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WO2025160032A2 - Plantes ayant une tolérance accrue aux herbicides - Google Patents

Plantes ayant une tolérance accrue aux herbicides

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Publication number
WO2025160032A2
WO2025160032A2 PCT/US2025/012342 US2025012342W WO2025160032A2 WO 2025160032 A2 WO2025160032 A2 WO 2025160032A2 US 2025012342 W US2025012342 W US 2025012342W WO 2025160032 A2 WO2025160032 A2 WO 2025160032A2
Authority
WO
WIPO (PCT)
Prior art keywords
plant
ppo
herbicide
plants
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/012342
Other languages
English (en)
Other versions
WO2025160032A3 (fr
Inventor
Aimone PORRI
Philipp Rudi JOHNEN
Ingo MEINERS
Jens Lerchl
Brian JENKS
Samuel D. WILLINGHAM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
North Dakota State University NDSU
BASF Agricultural Solutions Seed US LLC
Original Assignee
North Dakota State University NDSU
BASF Agricultural Solutions Seed US LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by North Dakota State University NDSU, BASF Agricultural Solutions Seed US LLC filed Critical North Dakota State University NDSU
Publication of WO2025160032A2 publication Critical patent/WO2025160032A2/fr
Publication of WO2025160032A3 publication Critical patent/WO2025160032A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention relates in general to methods for conferring on plants agricultural level tolerance to herbicides.
  • the invention refers to plants having an increased tolerance to PPO-inhibiting herbicides.
  • the present invention relates to methods and plants obtained by mutagenesis and crossbreeding and transformation that have an increased tolerance to PPO-inhibiting herbicides.
  • Protox or PPO Herbicides that inhibit protoporphyrinogen oxidase (hereinafter referred to as Protox or PPO; EC: 1.3.3.4), a key enzyme in the biosynthesis of protoporphyrin IX, have been used for selective weed control since the 1960s.
  • PPO catalyzes the last common step in chlorophyll and heme biosynthesis which is the oxidation of protoporphyrinogen IX to protoporphyrin IX. (Matringe et al. 1989. Biochem. 1. 260: 231).
  • PPO-inhibiting herbicides include many different structural classes of molecules (Duke et al. 1991 . Weed Sci. 39: 465;
  • lactofen (+-)-2-ethoxy-1-methyl-2-oxoethyl 5- ⁇ 2-chloro-4-(trifluoromethyl)phenoxy ⁇ -2-nitrobenzoate; acifluorfen, 5- ⁇ 2-chloro-4-(trifluoromethyl)phenoxy ⁇ -2-nitrobenzoic acid; its methyl ester; or oxyfluorfen, 2-chloro- 1-(3-ethoxy-4-nitrophenoxy)-4-(trifluorobenzene) ⁇ , oxidiazoles, (e.g.
  • oxidiazon 3- ⁇ 2,4-dichloro-5-(1- methylethoxy)phenyl ⁇ -5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one
  • cyclic imides e.g. S-23142, N-(4- chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide; chlorophthalim, N-(4-chlorophenyl)- 3,4,5,6-tetrahydrophthalimide
  • phenyl pyrazoles e.g.
  • protoporphyrinogen IX in rganelles containing PPO enzymel, which is believed to leak into the cytosol where it is oxidized by a peroxidase.
  • protoporphyrin IX When exposed to light, protoporphyrin IX causes formation of singlet oxygen in the cytosol and the formation of other reactive oxygen species, which can cause lipid peroxidation and membrane disruption leading to rapid cell death (Lee et al. 1993. Plant Physiol. 102: 881).
  • PPO enzymes are sensitive to herbicides which inhibit plant PPO enzymes. Both the Escherichia coli and Bacillus subtilis PPO enzymes (Sasarmen et al. 1993. Can. J. Microbiol. 39: 1155; Dailey et al. 1994. J. Biol. Chem. 269: 813) are resistant to these herbicidal inhibitors. Also many fungi including yeasts are insensitive to PPO type herbicides. Mutants of the unicellular alga Chlamydomonas reinhardtii resistant to the phenylimide herbicide S-23142 have been reported (Kataoka et al. 1990. J. Pesticide Sci. 15: 449; Shibata et al. 1992.
  • WO 2012/080975 discloses plants the tolerance of which to a PPO-inhibiting herbicide named (1 ,5-dimethyl-6- thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4- dione) had been increased by transforming said plants with nucleic acids encoding mutated PPO mutated enzymes.
  • WO 2012/080975 discloses that the introduction of nucleic acids which code for a mutated PPO of an Amaranthus type II PPO in which the Arginine at position 128 had been replaced by a leucine, alanine, or valine, and the phenylalanine at position 420 had been replaced by a methionine, cysteine, isoleucine, leucine, or threonine, confers increased tolerance/resistance to a benzoxazinone-derivative herbicide.
  • WO 2013/189984 discloses plants the tolerance of which to PPO inhibitors had been increased by transforming said plants with nucleic acids encoding mutated PPO enzymes, in which the Leucine corresponding to position 397 of an Amaranthus type II PPO had been replaced, and the phenylalanine corresponding to position 420 of an Amaranthus type II PPO had been replaced.
  • WO2015/022636 discloses plants the tolerance of which to PPO inhibitors had been increased by transforming said plants with nucleic acids encoding mutated PPO enzymes, in which the Arginine corresponding to position 128 of an Amaranthus type II PPO had been replaced, and the phenylalanine corresponding to position 420 of an Amaranthus type II PPO had been replaced, but the replacement occurred with amino acids, which are different from those disclosed in WO 2012/080975.
  • WO2015/092706 describes PPO polypeptides from a plurality of organisms, which PPO polypeptides had been mutated to comprise the advantageous mutations employed for the Amaranthus type II PPO.
  • W02015/022640 discloses PPO polypeptides from Alopecurus myosuroides and mutants thereof, which confer tolerance to a broad spectrum of PPO inhibiting herbicides.
  • WO2019/106568 describes that those types of mutants confer increased tolerance/resistance to a new class of PPO inhibitors, known as uracilpyridines or uracilpyridine herbicides.
  • a mutated PPO polypeptide namely a PPO1 popypeptide from the species Kochia (Bassia scoparia)
  • Kochia Bath scoparia
  • the problem is solved by the present invention which refers to a method for controlling undesired vegetation at a plant cultivation site, the method comprising the steps of: a) providing, at said site, a plant that comprises at least one nucleic acid comprising a nucleotide sequence encoding a protoporphyrinogen oxidase (PPO) polypeptide which is resistant or tolerant to a “PPO inhibiting herbicide”; b) applying to said site an effective amount of said herbicide, wherein the resistant or tolerant PPO polypeptide is characterized in that the amino acid at a position corresponding to position 454 of SEQ ID NO: 2 is substituted by lie, Leu, or Vai; and wherein the effective amount of said herbicide does not kill or inhibit the growth of the herbicide-tolerant plant of a).
  • PPO protoporphyrinogen oxidase
  • the herbicide resistant or tolerant PPO polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8, or a variant, homologue, or orthologue thereof.
  • the herbicide resistant or tolerant PPO polypeptide is encoded by a nucleic acid molecule comprising a sequence being, over the full length, at least 80% identical to the nucleic acid sequence of SEQ ID NO: 1, 3, 5, or 7, or a variant, homologue, or orthologue thereof thereof.
  • Another object refers to a method of identifying a nucleotide sequence encoding a mutated PPO which is resistant or tolerant to a PPO inhibiting herbicide, the method comprising: a) generating a library of mutated PPO-encoding nucleic acids, b) screening a population of the resulting mutated PPO-encoding nucleic acids by expressing each of said nucleic acids in a cell or plant and treating said cell or plant with a PPO inhibiting, c) comparing the PPO inhibiting herbicide-tolerance levels provided by said population of mutated PPO encoding nucleic acids with the PPO inhibitor-tolerance level provided by a control PPO-encoding nucleic acid, d) selecting at least one mutated PPO-encoding nucleic acid that provides a significantly increased level of tolerance to a PPO inhibitor as compared to that provided by the control PPO-encoding nucleic acid.
  • the mutated PPO-encoding nucleic acid selected in step d) provides at least 2-fold as much tolerance to a PPO inhibitor as compared to that provided by the control PPO-encoding nucleic acid.
  • the resistance or tolerance can be determined by generating a transgenic plant population comprising a nucleic acid sequence of the library of step a) and comparing said transgenic plant population with a control plant.
  • the invention refers to a plant cell transformed by and expressing a PPO nucleic acid according to the present invention or a plant which has been mutated to obtain a plant expressing, preferably over-expressing a wild-type or a mutated PPO nucleic acid according to the present invention, wherein expression of said nucleic acid in the plant cell results in increased resistance or tolerance to a PPO inhibitor as compared to a wild type variety of the plant cell.
  • the invention refers to a plant that expresses a mutagenized or recombinant mutated PPO polypeptide, and wherein said mutated PPO polypeptide confers upon the plant increased PPO inhibitor tolerance as compared to the corresponding wild-type variety of the plant when expressed therein.
  • the invention refers to a transgenic plant comprising a plant cell according to the present invention, wherein expression of the nucleic acid in the plant results in the plant’s increased resistance to PPO inhibitor herbicide as compared to a wild type variety of the plant.
  • the expression of the nucleic acid of the invention in the plant results in the plant’s increased resistance to PPO inhibitor herbicides as compared to a wild type variety of the plant.
  • the invention refers to a method for growing the plant according to the present invention while controlling weeds in the vicinity of said plant, said method comprising the steps of: a) growing said plant ; and b) applying a herbicide composition comprising a PPO inhibitor herbicide to the plant and weeds, wherein the herbicide normally inhibits protoporphyrinogen oxidase, at a level of the herbicide that would inhibit the growth of a corresponding wild-type plant.
  • the invention refers to a seed produced by a transgenic plant comprising a plant cell of the present invention, or to a seed produced by the non-transgenic plant that expresses a mutagenized PPO polypeptide, wherein the seed is true breeding for an increased resistance to a PPO inhibitor herbicide as compared to a wild type variety of the seed.
  • the invention refers to a method of producing a transgenic plant cell with an increased resistance to a PPO inhibitor herbicide as compared to a wild type variety of the plant cell comprising, transforming the plant cell with an expression cassette comprising a wild-type or a mutated PPO nucleic acid.
  • the invention refers to a method of producing a transgenic plant comprising, (a) transforming a plant cell with an expression cassette comprising a wild-type or a mutated PPO nucleic acid, and (b) generating a plant with an increased resistance to PPO inhibitor herbicide from the plant cell.
  • the expression cassette further comprises a transcription initiation regulatory region and a translation initiation regulatory region that are functional in the plant.
  • the invention relates to using the mutated PPO of the invention as selectable marker.
  • the invention provides a method of identifying or selecting a transformed plant cell, plant tissue, plant or part thereof comprising a) providing a transformed plant cell, plant tissue, plant or part thereof, wherein said transformed plant cell, plant tissue, plant or part thereof comprises an isolated nucleic acid encoding a mutated PPO polypeptide of the invention as described hereinafter, wherein the polypeptide is used as a selection marker, and wherein said transformed plant cell, plant tissue, plant or part thereof may optionally comprise a further isolated nucleic acid of interest; b) contacting the transformed plant cell, plant tissue, plant or part thereof with at least one PPO-inhibiting inhibiting compound, preferably a PPO inhibitor; c) determining whether the plant cell, plant tissue, plant or part thereof is affected by the inhibitor or inhibiting compound; and d) identifying or selecting the transformed plant cell, plant tissue, plant or part thereof.
  • the invention is also embodied in purified mutated PPO proteins that contain the mutations described herein, which are useful in molecular modeling studies to design further improvements to herbicide tolerance.
  • Methods of protein purification are well known, and can be readily accomplished using commercially available products or specially designed methods, as set forth for example, in Protein Biotechnology, Walsh and Headon (Wiley, 1994).
  • the invention in another embodiment, relates to a combination useful for weed control, comprising (a) a polynucleotide encoding a mutated PPO polypeptide according to the present invention, which polynucleotide is capable of being expressed in a plant to thereby provide to that plant tolerance to a PPO inhibitor herbicide; and (b) a PPO inhibitor herbicide.
  • the invention in another embodiment, relates to a process for preparing a combination useful for weed control comprising (a) providing a polynucleotide encoding a mutated PPO polypeptide according to the present invention, which polynucleotide is capable of being expressed in a plant to thereby provide to that plant tolerance to a PPO inhibitor herbicide; and (b) providing a PPO inhibitor herbicide.
  • said step of providing a polynucleotide comprises providing a plant containing the polynucleotide.
  • said step of providing a polynucleotide comprises providing a seed containing the polynucleotide.
  • said process further comprises a step of applying the PPO inhibitor herbicide to the seed.
  • the invention relates to the use of a combination useful for weed control, comprising (a) a polynucleotide encoding a mutated PPO polypeptide according to the present invention, which polynucleotide is capable of being expressed in a plant to thereby provide to that plant tolerance to a PPO inhibitor herbicide; and (b) a PPO inhibitor herbicide, to control weeds at a plant cultivation site.
  • Figure 1 shows the ectopic expression of PPO1 F454I (A), F454L (B) and F454V (C), expressed under the control of the Pcllbi promoter (Petroselinum crispum Ubiquitin), leading to tolerance to Saflufenacil in postemergence; two plants at the bottom in the white square represent WT non transgenics. In the upper part, 5 transgenics events are shown 21 days after herbicide treatment. Amounts of saflufenacil are indicated in g/ha.
  • Figure 2 shows the ectopic expression of PPO1 F454I (A), F454L (B) and F454V (C), expressed under the control of the PcUbi promoter, leading to tolerance to carfentrazone in postemergence; two plants at the bottom in the white square represent WT non transgenics. In the upper part, 5 transgenics events are shown 21 days after herbicide treatment. Amounts of carfentrazone are indicated in g/ha.
  • Figure 3 shows the ectopic expression of PPO1 F454I (A), F454L (B) and F454V (C), expressed under the control of the Pcllbi promoter leading to tolerance to flumioxazin in postemergence; two plants at the bottom in the white square represent WT non transgenics. In the upper part, 5 transgenics events are shown 21 days after herbicide treatment. Amounts of flumioxazin are indicated in g/ha.
  • an element means one or more elements.
  • the inventors of the present invention have found, that the tolerance or resistance of a plant to a PPO inhibitor herbicide could be remarkably increased by overexpressing a nucleic acid encoding PPO polypeptides described hereinafter.
  • the present invention refers to a method for controlling undesired vegetation at a plant cultivation site, the method comprising the steps of: a) providing, at said site, a plant that comprises at least one nucleic acid comprising a nucleotide sequence encoding a protoporphyrinogen oxidase (PPO) polypeptide which is resistant or tolerant to a “PPO inhibiting herbicide”; b) applying to said site an effective amount of said herbicide, wherein the resistant or tolerant PPO polypeptide is characterized in that the amino acid at a position corresponding to position 454 of SEQ ID NO: 2 is substituted by lie, Leu, or Vai; and wherein the effective amount of said herbicide does not kill or inhibit the growth of the herbicide-tolerant plant of a).
  • PPO protoporphyrinogen oxidase
  • control of undesired vegetation is to be understood as meaning the killing of weeds and/or otherwise retarding or inhibiting the normal growth of the weeds.
  • Weeds in the broadest sense, are understood as meaning all those plants which grow in locations where they are undesired, e.g. (crop) plant cultivation sites.
  • the weeds of the present invention include, for example, dicotyledonous and monocotyledonous weeds.
  • Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum.
  • Monocotyledonous weeds include, but are not limited to, weeds of of the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera.
  • the weeds of the present invention can include, for example, crop plants that are growing in an undesired location.
  • a volunteer maize plant that is in a field that predominantly comprises soybean plants can be considered a weed, if the maize plant is undesired in the field of soybean plants.
  • plant is used in its broadest sense as it pertains to organic material and is intended to encompass eukaryotic organisms that are members of the Kingdom Plantae, examples of which include but are not limited to vascular plants, vegetables, grains, flowers, trees, herbs, bushes, grasses, vines, ferns, mosses, fungi and algae, etc, as well as clones, offsets, and parts of plants used for asexual propagation (e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.).
  • asexual propagation e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.
  • plant further encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, florets, fruits, pedicles, peduncles, stamen, anther, stigma, style, ovary, petal, sepal, carpel, root tip, root cap, root hair, leaf hair, seed hair, pollen grain, microspore, cotyledon, hypocotyl, epicotyl, xylem, phloem, parenchyma, endosperm, a companion cell, a guard cell, and any other known organs, tissues, and cells of a plant, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
  • Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp.
  • Avena sativa e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida
  • Averrhoa carambola e.g. Bambusa sp.
  • Benincasa hispida Bertholletia excelsea
  • Beta vulgaris Brassica spp.
  • Brassica napus e.g. Brassica napus, Brassica rapa ssp.
  • the plant is a crop plant.
  • crop plants include inter alia soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco.
  • the plant is a monocotyledonous plant, such as sugarcane.
  • the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.
  • the plant has been previously produced by a process comprising recombinantly preparing a plant by introducing and over-expressing a wild-type or mutated PPG transgene according to the present invention, as described in greater detail hereinafter.
  • the nucleic acids of the invention find use in enhancing the herbicide tolerance of plants that comprise in their genomes a gene encoding a herbicide-tolerant wild-type or mutated PPO protein.
  • a gene may be an endogenous gene or a transgene, as described hereinafter.
  • the present invention refers to a method of increasing or enhancing the PPO inhibitor herbicide tolerance or resistance of a plant, the method comprising overexpressing a nucleic acid encoding a herbicide resistant or tolerant PPO polypeptide which comprises comprises a sequence being, over the full length, at least 80% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8, or a variant, homologue or orthologue thereof.
  • polynucleotides that may be stacked with the nucleic acids of the present invention include nucleic acids encoding polypeptides conferring resistance to pests/pathogens such as viruses, nematodes, insects or fungi, and the like.
  • Exemplary polynucleotides that may be stacked with nucleic acids of the invention include polynucleotides encoding: polypeptides having pesticidal and/or insecticidal activity, such as other Bacillus thuringiensis toxic proteins (described in U.S. Pat. Nos.
  • acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations
  • glyphosate resistance e.g., 5-enol-pyrovyl-shikimate-3-phosphate-synthase (EPSPS) gene, described in U.S. Pat. Nos. 4,940,935 and 5,188,642; or the glyphosate N-acetyltransferase (GAT) gene, described in Castle et al. (2004) Science, 304:1151-1154; and in U.S. Patent App. Pub. Nos.
  • EPSPS 5-enol-pyrovyl-shikimate-3-phosphate-synthase
  • GAT glyphosate N-acetyltransferase
  • glufosinate resistance e.g, phosphinothricin acetyl transferase genes PAT and BAR, described in U.S. Pat. Nos.
  • the plant comprises at least one additional heterologous nucleic acid comprising a nucleotide sequence encoding a herbicide tolerance enzyme selected, for example, from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), cytochrome P450 monooxygenase, phosphinothricin acetyltransferase (PAT), Acetohydroxyacid synthase (AHAS; EC 4.1.3.18, also known as acetolactate synthase or ALS), hydroxyphenyl pyruvate dioxygenase (HPPD), Phytoene desaturase (PD) and dicamba degrading enzymes as disclosed in WO 02/068607, or phenoxyaceticacid- and phenoxypropionicacid-derivative degrading enzymes as disclosed in WO 2008141154 or WO 2005107437
  • EPSPS
  • the term “herbicide” is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies the growth of plants.
  • the preferred amount or concentration of the herbicide is an "effective amount” or “effective concentration.”
  • effective amount and concentration is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a similar, wild-type, plant, plant tissue, plant cell, or host cell, but that said amount does not kill or inhibit as severely the growth of the herbicideresistant plants, plant tissues, plant cells, and host cells of the present invention.
  • the effective amount of a herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest. Such an amount is known to those of ordinary skill in the art.
  • Herbicidal activity is exhibited by herbicides useful for the present invention when they are applied directly to the plant or to the locus of the plant at any stage of growth or before planting or emergence. The effect observed depends upon the plant species to be controlled, the stage of growth of the plant, the application parameters of dilution and spray drop size, the particle size of solid components, the environmental conditions at the time of use, the specific compound employed, the specific adjuvants and carriers employed, the soil type, and the like, as well as the amount of chemical applied. These and other factors can be adjusted as is known in the art to promote non-selective or selective herbicidal action. Generally, it is preferred to apply the herbicide postemergence to relatively immature undesirable vegetation to achieve the maximum control of weeds.
  • a herbicide-tolerant or “herbicide-resistant” plant it is intended that a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill, or inhibit the growth of, a normal or wild-type plant.
  • herbicide-tolerant wildtype or mutated PPO protein or “herbicide -resistant wildtype or mutated PPO protein”
  • PPO activity of such a herbicide-tolerant or herbicide-resistant mutated PPO protein may be referred to herein as "herbicide-tolerant” or “herbicide-resistant” PPO activity.
  • the herbicides useful for the present invention refer to the following PPO inhibitors: acifluorfen, acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxyfen, chlorphthalim, cinidon-ethyl, cyclopyranil, fluazolate, flufenoximacil, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoroglycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, profluazol,
  • the PPO inhibitors useful for the present invention may be mixed with a large number of representatives of other herbicidal or growth-regulating active ingredient groups and then applied concomitantly.
  • Suitable components for mixtures are, for example, herbicides from the classes of the acetamides, amides, aryloxyphenoxypropionates, benzamides, benzofuran, benzoic acids, benzothiadiazinones, bipyridylium, carbamates, chloroacetamides, chlorocarboxylic acids, cyclohexanediones, dinitroanilines, dinitrophenol, diphenyl ether, glycines, imidazolinones, isoxazoles, isoxazolidinones, nitriles, N-phenylphthalimides, oxadiazoles, oxazolidinediones, oxyacetamides, phenoxycarboxylic acids,
  • PPO inhibitors alone or in combination with other herbicides, or else in the form of a mixture with other crop protection agents, for example together with agents for controlling pests or phytopathogenic fungi or bacteria.
  • miscibility with mineral salt solutions which are employed for treating nutritional and trace element deficiencies.
  • Other additives such as non-phytotoxic oils and oil concentrates may also be added.
  • the further herbicidal compound B is preferably selected from the herbicides of class b1) to b15): b1) lipid biosynthesis inhibitors; b2) acetolactate synthase inhibitors (ALS inhibitors); b3) photosynthesis inhibitors; b4) protoporphyrinogen-IX oxidase inhibitors, b5) bleacher herbicides; b6) enolpyruvyl shikimate 3-phosphate synthase inhibitors (EPSP inhibitors); b7) glutamine synthetase inhibitors; b8) 7,8-dihydropteroate synthase inhibitors (DHP inhibitors); b9) mitosis inhibitors; b10) inhibitors of the synthesis of very long chain fatty acids (VLCFA inhibitors); b11) cellulose biosynthesis inhibitors; b12) decoupler herbicides; b13) auxinic herbicides; b14) auxin transport inhibitors; and b15) other herbicides selected from the herbicides
  • herbicides B which can be used in combination with the PPO inhibitors useful for the present invention are: b1) from the group of the lipid biosynthesis inhibitors:
  • ACC-herbicides such as alloxydim, alloxydim-sodium, butroxydim, clethodim, clodinafop, clodinafop-propargy I, cycloxydim, cyhalofop, cyhalofop-butyl, diclofop, diclofop-methyl, fenoxaprop, fenoxaprop-ethyl, fenoxaprop-P, fenoxaprop-P-ethyl, fluazifop, fluazifop-butyl, fluazifop-P, fluazifop-P-butyl, haloxyfop, haloxyfop-methyl, haloxyfop-P, haloxyfop-P-methyl, metamifop, pinoxaden, profoxydim, propaquizafop, quizalofop, quizalofop- eth
  • a preferred embodiment of the invention relates to those compositions comprising at least one aryl urea herbicide.
  • a preferred embodiment of the invention relates to those compositions comprising at least one triazine herbicide.
  • a preferred embodiment of the invention relates to those compositions comprising at least one nitrile herbicide; b4) from the group of the protoporphyrinogen-IX oxidase inhibitors: acifluorfen, acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxyfen, chlorphtalim, cinidon-ethyl, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin,
  • PDS inhibitors beflubutamid, diflufenican, fluridone, flurochloridone, flurtamone, norflurazon, picolinafen, and 4- (3-trifluoromethylphenoxy)-2-(4-trifluoromethylphenyl)pyrimidine (CAS 180608-33-7), HPPD inhibitors: benzobicyclon, benzofenap, bicyclopyrone, clomazone, fenquinotrione, isoxaflutole, mesotrione, oxotrione (CAS 1486617-21-3), pyrasulfotole, pyrazolynate, pyrazoxyfen, sulcotrione, tefuryltrione, tembotrione, tolpyralate, topramezone , bleacher, unknown target: aclonifen, amitrole flumeturon and 2-chloro-3-methylsulfanyl-N-(1 - methyltetrazol-5-y
  • chloroacetamides and oxyacetamides preference is given to chloroacetamides and oxyacetamides; b11) from the group of the cellulose biosynthesis inhibitors: chlorthiamid, dichlobenil, flupoxam, indaziflam, isoxaben, triaziflam and 1-cyclohexyl-5-pentafluorphenyloxy-1 4 - [1 ,2,4,6]thiatriazin-3-ylamine (CAS 175899-01-1); b12) from the group of the decoupler herbicides: dinoseb, dinoterb and DNOC and its salts; b13) from the group of the auxinic herbicides:
  • 2,4-D and its salts and esters such as clacyfos, 2,4-DB and its salts and esters, aminocyclopyrachlor and its salts and esters, aminopyralid and its salts such as aminopyralid-dimethylammonium, aminopyralid-tris(2- hydroxypropyl)ammonium and its esters, benazolin, benazolin-ethyl, chloramben and its salts and esters, clomeprop, clopyralid and its salts and esters, dicamba and its salts and esters, dichlorprop and its salts and esters, dichlorprop-P and its salts and esters, flopyrauxifen, fluroxypyr, fluroxypyr-butometyl, fluroxypyr-mepty I, halauxifen and its salts and esters (CAS 943832-60-8); MCPA and its salts and esters, MCPA-thioethyl, MCPB and its
  • Safeners are chemical compounds which prevent or reduce damage on useful plants without having a major impact on the herbicidal action of the PPO inhibitors towards unwanted plants. They can be applied either before sowings (e.g. on seed treatments, shoots or seedlings) or in the pre-emergence application or post-emergence application of the useful plant.
  • the safeners and the PPO inhibitors and optionally the herbicides B can be applied simultaneously or in succession.
  • Suitable safeners are e.g. (quinolin-8-oxy)acetic acids, 1 -phenyl-5-haloalkyl-1 H-1 ,2,4-triazol-3-carboxylic acids, 1 -phenyl-4,5-dihydro-5-alkyl-1 H-pyrazol-3,5-dicarboxylic acids, 4,5-dihydro-5,5-diaryl-3-isoxazol carboxylic acids, dichloroacetamides, alpha-oximinophenylacetonitriles, acetophenonoximes, 4,6-dihalo-2- phenylpyrimidines, N-[[4-(aminocarbonyl)phenyl]sulfonyl]-2-benzoic amides, 1 ,8-naphthalic anhydride, 2-halo-4- (haloalkyl)-5-thiazol carboxylic acids, phosphorthiolates and N-alkyl-O-phenylc
  • Examples of preferred safeners C are benoxacor, cloquintocet, cyometrinil, cyprosulfamide, dichlormid, dicyclonon, dietholate, fenchlorazole, fenclorim, flurazole, fluxofenim, furilazole, isoxadifen, mefenpyr, mephenate, naphthalic anhydride, oxabetrinil, 4-(dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane (MON4660, CAS 71526-07-3), 2,2,5-trimethyl-3-(dichloroacetyl)-1 ,3-oxazolidine (R-29148, CAS 52836-31-4), metcamifen and BPCMS (CAS 54091-06-4); especially preferred benoxacor, cloquintocet, cyometrinil, cyprosulfamide, dichlormid, dicyclonon, dietholate
  • safeners C which, as component C, are constituent of the composition according to the invention are the safeners C as defined above; in particular the safeners C.1 - C.17 listed below in table C:
  • the active compounds B of groups b1) to b15) and the active compounds C are known herbicides and safeners, see, for example, The Compendium of Pesticide Common Names (http://www.alanwood.net/pesticides/); Farm Chemicals Handbook 2000 volume 86, Meister Publishing Company, 2000; B. Hock, C. Fedtke, R. R. Schmidt, Herbizide [Herbicides], Georg Thieme Verlag, Stuttgart 1995; W. H. Ahrens, Herbicide Handbook, 7th edition, Weed Science Society of America, 1994; and K. K. Hatzios, Herbicide Handbook, Supplement for the 7th edition, Weed Science Society of America, 1998.
  • the assignment of the active compounds to the respective mechanisms of action is based on current knowledge. If several mechanisms of action apply to one active compound, this substance was only assigned to one mechanism of action.
  • the compounds of the invention in combination with herbicides that are selective for the crop being treated and which complement the spectrum of weeds controlled by these compounds at the application rate employed. It is further generally preferred to apply the compounds of the invention and other complementary herbicides at the same time, either as a combination formulation or as a tank mix.
  • polypeptides and polypeptides of the invention encompass polypeptides comprising an amino acid sequence that is sufficiently identical to the amino acid sequences set forth in SEQ ID Nos: 2, 4, 6, or 8.
  • the term "sufficiently identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity.
  • sequence identity refers to the extent to which two optimally aligned DNA or amino acid sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids.
  • 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 reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. "Percent identity” is the identity fraction times 100.
  • 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 preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG. Wisconsin Package. (Accelrys Inc. Burlington, Mass.)
  • an “isolated polynucleotide”, including DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, dsRNA or otherwise we mean a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state. That means other nucleic acid molecules are present in an amount less than 5% based on weight of the amount of the desired nucleic acid, preferably less than 2% by weight, more preferably less than 1 % by weight, most preferably less than 0.5% by weight.
  • an “isolated” nucleic acid is free of some of the sequences that naturally flank the nucleic acid (i.e., sequences located at the 5’ and 3’ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated herbicide resistance and/or tolerance related protein encoding nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an “isolated” nucleic acid molecule such as a cDNA molecule
  • the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
  • an isolated polynucleotide can be an exogenous polynucleotide present in, for example, a transgenic organism which does not naturally comprise the polynucleotide.
  • nucleic acid sequence(s) refers to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.
  • mutated PPG nucleic acid refers to a PPO nucleic acid having a sequence that is mutated from a wild-type PPO nucleic acid; and that confers increased PPO inhibitor herbicide tolerance to a plant in which it is expressed.
  • mutated protoporphyrinogen oxidase refers to the replacement of an amino acid of the wild-type primary sequence of SEQ ID NO: 2, depicted in SEQ ID NO:s 4, or 6, or a variant, a derivative, a homologue, an orthologue, or paralogue thereof, with another amino acid.
  • the expression "mutated amino acid” will be used below to designate the amino acid which is replaced by another amino acid, thereby designating the site of the mutation in the primary sequence of the protein.
  • the PPO nucleotide sequence encoding a mutated PPO comprises the sequence of SEQ ID NO: 1, 3, 5, or 7, or a variant or derivative thereof.
  • PPO nucleotide sequences encompasse homologues, paralogues and and orthologues of SEQ ID NO: 1, 3, 5, or 7, as defined hereinafter.
  • variants with respect to a sequence (e.g., a polypeptide or nucleic acid sequence such as - for example - a transcription regulating nucleotide sequence of the invention) is intended to mean substantially similar sequences.
  • variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein, e.g. the mutated PPO according to the present invention as disclosed herein.
  • nucleotide sequence variants of the invention will have at least 30, 40, 50, 60, to 70%, e.g., preferably 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide “sequence identity” to the nucleotide sequence of : 1, 3, 5, or 7, or to a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2, 4, 6, or 8.
  • the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides.
  • the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides.
  • the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
  • substantially purified polypeptide or “purified” a polypeptide is meant that has been separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules with which it is associated in its native state. It is preferred that the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. As the skilled addressee will appreciate, the purified polypeptide can be a recombinantly produced polypeptide.
  • polypeptide and “protein” are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups.
  • polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors.
  • proteins and “polypeptides” as used herein also include variants, mutants, modifications, analogous and/or derivatives of the polypeptides of the invention as described herein.
  • the query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
  • the PPG polypeptide of the invention comprises an amino acid sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%,
  • variant polypeptide is intended a polypeptide derived from the protein of SEQ ID NO: 2, 4, 6, or 8, by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
  • “Derivatives” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
  • “Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
  • a deletion refers to removal of one or more amino acids from a protein.
  • Insertions refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues.
  • N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag*100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
  • a transcriptional activator as used in the yeast two-hybrid system
  • phage coat proteins phage coat proteins
  • glutathione S-transferase-tag glutathione S-transferase-tag
  • protein A maltose-binding protein
  • dihydrofolate reductase Tag*100 epitope
  • c-myc epitope
  • a substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or p-sheet structures).
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids; insertions will usually be of the order of about 1 to 10 amino acid residues.
  • the amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds).
  • Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
  • “Derivatives” further include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the protein of interest, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. “Derivatives” of a protein also encompass peptides, oligopeptides, polypeptides which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide.
  • a derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
  • “derivatives” also include fusions of the naturally-occurring form of the protein with tagging peptides such as FLAG, HIS6 or thioredoxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).
  • orthologues and “paralogues” encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.
  • domains refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
  • motif or “consensus sequence” refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).
  • GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
  • the BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI).
  • Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10;4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used.
  • sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters.
  • Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1); 195-7).
  • the proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) PNAS, 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.
  • variant nucleotide sequences can be made by introducing mutations randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened to identify mutants that encode proteins that retain activity. For example, following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.
  • the inventors of the present invention have found that by substituting one or more of the key amino acid residues of the PPO1 polypeptide of SEQ ID NO: 2 e.g. by employing one of the above described methods to mutate the PPO1 encoding nucleic acids, the tolerance or resistance to particular PPO-inhibiting herbicides, could be remarkably increased.
  • Preferred substitutions of mutated PPO1 are those that increase the herbicide tolerance of the plant, but leave the biological activitiy of the cellulose synthase activity substantially unaffected
  • PPO1 polypeptide comprising the sequence of SEQ ID NO: 2, 4, 6, or 8, a variant, derivative, orthologue, paralogue or homologue thereof, the key amino acid residues of which is substituted by any other amino acid.
  • a variant, derivative, orthologue, paralogue or homologue thereof comprises a mutated PPO1, wherein an amino acid ⁇ 3, ⁇ 2 or ⁇ 1 amino acid positions from a key amino acid is substituted by any other amino acid.
  • a highly characteristic sequence pattern can be developed, by means of which further of mutated PPO1 candidates with the desired activity may be searched.
  • the present sequence pattern is not limited by the exact distances between two adjacent amino acid residues of said pattern.
  • Each of the distances between two neighbours in the above patterns may, for example, vary independently of each other by up to ⁇ 10, ⁇ 5, ⁇ 3, ⁇ 2 or ⁇ 1 amino acid positions without substantially affecting the desired activity.
  • site directed mutagenesis in particular saturation mutagenes (see e.g. Schenk et al., Biospektrum 03/2006, pages 277-279) PCR based site-directed mutagenesis (e.g.
  • the inventors of the present invention have identified and generated specific amino acid subsitutions and combinations thereof, which - when introduced into a plant by transforming and expressing the respective mutated PPO1 encoding nucleic acid - confer increased herbicide resistance or tolerance to a PPO1 inhibiting herbicide to said plant.
  • the variant or derivative of the PPO1 polypeptide refers to a PPO1 polypeptide comprising SEQ ID NO: 2, a orthologue, paralogue, or homologue thereof, wherein the amino acid sequence differs from the wildtype amino acid sequence of a PPO1 polypeptide at position corresponding to position 454 of SEQ ID NO:2.
  • amino acid at or corresponding to position 454 of SEQ ID NO: 2 is substituted lie, Leu, or Vai.
  • amino acids can be chosen to be subsituted by any other amino acid.
  • PPO1 orthologues are for example those depicted in SEQ ID Nos: 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.
  • the present invention refers to a method for identifying a PPO inhibitor herbicide by using a herbicide tolerant PPO polypeptide as defined SUPRA.
  • Said method comprises the steps of: a) generating a transgenic cell or plant comprising a nucleic acid encoding a mutated PPO, wherein the mutated PPO is expressed; b) applying a PPO inhibitor herbicide to the transgenic cell or plant of a) and to a control cell or plant of the same variety; c) determining the growth or the viability of the transgenic cell or plant and the control cell or plant after application of said PPO inhibitor herbicide, and d) selecting “PPO inhibitor herbicides” which confer reduced growth to the control cell or plant as compared to the growth of the transgenic cell or plant.
  • control cell or “similar, wild-type, plant, plant tissue, plant cell or host cell” is intended a plant, plant tissue, plant cell, or host cell, respectively, that lacks the herbicide-resistance characteristics and/or particular polynucleotide of the invention that are disclosed herein.
  • wild-type is not, therefore, intended to imply that a plant, plant tissue, plant cell, or other host cell lacks recombinant DNA in its genome, and/or does not possess herbicide-resistant characteristics that are different from those disclosed herein.
  • Another object refers to a method of identifying a nucleotide sequence encoding a mutated PPO which is resistant or tolerant to a PPO inhibitor herbicide, the method comprising: a) generating a library of mutated PPO-encoding nucleic acids, b) screening a population of the resulting mutated PPO-encoding nucleic acids by expressing each of said nucleic acids in a cell or plant and treating said cell or plant with a PPO inhibitor herbicide, c) comparing the PPO inhibitor herbicide-tolerance levels provided by said population of mutated PPO encoding nucleic acids with the PPO inhibitor herbicide-tolerance level provided by a control PPO- encoding nucleic acid, d) selecting at least one mutated PPO-encoding nucleic acid that provides a significantly increased level of tolerance to a PPO inhibitor herbicide as compared to that provided by the control PPO-encoding nucleic acid.
  • the mutated PPO-encoding nucleic acid selected in step d) provides at least 2-fold as much resistance or tolerance of a cell or plant to a PPO inhibitor herbicide as compared to that provided by the control PPO-encoding nucleic acid.
  • the mutated PPO-encoding nucleic acid selected in step d) provides at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, as much resistance or tolerance of a cell or plant to a PPO inhibitor herbicide as compared to that provided by the control PPO-encoding nucleic acid.
  • the resistance or tolerance can be determined by generating a transgenic plant or host cell, preferably a plant cell, comprising a nucleic acid sequence of the library of step a) and comparing said transgenic plant with a control plant or host cell, preferably a plant cell.
  • Another object refers to a method of identifying a plant or algae containing a nucleic acid comprising a nucleotide sequence encoding a wild-type or mutated PPO which is resistant or tolerant to a PPO inhibitor herbicide, the method comprising: a) identifying an effective amount of a PPO inhibitor herbicide in a culture of plant cells or green algae that leads to death of said cells.
  • said mutagenizing agent is ethylmethanesulfonate (EMS).
  • Suitable candidate nucleic acids for identifying a nucleotide sequence encoding a mutated PPO from a variety of different potential source organisms including microbes, plants, fungi, algae, mixed cultures etc. as well as environmental sources of DNA such as soil.
  • These methods include inter alia the preparation of cDNA or genomic DNA libraries, the use of suitably degenerate oligonucleotide primers, the use of probes based upon known sequences or complementation assays (for example, for growth upon tyrosine) as well as the use of mutagenesis and shuffling in order to provide recombined or shuffled mutated PPO-encoding sequences.
  • Nucleic acids comprising candidate and control PPO encoding sequences can be expressed in yeast, in a bacterial host strain, in an alga or in a higher plant such as tobacco or Arabidopsis and the relative levels of inherent tolerance of the PPO encoding sequences screened according to a visible indicator phenotype of the transformed strain or plant in the presence of different concentrations of the selected PPO inhibitor herbicide.
  • Dose responses and relative shifts in dose responses associated with these indicator phenotypes are conveniently expressed in terms, for example, of GR50 (concentration for 50% reduction of growth) or MIC (minimum inhibitory concentration) values where increases in values correspond to increases in inherent tolerance of the expressed PPO.
  • each mutated PPO encoding sequence may be expressed, for example, as a DNA sequence under expression control of a controllable promoter such as the lacZ promoter and taking suitable account, for example by the use of synthetic DNA, of such issues as codon usage in order to obtain as comparable a level of expression as possible of different PPO sequences.
  • a controllable promoter such as the lacZ promoter
  • suitable account for example by the use of synthetic DNA, of such issues as codon usage in order to obtain as comparable a level of expression as possible of different PPO sequences.
  • Such strains expressing nucleic acids comprising alternative candidate PPO sequences may be plated out on different concentrations of the selected PPO inhibitor herbicide in, optionally, a tyrosine supplemented medium and the relative levels of inherent tolerance of the expressed PPO enzymes estimated on the basis of the extent and MIC for inhibition of the formation of the brown, ochronotic pigment. Additionally, the enodenous form of the PPO enzyme in a bacterial host can be inactivated.
  • candidate nucleic acids are transformed into plant material to generate a transgenic plant, regenerated into morphologically normal fertile plants which are then measured for differential tolerance to selected PPO inhibitor herbicides as described in the Example section hereinafter.
  • suitable selection markers such as kanamycin, binary vectors such as from Agrobacterium and plant regeneration as, for example, from tobacco leaf discs are well known in the art.
  • a control population of plants is likewise transformed with a nucleic acid expressing the control PPO.
  • an untransformed dicot plant such as Arabidopsis or Tobacco can be used as a control since this, in any case, expresses its own endogenous PPO.
  • the average, and distribution, of herbicide tolerance levels of a range of primary plant transformation events or their progeny to PPO inhibitor herbicides described supra are evaluated in the normal manner based upon plant damage, meristematic bleaching symptoms etc. at a range of different concentrations of herbicides.
  • These data can be expressed in terms of, for example, GR50 values derived from dose/response curves having "dose” plotted on the x-axis and “percentage kill", “herbicidal effect”, “numbers of emerging green plants” etc. plotted on the y-axis where increased GR50 values correspond to increased levels of inherent tolerance of the expressed PPO.
  • Herbicides can suitably be applied pre-emergence or postemergence.
  • the invention refers to a plant cell transformed by a nucleic acid encoding a herbicide tolerant PPO polypeptide disclosed herein or to a plant cell which has been mutated to obtain a plant expressing a nucleic acid encoding a mutated PPO polypeptide according to the present invention, wherein expression of the nucleic acid in the plant cell results in increased resistance or tolerance to a PPO inhibitor herbicide as compared to a wild type variety of the plant cell.
  • expression/expressing means the transcription of a specific gene or specific genes or specific genetic construct.
  • expression in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
  • the at least one nucleic acid is “over-expressed” by methods and means known to the person skilled in the art.
  • the term “increased expression” or “overexpression” as used herein means any form of expression that is additional to the original wild-type expression level. Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers.
  • Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non- heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the polypeptide of interest.
  • endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, US 5,565,350; Zarling et al., WO9322443), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
  • polypeptide expression it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
  • An intron sequence may also be added to the 5' untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol.
  • UTR 5' untranslated region
  • coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol.
  • Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1 :1183-1200).
  • Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit.
  • Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. For general information see: The Maize Handbook, Chapter 116, Freeling and Walbot,
  • introduction or “transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a genetic construct of the present invention and a whole plant regenerated there from.
  • the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • the polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome.
  • the resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
  • Transformation of plant species is now a fairly routine technique.
  • any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transforrmation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., (1982) Nature 296, 72-74; Negrutiu I et al.
  • Transgenic plants including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation.
  • An advantageous transformation method is the transformation in planta.
  • agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743).
  • Methods for Agrobacterium- mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.
  • nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin 19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
  • Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media.
  • plants used as a model like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media.
  • the transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White,
  • plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
  • the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
  • the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying.
  • a further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants.
  • the transformed plants are screened for the presence of a selectable marker such as the ones described above.
  • putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation.
  • expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
  • PPO nucleic acid comprises a polynucleotide sequence selected from the group consisting of: a) a polynucleotide comprising the sequence as shown in SEQ ID NO:1, 3, 5, or 7; b) a polynucleotide encoding a polypeptide as shown in SEQ ID NO:2, 4, 6, or 8, or a variant or derivative thereof; c) a polynucleotide comprising at least 60 consecutive nucleotides of any of a); and b) and d) a polynucleotide complementary to the polynucleotide of any of a) through c).
  • the expression of the nucleic acid in the plant results in the plant's increased resistance to PPO inhibitor herbicide as compared to a wild type variety of the plant.
  • the invention refers to a plant, preferably a transgenic plant, comprising a plant cell according to the present invention, wherein expression of the nucleic acid in the plant results in the plant's increased resistance to PPO inhibitor herbicide as compared to a wild type variety of the plant.
  • transgenic means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
  • genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or
  • (c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues in order to allow for the expression of the mutated PPO of the present invention.
  • the natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part.
  • the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp.
  • a naturally occurring expression cassette for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above - becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815.
  • transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously.
  • transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified.
  • Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place.
  • transgenic plants are mentioned herein.
  • transgenic refers to any plant, plant cell, callus, plant tissue, or plant part, that contains all or part of at least one recombinant polynucleotide. In many cases, all or part of the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations.
  • recombinant polynucleotide refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering.
  • Examples include any cloned polynucleotide, or polynucleotides, that are linked or joined to heterologous sequences.
  • the term “recombinant’ does not refer to alterations of polynucleotides that result from naturally occurring events, such as spontaneous mutations, or from non-spontaneous mutagenesis followed by selective breeding.
  • non-transgenic plants Plants containing mutations arising due to non-spontaneous mutagenesis and selective breeding are referred to herein as non-transgenic plants and are included in the present invention.
  • the nucleic acids can be derived from different genomes or from the same genome.
  • the nucleic acids are located on different genomes or on the same genome.
  • mutant refers to an organism or DNA thereof having alteration(s) in the biomolecular sequence of its native genetic material as compared to the sequence of the genetic material of a corresponding wild-type organism or DNA, wherein the alteration(s) in genetic material were induced and/or selected by human action.
  • Methods of inducing mutations can induce mutations in random positions in the genetic material or can induce mutations in specific locations in the genetic material (i.e., can be directed mutagenesis techniques), such as by use of a genoplasty technique.
  • the present invention involves herbidicide-resistant plants that are produced by mutation breeding.
  • Such plants comprise a polynucleotide encoding a mutated PPO and are tolerant to one or more PPO inhibitor herbicides.
  • Such methods can involve, for example, exposing the plants or seeds to a mutagen, particularly a chemical mutagen such as, for example, ethyl methanesulfonate (EMS) and selecting for plants that have enhanced tolerance to at least one or more PPO inhibitor herbicide.
  • EMS ethyl methanesulfonate
  • the present invention is not limited to herbicide-tolerant plants that are produced by a mutagenesis method involving the chemical mutagen EMS. Any mutagenesis method known in the art may be used to produce the herbicide-resistant plants of the present invention. Such mutagenesis methods can involve, for example, the use of any one or more of the following mutagens: radiation, such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (e.g., product of nuclear fission by uranium 235 in an atomic reactor), Beta radiation (e.g., emitted from radioisotopes such as phosphorus 32 or carbon 14), and ultraviolet radiation (preferably from 2500 to 2900 nm), and chemical mutagens such as base analogues (e.g., 5-bromo-uracil), related compounds (e.g., 8-ethoxy caffeine), antibiotics (e.g., streptonigrin), alkylating agents (e.
  • Herbicide-resistant plants can also be produced by using tissue culture methods to select for plant cells comprising herbicide-resistance mutations and then regenerating herbicide-resistant plants therefrom. See, for example, U.S. Patent Nos. 5,773,702 and 5,859,348, both of which are herein incorporated in their entirety by reference. Further details of mutation breeding can be found in "Principals of Cultivar Development” Fehr, 1993 Macmillan Publishing Company the disclosure of which is incorporated herein by reference
  • herbicide-resistant plants according to the present invention can also be produced by using genome editing methods to select for plant cells comprising herbicide-resistance mutations and then regenerating herbicide-resistant plants therefrom.
  • Gene Editing refers to a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using engineered nucleases. These nucleases are known to the skilled artisan to create site-specific double-strand breaks at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining or homologous recombination, resulting in targeted mutations.
  • the invention refers to a non-transgenic plant, comprising a plant cell according to the present invention, wherein expression of the nucleic acid encoding a mutated PPO in the plant results in the plant's increased resistance to PPO inhibitor herbicide as compared to a wild type variety of the plant.
  • plant is intended to encompass crop plants at any stage of maturity or development, as well as any tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context.
  • Plant parts include, but are not limited to, stems, roots, flowers, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, and the like.
  • the plant of the present invention comprises at least one mutated PPO nucleic acid or over-expressed wild-type PPO nucleic acid, and has increased tolerance to a PPO inhibitor herbicide as compared to a wild-type variety of the plant. It is possible for the plants of the present invention to have multiple wild-type or mutated PPO nucleic acids from different genomes since these plants can contain more than one genome. For example, a plant contains two genomes, usually referred to as the A and B genomes. Because PPO is a required metabolic enzyme, it is assumed that each genome has at least one gene coding for the PPO enzyme (i.e. at least one PPO gene).
  • PPO gene locus refers to the position of an PPO gene on a genome
  • PPO gene and PPO nucleic acid refer to a nucleic acid encoding the PPO enzyme.
  • the PPO nucleic acid on each genome differs in its nucleotide sequence from an PPO nucleic acid on another genome.
  • One of skill in the art can determine the genome of origin of each PPO nucleic acid through genetic crossing and/or either sequencing methods or exonuclease digestion methods known to those of skill in the art.
  • the present invention includes plants comprising one, two, three, or more mutated PPO alleles, wherein the plant has increased tolerance to a PPO inhibitor herbicide as compared to a wild-type variety of the plant.
  • the mutated PPO alleles can comprise a nucleotide sequence selected from the group consisting of a polynucleotide encoding a polypeptide as defined in SEQ ID NO: 2, 4, 6, or 8, or a variant or derivative, homologue, orthologue, paralogue thereof, a polynucleotide comprising at least 60 consecutive nucleotides of any of the aforementioned polynucleotides; and a polynucleotide complementary to any of the aforementioned polynucleotides.
  • Allelic variants are alternative forms of a given gene, located at the same chromosomal position. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small I nsertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms
  • cultivar or variety refers to a group of plants within a species defined by the sharing of a common set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one cultivar or variety from another cultivar or variety. There is no implication in either term that all plants of any given cultivar or variety will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci. A cultivar or variety is considered “true breeding” for a particular trait if, when the true-breeding cultivar or variety is self-pollinated, all of the progeny contain the trait.
  • breeding line or “line” refer to a group of plants within a cultivar defined by the sharing of a common set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one breeding line or line from another breeding line or line. There is no implication in either term that all plants of any given breeding line or line will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci.
  • a breeding line or line is considered “true breeding” for a particular trait if, when the true-breeding line or breeding line is self-pollinated, all of the progeny contain the trait. In the present invention, the trait arises from a mutation in a PPO gene of the plant or seed.
  • the present invention provides a method for producing a PPO inhibitor herbicides-tolerant progeny plant, the method comprising: crossing a parent plant with a PPO inhibitor herbicides-tolerant plant to introduce the PPO inhibitor herbicides-tolerance characteristics of the PPO inhibitor herbicides-tolerant plant into the germplasm of the progeny plant, wherein the progeny plant has increased tolerance to the PPO inhibitor herbicides relative to the parent plant.
  • the method further comprises the step of introgressing the PPO inhibitor herbicides-tolerance characteristics through traditional plant breeding techniques to obtain a descendent plant having the PPO inhibitor herbicides-tolerance characteristics.
  • the herbicide-resistant plants of the invention that comprise polynucleotides encoding mutated PPO polypeptides also find use in methods for increasing the herbicide-resistance of a plant through conventional plant breeding involving sexual reproduction.
  • the methods comprise crossing a first plant that is a herbicideresistant plant of the invention to a second plant that may or may not be resistant to the same herbicide or herbicides as the first plant or may be resistant to different herbicide or herbicides than the first plant.
  • the second plant can be any plant that is capable of producing viable progeny plants (i.e., seeds) when crossed with the first plant.
  • the first and second plants are of the same species.
  • the methods can optionally involve selecting for progeny plants that comprise the mutated PPO polypeptides of the first plant and the herbicide resistance characteristics of the second plant.
  • the progeny plants produced by this method of the present invention have increased resistance to a herbicide when compared to either the first or second plant or both.
  • the progeny plants will have the combined herbicide tolerance characteristics of the first and second plants.
  • the methods of the invention can further involve one or more generations of backcrossing the progeny plants of the first cross to a plant of the same line or genotype as either the first or second plant.
  • the progeny of the first cross or any subsequent cross can be crossed to a third plant that is of a different line or genotype than either the first or second plant.
  • the present invention also provides plants, plant organs, plant tissues, plant cells, seeds, and non-human host cells that are transformed with the at least one polynucleotide molecule, expression cassette, or transformation vector of the invention.
  • Such transformed plants, plant organs, plant tissues, plant cells, seeds, and non-human host cells have enhanced tolerance or resistance to at least one herbicide, at levels of the herbicide that kill or inhibit the growth of an untransformed plant, plant tissue, plant cell, or non-human host cell, respectively.
  • the transformed plants, plant tissues, plant cells, and seeds of the invention are Arabidopsis thaliana and crop plants.
  • plants of the invention include those plants which, in addition to being tolerant to PPO inhibitor herbicides, have been subjected to further genetic modifications by breeding, mutagenesis or genetic engineering, e.g. have been rendered tolerant to applications of specific other classes of herbicides, such as AHAS inhibitors; auxinic herbicides; bleaching herbicides such as hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors or phytoene desaturase (PDS) inhibitors; EPSPS inhibitors such as glyphosate; glutamine synthetase (GS) inhibitors such as glufosinate; lipid biosynthesis inhibitors such as acetyl CoA carboxylase (ACCase) inhibitors; or oxynil ⁇ i.e.
  • AHAS inhibitors such as AHAS inhibitors
  • auxinic herbicides such as hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors or phytoene desaturase (PDS) inhibitors
  • PPO inhibitor herbicides-tolerant plants of the invention can be made resistant to multiple classes of herbicides through multiple genetic modifications, such as resistance to both glyphosate and glufosinate or to both glyphosate and a herbicide from another class such as HPPD inhibitors, AHAS inhibitors, or ACCase inhibitors.
  • PPO inhibitor herbicides-tolerant plants of the invention may be tolerant to ACCase inhibitors, such as "dims” ⁇ e.g., cycloxydim, sethoxydim, clethodim, or tepraloxydim), "fops” ⁇ e.g. , clodinafop, diclofop, fluazifop, haloxyfop, or quizalofop), and "dens” (such as pinoxaden); to auxinic herbicides, such as dicamba; to EPSPS inhibitors, such as glyphosate; to other PPO inhibitors; and to GS inhibitors, such as glufosinate.
  • ACCase inhibitors such as "dims” ⁇ e.g., cycloxydim, sethoxydim, clethodim, or tepraloxydim
  • fops ⁇ e.g. , clodinafop, diclo
  • Such tolerance traits may be expressed, e.g. : as mutant or wildtype PPO proteins, as mutant AHASL proteins, mutant ACCase proteins, mutant EPSPS proteins, or mutant glutamine synthetase proteins; or as mutant native, inbred, or transgenic aryloxyalkanoate dioxygenase (AAD or DHT), haloarylnitrilase (BXN), 2,2- dichloropropionic acid dehalogenase (DEH), glyphosate-N- acetyltransferase (GAT), glyphosate decarboxylase (GDC), glyphosate oxidoreductase (GOX), glutathione-S-transferase (GST), phosphinothricin acetyltransferase (PAT or bar), or CYP450s proteins having an herbicide-degrading activity.
  • AAD or DHT transgenic aryloxyalkanoate dioxygenas
  • PPO inhibitor herbicides- tolerant plants hereof can also be stacked with other traits including, but not limited to, pesticidal traits such as Bt Cry and other proteins having pesticidal activity toward coleopteran, lepidopteran, nematode, or other pests; nutrition or nutraceutical traits such as modified oil content or oil profile traits, high protein or high amino acid concentration traits, and other trait types known in the art.
  • pesticidal traits such as Bt Cry and other proteins having pesticidal activity toward coleopteran, lepidopteran, nematode, or other pests
  • nutrition or nutraceutical traits such as modified oil content or oil profile traits, high protein or high amino acid concentration traits, and other trait types known in the art.
  • PPO inhibitor herbicides-tolerant plants are also covered which are, by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such characteristics, rendered able to synthesize one or more insecticidal proteins, especially those known from the bacterial genus Bacillus, particularly from Bacillus thuringiensis, such as [delta]-endotoxins, e.g. CrylA(b), CrylA(c), CrylF, CrylF(a2), CryllA(b), CrylllA, CrylllB(bl) or Cry9c; vegetative insecticidal proteins (VIP), e.g.
  • VIP vegetative insecticidal proteins
  • insecticidal proteins contained in the genetically modified plants impart to the plants producing these proteins tolerance to harmful pests from all taxonomic groups of arthropods, especially to beetles (Coeloptera), two-winged insects (Diptera), and moths (Lepidoptera) and to nematodes (Nematoda).
  • expression of one or more protein toxins (e.g., insecticidal proteins) in the PPO inhibitor herbicides-tolerant plants is effective for controlling organisms that include, for example, members of the classes and orders: Coleoptera such as the American bean weevil Acanthoscelides obtectus; the leaf beetle Agelastica alni; click beetles (Agriotes lineatus, Agriotes obscurus, Agriotes bicolor); the grain beetle Ahasverus advena; the summer schafer Amphimallon solstitialis; the furniture beetle Anobium punctatum; Anthonomus spp.
  • Coleoptera such as the American bean weevil Acanthoscelides obtectus
  • click beetles Agriotes lineatus, Agriotes obscurus, Agriotes bicolor
  • weevils the Pygmy mangold beetle Atomaria linearis; carpet beetles (Anthrenus spp., Attagenus spp.); the cowpea weevil Callosobruchus maculates; the fried fruit beetle Carpophilus hemipterus; the cabbage seedpod weevil Ceutorhynchus assimilis; the rape winter stem weevil Ceutorhynchus picitarsis; the wireworms Conoderus vespertinus and Conoderus falli; the banana weevil Cosmopolites sordidus; the New Zealand grass grub Costelytra zealandica; the June beetle Cotinis nitida; the sunflower stem weevil Cylindrocopturus adspersus; the larder beetle Dermestes lardarius; the corn rootworms Diabrotica virgifera, Diabrotica virgifera virgifera, and Diabro
  • the pollen beetle Meligethes aeneus the common cockshafer Melolontha melolontha; the American spider beetle Mezium americanum; the golden spider beetle Niptus hololeuc s; the grain beetles Oryzaephilus surinamensis and Oryzaephilus Mercator; the black vine weevil Otiorhynchus sulcatus; the mustard beetle Phaedon cochleariae, the crucifer flea beetle Phyllotreta cruciferae; the striped flea beetle Phyllotreta striolata; the cabbage steam flea beetle Psy Diodes chrysocephala; Ptinus spp.
  • expression of one or more protein toxins (e.g., insecticidal proteins) in the PPO inhibitor herbicides-tolerant plants is effective for controlling flea beetles, i.e. members of the flea beetle tribe of family Chrysomelidae, preferably against Phyllotreta spp., such as Phyllotreta cruciferae and/or Phy llotreta triolata.
  • expression of one or more protein toxins ⁇ e.g., insecticidal proteins) in the PPO inhibitor herbicides- tolerant plants is effective for controlling cabbage seedpod weevil, the Bertha armyworm, Lygus bugs, or the diamondback moth.
  • PPO inhibitor herbicides-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, rendered able to synthesize one or more proteins to increase the resistance or tolerance of those plants to bacterial, viral or fungal pathogens.
  • the methods for producing such genetically modified plants are generally known to the person skilled in the art.
  • PPO inhibitor herbicides-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, rendered able to synthesize one or more proteins to increase the productivity (e.g. oil content), tolerance to drought, salinity or other growth- limiting environmental factors or tolerance to pests and fungal, bacterial or viral pathogens of those plants.
  • PPO inhibitor herbicides-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, altered to contain a modified amount of one or more substances or new substances, for example, to improve human or animal nutrition, e.g. oil crops that produce health-promoting long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids (e.g. Nexera(R) rape, Dow Agro Sciences, Canada).
  • PPO inhibitor herbicides-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, altered to contain increased amounts of vitamins and/or minerals, and/or improved profiles of nutraceutical compounds.
  • PPO inhibitor herbicides-tolerant plants of the present invention relative to a wild-type plant, comprise an increased amount of, or an improved profile of, a compound selected from the group consisting of: glucosinolates (e.g., glucoraphanin (4-methylsulfinylbutyl-glucosinolate), sulforaphane, 3- indolylmethyl-glucosinolate(glucobrassicin), I -methoxy-3-indolylmethyl-glucosinolate (neoglucobrassicin)); phenolics (e.g., flavonoids (e.g., quercetin, kaempferol), hydroxycinnamoyl derivatives (e.g., 1 ,2,2'- trisinapoylgentiobiose, 1 ,2-diferuloylgentiobiose, 1 ,2'-disinapoyl-2-fer
  • PPO inhibitor herbicides-tolerant plants of the present invention relative to a wild-type plant, comprise an increased amount of, or an improved profile of, a compound selected from the group consisting of: progoitrin; isothiocyanates; indoles (products of glucosinolate hydrolysis); glutathione; carotenoids such as beta-carotene, lycopene, and the xanthophyll carotenoids such as lutein and zeaxanthin; phenolics comprising the flavonoids such as the flavonols (e.g.
  • flavans/tannins such as the procyanidins comprising coumarin, proanthocyanidins, catechins, and anthocyanins
  • flavones such as the procyanidins comprising coumarin, proanthocyanidins, catechins, and anthocyanins
  • flavones phytoestrogens such as coumestans, lignans, resveratrol, isoflavones e.g. genistein, daidzein, and glycitein
  • resorcyclic acid lactones organosulphur compounds
  • phytosterols terpenoids such as carnosol, rosmarinic acid, glycyrrhizin and saponins
  • chlorophyll chlorphyllin, sugars, anthocyanins, and vanilla.
  • PPO inhibitor herbicides-tolerant plants of the present invention relative to a wild-type plant, comprise an increased amount of, or an improved profile of, a compound selected from the group consisting of: vincristine, vinblastine, taxanes (e.g., taxol (paclitaxel), baccatin III, 10-desacetylbaccatin III, 10-desacetyl taxol, xylosyl taxol, 7- epitaxol, 7- epibaccatin III, 10-desacetylcephalomannine, 7-epicephalomannine, taxotere, cephalomannine, xylosyl cephalomannine, taxagifine, 8-benxoyloxy taxagifine, 9-acetyloxy taxusin, 9-hydroxy taxusin, taiwanxam, taxane la, taxane lb, taxane Ic, taxane Id, GMP paclitaxel, 9-dihydr
  • the plant of the present invention can comprise a wild type PPO nucleic acid in addition to a mutated PPO nucleic acid. It is contemplated that the PPO inhibitor herbicide tolerant lines may contain a mutation in only one of multiple PPO isoenzymes. Therefore, the present invention includes a plant comprising one or more mutated PPO nucleic acids in addition to one or more wild type PPO nucleic acids.
  • the invention refers to a seed produced by a transgenic plant comprising a plant cell of the present invention, wherein the seed is true breeding for an increased resistance to a PPO inhibitor herbicide as compared to a wild type variety of the seed.
  • the invention refers to a method of producing a transgenic plant cell with an increased resistance to a PPO inhibitor herbicide as compared to a wild type variety of the plant cell comprising, transforming the plant cell with an expression cassette comprising a mutated PPO nucleic acid.
  • the invention refers to a method of producing a transgenic plant comprising, (a) transforming a plant cell with an expression cassette comprising a mutated PPO nucleic acid, and (b) generating a plant with an increased resistance to PPO inhibitor herbicide from the plant cell.
  • mutated PPO nucleic acids of the invention are provided in expression cassettes for expression in the plant of interest.
  • the cassette will include regulatory sequences operably linked to a mutated PPO nucleic acid sequence of the invention.
  • regulatory element refers to a polynucleotide that is capable of regulating the transcription of an operably linked polynucleotide. It includes, but not limited to, promoters, enhancers, introns, 5' UTRs, and 3' UTRs.
  • operably linked is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the organism.
  • the additional gene(s) can be provided on multiple expression cassettes.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the mutated PPO nucleic acid sequence to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the expression cassette of the present invention will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a mutated PPO encoding nucleic acid sequence of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants.
  • the promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the mutated PPO nucleic acid sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is "foreign" or "heterologous" to the plant host, it is intended that the promoter is not found in the native plant into which the promoter is introduced.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • the native promoter sequences may be used. Such constructs would change expression levels of the mutated PPO protein in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked mutated PPO sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the mutated PPO nucleic acid sequence of interest, the plant host, or any combination thereof).
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al.
  • the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage.
  • Nucleotide sequences for enhancing gene expression can also be used in the plant expression vectors. These include the introns of the maize Adhl, intronl gene (Callis et al. Genes and Development 1: 1183-1200, 1987), and leader sequences, (W- sequence) from the Tobacco Mosaic virus (TMV), Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al. Nucleic Acid Res. 15:8693-8711, 1987 and Skuzeski et al. Plant Mol. Biol. 15:65-79, 1990).
  • TMV Tobacco Mosaic virus
  • Maize Chlorotic Mottle Virus Maize Chlorotic Mottle Virus
  • Alfalfa Mosaic Virus Alfalfa Mosaic Virus
  • the first intron from the shrunken- 1 locus of maize has been shown to increase expression of genes in chimeric gene constructs.
  • U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallie et al. (Plant Physiol. 106:929-939, 1994) also have shown that introns are useful for regulating gene expression on a tissue specific basis.
  • the plant expression vectors of the invention may also contain DNA sequences containing matrix attachment regions (MARs). Plant cells transformed with such modified expression systems, then, may exhibit overexpression or constitutive expression of a nucleotide sequence of the invention.
  • MARs matrix attachment regions
  • the expression cassettes of the present invention may additionally contain 5' leader sequences in the expression cassette construct.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. ScL USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al.
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and trans versions may be involved.
  • a number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome.
  • the nucleic acids can be combined with constitutive, tissue -preferred, or other promoters for expression in plants.
  • Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163- 171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.
  • Tissue-preferred promoters can be utilized to target enhanced mutated PPO expression within a particular plant tissue.
  • tissue-preferred promoters include, but are not limited to, leaf -preferred promoters, root-preferred promoters, seed- preferred promoters, and stem-preferred promoters.
  • Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7)792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol.
  • the nucleic acids of interest are targeted to the chloroplast for expression.
  • the expression cassette will additionally contain a chloroplast-targeting sequence comprising a nucleotide sequence that encodes a chloroplast transit peptide to direct the gene product of interest to the chloroplasts.
  • a chloroplast-targeting sequence comprising a nucleotide sequence that encodes a chloroplast transit peptide to direct the gene product of interest to the chloroplasts.
  • transit peptides are known in the art.
  • "operably linked" means that the nucleic acid sequence encoding a transit peptide (i.e., the chloroplast-targeting sequence) is linked to the mutated PPO nucleic acid of the invention such that the two sequences are contiguous and in the same reading frame.
  • the mutated PPO proteins of the invention may include a native chloroplast transit peptide, but any chloroplast transit peptide known in the art can be fused to the amino acid sequence of a mature mutated PPO protein of the invention by operably linking a choloroplast-targeting sequence to the 5'-end of a nucleotide sequence encoding a mature mutated PPO protein of the invention.
  • Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem.
  • EPSPS 5 -(enolpyruvyl)shikimate-3 -phosphate synthase
  • EPSPS 5 -(enolpyruvyl)shikimate-3 -phosphate synthase
  • EPSPS 5 -(enolpyruvyl)shikimate-3 -phosphate synthase
  • tryptophan synthase Zhao et al. (1995) J. Biol. Chem. 270(11 ):6081 -6087
  • plastocyanin (1997) J. Biol. Chem. 272(33):20357-20363); chorismate synthase (Schmidt et al. (1993) J. Biol. Chem.
  • plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase.
  • tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase.
  • the nucleic acids of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831 , herein incorporated by reference.
  • the mutated PPO nucleic acid comprises a polynucleotide sequence selected from the group consisting of: a) a polynucleotide comprising a sequence as shown in SEQ ID NO: 1, 3, 5, or 7; b) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 2, 4, 6, or 8, or a variant or derivative thereof; c) a polynucleotide comprising at least 60 consecutive nucleotides of any of a) and b); and d) a polynucleotide complementary to the polynucleotide of any of a) through c)
  • the expression cassette of the present invention further comprises a transcription initiation regulatory region and a translation initiation regulatory region that are functional in the plant.
  • the expression cassettes of the invention can include another selectable marker gene for the selection of transformed cells.
  • Selectable marker genes including those of the present invention, are utilized for the selection of transformed cells or tissues.
  • Marker genes include, but are not limited to, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • selectable marker genes are not meant to be limiting. Any selectable marker gene can be used in the present invention.
  • the invention further provides an isolated recombinant expression vector comprising the expression cassette containing a mutated PPO nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to a PPO inhibitor herbicide as compared to a wild type variety of the host cell.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein (e.g., mutated PPO polypeptides, fusion polypeptides, etc.).
  • the mutated PPO polypeptides are expressed in plants and plants cells such as unicellular plant cells (such as algae) (See Falciatore et al., 1999, Marine Biotechnology 1 (3):239-251 and references therein) and plant cells from higher plants (e.g., the spermatophytes, such as crop plants).
  • a mutated PPO polynucleotide may be “introduced” into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agroinfection, biolistics, and the like.
  • Suitable methods for transforming or transfecting host cells including plant cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa, New Jersey.
  • Forage crops include, but are not limited to, Wheatgrass, Canarygrass, Bromegrass, Wildrye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin, Birdsfoot Trefoil, Alsike Clover, Red Clover, and Sweet Clover.
  • transfection of a mutated PPO polynucleotide into a plant is achieved by Agrobacterium mediated gene transfer.
  • Agrobacterium mediated gene transfer One transformation method known to those of skill in the art is the dipping of a flowering plant into an Agrobacteria solution, wherein the Agrobacteria contains the mutated PPO nucleic acid, followed by breeding of the transformed gametes.
  • Agrobacterium mediated plant transformation can be performed using for example the GV3101(pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain.
  • Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994, Nucl. Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd Ed. - Dordrecht : Kluwer Academic Publ., 1995. - in Sect., Ringbuc Biology Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R. and Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton : CRC Press, 1993 360 S., ISBN 0-8493-5164-2).
  • rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant Cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91 :694-701).
  • Use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker.
  • Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994, Plant Cell Report 13:282-285.
  • transformation of soybean can be performed using for example a technique described in European Patent No.
  • Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake, or via the silicon carbide fiber technique. (See, for example, Freeling and Walbot “The maize handbook” Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific example of maize transformation is found in U.S. Patent No. 5,990,387, and a specific example of wheat transformation can be found in PCT Application No. WO 93/07256.
  • the introduced mutated PPO polynucleotide may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes.
  • the introduced mutated PPO polynucleotide may be present on an extra- chromosomal non-replicating vector and be transiently expressed or transiently active.
  • a homologous recombinant microorganism can be created wherein the mutated PPO polynucleotide is integrated into a chromosome, a vector is prepared which contains at least a portion of an PPO gene into which a deletion, addition, or substitution has been introduced to thereby alter, e.g., functionally disrupt, the endogenous PPO gene and to create a mutated PPO gene.
  • DNA-RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et al., 1999, Nucleic Acids Research 27(5): 1323-1330 and Kmiec, 1999, Gene therapy American Scientist 87(3):240-247).
  • Other homologous recombination procedures in Triticum species are also well known in the art and are contemplated for use herein.
  • the mutated PPO gene can be flanked at its 5’ and 3’ ends by an additional nucleic acid molecule of the PPO gene to allow for homologous recombination to occur between the exogenous mutated PPO gene carried by the vector and an endogenous PPO gene, in a microorganism or plant.
  • the additional flanking PPO nucleic acid molecule is of sufficient length for successful homologous recombination with the endogenous gene.
  • several hundreds of base pairs up to kilobases of flanking DNA are included in the vector (see e.g., Thomas, K. R., and Capecchi, M.
  • the homologous recombination vector is introduced into a microorganism or plant cell (e.g., via polyethylene glycol mediated DNA), and cells in which the introduced mutated PPO gene has homologously recombined with the endogenous PPO gene are selected using art-known techniques.
  • recombinant microorganisms can be produced that contain selected systems that allow for regulated expression of the introduced gene. For example, inclusion of a mutated PPO gene on a vector placing it under control of the lac operon permits expression of the mutated PPO gene only in the presence of IPTG.
  • Such regulatory systems are well known in the art.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but they also apply to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a mutated PPO polynucleotide can be expressed in bacterial cells such as C.
  • glutamicum insect cells, fungal cells, or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), algae, ciliates, plant cells, fungi or other microorganisms like C. glutamicum.
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells
  • algae ciliates
  • plant cells fungi or other microorganisms like C. glutamicum.
  • Other suitable host cells are known to those skilled in the art.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a mutated PPO polynucleotide.
  • the invention further provides methods for producing mutated PPO polypeptides using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a mutated PPO polypeptide has been introduced, or into which genome has been introduced a gene encoding a wild-type or mutated PPO polypeptide) in a suitable medium until mutated PPO polypeptide is produced.
  • the method further comprises isolating mutated PPO polypeptides from the medium or the host cell.
  • Another aspect of the invention pertains to isolated mutated PPO polypeptides, and biologically active portions thereof.
  • An “isolated” or “purified” polypeptide or biologically active portion thereof is free of some of the cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of mutated PPO polypeptide in which the polypeptide is separated from some of the cellular components of the cells in which it is naturally or recombinantly produced.
  • the language “substantially free of cellular material” includes preparations of a mutated PPO polypeptide having less than about 30% (by dry weight) of non-mutated PPO material (also referred to herein as a “contaminating polypeptide”), more preferably less than about 20% of non-mutated PPO material, still more preferably less than about 10% of non-mutated PPO material, and most preferably less than about 5% non-mutated PPO material.
  • a mutated PPO polypeptide having less than about 30% (by dry weight) of non-mutated PPO material (also referred to herein as a “contaminating polypeptide”), more preferably less than about 20% of non-mutated PPO material, still more preferably less than about 10% of non-mutated PPO material, and most preferably less than about 5% non-mutated PPO material.
  • the mutated PPO polypeptide, or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation.
  • culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation.
  • substantially free of chemical precursors or other chemicals includes preparations of mutated PPO polypeptide in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of a mutated PPO polypeptide having less than about 30% (by dry weight) of chemical precursors or non-mutated PPO chemicals, more preferably less than about 20% chemical precursors or non-mutated PPO chemicals, still more preferably less than about 10% chemical precursors or non-mutated PPO chemicals, and most preferably less than about 5% chemical precursors or non-mutated PPO chemicals.
  • isolated polypeptides, or biologically active portions thereof lack contaminating polypeptides from the same organism from which the mutated PPO polypeptide is derived.
  • such polypeptides are produced by recombinant expression of, for example, a mutated PPO polypeptide in plants other than, or in microorganisms such as C. glutamicum, ciliates, algae, or fungi.
  • the method comprises contacting the plant with an agronomically acceptable composition.
  • the present invention provides a method for preparing a descendent seed.
  • the method comprises planting a seed of or capable of producing a plant of the present invention.
  • the method further comprises growing a descendent plant from the seed; and harvesting a descendant seed from the descendent plant.
  • the method further comprises applying a PPO inhibitor herbicides herbicidal composition to the descendent plant.
  • the invention refers to harvestable parts of the transgenic plant according to the present invention.
  • the harvestable parts comprise the PPO nucleic acid or PPO protein of the present invention.
  • the harvestable parts may be seeds, roots, leaves and/or flowers comprising the PPO nucleic acid or PPO protein or parts thereof.
  • Preferred parts of soy plants are soy beans comprising the PPO nucleic acid or PPO protein.
  • the invention refers to products derived from a plant according to the present invention, parts thereof or harvestable parts thereof.
  • a preferred plant product is fodder, seed meal, oil, or seed-treatment- coated seeds.
  • the meal and/or oil comprises the mutated PPO nucleic acids or PPO proteins of the present invention.
  • the invention refers to a method for the production of a product, which method comprises a) growing the plants of the invention or obtainable by the methods of invention and b) producing said product from or by the plants of the invention and/or parts, e.g. seeds, of these plants.
  • the method comprises the steps a) growing the plants of the invention, b) removing the harvestable parts as defined above from the plants and c) producing said product from or by the harvestable parts of the invention.
  • the product may be produced at the site where the plant has been grown, the plants and/or parts thereof may be removed from the site where the plants have been grown to produce the product.
  • the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant.
  • the step of growing the plant may be performed only once each time the methods of the invention is performed, while allowing repeated times the steps of product production e.g. by repeated removal of harvestable parts of the plants of the invention and if necessary further processing of these parts to arrive at the product. It is also possible that the step of growing the plants of the invention is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts.
  • the steps of growing the plants and producing the product may be performed with an overlap in time, even simultaneously to a large extend or sequentially. Generally the plants are grown for some time before the product is produced.
  • the products produced by said methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fiber, cosmetic and/or pharmaceutical.
  • Foodstuffs are regarded as compositions used for nutrition and/or for supplementing nutrition.
  • Animal feedstuffs and animal feed supplements, in particular, are regarded as foodstuffs.
  • inventive methods for the production are used to make agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.
  • a plant product consists of one or more agricultural products to a large extent.
  • the present invention teaches compositions and methods for increasing the PPO-inhibiting tolerance of a crop plant or seed as compared to a wild-type variety of the plant or seed.
  • the PPO-inhibiting tolerance of a crop plant or seed is increased such that the plant or seed can withstand a PPO inhibitor herbicide application of preferably approximately 1-1000 g ai ha 1 , more preferably 1- 200 g ai ha 1 , even more preferably 5-150 g ai ha 1 , and most preferably 10-100 g ai ha 1 .
  • to “withstand” a PPO inhibitor herbicide application means that the plant is either not killed or only moderately injured by such application. It will be understood by the person skilled in the art that the application rates may vary, depending on the environmental conditions such as temperature or humidity, and depending on the chosen kind of herbicide (active ingredient ai).
  • the present invention provides methods that involve the use of at least one PPO inhibitor herbicide, optionally in combination with one or more herbicidal compounds B, and, optionally, a safener C, as described in detail supra.
  • the PPO inhibitor herbicide can be applied by any method known in the art including, but not limited to, seed treatment, soil treatment, and foliar treatment.
  • the PPO inhibitor herbicide Prior to application, can be converted into the customary formulations, for example solutions, emulsions, suspensions, dusts, powders, pastes and granules.
  • the use form depends on the particular intended purpose; in each case, it should ensure a fine and even distribution of the compound according to the invention.
  • PPO inhibitor herbicide By providing plants having increased tolerance to PPO inhibitor herbicide, a wide variety of formulations can be employed for protecting plants from weeds, so as to enhance plant growth and reduce competition for nutrients.
  • a PPO inhibitor herbicide can be used by itself for pre-emergence, post-emergence, pre-planting, and at- planting control of weeds in areas surrounding the crop plants described herein, or a PPO inhibitor herbicide formulation can be used that contains other additives.
  • the PPO inhibitor herbicide can also be used as a seed treatment.
  • Additives found in a PPO inhibitor herbicide formulation include other herbicides, detergents, adjuvants, spreading agents, sticking agents, stabilizing agents, or the like.
  • the PPO inhibitor herbicide formulation can be a wet or dry preparation and can include, but is not limited to, flowable powders, emulsifiable concentrates, and liquid concentrates.
  • the PPO inhibitor herbicide and herbicide formulations can be applied in accordance with conventional methods, for example, by spraying, irrigation, dusting, or the like.
  • the PPO1 cDNA sequence from Bassia scoparia (kochia) carrying the mutations F454 L, I and V were inserted into a RTP6557 transformation vector, which was then inserted into Agrobacterium tumefaciens strain C58C1pMP90.
  • the gene insert also included an acetolactate synthase herbicide-resistance trait as a selectable marker to identify transformed Arabidopsis seedlings. This would ensure that plants eventually tested for tolerance to PPOi all expressed the transgene.
  • the Agrobacterium culture containing the plasmid was prepared a day before dipping by inoculating 1 ml of glycerol stock into 250 ml of YEB medium (1 g L-1 yeast, 5 g L-1 beef extract, 5 g L-1 peptone, 5 g L-1 sucrose, 0.49 g L-1 MgSO47H2O) plus the appropriate antibiotic. Bacteria were cultured for 12 h at 28°C with continuous agitation at 150 rpm.
  • the culture was collected by centrifugation at 1600 g for 10 min and resuspended in 150 ml of infiltration medium composed of 2.2 g L-1 Murashige & Skoog (MS) medium, 50 g L-1 sucrose, 0.5 g L-1 MES hydrate, 10 pl L-1 BAP (benzylaminopurin, 1 mg ml-1). The pH was then adjusted to 5.7-5.8.
  • Plants with immature floral buds were dipped in the bacterial suspension for 10 s after adding 75 pl of Silwet- L77 per 150 ml of infiltration medium to ajar. After dipping, plants were kept overnight in a cabinet under high humidity and low light intensity and were grown under long-day conditions until maturity. When siliques turned yellow, plants were placed in paper bags to collect the seeds. T 1 seeds were then transferred to falcon tubes and stored at 4°C.
  • T 1 seeds were sown to select putative transgenic Arabidopsis plants. Seeds were then treated with a 20 ppm imazamox (technical grade) solution and cultivated under short-day growth conditions for 12-14 days, when resistant seedlings (four-leaf stage) were transplanted into 6 x 6 cm pots filled with GS90 soil and grown for another 10 days. One day prior to herbicide application, growth conditions were set to ‘long-day’ and maintained throughout the duration of the test.
  • Herbicide treatments consisted of three concentrations of Flumioxazin at (428, 214, 107)g of active ingredient (a.i.) ha-1 ], Carfentrazone at (1680, 840, 420)g of active ingredient (a.i.) ha- 1 ] and Saflufenacil at (100, 50, 25)g of active ingredient (a.i.) ha- 1 ], all applied when plants reached the ten-leaf stage, using a spray chamber calibrated to deliver 375 L ha- 1 of spray solution. Herbicide efficacy was visually assessed after 21 days from herbicide treatments.
  • Herbicides spray solution and empty control contain the adjuvant Dash HC containing 349 g/l oil (fatty acid esters) and 209 g/l alkoxylated alcohols-phosphate esters. WT non transgenics Arabidopsis was used as control.
  • Clones in pRSET vector are transformed into BL21(DE3)-pLysS strain of E. coli. Cells are grown in 250 mL of LB with 100 pgmL-1 of carbenicillin, shaking overnight at 37 °C.
  • Cultures are diluted in 1 L of LB with antibiotic and grown at 37 °C shaking for 2 h, induced with 1 mM IPTG and grown at 25 °C shaking for 5 more hours. The cells are harvested by centrifugation at 1600xg, washed with 0.09% NaCI, and stored at -80 °C. Cells are lysed using a French press at 140 MPa in 50 mM sodium phosphate pH 7.5, 1 M NaCI, 5 mM imidazole, 5% glycerol, and 1 pg mL-1 leupeptin.
  • benzonase Novagen, EMD Chemicals, Inc., Gibbstown, NJ
  • PMSF final concentration of 1 mM
  • Cell debris is removed by centrifugation at 3000xg.
  • His-tagged PPO proteins are purified on a nickel activated Hitrap Chelating HP column (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) equilibrated with 20 mM sodium phosphate pH 8.0, 50 mM NaCI, 5 mM imidazole, 5 mM MgCI2, 0.1 mM EDTA, and 17% glycerol.
  • PPO is eluted with 250 mM imidazole.
  • the active protein is desalted on a PD-10 column (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) equilibrated with a 20 mM sodium phosphate buffer, pH 7.5, 5 mM MgCI2, 1 mM EDTA and 17% glycerol. Each litre of culture provided approximately 10 mg of pure PPO, which is stored at -20 °C until being used in assays.
  • PPO protein (EC 1 .3.3.4) is extracted from coleoptiles or shoots (150 g fresh weight) of dark-grown corn, black nightshade, morning glory, and velvetleaf seedlings as described previously (Grossmann et al. 2010). Before harvesting, the seedlings are allowed to green for 2 hours in the light in order to achieve the highest specific enzyme activities in the thylakoid fractions at low chlorophyll concentrations. At high chlorophyll concentrations significant quenching of fluorescence occurs, which limits the amount of green thylakoids that can be used in the test. Plant materials are homogenized in the cold with a Braun blender using a fresh-weight-to-volume ratio of 1 :4.
  • Homogenization buffer consisted of tris(hydroxymethyl)aminomethane (Tris)-HCI (50 mM; pH 7.3), sucrose (0.5 M), magnesium chloride (1 mM), ethylenediaminetetraacetic acid (EDTA) (1 mM) and bovine serum albumin (2 g L 1 ). After filtration through four layers of Miracloth, crude plastid preparations are obtained after centrifugation at 10 000 x g for 5 min and resuspension in homogenization buffer before centrifugation at 150 x g for 2 min to remove crude cell debris.
  • Tris tris(hydroxymethyl)aminomethane
  • EDTA ethylenediaminetetraacetic acid
  • the supernatant is centrifuged at 4000 x g for 15 min and the pellet fraction is resuspended in 1 ml of a buffer containing Tris-HCI (50 mM; pH 7.3), EDTA (2 mM), leupeptin (2 pM), pepstatin (2 pM) and glycerol (200 ml L 1 ) and stored at -80°C until use.
  • Protein is determined in the enzyme extract with bovine serum albumin as a standard.
  • PPO activity is assayed fluorometrically by monitoring the rate of Proto formation from chemically reduced protoporphyrinogen IX under initial velocity conditions.
  • the assay mixture consisted of Tris-HCI (100 mM; pH 7.3), EDTA (1 mM), dithiothreitol (5 mM), Tween 80 (0.085%), protoporphyrinogen IX (2 pM), and 40 pg extracted protein in a total volume of 200 pl.
  • the reaction is initiated by addition of substrate protoporphyrinogen IX at 22°C.
  • the PPO inhibitors disclosed SUPRA, and photosynthesis inhibitor diuron as negative control are prepared in dimethyl sulfoxide (DMSO) solution (0.1 mM concentration of DMSO in the assay) and added to the assay mixture in concentrations of 0.005 pM to 5 pM before incubation.
  • DMSO dimethyl sulfoxide
  • Proto is purchased from Sigma-Aldrich (Milwaukee, Wl).
  • Protogen is prepared according to Jacobs and Jacobs (N.J. Jacobs, J.M. Jacobs, Assay for enzymatic protoporphyrinogen oxidation, a late step in heme synthesis, Enzyme 28 (1982) 206-219) .
  • Assays are conducted in 100 mM sodium phosphate pH 7.4 with 0.1 mM EDTA, 0.1% Tween 20, 5 pM FAD, and 500mM imidazole.
  • Dose-response curves with the PPO inhibitors disclosed SUPRA, and photosynthesis inhibitor diuron as negative control, and MC-15608 are obtained in the presence of 150 pM Protogen.
  • excitation and emission bandwidths are set at 1.5 and 30 nm, respectively. All assays are made in duplicates or triplicates and measured using a POLARstar Optima / Galaxy (BMG) with excitation at 405 nm and emission monitored at 630 nm. Molar concentrations of compound required for 50% enzyme inhibition (IC50 values) are calculated by fitting the values to the dose-response equation using non-linear regression analysis.
  • EXAMPLE 9 Engineering PPO-derivative herbicide tolerant plants having wildtype or mutated PPO sequences.
  • PPO-derivative herbicide tolerant soybean plants are produced by a method as described by Olhoft ef al. (US patent 2009/0049567).
  • Wildtype or Mutated PPO sequences encoding herbicide tolerant PPO polypeptides are cloned with standard cloning techniques as described in Sambrook et al. (Molecular cloning (2001) Cold Spring Harbor Laboratory Press) in a binary vector containing resistance marker gene cassette (AHAS) and mutated PPO sequence (marked as GOI) in between ubiquitin promoter (PcUbi) and nopaline synthase terminator (NOS) sequence.
  • AHAS resistance marker gene cassette
  • mutated PPO sequence marked as GOI
  • Wildtype or Mutated PPO sequences are cloned with standard cloning techniques as described in Sambrook et al. (Molecular cloning (2001) Cold Spring Harbor Laboratory Press) in a binary vector containing resistance marker gene cassette (AHAS) and mutated PPO sequence (marked as GOI) in between corn ubiquitin promoter (ZmUbi) and nopaline synthase terminator (NOS) sequence.
  • Binary plasmids are introduced to Agrobacterium tumefaciens for plant transformation. Plasmid constructs are introduced into soybean’s axillary meristem cells at the primary node of seedling explants via Agrobacterium-mediated transformation.
  • the explants After inoculation and co-cultivation with Agrobacteria, the explants are transferred to shoot introduction media without selection for one week. The explants are subsequently transferred to a shoot induction medium with 1-3 pM imazapyr (Arsenal) for 3 weeks to select for transformed cells. Explants with healthy callus/shoot pads at the primary node are then transferred to shoot elongation medium containing 1-3 pM imazapyr until a shoot elongated or the explant died. Transgenic plantlets are rooted, subjected to TaqMan analysis for the presence of the transgene, transferred to soil and grown to maturity in greenhouse. Plant transformation vector constructs containing mutated PPO sequences are introduced into maize immature embryos via Agrobacterium-mediated transformation according to the procedure outlined in Peng et al.( W02006/136596). .
  • Transformed cells are selected in selection media supplemented with 0.5-1 .5 pM imazethapyr for 3-4 weeks.
  • Transgenic plantlets are regenerated on plant regeneration media and rooted afterwards.
  • Transgenic plantlets are subjected to TaqMan analysis for the presence of the transgene before being transplanted to potting mixture and grown to maturity in greenhouse.
  • Arabidopsis thaliana are transformed with wildtype or mutated PPO sequences by floral dip method as decribed by McElver and Singh (WO 2008/124495).
  • Transgenic Arabidopsis plants are subjected to TaqMan analysis for analysis of the number of integration loci. Transformation of Oryza sativa (rice) are done by protoplast transformation as decribed by Peng et al.
  • An in vitro tissue culture mutagenesis assay has been developed to isolate and characterize plant tissue (e.g., maize, rice tissue) that is tolerant to protoporphyrinogen oxidase inhibiting herbicides, e.g. the PPO inhibitors disclosed SUPRA, and photosynthesis inhibitor diuron as negative control).
  • the assay utilizes the somaclonal variation that is found in in vitro tissue culture.
  • Spontaneous mutations derived from somaclonal variation can be enhanced by treatment with a chemical mutagen (e.g. Ethyl methanesulfonate, N-ethyl-N-nitrosourea, N- Nitroso-N-methylurea) and subsequent selection in a stepwise manner, on increasing concentrations of herbicide.
  • a chemical mutagen e.g. Ethyl methanesulfonate, N-ethyl-N-nitrosourea, N- Nitroso-N-methylurea
  • the present invention provides tissue culture conditions for encouraging growth of friable, embryogenic maize or rice callus that is regenerable. Calli are initiated from rice cultivar Indica (Indica I). Seeds are surface sterilized in 70% ethanol for approximately I min followed by 20% commercial Clorox bleach for 20 minutes. Seeds are rinsed with sterile water and plated on R001 M media. The ingredient lists for the media tested are presented in Table y.
  • ROO 1 M callus induction media is selected after testing numerous variations. Cultures are kept in the dark at 30°C. Embryogenic callus is subcultured to fresh media after 10-14 days.
  • tissue culture conditions are determined, further establishment of selection conditions are established through the analysis of tissue survival in kill curves with saflufenacil, trifludimoxazin, sulfentrazone, and the PPO inhibitors disclosed SUPRA, and photosynthesis inhibitor diuron as negative control. Careful consideration of accumulation of the herbicide in the tissue, as well as its persistence and stability in the cells and the culture media is performed. Through these experiments, a sub-lethal dose has been established for the initial selection of mutated material.
  • the tissues are selected in a step- wise fashion by increasing the concentration of the PPO inhibitor with each transfer until cells are recovered that grew vigorously in the presence of toxic doses.
  • the resulting calli are further subcultured every 3-4 weeks to R001M with selective agent. Over 26,000 calli are subjected to selection for 4-5 subcultures until the selective pressure is above toxic levels as determined by kill curves and observations of continued culture.
  • liquid cultures initiated from calli in MS711 R with slow shaking and weekly subcultures. Once liquid cultures are established, selection agent is added directly to the flask at each subculture. Following 2-4 rounds of liquid selection, cultures are transferred to filters on solid R001M media for further growth.
  • Tolerant tissue e.g. rice tissue
  • PPO gene e.g. Oryza sative PPO2 sequence mutations and/or biochemically for altered PPO activity in the presence of the selective agent.
  • genes involved directly and/or indirectly in tetrapyrrole biosynthesis and/or metabolism pathways are also sequenced to characterize mutations.
  • enzymes that change the fate e.g. metabolism, translocation, transportation
  • calli are regenerated using a media regime of R025M for 10 - 14 days, R026M for ca.
  • Leaf tissue is collected from clonal plants separated for transplanting and analyzed as individuals. Genomic DNA is extracted using a Chloropure Nucleic acid extraction kit (Agencourt, US patents Nos. 5,898,071; 5,705,628; 6,534,262) as directed by the manufacturer. Isolated DNA is PCR amplified using the appropriate forward and reverse primer.
  • PCR amplification is performed using LongAmp HotStart Taq DNA Polymerase Mix (New England Biolabs) using thermocycling program as follows: 94°C for 30 sec, followed by 35 cycles (94°C, 30 sec; 54°C, 30 sec; 65°C, 300 sec), 10 min at 65°C.
  • PCR products are verified for concentration and fragment size via agarose gel electrophoresis.
  • Dephosphorylated PCR products are analyzed by direct sequence using the PCR primers (Genewiz or GenScript).
  • Chromatogram trace files (.scf) are analyzed for mutation relative to the wild-type gene using Sequencher (Gene Codes) or Vector NTI Advance 10TM (Invitrogen). Based on sequence information, mutations are identified in several individuals. Sequence analysis is performed on the representative chromatograms and corresponding alignment with default settings and edited to call secondary peaks.
  • TO or T1 transgenic plant of soybean, corn, Canola varieties and rice containing PPO1 and or PPO2 sequences are tested for improved tolerance to herbicides in greenhouse studies and mini-plot studies with the PPO inhibitors disclosed SUPRA, and photosynthesis inhibitor diuron as negative control.
  • the herbicides are applied directly after sowing by means of finely distributing nozzles.
  • the containers are irrigated gently to promote germination and growth and subsequently covered with transparent plastic hoods until the plants have rooted. This cover causes uniform germination of the test plants, unless this has been impaired by the herbicides.
  • the test plants are first grown to a height of 3 to 15 cm, depending on the plant habit, and only then treated with the herbicides. For this purpose, the test plants are either sown directly, and grown in the same containers or they are first grown separately and transplanted into the test containers a few days prior to treatment.
  • cuttings can be used.
  • an optimal shoot for cutting is about 7.5 to 10 cm tall, with at least two nodes present.
  • Each cutting is taken from the original transformant (mother plant) and dipped into rooting hormone powder (indole-3-butyric acid, IBA). The cutting is then placed in oasis wedges inside a bio-dome. Wild type cuttings are also taken simultaneously to serve as controls.
  • the cuttings are kept in the bio-dome for 5-7 days and then transplanted to pots and then acclimated in the growth chamber for two more days. Subsequently, the cuttings are transferred to the greenhouse, acclimated for approximately 4 days, and then subjected to spray tests as indicated.
  • the plants are kept at 10-25°C or 20-35°C.
  • the test period extends over 3 weeks. During this time, the plants are tended and their response to the individual treatments is evaluated. Herbicide injury evaluations are taken at 2 and 3 weeks after treatment. Plant injury is rated on a scale of 0% to 100%, 0% being no injury and 100% being complete death.
  • Transgenic Arabidopsis thaliana plants are assayed for improved tolerance to the PPO inhibitors disclosed SUPRA, in 48-well plates. Therefore, T2 seeds are surface sterilized by stirring for 5 min in ethanol + water (70+30 by volume), rinsing one time with ethanol + water (70+30 by volume) and two times with sterile, deionized water. The seeds are resuspended in 0.1% agar dissolved in water (w/v) Four to five seeds per well are plated on solid nutrient medium consisting of half-strength murashige skoog nutrient solution, pH 5.8 (Murashige and Skoog (1962) Physiologia Plantarum 15: 473-497).
  • DMSO dimethylsulfoxid
  • Multi well plates are incubated in a growth chamber at 22°C, 75% relative humidity and 110 mol Phot * nr 2 * s 1 with 14 : 10 h light : dark photoperiod. Growth inhibition is evaluated seven to ten days after seeding in comparison to wild type plants.
  • EXAMPLE 15 Herbicide Selection Using Tissue Culture.
  • Media is selected for use and kill curves developed as specified above. For selection, different techniques are utilized. Either a step wise selection is applied, or an immediate lethal level of herbicide is applied. In either case, all of the calli are transferred for each new round of selection. Selection is 4-5 cycles of culture with 3-5 weeks for each cycle. Cali are placed onto nylon membranes to facilitate transfer (200 micron pore sheets, Biodesign, Saco, Maine). Membranes are cut to fit 100x20 mm Petri dishes and are autoclaved prior to use 25- 35 calli (average weight/calli being 22mg) are utilized in every plate. In addition, one set of calli are subjected to selection in liquid culture media with weekly subcultures followed by further selection on semi-solid media.
  • Mutant lines are selected using the PPO inhibitors as disclosed SUPRA, and photosynthesis inhibitor diuron as negative control. Efficiencies of obtaining mutants is high either based on a percentage of calli that gave rise to a regenerable, mutant line or the number of lines as determined by the gram of tissue utilized.
  • EXAMPLE 16 Maize whole plant transformation and PPO inhibitor tolerance testing.
  • Immature embryos are transformed according to the procedure outlined in Peng et al. (W02006/136596). Plants are tested for the presence of the T-DNA by Taqman analysis with the target being the nos terminator which is present in all constructs. Healthy looking plants are sent to the greenhouse for hardening and subsequent spray testing. The plants are individually transplanted into MetroMix 360 soil in 4” pots. Once in the greenhouse (day/night cycle of 27oC /21oC with 14 hour day length supported by 600W high pressure sodium lights), they are allowed to grow for 14 days. TO or T1 plants are sprayed with a treatment of the PPO inhibitors as disclosed SUPRA and photosynthesis inhibitor diuron as negative control. Herbicide injury evaluations are taken at 7, 14 and 21 days after treatment. Herbicide injury evaluations are taken 2, 7, 14 and 21 days post-spray to look for injury to new growth points and overall plant health. The top survivors are transplanted into gallon pots filled with MetroMix 360 for seed production.
  • EXAMPLE 17 Soybean transformation and PPO Inhibitor tolerance testing.
  • Soybean cv Jake is transformed as previously described by Siminszky et al., Phytochem Rev. 5:445-458 (2006). After regeneration, transformants are transplanted to soil in small pots, placed in growth chambers (16 hr day/ 8 hr night; 25°C day/ 23°C night; 65% relative humidity; 130-150 microE m-2 s-1) and subsequently tested for the presence of the T-DNA via Taqman analysis. After a few weeks, healthy, transgenic positive, single copy events are transplanted to larger pots and allowed to grow in the growth chamber. An optimal shoot for cutting is about 3-4 inches tall, with at least two nodes present.
  • Each cutting is taken from the original transformant (mother plant) and dipped into rooting hormone powder (indole-3-butyric acid, IBA). The cutting is then placed in oasis wedges inside a bio-dome. The mother plant is taken to maturity in the greenhouse and harvested for seed. Wild type cuttings are also taken simultaneously to serve as negative controls. The cuttings are kept in the bio-dome for 5-7 days and then transplanted to 3 inch pots and then acclimated in the growth chamber for two more days. Subsequently, the cuttings are transferred to the greenhouse, acclimated for approximately 4 days prior to spray. TO or 2 -week old T 1 plants are sprayed with a treatment of the PPO inhibitors as disclosed SUPRA and photosynthesis inhibitor diuron as negative control. Herbicide injury evaluations are taken at 2, 7, 14 and 21 days after treatment.

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Abstract

La présente invention concerne un procédé de lutte contre la végétation indésirable au niveau d'un site de culture de plantes, le procédé comprenant les étapes consistant à obtenir, au niveau de ce site, une plante qui comprend au moins un acide nucléique comportant une séquence nucléotidique codant pour la protoporphyrinogène oxydase (PPO) qui est résistante ou tolérante à un herbicide inhibant la PPO par l'application sur ce site d'une quantité efficace dudit herbicide. L'invention concerne en outre des plantes comprenant des enzymes PPO mutées, et des procédés d'obtention de telles plantes.
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