 S.
  • tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the Sequence Analysis software package of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA), MEGAlign (DNAStar Inc., 1228 S.
  • An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in the portion of the reference sequence segment being aligned, that is, the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more sequences may be to a full-length sequence or a portion thereof, or to a longer sequence.
  • a “DNA construct” is a recombinant DNA molecule comprising two or more heterologous DNA sequences.
  • DNA constructs are useful for transgene expression and may be comprised in vectors and plasmids.
  • DNA constructs may be used in vectors for transformation (that is, the introduction of heterologous DNA into a host cell) to produce recombinant bacteria or transgenic plants and cells (and as such may also be contained in the plastid DNA or genomic DNA of a transgenic plant, seed, cell, or plant part).
  • a “vector” means any recombinant DNA molecule that may be used for bacterial or plant transformation.
  • DNA molecules provided by the present disclosure can, for example, be inserted into a vector as part of a DNA construct having the DNA molecule operably linked to a heterologous gene expression element that functions in a plant to affect expression of the engineered protein encoded by the DNA molecule.
  • Methods for making and using DNA constructs and vectors are well known in the art and described in detail in, for example, handbooks and laboratory manuals including Green and Sambrook, “Molecular Cloning: A Laboratory Manual” Vol. 1, 4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012.
  • the components for a DNA construct, or a vector comprising a DNA construct include one or more gene expression elements operably linked to a transcribable nucleic acid sequence, such as the following: a promoter for the expression of an operably linked DNA, an operably linked protein-coding DNA molecule, and an operably linked 3’ untranslated region (UTR).
  • Gene expression elements useful in practicing the present disclosure include, but are not limited to, one or more of the following type of elements: promoter, 5’ UTR, enhancer, leader, cis-acting element, intron, transit sequence, 3’ UTR, and one or more selectable marker transgenes.
  • transgenic refers to a DNA molecule artificially incorporated into the genome of an organism as a result of human intervention, such as by plant transformation methods.
  • transgenic means comprising a transgene, for example a “transgenic plant” refers to a plant comprising a transgene in its genome and a “transgenic trait” refers to a characteristic or phenotype conveyed or conferred by the presence of a transgene incorporated into the plant genome.
  • a transgenic plant refers to a plant comprising a transgene in its genome
  • a “transgenic trait” refers to a characteristic or phenotype conveyed or conferred by the presence of a transgene incorporated into the plant genome.
  • Transgenic plants of the present disclosure comprise the recombinant DNA molecules and proteins described herein.
  • heterologous refers to the relationship between two or more things not normally associated in nature, for instance that are derived from different sources or not normally found in nature together in any other manner.
  • a DNA molecule or protein may be heterologous with respect to another DNA molecule, protein, cell, plant, seed, or organism if not normally found in nature together or in the same context.
  • a first DNA molecule is heterologous to a second DNA molecule if the two DNA molecules are not normally found in nature together in the same context.
  • a protein-coding recombinant DNA molecule is heterologous with respect to an operably linked promoter if such a combination is not normally found in nature.
  • a protein is heterologous with respect to a second operably linked protein, such as a transit peptide, if such combination is not normally found in nature.
  • a recombinant DNA molecule encoding a PPO enzyme is heterologous with respect to an operably linked promoter that is functional in a plant cell if such combination is not normally found in nature.
  • a recombinant DNA molecule also may be heterologous with respect to a cell, seed, or organism into which it is inserted when it would not naturally occur in that cell, seed, or organism.
  • heterologous protein is a protein present in a plant, seed, cell, tissue, or organism in which it does not naturally occur or operably linked to a protein with which it is not naturally linked.
  • heterologous proteins are the PPO enzymes described herein that is expressed in any plant, seed, cell, tissue, or organism.
  • Another example is a protein operably linked to a second protein, such as a transit peptide or herbicide-tolerant protein, with which it is not naturally linked, or a protein introduced into a plant cell in which it does not naturally occur using the techniques of genetic engineering.
  • operably linked means two or more DNA molecules or two or more proteins linked in manner so that one may affect the function of the other.
  • Operably linked DNA molecules or operably linked proteins may be part of a single contiguous molecule and may or may not be adjacent.
  • a promoter is operably linked with a protein-coding DNA molecule in a DNA construct where the two DNA molecules are so arranged that the promoter may affect the expression of the transgene.
  • the DNA constructs described herein may include a promoter operably linked to a protein-coding DNA molecule provided herein, whereby the promoter drives expression of the protein.
  • Useful promoters include those that function in a cell for expression of an operably linked DNA molecule, such as a bacterial or plant promoter. Plant promoters are varied and well known in the art and include, for instance, those that are inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, or spatio-temporally regulated.
  • a DNA construct provided herein includes a DNA sequence encoding a transit sequence that is operably linked to a heterologous DNA sequence encoding a PPO enzyme, whereby the transit sequence facilitates localizing the protein molecule within the cell.
  • Transit sequences are known in the art as signal sequences, targeting peptides, targeting sequences, localization sequences, and transit peptides.
  • An example of a transit sequence is a chloroplast transit peptide (CTP), a mitochondrial transit sequence (MTS), or a dual chloroplast and mitochondrial transit peptide.
  • the transit sequence may increase the accumulation of recombinant protein, protect the protein from proteolytic degradation, or enhance the level of herbicide tolerance, and thereby reduce levels of injury in the cell, seed, or organism after herbicide application.
  • CTPs and other targeting molecules that may be used in connection with the present disclosure are well known in the art.
  • transgene expression means the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into polypeptide chains, which are ultimately folded into proteins.
  • mRNA messenger RNA
  • a protein-coding DNA molecule may be operably linked to a heterologous promoter in a DNA construct for use in expressing the protein in a cell transformed with the recombinant DNA molecule.
  • cells, tissues, plants, and seeds that comprising the recombinant DNA molecules or proteins are provided herein. These cells, tissues, plants, and seeds comprising the recombinant DNA molecules or proteins exhibit tolerance to one or more PPO inhibiting herbicide(s).
  • weeds unwanted plants
  • An ideal treatment would be one which could be applied to an entire field but which would eliminate only the unwanted plants while leaving the crop plants unaffected.
  • One such treatment system would involve the use of crop plants which are tolerant to an herbicide so that when the herbicide is sprayed on a field of herbicide-tolerant crop plants, the crop plants would continue to thrive while non-herbicide-tolerant weeds are killed or severely damaged.
  • such treatment systems would take advantage of varying herbicide properties so that weed control could provide the best possible combination of flexibility and economy.
  • herbicides have different longevities in the field, and some herbicides persist and are effective for a relatively long time after they are applied to a field while other herbicides are quickly broken down into other and/or nonactive compounds.
  • An ideal treatment system would allow the use of different herbicides so that growers could tailor the choice of herbicides for a particular situation.
  • While a number of herbicide-tolerant crop plants are presently commercially available, one issue that has arisen for many commercial herbicides and herbicide/crop combinations is that individual herbicides typically have incomplete spectrum of activity against common weed species.
  • One method of producing such cells, tissues, plants, and seeds is through plant transformation.
  • Suitable methods for transformation of host plant cells for use with the current disclosure include any method by which DNA can be introduced into a cell (for example, where a recombinant DNA construct is stably integrated into a plant chromosome) and are well known in the art.
  • Two effective, and widely utilized, methods for cell transformation are Agrobacterium- mediated transformation and microprojectile bombardment-mediated transformation. Microprojectile bombardment methods are illustrated, for example, in US Patent Nos. 5,550,318; 5,538,880; 6,160,208; and 6,399,861.
  • Agrobacterium-mediated transformation methods are described, for example in US Patent No. 5,591,616.
  • a cell with a recombinant DNA molecule or protein of the present disclosure may be selected for the presence of the recombinant DNA molecule or protein, for instance through its encoded enzymatic activity, before or after regenerating such a cell into a plant.
  • Another method of producing the cells, plants, and seeds of the present disclosure is through genome modification using site-specific integration or genome editing.
  • Targeted modification of plant genomes through the use of genome editing methods can be used to create improved plant lines through modification of plant genomic DNA.
  • site-directed integration refers to genome editing methods the enable targeted insertion of one or more nucleic acids of interest into a plant genome. Suitable methods for altering a wild-type DNA sequence or a preexisting transgenic sequence or for inserting DNA into a plant genome at a pre-determined chromosomal site include any method known in the art.
  • Exemplary methods include the use of sequence specific nucleases, such as zinc-finger nucleases, engineered or native meganucleases, TALE-endonucleases, or an RNA-guided endonucleases (for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)/Cas9 system, a CRISPR/Cpfl system, a CRISPR/CasX system, a CRISPR/CasY system, a CRISPR/Cascade system).
  • sequence specific nucleases such as zinc-finger nucleases, engineered or native meganucleases, TALE-endonucleases, or an RNA-guided endonucleases (for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)/Cas9 system, a CRISPR/Cpfl system, a CRISPR/CasX system, a CRISPR/CasY
  • the present disclosure provides modification or replacement of an existing coding sequence, such as a PPO coding sequence or another existing transgenic insert, within a plant genome with a sequence encoding a protein, such as a PPO coding sequence of the present disclosure, or an expression cassette comprising such a protein.
  • an existing coding sequence such as a PPO coding sequence or another existing transgenic insert
  • RNA-guided endonucleases for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)/Cas9 system, a CRISPR/Cpfl system, a CRISPR/CasX system, a CRISPR/CasY system, a CRISPR/Cascade system.
  • CRISPR Clustered Regularly Interspersed Short Palindromic Repeat
  • RNA construct comprising an expression cassette(s) encoding a site-specific nuclease and, optionally, any associated protein(s) to carry out genome modification.
  • These nuclease-expressing cassette(s) may be present in the same molecule or vector as a donor template for templated editing or an expression cassette comprising nucleic acid sequence encoding a PPO protein as described herein (in cis) or on a separate molecule or vector (in trans).
  • DSB double strand break
  • nick a desired genomic site or locus.
  • the donor template DNA, transgene, or expression cassette may become integrated into the genome at the site of the DSB or nick.
  • the presence of the homology arm(s) in the DNA to be integrated may promote the adoption and targeting of the insertion sequence into the plant genome during the repair process through homologous recombination, although an insertion event may occur through non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • double-strand break inducing agent refers to any agent that can induce a double-strand break (DSB) in a DNA molecule.
  • the doublestrand break inducing agent is a site-specific genome modification enzyme.
  • site-specific genome modification enzyme refers to any enzyme that can modify a nucleotide sequence in a sequence-specific manner.
  • a site-specific genome modification enzyme modifies the genome by inducing a single-strand break.
  • a site-specific genome modification enzyme modifies the genome by inducing a double-strand break.
  • a site-specific genome modification enzyme comprises a cytidine deaminase.
  • a site-specific genome modification enzyme comprises an adenine deaminase.
  • sitespecific genome modification enzymes include endonucleases, recombinases, transposases, deaminases, helicases and any combination thereof.
  • the site-specific genome modification enzyme is a sequence-specific nuclease.
  • the endonuclease is selected from a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nucleases (TALEN), an Argonaute (non-limiting examples of Argonaute proteins include Therrnus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), an RNA-guided nuclease, such as a CRISPR associated nuclease (non-limiting examples of CRISPR associated nucleases include Cast, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, C
  • the site-specific genome modification enzyme is a recombinase.
  • recombinases include a tyrosine recombinase attached to a DNA recognition motif and is selected from the group consisting of a Cre recombinase, a Gin recombinase, a Flp recombinase, and a Tnpl recombinase.
  • a Cre recombinase or a Gin recombinase provided herein is tethered to a zinc-finger DNA-binding domain, or a TALE DNA-binding domain, or a Cas9 nuclease.
  • a serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • a DNA transposase attached to a DNA binding domain provided herein is selected from the group consisting of a TALE-piggyBac and TALE- Mutator.
  • Any of the DNA of interest provided herein can be integrated into a target site of a chromosome sequence by introducing the DNA of interest and the provided site-specific genome modification enzymes. Any method provided herein can utilize any site-specific genome modification enzyme provided herein.
  • a “weed” is any undesired plant.
  • a plant may be considered generally undesirable for agriculture or horticulture purposes (for example, Amaranthus species) or may be considered undesirable in a particular situation (for example, a crop plant of one species in a field of a different species, also known as a volunteer plant).
  • transgenic plants, progeny, seeds, plant cells, and plant parts described herein may also contain one or more additional traits. Additional traits may be introduced by crossing a plant containing a transgene comprising the recombinant DNA molecules provided herein with another plant containing one or more additional trait(s). As used herein, “crossing” means breeding two individual plants to produce a progeny plant. Two plants may thus be crossed to produce progeny that contain the desirable traits from each parent. As used herein “progeny” means the offspring of any generation of a parent plant, and transgenic progeny comprise a DNA construct provided herein and inherited from at least one parent plant.
  • Additional trait(s) also may be introduced by co-transforming a DNA construct for that additional transgenic trait(s) with a DNA construct comprising the recombinant DNA molecules provided herein (for example, with all the DNA constructs present as part of the same vector used for plant transformation) or by inserting the additional trait(s) into a transgenic plant comprising a DNA construct provided by the herein or vice versa (for example, by using any of the methods of plant transformation or genome editing on a transgenic plant or plant cell).
  • additional traits include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, and herbicide tolerance, in which the trait is measured with respect to a wild-type plant.
  • Illustrative additional herbicide-tolerance traits may include transgenic or non-transgenic tolerance to one or more herbicides such as ACCase inhibitors (for example aryloxyphenoxy propionates and cyclohexanediones), ALS inhibitors (for example sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones) EPSPS inhibitors (for example glyphosate), synthetic auxins (for example phenoxys, benzoic acids, carboxylic acids, semicarbazones), photosynthesis inhibitors (for example triazines, triazinones, nitriles, benzothiadiazoles, and ureas), glutamine synthesis inhibitors (for example glufosinate), HPPD inhibitors (for example isoxazoles, pyrazolones, and triketones), PPO inhibitors (for example diphenylethers, N-phenylphthalimide, aryl
  • herbicide-tolerance proteins useful for producing additional herbicidetolerance traits are well known in the art and include, but are not limited to, glyphosate-tolerant 5- enolypyruvyl shikimate 3-phosphate synthases (e.g., CP4 EPSPS, 2mEPSPS), glyphosate oxidoreductases (GOX), glyphosate N-acetyltransferases (GAT), herbicide-tolerant acetolactate synthases (ALS) / acetohydroxyacid synthases (AHAS), herbicide-tolerant 4- hydroxyphenylpyruvate dioxygenases (HPPD), dicamba monooxygenases (DMO), phosphinothricin acetyl transferases (PAT), herbicide-tolerant glutamine synthetases (GS), 2,4- dichlorophenoxyproprionate dioxygenases (TfdA), R-2,4-dichlorophenoxypropionate dioxygenases (R
  • Exemplary insect resistance traits may include resistance to one or more insect members within one or more of the orders of Lepidoptera, Coleoptera, Hemiptera, Thysanoptera, Diptera, Hymenoptera, and Orthoptera, among others.
  • additional traits are well known to one of skill in the art; for example, and a list of such transgenic traits is provided by the United States Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS).
  • Transgenic plants and progeny that are tolerant to PPO inhibiting herbicides may be used with any breeding methods that are known in the art.
  • the traits may be independently segregating, linked, or a combination of both in plant lines comprising three or more transgenic traits.
  • Backcrossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of breeding methods that are commonly used for different traits and crops are well known to those of skill in the art.
  • Such assays include, for example, molecular biology assays, such as Southern and Northern blotting, PCR, and DNA sequencing; biochemical assays, such as detecting the presence of a protein product, for example, by immunological means (ELISAs and western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole plant.
  • molecular biology assays such as Southern and Northern blotting, PCR, and DNA sequencing
  • biochemical assays such as detecting the presence of a protein product, for example, by immunological means (ELISAs and western blots) or by enzymatic function
  • plant part assays such as leaf or root assays
  • analyzing the phenotype of the whole plant include, for example, molecular biology assays, such as Southern and Northern blotting, PCR, and DNA sequencing; biochemical assays, such as detecting the presence of a protein product, for example,
  • Introgression of a transgenic trait into a plant genotype is achieved as the result of the process of backcross conversion.
  • a plant genotype into which a transgenic trait has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid.
  • a plant genotype lacking the desired transgenic trait may be referred to as an unconverted genotype, line, inbred, or hybrid.
  • Novel microbial HemG protoporphyrinogen oxidases that are tolerant to PPO inhibitor herbicides were previously identified from microbial sequence databases using bioinformatic methods and an herbicide bacterial screening system and are provided as SEQ ID NOs:l-20 and recombinant variants of these microbial HemG protoporphyrinogen oxidases are provided as SEQ ID NOs:65-193.
  • DNA sequences encoding microbial HemG protoporphyrinogen oxidases and their variants, along with DNA sequences that are optimized for expression in a monocot or dicot can optionally be synthesized and are provided as SEQ ID NOs:22-64 and 194-322.
  • a codon for a methionine commonly known as a start codon
  • this codon (and optionally a few amino-terminal amino acids, for example 2 to 7), can be eliminated to facilitate operable linkage of a transit peptide sequence to the 5’ end of the coding sequence.
  • Novel transit peptides were previously identified by using bioinformatic methods and tools, such as hidden Markov models (HMM), the Pfam database, and basic local alignment search tool (BLAST), to identify thousands of EST and genomic sequences predicted to encode proteins known to be localized to the chloroplast and mitochondria in plant cells and are provided herein as SEQ ID NOs: 323-328, 340, and 342-407, along with their corresponding nucleotide sequences, provided herein as SEQ ID NOs:329-339, 341, and 408-483.
  • HMM hidden Markov models
  • BLAST basic local alignment search tool
  • Protoporphyrinogen oxidases operably linked to transit peptides are tested in transgenic soybean, com, and cotton plants for tolerance to PPO inhibiting herbicides and for weed control in the field.
  • Plant transformation vectors were constructed for expressing a chloroplast transit peptide operably linked to the PPO H_N90 (SEQ ID NO:1) in transgenic soybean, inbred com, hybrid com, and cotton plants, and introduced into seed-derived explants of soybean, inbred and hybrid com, and cotton, respectively, through Agrobacterium tumefaciens-mQdi transformation using standard methods known in the art.
  • the regenerated Ro plants were analyzed to select for events with a single copy insertion for advancement to Ri nursery for Ri seed production.
  • Plants were visually assessed for herbicide injury at the following time points: 7 days, 14 days, 21 days, and 28 days after application. Unsprayed transgenic plants were used for phenotypic comparison with unsprayed wild-type plants. Injury rating was determined as the percentage of leaf area of a plant exhibiting damage such as necrosis (brown or dead tissue), chlorosis (yellow tissue or yellow spotting), and malformation (misshapen leaves or plant structures, epinasty or twisting of stem, cupping of leaves) caused by herbicide application and was measured on a scale of 0-100, with zero being no injury and 100 being complete crop death. The data obtained at 14 days after application are shown in Table 1 below.
  • H_N90 conferred to transgenic soybean, inbred com, hybrid com, and cotton plants
  • Protoporphyrinogen oxidases operably linked to transit peptides are tested in transgenic soybean, com, and cotton plants for tolerance to PPO inhibiting herbicides and for weed control in the field.
  • Plant transformation vectors are constructed for expressing a chloroplast transit peptide operably linked to a HemG PPO enzyme, such as H_N90 (SEQ ID NO:1) in transgenic soybean, com, and cotton plants, and introduced into seed-derived explants of soybean, com, and cotton, respectively, through Agrobacterium tumefaciens-mediated transformation using standard methods known in the art.
  • the regenerated Ro plants are analyzed to select for events with a single copy insertion for advancement to Ri nursery for Ri seed production. Homozygous Ri or later generation events are tested under field conditions to confirm their tolerance to PPO inhibitor herbicides.
  • substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, such as any of the compounds of Al, A2, and A3 as described herein, and a commercial PPO inhibitor herbicide (such as saflufenacil) are applied at emergence (VE), V3, and RI developmental stages at one of two rates. Plants are visually assessed for herbicide injury 14 days after treatment for VE, and 7 days after treatment for V3 and RI stages. Unsprayed transgenic plants are used for phenotypic comparison with unsprayed wild-type plants.
  • substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, such as any of the compounds of Al, A2, and A3 as described herein, and a commercial PPO inhibitor herbicide (such as fomesafen) are applied to transgenic inbred or hybrid plants at emergence (VE), V2, V6, and VT developmental stages at one of two rates. Plants are visually assessed for herbicide injury 10-14 days after herbicide treatment. Unsprayed transgenic plants are used for phenotypic comparison with unsprayed wild-type plants.
  • substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, such as any of the compounds of Al, A2, and A3 as described herein, and a commercial PPO inhibitor herbicide (such as fomesafen) are applied at pre-emergence (PRE), V4, and V8 developmental stages at one of two rates. Plants are visually assessed for herbicide injury 10-14 days after herbicide treatment. Unsprayed transgenic plants are used for phenotypic comparison with unsprayed wild-type plants.
  • PPO inhibitor herbicide such as fomesafen
  • the injury rating is determined as the percentage of leaf area of a plant exhibiting damage such as necrosis (brown or dead tissue), chlorosis (yellow tissue or yellow spotting), and malformation (misshapen leaves or plant structures, epinasty or twisting of stem, cupping of leaves) caused by herbicide application and is measured on a scale of 0-100, with zero being no injury and 100 being complete crop death.
  • Transgenic crop seeds conferring PPO inhibiting herbicide tolerance prepared as described above are planted in a field or crop growing area.
  • PPO inhibiting herbicide formulations such as substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, such as any of the compounds of Al, A2, and A3 as described herein, are applied to the field or crop growing area before or/and after planting the seeds to control weed growth.
  • the herbicide application comprises an effective amount of at least one PPO inhibiting herbicide that prevents or controls the growth of weeds, but does not damage or injure the transgenic soybean, com, or cotton crop plants comprising a recombinant DNA molecule encoding a chloroplast-targeted heterologous HemG protein.
  • the PPO inhibiting herbicides can be applied, once or more than once, at about IX application rate. However, the rate may be adjusted or varied depending on environmental conditions (such as temperature and humidity) and the type of weeds being controlled, as is known in the art.
  • the application rate may vary within wide limits, and may consist of a range from about 0.02 g a.i./ha to about 750 g a.i./ha, about 0.05 g a.i./ha to about 400 g a.i./ha, about 0.25 g a.i./ha to about 300 g a.i./ha, about 0.3 g a.i./ha to about 250 g a.i./ha, about 0.4 g a.i./ha to about 150 g a.i./ha, or about 0.5 g a.i./ha to about 120 g a.i./ha.
  • a desired application rate in any particular environment or in the context of a particular weed can be determined empirically by one of skill in the art in view of the present disclosure.
  • the herbicide rate is carefully selected to avoid 1) over use than what is needed, which could lead to injury to the herbicide tolerant crop(s); 2) under use, resulting in poor weed control, which could lead to development of herbicide tolerant weeds.
  • the PPO inhibiting herbicides may be applied pre-emergence and/or post emergence. In the case of post emergence application, the PPO inhibiting herbicides may be applied over the top of the crop growing area.
  • an effective amount of at least a second herbicide can be applied for weed control.
  • a second herbicide include, but are not limited to, an ACCase inhibitor (such as an aryloxyphenoxy propionate or a cyclohexanedione), an ALS inhibitor (such as sulfonylurea, imidazolinone, triazoloyrimidine, or a triazolinone), an EPSPS inhibitor (such as glyphosate), a synthetic auxin (such as a phenoxy herbicide, a benzoic acid, a carboxylic acid, or a semicarbazone), a photosynthesis inhibitor (such as a triazine, a triazinone, a nitrile, a benzothiadiazole, or a urea), a glutamine synthetase inhibitor (such as glufosinate), a HPPD inhibitor (such as an iso

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Abstract

La présente invention concerne l'utilisation de certaines uracile phényle substituées comprenant une chaîne latérale à base d'acide cyclopropylcarboxylique, ou des sels agrochimiquement acceptables de celle-ci, pour commander ou prévenir la croissance de mauvaises herbes dans des zones de croissance de plantes de plantes cultivées transgéniques qui sont tolérantes aux herbicides inhibiteurs de PPO. L'invention concerne également des plantes transgéniques tolérantes aux herbicides, des graines, des cellules et des parties de plante contenant les molécules d'ADN recombinant, ainsi que des procédés d'utilisation de celles-ci.
PCT/US2023/070191 2022-07-22 2023-07-14 Procédés et compositions pour la tolérance aux herbicides ppo WO2024020316A2 (fr)

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