WO2014030090A1 - Herbicide-metabolizing cytochrome p450 monooxygenase - Google Patents

Herbicide-metabolizing cytochrome p450 monooxygenase Download PDF

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WO2014030090A1
WO2014030090A1 PCT/IB2013/056519 IB2013056519W WO2014030090A1 WO 2014030090 A1 WO2014030090 A1 WO 2014030090A1 IB 2013056519 W IB2013056519 W IB 2013056519W WO 2014030090 A1 WO2014030090 A1 WO 2014030090A1
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Prior art keywords
plant
polypeptide
sequence
ppo
polynucleotide
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PCT/IB2013/056519
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French (fr)
Inventor
Lutz Petzke
Andrea Herold
Robert THUMMER
Oskar Zelder
Dale R. Carlson
Jill Marie Paulik
Alexander Seifert
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Basf Se
Basf (China) Company Limited
Basf Schweiz Ag
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Publication of WO2014030090A1 publication Critical patent/WO2014030090A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)

Definitions

  • the present invention relates to methods of using a bacterial cytochrome P450 monooxygenase which is able to metabolize PPO inhibiting herbicides such as Saflufenacil, butafenacil, S-3100, or benzfendizone, as well as polynucleotides encoding this enzyme.
  • the invention also relates to transgenic plants producing these enzymes which are resistant and/or tolerant to PPO inhibiting herbicide activity.
  • Cytochrome P450 monooxygenases form a large diverse gene family with about 246 isoforms in Arabidopsis and 372 identified in rice. CytP450s are hemoproteins that convert a broad range of substrates to more or less bioactive products. The reaction cycle catalyzed by CytP450s requires the sequential input of two reducing equivalents (i.e., two electrons and two protons).
  • the reducing equivalents for the CytP450-catalyzed reaction are supplied by either NADPH or NADH, depending on the type of redox system concerned, and electron transfer is mediated by two co-factors, one of which is FAD; the other being either FMN or an iron-sulfur Fe2S2 redoxin (ferredoxin) or, in the microsomal system, cytochrome b5.
  • FAD iron-sulfur Fe2S2 redoxin
  • cytochrome b5 the majority of bacterial CytP450s utilize an electron transport chain which consists of an FAD-containing NADH-dependent oxidoreductase, and reduction is mediated by ferredoxin.
  • the mitochondrial system in mammalia bears many similarities with the prokaryotic P450 electron transport chain, except that NADPH is the source of reducing equivalents, and both systems are generally referred to as Class I (see Lewis and Hlavica, Biochimica et Biophysica Acta 1460 (2000) 353- 374, as well as references contained therein).
  • CytP450s are critical in numerous metabolic pathways, including lignin and pigment biosynthesis, detoxification of harmful compounds, and are considered important in the evolution of land plants.
  • Inhibitors of CytP450 activity include 1-aminobenzo-triazole, tetcyclacis, piperonyl butox- ide, cinnamonic acid, and tridiphane.
  • Saflufenacil is an herbicide active ingredient (A.I.) of the pyrimidinedione chemical class that is similar to flumioxazin and sulfentrazone and is readily absorbed by foliage, root, and shoot tissue of plants.
  • Saflufenacil inhibits the pigment biosynthesis pathway at protoporphyrinogen oxidase (PPO), which causes an accumulation of photodynamic, toxic compounds that rapidly damage cell membranes and results in cell death.
  • PPO- inhibiting herbicides such as Saflufenacil have been labeled for pre-plant or pre-emergence treatment in corn, sorghum, wheat, barley, oats, rye, triticale, soybean, and tree/nut/vine cropping systems.
  • PPO inhibitor-containing herbicidal compositions have good foliar and residual activity on broadleaf weeds in both no-till and tilled cropping systems.
  • a) modification of the target molecule of the herbicide b) metabolic approach, i. e. making the compound non-hazardous.
  • one or more enzymes are needed, that catalyze the conversion of the herbicide to a non toxic compound.
  • One source of such enzymes can be microorganisms isolated from nature. Bacteria, especially those of the order Actinomycetales, are known for their potential to detoxify soil by metabolizing xenobiotics, including herbicides (Cork et al, 1991 ; Schrijver et al., 1999; Caracciolo et al., 2010).
  • CytP450s In many other cases those reactions are catalyzed by CytP450s. Those can be plant derived (Pan et al., 2006), from algal origin (Thies et al., 1996) or microbial (O'Keefe et al., 1991 ).
  • actinomycetes offer a broad spectrum of CytP450s.
  • the genome analysis of Streptomyes coelicolor revealed 18 CytP450s (Lamb et al., 2002), the Streptomyes avermitilis genome revealed 33 (Lamb et al., 2003), respectively.
  • Nelson 201 1
  • actinobacteria hold the largest number of CytP450s per genome.
  • the present invention provides a method for metabolizing a PPO inhibiting herbicide, the method comprising contacting a PPO inhibiting herbicide with a
  • CytP450 polypeptide comprises amino acids having a sequence selected from: i) SEQ ID NO:1 , and
  • ii an amino acid sequence which is at least 85% identical to i).
  • PPO inhibiting herbicides include, but are not limited to Saflufenacil, flumioxazin, butafenacil, S-3100, benzfendizone, and sulfentrazone.
  • the PPO inhibiting herbicide is Saflufenacil.
  • the CytP450 polypeptide is produced by a host cell of the invention.
  • the PPO inhibiting herbicide is Saflufenacil.
  • Polypeptides provided herein can be produced in plants to enhance the host plants ability to grow when exposed to a PPO inhibiting herbicide such as Saflufenacil, flumioxazin, butafenacil, S-3100, benzfendizone, and sulfentrazone.
  • a PPO inhibiting herbicide such as Saflufenacil, flumioxazin, butafenacil, S-3100, benzfendizone, and sulfentrazone.
  • the present invention provides a transgenic plant comprising an exogenous polynucleotide, the polynucleotide encoding at least one CytP450 polypeptide of the invention.
  • the PPO inhibiting herbicide is Saflufenacil, flumioxazin, butafenacil, S-3100, benzfendizone, and sulfentrazone.
  • the polynucleotide is stably incorporated into the genome of the plant.
  • Also provided is a method of producing plants with enhanced resistance to a PPO inhibiting herbicide comprising the steps of: a) inserting into the genome of a plant cell a CytP450 polynucleotide comprising: a promoter that functions in plant cells to cause the production of a RNA sequence, operably linked to; a structural DNA sequence that caused the production of a RNA sequence that encodes a polypeptide of the invention, operably linked to; a 3' non-translated region that functions in plant cells to cause the addition of polyadenyl nucleotides at the 3' end of the RNA sequence; where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the polypeptide to enhance resistance to a PPO inhibiting herbicide of a plant cell transformed with the DNA molecule; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which has increased resistance to a PPO inhibiting herbicide,
  • CytP450 polynucleotide comprises nucleotides having a sequence selected from: i) SEQ ID NO:2
  • iv a sequence of nucleotides which hybridizes to i) under low stringency conditions, v) a sequence of nucleotides complementary to i) to iv).
  • the polynucleotide encodes a CytP450 polypeptide that metabolizes a PPO inhibiting herbicide. More preferably, the PPO inhibiting herbicide is Saflufenacil.
  • the CytP450 polynucleotide comprises a sequence which hybridizes to i) under moderate stringency conditions. More preferably, the polynucleotide comprises a sequence which hybridizes to i) under stringent conditions.
  • the present invention provides a transgenic plant produced using a method of the invention.
  • the present invention provides a method for metabolizing a PPO inhibiting herbicide in a sample, the method comprising exposing the sample to a transgenic plant of the invention.
  • the sample is soil.
  • Such soil can be in a field.
  • the present invention provides a recombinant CytP450 polynucleotide comprising a promoter that functions in a plant cell, operably linked to a structural DNA sequence that encodes a polypeptide of the invention, operably linked to a 3' polyadenylation sequence that functions in the cell, wherein the promoter is heterologous with respect to the structural DNA sequence and capable of expressing the structural DNA sequence to enhance resistance of the cell to a PPO inhibiting herbicide.
  • the present invention provides a vector comprising the CytP450 polynucleotide of the invention.
  • the CytP450 polynucleotide is operably linked to a promoter.
  • the present invention provides a host cell comprising at least one polynucleotide of the invention, and/or at least one vector of the invention.
  • the host cell can be any type of cell.
  • the host cell is a plant cell.
  • the present invention provides a recombinant cell that metabolizes a PPO inhibiting herbicide. .
  • the cell comprises a polynucleotide of the invention, wherein the cell does not naturally comprise said polynucleotide.
  • the present invention provides a recombinant cell comprising an introduced CytP450 polypeptide of the invention that metabolizes a PPO inhibiting herbicide.
  • the polypeptide is produced by the cell by the expression of a CytP450 polynucleotide of the invention.
  • the present invention provides a process for preparing a CytP450 polypeptide of the invention, the process comprising cultivating a host cell of the invention encoding said polypeptide, or a vector of the invention encoding said polypeptide, under conditions which allow expression of the polynucleotide encoding the polypeptide, and recovering the expressed polypeptide.
  • CytP450 polypeptide produced using a method of the invention.
  • the present invention provides a composition comprising at least one polypeptide of the invention, at least one polynucleotide of the invention, a vector of the invention, a host cell of the invention, and/or a recombinant cell of the invention of the invention, and one or more acceptable carriers.
  • polypeptides useful for the invention can be used as a selectable marker to detect a recombinant cell.
  • a polypeptide of the invention, or a polynucleotide encoding said polypeptide as a selectable marker for detecting and/or selecting a recombinant cell.
  • the present invention provides a method for detecting a recombinant cell, the method comprising
  • a suitable cell examples include, but are not limited to, a plant cell, bacterial cell, fungal cell.
  • the cell is a plant cell.
  • the PPO inhibiting herbicide is Saflufenacil.
  • the present invention provides an extract of host cell of the invention, a recombinant cell of the invention, a transgenic plant of the invention, or a strain of the invention, comprising a polypeptide of the invention.
  • the present invention provides a product produced from a plant of the invention.
  • Examples of products include, but are not limited to, starch, oil, vegetables plant fibres such as cotton, malt and flour.
  • the present invention provides a part of a plant of the invention.
  • examples include, but are not limited to, seeds, fruit and nuts.
  • the present invention provides a method for controlling undesired vegetation at a plant cultivation site, the method comprising the steps of
  • the CytP450 polypeptides of the present invention can be mutated, and the resulting mutants screened for altered activity such as enhanced enzymatic activity.
  • Such mutations can be site- directed or random and can be performed using any technique known in the art including, but not limited to, in vitro or in vivo mutagenesis methods, UV radiation, NTG, transposon-mediated mutagenesis and DNA shuffling.
  • the present invention provides a method of producing a polypeptide with enhanced ability to metabolize a PPO inhibiting herbicide, the method comprising
  • step (ii) determining the ability of the altered CytP450 polypeptide obtained from step (i) to metabolize a PPO inhibiting herbicide
  • polypeptide produced by the method of the invention.
  • the present invention provides a method for screening for a microorganism capable of metabolizing a PPO inhibiting herbicide, the method comprising
  • the microorganism is a bacterium, fungus or protozoa.
  • the microorganism is a recombinant microorganism. Furthermore, it is preferred that a population of recombinant microorganisms is screened, wherein the recombinant microorganisms comprise a plurality of different foreign DNA molecules. Examples of such foreign DNA molecules include plasmid or cosmid genomic DNA libraries.
  • the PPO inhibiting herbicide is Saflufenacil.
  • microorganism isolated using a method of the invention Also provided is a microorganism isolated using a method of the invention.
  • FIG. 1 Influence of P450 inhibitors on S. coelicolor strain analyzed by HPLC measurements of Saflufenacil concentrations; solely medium (negative control), Saflufenacil after incubation with S. coelicolor strain and: 20 ⁇ , 50 ⁇ and 100 ⁇ ABT, 20 ⁇ , 50 ⁇ and 100 ⁇ tetcyclasis, 20 ⁇ , 50 ⁇ and 100 ⁇ ⁇ and medium with 300 ⁇ Saflufenacil and S. coelicolor strain (positive control)
  • FIG. 2 Time dependent degradation pattern of Saflufenacil through S. coelicolor; shown are concentrations of Saflufenacil (line with dots), Metabolite 1 (line with asterisks), Metabolite 2 (line with crossed-out dots) and Metabolite 3 (line with triangles), measured by HPLC.
  • FIG. 3. Plant transformation vector map for RTP8830-3. A.) RTP8830-3 contains the
  • the CYP450-657 Frdxn-656 Plast targ_Gm cassette contains the S. ceolicolor CYP450 gene and ferredoxin 656 fused together and driven by a Parsley ubiquitin promoter (p-PcUbi4-2).
  • the S. ceolicolor CYP105D5 reductase expression is driven by the Super promoter (p-Super). Both encoded sequences are fused to the ferredoxin transit peptide from Silene pratensis for subcellular targeting to plastids (tp-SpFdx).
  • B. A schematic of the protein fusion of the cytochrome P450 CYP657 and the ferredoxin 656 from S. ceolicolor.
  • FIG. 4 Kixor treatment of wild type and transformed soybean cuttings.
  • A. Herbicide injury scores for cuttings of independent TO soybean events (LJ393, HT2106, HT2108, HT2145) transformed with RTP8830-3 (8830-3 in legend) and treated with 0, 6.25, 12.5, 25, 50, 100 g ai/ha Kixor + 1 % MSO.
  • Injury scores are from 0-9, 0 being no injury and 9 being death. Injury evaluation was done 1 week after treatment.
  • B. Photograph of wild type (Williams 82) and RTP8830-3 event HT2145 sprayed with the indicated rates of Kixor. Photos were taken one week after treatment
  • SEQ ID NO:1 Amino acid sequence of Cyp450 polypeptide of the invention .
  • SEQ ID NO:10 Streptomyces coelicolor CYP105D5reductase DNA DETAILED DESCRIPTION OF THE INVENTION
  • 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 herbicide-resistant 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 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.
  • 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.
  • PPO inhibiting herbicides which can be used according to the present invention are acifluorfen, acifluorfen-sodium, aclonifen, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxyfen, cinidon-ethyl, fluazo- late, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoro- glycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadia- zon, oxyfluorfen, pentoxazone, profluazol, pyraclonil, pyraflufen,
  • R 30 and R 31 independently of one another are F, CI or CN;
  • R 32 is O or S
  • R 33 is H, F, CI, CH 3 or OCH 3 ;
  • R 34 is CH or N;
  • R 35 is O or S
  • R 36 is H, CN, CH 3 , CF 3 , OCH 3 , OC 2 H 5 , SCH 3 , SC 2 H 5 , (CO)OC 2 H 5 or CH 2 R 38 ,
  • R 38 is F, CI, OCH 3 , SCH 3 , SC 2 H 5 , CH 2 F, CH 2 Br or CH 2 OH;
  • R 37 is (Ci-C 6 -alkyl)amino, (Ci-C 6 -dialkyl)amino, (NH)OR 39 , OH, OR 40 or SR 40
  • R 39 is CH 3 , C 2 H 5 or phenyl
  • R 40 is independently of one another Ci-C6-alkyl, C 2 -C6-alkenyl, C 3 -C6- alkynyl, Ci-C6-haloalkyl, Ci-C6-alkoxy-Ci-C6-alkyl, Ci-C6-alkoxy-Ci- C6-alkoxy-Ci-C6-alkyl, C 2 -C6-cyanoalkyl, Ci-C4-alkoxy-carbonyl-Ci-C4- alkyl, Ci-C4-alkyl-carbonyl-amino, Ci-C6-alkylsulfinyl-Ci-C6-alkyl, Ci- C6-alkyl-sulfonyl-Ci-C6-alkyl, Ci-C6-dialkoxy-Ci-C6-alkyl, Ci-C6-alkyl- carbonyloxy-Ci-C6-alkyl, phenyl-carbony
  • cycloalkyls indenpently of one another are unsubstituted or substituted by one to five substituents selected from the group consisting of halogen, Ci-C 3 -alkyl and Ci-C 2 -haloalkyl; including their agriculturally acceptable alkali metal salts or ammonium salts.
  • PPO-inhibiting herbicides are the PPO-inhibiting herbicides.1 to A.14 listed below in table A:
  • PPO-inhibiting herbicides described above that are useful to carry out the present invention are often best applied in conjunction with one or more other herbicides to obtain control of a wider variety of undesirable vegetation.
  • PPO-inhibiting herbicides may further be used in conjunction with additional herbicides to which the crop plant is naturally tolerant, or to which it is resistant via expression of one or more additional transgenes as mentioned hereinafter.
  • the PPO-inhibiting herbicides, to which the plant of the present invention had been made resistant or tolerant can be formulated with the other herbicide or herbicides, tank mixed with the other herbicide or herbicides, or applied sequentially with the other herbicide or herbicides.
  • Suitable components for mixtures are, for example, selected from the herbicides of class b1 ) to b15)
  • ALS inhibitors acetolactate synthase inhibitors
  • EBP inhibitors enolpyruvyl shikimate 3-phosphate synthase inhibitors
  • DHP inhibitors 7,8-dihydropteroate synthase inhibitors
  • VLCFA inhibitors inhibitors of the synthesis of very long chain fatty acids
  • herbicides selected from the group consisting of bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, dalapon, dazomet, difenzoquat, dif- enzoquat-metilsulfate, dimethipin, DSMA, dymron, endothal and its salts, etoben- zanid, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flam- prop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine- ammonium, indanofan, indaziflam, maleic hydrazide, mefluidide, metam, methi- ozolin (CAS 403640-27-7), methyl azide, methyl bromide, methyl-dymron, methyl
  • herbicides B which can be used in combination with the PPO-inhibiting herbicides according to the present invention are: b1 ) from the group of the lipid biosynthesis inhibitors:
  • ACC-herbicides such as alloxydim, alloxydim-sodium, butroxydim, clethodim, clodinafop, clodinafop-propargyl, cycloxydim, cyhalofop, cyhalofop-butyl, diclofop, diclofop-methyl, fenoxa- prop, 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
  • sulfonylureas such as amidosulfuron, azimsulfuron, bensulfuron, bensulfuron-methyl, chlo- rimuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, flupyrsul- furon-methyl-sodium, foramsulfuron, halosulfuron, halosulfuron-methyl, imazosulfuron, iodosul- furon, iodosulfuron-methyl-sodium, iofensulfuron, iofensulfuron-sodium, mesosulfuron, met- azosulfuron, metsulfuron, met-
  • imidazolinones such as imazamethabenz, imazamethabenz-methyl, imazamox, imazapic, ima- zapyr, imazaquin and imazethapyr, triazolopyrimidine herbicides and sulfonamides such as cloransulam, cloransulam-methyl, diclosulam, flumetsulam, florasulam, metosulam, penoxsu- lam, pyrimisulfan and pyroxsulam,
  • pyrimidinylbenzoates such as bispyribac, bispyribac-sodium, pyribenzoxim, pyriftalid, pyrimino- bac, pyriminobac-methyl, pyrithiobac, pyrithiobac-sodium, 4-[[[2-[(4,6-dimethoxy-2- pyrimidinyl)oxy]phenyl]methyl]amino]-benzoic acid-1 -methylethyl ester (CAS 420138-41 -6), 4- [[[2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]phenyl]methyl]amino]-benzoic acid propyl ester (CAS 420138-40-5), N-(4-bromophenyl)-2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzenemethanamine (CAS 420138-01 -8),
  • sulfonylaminocarbonyl-triazolinone herbicides such as flucarbazone, flucarbazone-sodium, propoxycarbazone, propoxycarbazone-sodium, thiencarbazone and thiencarbazone-methyl; and triafamone;
  • compositions comprising at least one imidazolinone herbicide; b3) from the group of the photosynthesis inhibitors:
  • inhibitors of the photosystem II e.g. triazine herbicides, including of chlorotria- zine, triazinones, triazindiones, methylthiotriazines and pyridazinones such as ametryn, atra- zine, chloridazone, cyanazine, desmetryn, dimethametryn,hexazinone, metribuzin, prometon, prometryn, propazine, simazine, simetryn, terbumeton, terbuthylazin, terbutryn and trietazin, aryl urea such as chlorobromuron, chlorotoluron, chloroxuron, dimefuron, diuron, fluometuron, isoproturon, isouron, linuron, metamitron, methabenzthiazuron, metobenzuron, metoxuron, monolinuron, neburon, si
  • a preferred embodiment of the invention relates to those compositions comprising at least one aryl urea herbicide. Among these, likewise a preferred embodiment of the invention relates to those compositions comprising at least one triazine herbicide. Among these, likewise 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, cinidon-ethyl, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoroglycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, profluazol, pyraclonil, pyraflufen, pyraf I uf en-ethyl, saflufenacil, sulf
  • 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, clomazone, isoxaflutole, mesotri- one, pyrasulfotole, pyrazolynate, pyrazoxyfen, sulcotrione, tefuryltrione, tembotrione, toprame- zone and bicyclopyrone, bleacher, unknown target: aclonifen, amitrole and flumeturon; b6) from the group of the EPSP synthase inhibitors:
  • bilanaphos (bialaphos), bilanaphos-sodium, glufosinate, glufosinate-P and glufosinate- ammonium; b8) from the group of the DHP synthase inhibitors:
  • compounds of group K1 dinitroanilines such as benfluralin, butralin, dinitramine, ethalfluralin, fluchloralin, oryzalin, pendimethalin, prodiamine and trifluralin, phosphoramidates such as ami- prophos, amiprophos-methyl, and butamiphos, benzoic acid herbicides such as chlorthal, chlor- thal-dimethyl, pyridines such as dithiopyr and thiazopyr, benzamides such as propyzamide and tebutam; compounds of group K2: chlorpropham, propham and carbetamide, among these, compounds of group K1 , in particular dinitroanilines are preferred; b10) from the group of the VLCFA inhibitors: chloroacetamides such as acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, dime- thenamid-P, metazachlor, metolachlor, metolachlor,
  • the isoxazoline compounds of the formula (l)l are known in the art, e.g. from WO 2006/024820, WO 2006/037945, WO 2007/071900 and WO 2007/096576; among the VLCFA inhibitors, preference is given to chloroacetamides and oxyacetamides; b1 1 ) from the group of the cellulose biosynthesis inhibitors:
  • 2,4-D and its salts and esters such as clacyfos, 2,4-DB and its salts and esters, aminocyclopy- rachlor and its salts and esters, aminopyralid and its salts such as 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, fluroxypyr, fluroxy- pyr-butometyl, fluroxypyr-meptyl, halauxifen and its salts and esters (CAS 943832-60-8); MCPA and its salts and esters, MCPA-thioethyl, MCPB and its salts and esters, mecoprop and its salts
  • 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.
  • the weeds of the present invention include, for example, dicotyledonous and mono- cotyledonous 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, Alopecu- rus, 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.
  • 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 or synthetically manufactured 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. It would be understood that such 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.
  • biologically active fragment is a portion of a polypeptide of the invention which maintains a defined activity of the full-length polypeptide, namely be able to metabolize a PPO inhibiting herbicide, especially Saflufenacil.
  • Biologically active fragments can be any size as long as they maintain the defined activity.
  • biologically active fragments are at least 100, more preferably at least 200, and even more preferably at least 350 amino acids in length, most preferably at least 400 amino acids of SEQ ID NO:1
  • the CytP450 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
  • Amino acid sequence mutants of the polypeptides of the present invention can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the present invention, or by in vitro synthesis of the desired polypeptide.
  • Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence.
  • a combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.
  • Mutant (altered) polypeptides can be prepared using any technique known in the art.
  • a polynucleotide of the invention can be subjected to in vitro mutagenesis.
  • in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations.
  • the polynucleotides of the invention are subjected to DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they are able to metabolize a PPO inhibiting herbicide such as Saflufenacil.
  • the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified.
  • the sites for mutation can be modified individually or in series, e.g., by (1 ) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
  • Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
  • Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 .
  • 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 [0131].
  • unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the polypeptides of the present invention.
  • amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4- diaminobutyric acid, [alpha]-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6- amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, [beta]-alanine, fluoro-amino acids, designer amino acids such as [beta]-methyl amino acids, C[alpha]-
  • polypeptides of the present invention which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosyla- tion, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide of the invention.
  • Polypeptides of the present invention can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides.
  • an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide.
  • a preferred cell to culture is a recombinant cell of the present invention.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production.
  • An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention.
  • Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • 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.
  • 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.
  • polynucleotide is used interchangeably herein with the term "nucleic acid”.
  • 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. More preferably, 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.
  • a polynucleotide of the invention comprises a 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
  • the term "gene” is to be taken in its broadest context and includes the deoxyri- bonucleotide sequences comprising the protein coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of at least about 2 kb on either end.
  • the sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences.
  • the sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences.
  • the term “gene” encompasses both cDNA and genomic forms of a gene.
  • the term “gene” includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above.
  • stringent conditions refers to conditions under which a polynucleotide, probe, primer and/or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5[deg]C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.
  • Tm thermal melting point
  • stringent conditions will be those in which the salt concentration is less than about 1 .0 M sodium ion, typically about 0.01 to 1 .0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30[deg.] C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60[deg.] C. for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • a non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6 * SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65[deg.] C, followed by one or more washes in 0.2. * SSC, 0.01 % BSA at 50[deg.] C.
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2 under conditions of moderate stringency is provided.
  • moderate stringency hybridization conditions are hybridization in 6 * SSC, 5 * Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55[deg.] C, followed by one or more washes in 1 * SSC, 0.1 % SDS at 37[deg.] C.
  • Other conditions of moderate stringency that may be used are well-known within the art, see, e.g., Ausubel et al. (supra), and Kriegler, 1990; Gene Transfer And Expression, A Laboratory Manual, Stockton Press, NY.
  • a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NO:2, under conditions of low stringency is provided.
  • a non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5 * SSC, 50 mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt vol) dextran sulfate at 40[deg.] C, followed by one or more washes in 2 * SSC, 25 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1 % SDS at 50[deg.] C.
  • Polynucleotides of the present invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions, of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the nucleic acid).
  • Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. Although the terms polynucleotide and oligonucleotide have overlapping meaning, oligonucleotide are typically relatively short single stranded molecules. The minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a target nucleic acid molecule. Preferably, the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 25 nucleotides in length.
  • monomers of a polynucleotide or oligonucleotide are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from relatively short monomeric units, e.g., 12-18, to several hundreds of monomeric units.
  • Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate.
  • the present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, or primers to produce nucleic acid molecules.
  • Oligonucleotides of the present invention used as a probe are typically conjugated with a detectable label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.
  • Probes and/or primers can be used to clone homologues of the polynucleotides of the invention from other species. Furthermore, hybridization techniques known in the art can also be used to screen genomic or cDNA libraries for such homologues.
  • One embodiment of the present invention includes a recombinant vector, which comprises at least one isolated polynucleotide molecule of the present invention, inserted into any vector capable of delivering the polynucleotide molecule into a host cell.
  • a vector contains heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules of the present invention and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a trans- poson (such as described in U.S. Pat. No. 5,792,294), a virus or a plasmid.
  • One type of recombinant vector comprises a polynucleotide molecule of the present invention operably linked to an expression vector.
  • the phrase operably linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, arthropod, and plant cells.
  • Vectors of the invention can also be used to produce the polypeptide in a cell-free expression system, such systems are well known in the art.
  • Operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element to a transcribed sequence.
  • a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell and/or in a cell-free expression system.
  • promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis- acting.
  • some transcriptional regulatory elements, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention.
  • recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
  • Preferred transcription control sequences include those which function in bacterial, yeast, arthropod, nematode, plant or mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71 ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01 , metallothionein, alpha- mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous
  • Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention, or progeny cells thereof. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
  • Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention.
  • Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptides of the present invention or can be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule of the present invention.
  • Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite, nematode, arthropod, and plant cells.
  • host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS (e.g., COS-7) cells, and Vero cells.
  • E. coli including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
  • Particularly preferred host cells are plant cells.
  • Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, opera- tors, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
  • transcription control signals e.g., promoters, opera- tors, enhancers
  • translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
  • plant as used herein as a noun refers to whole plants, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plant cells and the like.
  • plant organs e.g. leaves, stems, roots, flowers
  • single cells e.g. pollen
  • Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons.
  • Examples of plant species of interest include, but are not limited to, corn or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
  • juncea particularly those Brassi- ca species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Se- cale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennise- tum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annu ), saffiower (Carthamus tinctorius), wheat (Triticum aestivum, T. turgidum ssp.
  • millet e.g., pearl millet (Pennise- tum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine cora
  • plants of the present invention are crop plants (for example, sunflower, Brassica sp., cotton, sugar, beet, soybean, peanut, alfalfa, saffiower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.).
  • crop plants for example, sunflower, Brassica sp., cotton, sugar, beet, soybean, peanut, alfalfa, saffiower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.
  • transgenic plant refers to a plant that comprises a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • Transgenic is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been so altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell.
  • a "recombinant" organism is a "transgenic” organism.
  • transgenic as used herein is not intended to encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods (e.g., crosses) or by naturally occurring events such as, e.g., self- fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacte- rial transformation, non- recombinant transposition, or spontaneous mutation.
  • the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not segregate for the desired pheno- type.
  • the transgenic plants may also be heterozygous for the introduced transgene(s), such as, for example, in F1 progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.
  • a polynucleotide of the present invention may be expressed constitutively in the transgenic plants during all stages of development. Depending on the use of the plant or plant organs, the polypeptides may be expressed in a stage-specific manner. Furthermore, the polynucleotides may be expressed tissue-specifically.
  • regulatory sequences which are known or are found to cause expression of a gene encoding a polypeptide of interest in plants may be used in the present invention.
  • the choice of the regulatory sequences used depends on the target plant and/or target organ of interest.
  • Such regulatory sequences may be obtained from plants or plant viruses, or may be chemically synthesized. Such regulatory sequences are well known to those skilled in the art.
  • plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker.
  • Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmen- tally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribo- some binding site, an RNA processing signal, a transcription termination site, and/or a polyad- enylation signal.
  • a promoter regulatory region e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmen- tally-regulated, or cell- or tissue-specific expression
  • a transcription initiation start site e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmen- tally-regulated, or cell- or tissue-specific expression
  • a transcription initiation start site e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmen- tally-regulated, or cell- or tissue-specific expression
  • RNA processing signal e.g., a transcription
  • 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.
  • 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 ); ubi- quitin (Christensen et al. (1989) Plant Mol. Biol.
  • 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.
  • the expression cassettes 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 (Encephalomyo- carditis 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.
  • MCMV chlorotic mottle virus leader
  • the termination of transcription is accomplished by a 3' non-translated DNA sequence operably linked in the chimeric vector to the polynucleotide of interest.
  • the 3' non-translated region of a recombinant DNA molecule contains a polyadenylation signal that functions in plants to cause the addition of adenylate nucleotides to the 3' end of the RNA.
  • 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.
  • 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.
  • bacterial cytochrome P450 monooxygenase systems are class I systems which require a flavin adenine dinucleotide-containing NADH-dependent reductase (ferredoxin reductase) and an iron-sulfur redoxin (ferredoxin) (Sasaki et al., Appl. Environ. Microbiol. December 2005 vol. 71 no. 12 8024-8030, as well as references contained therein).
  • the expression cassette of the present invention additionally comprises ferredoxin- and reductase encoding nucleic acids which are operably linked to the polynucleotide of the present invention.
  • the ferredoxin- and reductase encoding nucleic acids can be present on the same vector like the polynucleotide of the present invention, employing different promoters or can be present on two or more different vectors to be introduced into a host cell, preferably a plant cell.
  • the expression cassette of the present invention preferably contains the S. ceolicolor CYP450 gene comprising (i) SEQ ID NO: 2, (ii) a sequence of nucleotides encoding a polypeptide of SEQ ID NO:1 , or a polypeptide which is at least 85% identical to SEQ ID NO:1 , iii) a sequence of nucleotides which is at least 80% identical to i), iv) a sequence of nucleotides which hybridizes to i) under low stringency conditions, v) a sequence of nucleotides complementary to i) to iv) and the ferredoxin gene fused together and driven by a promoter, preferably a constitutive promoter, more preferably the Parsley ubiquitin promoter.
  • a promoter preferably a constitutive promoter, more preferably the Parsley ubiquitin promoter.
  • the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 8. Additionally, the expression cassette comprises the reductase-encoding polynucleotide, preferably the S. ceol- icolor CYP105D5 reductase (SEQ ID NO. 10), the expression of which expression can be driven by the Super promoter (p-Super).
  • the reductase-encoding polynucleotide preferably the S. ceol- icolor CYP105D5 reductase (SEQ ID NO. 10), the expression of which expression can be driven by the Super promoter (p-Super).
  • both encoded sequences are fused to a transit peptide, for subcellular targeting to plastids.
  • transit peptides are known in the art.
  • "operably linked" means that the nucleic acid sequence encoding a transit peptide (i.e., the plastid-targeting sequence) is linked to the CytP450 nucleic acid of the invention such that the two sequences are contiguous and in the same reading frame. See, for example, Von Heijne et al. (1991 ) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem.
  • said transit peptide is the ferredoxin transit peptide from Silene pratensis.
  • Acceleration methods include, for example, microprojectile bombardment and the like.
  • microprojectile bombardment One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994).
  • Non- biological particles that may be coated with nucleic acids and delivered into cells by a propelling force.
  • Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
  • An illustrative embodiment of a method for delivering DNA into Zea mays cells by acceleration is a biolistics [alpha]-particle delivery system that can be used to propel particles coated with DNA through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with corn cells cultured in suspension.
  • a particle delivery system suitable for use with the present invention is the helium acceleration PDS-1000/He gun is available from Bio-Rad Laboratories.
  • cells in suspension may be concentrated on filters.
  • Filters containing the cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the gun and the cells to be bombarded.
  • bombardment transformation one may optimize the pre-bombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids.
  • plastids can be stably transformed.
  • Methods disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (U.S. Pat. No. 5,451 ,513, U.S. Pat. No. 5,545,818, U.S. Pat. No. 5,877,402, U.S. Pat. No. 5,932,479, and WO 99/05265).
  • Transgenic plants are preferably produced via Agrobacterium- mediated transformation.
  • An advantageous transformation method is the transformation in planta.
  • Methods for Agrobacterium-medlated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP
  • the nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 871 1 ).
  • 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,
  • a transgenic plant formed using Agrobacterium transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene. More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair.
  • a homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants for the gene of interest.
  • the transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 (Nature Biotechnology 22 (2), 225-229). Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001 ) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21 ; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21 , 20-28.
  • Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. Application of these systems to different plant varieties depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).
  • Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos.
  • This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
  • the development or regeneration of plants containing the foreign, exogenous gene is well known in the art.
  • the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed- grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants.
  • a transgenic plant of the present invention containing a desired exogenous nucleic acid is cultivated using methods well known to one skilled in the art.
  • transgenic wheat or barley plants are produced by Agro bacterium tumefaciens mediated transformation procedures.
  • Vectors carrying the desired nucleic acid construct may be introduced into regenerable wheat cells of tissue cultured plants or explants, or suitable plant systems such as protoplasts.
  • PCR polymerase chain reaction
  • Southern blot analysis can be performed using methods known to those skilled in the art.
  • Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay.
  • One particularly useful way to quantitate protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS.
  • PPO inhibitor-tolerant, preferably PPO inhibitor-tolerant characteristics/traits of the present invention can be stacked with any combination of plant characteristics/trails) of interest to provide plants with a desired combination of characteristics/traits.
  • transgenic plants of the present invention include those plants which, in addition to being PPO inhibitor-tolerant, 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; protoporphyrinogen-IX oxidase (PPO) inhibitors to which the plant have been rendered tolerant by mechanisms other than inactivation or metabolization, e.g.
  • specific other classes of herbicides such as AHAS inhibitors; auxinic herbicides; bleaching herbicides such as hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor
  • PPO inhibitor-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.
  • herbicide resistance technologies are, for example, described in Pest Management Science (at volume, year, page): 61 , 2005, 246; 61 , 2005, 258; 61 , 2005, 277; 61 , 2005, 269; 61 , 2005, 286; 64, 2008, 326; 64, 2008, 332; Weed Science 57, 2009, 108; Australian Journal of Agricultural Research 58, 2007, 708; Science 316, 2007, 1 185; and references quoted therein.
  • PPO inhibitor-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 quizalo- fop), 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, diclofo
  • PPO inhibitor-tolerant plants of the invention may also be tolerant to herbicides having other modes of action, for example, chlorophyll/carotenoid pigment inhibitors, cell membrane disrupters, photosynthesis inhibitors, cell division inhibitors, root inhibitors, shoot inhibitors, and combinations thereof
  • tolerance traits may be expressed, e.g.
  • mutant AHASL proteins mutant ACCase proteins, mutant EPSPS proteins, or mutant glutamine synthetase proteins
  • AAD or DHT transgenic aryloxyalka- noate dioxygenase
  • PPO inhibitor- 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.
  • the present invention teaches compositions and methods for increasing the PPO-inhibiting tolerance of a crop plant or seed comprising a polynucleotide encoding a CYP450 polypeptide according to the present invention as compared to an untransformed 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-inhibiting 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 .
  • a PPO-inhibiting 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). Furthermore, the present invention provides methods that involve the use of at least one PPO- inhibiting herbicide, optionally in combination with one or more herbicidal compounds B, as described in detail supra.
  • the PPO-inhibiting 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-inhibiting herbicide 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-inhibiting herbicide By providing plants having increased tolerance to PPO-inhibiting 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-inhibiting 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-inhibiting herbicide formulation can be used that contains other additives.
  • the PPO-inhibiting herbicide can also be used as a seed treatment. Additives found in a PPO-inhibiting herbicide formulation include other herbicides, detergents, adjuvants, spreading agents, sticking agents, stabilizing agents, or the like.
  • the PPO-inhibiting 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-inhibiting herbicide and herbicide formulations can be applied in accordance with conventional methods, for example, by spraying, irrigation, dusting, or the like.
  • a polypeptide of the present invention can be provided in a composition which enhances the rate and/or degree of metabolization of a PPO inhibiting herbicide, or increases the stability of the polypeptide.
  • the polypeptide can be immobilized on a polyurethane matrix (Gordon et al., 1999), or encapsulated in appropriate liposomes (Petrikovics et al. 2000a and b).
  • One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a polypeptide of the present invention into a plant, plant material, or the environment (including soil and water samples).
  • a controlled release formulation comprises a composition of the present invention in a controlled release vehicle.
  • Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, micro- particles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres.
  • Preferred controlled release formulations are biodegradable (i.e., bioerodible).
  • Example 1 Screening for PPO inhibitor-metabolizing microorganisms
  • CytP450 monooxygenase inhibitors indicate CytP450 monooxygenase to be involved in Saflufenacil metabolization
  • CytP450 enzymes are known for their detoxifying potential (Thies et al., 1996; De Schrijver et al., 1999). To sustain the assumption, that a CytP450 demethylates Saflufenacil and thereby turns it into a non herbicidal derivative, several CytP450 monooxygenase inhibitors were tested. Five different inhibitors of CytP450 enzymes have been described (Thies et al., 1996) which are able to suppress the enzymatic activity of CytP450 monooxygenases. These substances were tested in a Streptomyces Saflufenacil degradation assay.
  • Figure 1 shows the results of this assay, including a negative control (solely medium), a positive control (a S. coelicolor strain with Saflufenacil) and the samples.
  • Example 3 Time-dependent metabolization of Saflufenacil by a S. coelicolor strain
  • CytP105D5 SEQ ID NO:1 as decribed by Chun et al.
  • the primers used for knock-out experiments are depicted in SEQ ID NOs: 3, 4, 5, and 6.
  • the knockout plasmid was constructed using two PCR products homologous to the Streptomyces genome, covering the locus of the CytP450 gene. The two PCR fragments with 1510 bp and 1557 bp, respectively, were cloned into the shuttle plasmid pKC1 132, flanking the hygromycin resistance conferring gene hph.
  • pKC1 132 is a plasmid not able to replicate in Streptomyces.
  • the resistance gene was cloned to the plasmid backbone, using BamHI and EcoRV restriction sites, followed by the homologous fragment covering the upstream region and the 5' end of SEQ ID NO: 2, utilizing Xbal and BamHI restriction sites.
  • the homologous fragment covering the 3' end, as well as a downstream region of SEQ ID NO: 2 was cloned to the plasmid using EcoRV restriction sites.
  • the resulting plasmid was named p62. Correct cloning was verified by restriction analysis and sequencing. The verified plasmid was introduced into E.
  • conjugation p62 was introduced into the respective S. coelicolor strain.
  • Exconjugands were plated out on MS plates (mannitol 20 g/L, soy flour 20 g/L) containing 100 ⁇ g/ ⁇ L hygromycin, for selection for crossover events.
  • Colonies resistant to hygromycin were transferred onto plates containing apramycin (for selection on the plasmid p62) and plates containing hygromycin. Such colonies that solely grew on agar containing only the latter antibiotic indicated a double-crossover event. Those colonies were chosen for further experiments.
  • Table 2 Data from HPLC; S. coelicolor wildtype (WT) cultivated for 5 d with -200 ⁇
  • Example 7 Plant transformation with Streptomyces coelicolor CYP105D5 cytochromo P450 system results in Kixor tolerance.
  • the first cassette contains the Parsley ubiq- uitin promoter driving the expression of a fusion of sequences (named CYP450-657 Frdxn-656 Plast targ_Gm, see SEQ ID NO: 8) ( Figure 3B) that encode the plastid targeting transit peptide from Silene pratensis ferredoxin (51 amino acids), the open reading frame of the Streptomyces coelicolor CYP105D5 (410 amino acids), a linker sequence from Methylococcus capsulatus MCCYP51 FX (21 amino acids) (see Jackson et al., J. Biol. Chem. 2002:277(49):46959-46965), and the open reading frame of a S.
  • Figure 3B that encode the plastid targeting transit peptide from Silene pratensis ferredoxin (51 amino acids), the open reading frame of the Streptomyces coelicolor CYP105D5 (410 amino acids), a
  • the second cassette contains the SUPER promoter (originating from Agrobacterium tumefaciens and contains 3 repeats of OCS enhancers upstream of mas2 promoter) driving the expression of the S. coelicolor 105D5 reductase (see SEQ ID NO: 10).
  • the coding sequences for each cassette were codon optimized for soybean based on frequency of codon usage and made via de novo DNA synthesis.
  • Soybean cv Williams 82 was transformed as previously described (see Hong HP, et al., In Vitro Cell. Dev. Biol. -Plant 2007: 43: 558-568). After regeneration, transformants were transplanted to soil in small pots, placed in growth chambers (18 hr day/ 6 hr night; 26°C constant; 65% relative humidity; 130-150 DE nr 2 s _1 ) and subsequently tested for the presence of the T-DNA via Taq- man analysis. After a few weeks, healthy, transgenic positive, 1 -2 copy events were transplanted to larger pots and allowed to grow in the growth chamber for subsequent generation of clones via shoot cutting. An optimal shoot for cutting was about 3-4" tall, with at least two nodes present.
  • Each cutting was taken from the original transformant (mother plant) and dipped into rooting hormone powder (indole-3-butyric acid, IBA). The cutting was then placed in oasis wedges inside a bio-dome. The mother plant was taken to maturity in the greenhouse and harvested for seed. Wild type cuttings were also taken simultaneously to serve as negative controls. The cuttings were kept in the bio-dome for 5-7 days and then transplanted to 3" pots and then acclimated in the growth chamber for two more days.
  • rooting hormone powder indole-3-butyric acid, IBA
  • the cuttings were transferred to the greenhouse, acclimated for approximately 4 days, and then subjected to spray tests containing 0, 6.25, 12.5, 25, and 50, 100 g ai/ha Saflufenacil (Kixor® or Sharpen®) plus 1 % MSO. Herbicide injury evaluations were taken at 7 days after treatment.
  • FIG. 3 A plant transformation vector, RTP8830-3, ( Figure 3) harboring two transgene expression cassettes in addition to the selectable marker was tested for the ability to confer tolerance to Saflufenacil in TO soybean cuttings.
  • Figures 4 and 5 indicate the level of injury of those plants treated with 0, 6.25, 12.5 25, 50, and 100 g ai/ha Saflufenacil + 1 % MSO. 1 out of 4 events showed increased tolerance to Saflufenacil at all spray rates tested.
  • Table 3 describes the health of the plant an type of injury observed on the 10 point injury rating scale indicated in Figure 4.
  • Table 3 Description of the 10 point injury rating scale.

Abstract

A method for metabolizing PPO-inhibiting herbicides, comprising contacting the PPO-inhibiting herbicide with a CytP450 monooxygenase, is provided. Transgenic plants comprising the CytP450 monooxygenase polypeptide of the invention, as well as methods for producing the same are provided. A method for controlling weeds, employing the CytP450 monooxygenase polypeptide of the invention, a method for producing a polypeptide with enhanced ability to metabolize PPO-inhibiting herbicide, and a method for screening for a microorganism capable of metabolizing PPO-inhibiting herbicide are also provided.

Description

HERBICIDE-METABOLIZING CYTOCHROME P450 MONOOXYGENASE
FIELD OF THE INVENTION
This application claims priority of applications with number 12181039.4 and 61/684831 , all of which are incorporated by reference in their entirety.
The present invention relates to methods of using a bacterial cytochrome P450 monooxygenase which is able to metabolize PPO inhibiting herbicides such as Saflufenacil, butafenacil, S-3100, or benzfendizone, as well as polynucleotides encoding this enzyme. The invention also relates to transgenic plants producing these enzymes which are resistant and/or tolerant to PPO inhibiting herbicide activity.
BACKGROUND OF THE INVENTION
Cytochrome P450 monooxygenases ("CytP450s") form a large diverse gene family with about 246 isoforms in Arabidopsis and 372 identified in rice. CytP450s are hemoproteins that convert a broad range of substrates to more or less bioactive products. The reaction cycle catalyzed by CytP450s requires the sequential input of two reducing equivalents (i.e., two electrons and two protons). The reducing equivalents for the CytP450-catalyzed reaction are supplied by either NADPH or NADH, depending on the type of redox system concerned, and electron transfer is mediated by two co-factors, one of which is FAD; the other being either FMN or an iron-sulfur Fe2S2 redoxin (ferredoxin) or, in the microsomal system, cytochrome b5. In particular, the majority of bacterial CytP450s utilize an electron transport chain which consists of an FAD-containing NADH-dependent oxidoreductase, and reduction is mediated by ferredoxin. The mitochondrial system in mammalia bears many similarities with the prokaryotic P450 electron transport chain, except that NADPH is the source of reducing equivalents, and both systems are generally referred to as Class I (see Lewis and Hlavica, Biochimica et Biophysica Acta 1460 (2000) 353- 374, as well as references contained therein).
CytP450s are critical in numerous metabolic pathways, including lignin and pigment biosynthesis, detoxification of harmful compounds, and are considered important in the evolution of land plants. Inhibitors of CytP450 activity include 1-aminobenzo-triazole, tetcyclacis, piperonyl butox- ide, cinnamonic acid, and tridiphane. Saflufenacil is an herbicide active ingredient (A.I.) of the pyrimidinedione chemical class that is similar to flumioxazin and sulfentrazone and is readily absorbed by foliage, root, and shoot tissue of plants.
It is believed that Saflufenacil inhibits the pigment biosynthesis pathway at protoporphyrinogen oxidase (PPO), which causes an accumulation of photodynamic, toxic compounds that rapidly damage cell membranes and results in cell death. Herbicidal compositions comprising PPO- inhibiting herbicides such as Saflufenacil have been labeled for pre-plant or pre-emergence treatment in corn, sorghum, wheat, barley, oats, rye, triticale, soybean, and tree/nut/vine cropping systems. PPO inhibitor-containing herbicidal compositions have good foliar and residual activity on broadleaf weeds in both no-till and tilled cropping systems. However, application of PPO-inhibiting herbicides such as Saflufenacil after emergence can result in rapid and signifi- cant crop injury. Thus, interest has been gained in the enzymatic degradation or modification of PPO-inhibiting herbicides such as Saflufenacil, both due to concern about the environmental fate of the molecule and as an additional complementation to the systems for engineering herbicide-tolerant plants by augmenting PPO levels in the plant, or replacing the native PPO with a modified PPO conferring tolerance to PPO-inhibiting herbicides such as Saflufenacil.
Several approaches can lead to herbicide tolerant plants: a) modification of the target molecule of the herbicide, b) metabolic approach, i. e. making the compound non-hazardous. For the metabolic solution, one or more enzymes are needed, that catalyze the conversion of the herbicide to a non toxic compound. One source of such enzymes can be microorganisms isolated from nature. Bacteria, especially those of the order Actinomycetales, are known for their potential to detoxify soil by metabolizing xenobiotics, including herbicides (Cork et al, 1991 ; Schrijver et al., 1999; Caracciolo et al., 2010). These detoxifying reactions can be catalyzed by O- demethylases from Pseudomonas maltophilia DI-6, like it was shown for the herbicide Dicamba (Chakraborty et al., 2005; Wang et al., 1997).
In many other cases those reactions are catalyzed by CytP450s. Those can be plant derived (Pan et al., 2006), from algal origin (Thies et al., 1996) or microbial (O'Keefe et al., 1991 ).
Among bacteria, especially actinomycetes offer a broad spectrum of CytP450s. The genome analysis of Streptomyes coelicolor revealed 18 CytP450s (Lamb et al., 2002), the Streptomyes avermitilis genome revealed 33 (Lamb et al., 2003), respectively. According to Nelson (201 1 ), actinobacteria hold the largest number of CytP450s per genome.
Current enzymes available for metabolizing PPO-inhibiting herbicides such as Saflufenacil, e.g. those described in WO2010/143743 particularly when expressed in plants, do not have particularly high activity. Thus, there is the need for the identification of further enzymes which can be used to degrade PPO inhibiting herbicides such as Saflufenacil.
The inventors of the present invention have now surprisingly found that another CytP450 which has been described and characterized by Chun et al. (The Journal of Biological Chemistry , 2007, Vol. 282, p 17486-17500) functions in metabolizing PPO inhibiting herbicides, particularly Saflufenacil.
SUMMARY OF THE INVENTION
Accordingly, in a first aspect, the present invention provides a method for metabolizing a PPO inhibiting herbicide, the method comprising contacting a PPO inhibiting herbicide with a
CytP450 polypeptide of the invention, as described hereinafter in greater detail,
wherein the CytP450 polypeptide comprises amino acids having a sequence selected from: i) SEQ ID NO:1 , and
ii) an amino acid sequence which is at least 85% identical to i).
Examples of PPO inhibiting herbicides include, but are not limited to Saflufenacil, flumioxazin, butafenacil, S-3100, benzfendizone, and sulfentrazone. In a preferred embodiment, the PPO inhibiting herbicide is Saflufenacil.
In one embodiment, the CytP450 polypeptide is produced by a host cell of the invention. Preferably, the PPO inhibiting herbicide is Saflufenacil.
Polypeptides provided herein can be produced in plants to enhance the host plants ability to grow when exposed to a PPO inhibiting herbicide such as Saflufenacil, flumioxazin, butafenacil, S-3100, benzfendizone, and sulfentrazone.
Thus, in yet a further aspect the present invention provides a transgenic plant comprising an exogenous polynucleotide, the polynucleotide encoding at least one CytP450 polypeptide of the invention.
Preferably, the PPO inhibiting herbicide is Saflufenacil, flumioxazin, butafenacil, S-3100, benzfendizone, and sulfentrazone.
Preferably, the polynucleotide is stably incorporated into the genome of the plant.
Also provided is a method of producing plants with enhanced resistance to a PPO inhibiting herbicide comprising the steps of: a) inserting into the genome of a plant cell a CytP450 polynucleotide comprising: a promoter that functions in plant cells to cause the production of a RNA sequence, operably linked to; a structural DNA sequence that caused the production of a RNA sequence that encodes a polypeptide of the invention, operably linked to; a 3' non-translated region that functions in plant cells to cause the addition of polyadenyl nucleotides at the 3' end of the RNA sequence; where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the polypeptide to enhance resistance to a PPO inhibiting herbicide of a plant cell transformed with the DNA molecule; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which has increased resistance to a PPO inhibiting herbicide,
Wherein the CytP450 polynucleotide comprises nucleotides having a sequence selected from: i) SEQ ID NO:2
ii) a sequence of nucleotides encoding a polypeptide of SEQ ID NO:1 , or a polypeptide
which is at least 85% identical to SEQ ID NO:1
iii) a sequence of nucleotides which is at least 80% identical to i),
iv) a sequence of nucleotides which hybridizes to i) under low stringency conditions, v) a sequence of nucleotides complementary to i) to iv).
Preferably, the polynucleotide encodes a CytP450 polypeptide that metabolizes a PPO inhibiting herbicide. More preferably, the PPO inhibiting herbicide is Saflufenacil.
In a preferred embodiment, the CytP450 polynucleotide comprises a sequence which hybridizes to i) under moderate stringency conditions. More preferably, the polynucleotide comprises a sequence which hybridizes to i) under stringent conditions.
In a further aspect, the present invention provides a transgenic plant produced using a method of the invention.
In another aspect, the present invention provides a method for metabolizing a PPO inhibiting herbicide in a sample, the method comprising exposing the sample to a transgenic plant of the invention.
Preferably, the sample is soil. Such soil can be in a field.
In another aspect, the present invention provides a recombinant CytP450 polynucleotide comprising a promoter that functions in a plant cell, operably linked to a structural DNA sequence that encodes a polypeptide of the invention, operably linked to a 3' polyadenylation sequence that functions in the cell, wherein the promoter is heterologous with respect to the structural DNA sequence and capable of expressing the structural DNA sequence to enhance resistance of the cell to a PPO inhibiting herbicide.
In a further aspect, the present invention provides a vector comprising the CytP450 polynucleotide of the invention.
Preferably, the CytP450 polynucleotide is operably linked to a promoter.
In yet a further aspect, the present invention provides a host cell comprising at least one polynucleotide of the invention, and/or at least one vector of the invention.
The host cell can be any type of cell. In one embodiment, the host cell is a plant cell.
In another aspect, the present invention provides a recombinant cell that metabolizes a PPO inhibiting herbicide. .
Preferably, the cell comprises a polynucleotide of the invention, wherein the cell does not naturally comprise said polynucleotide.
In another aspect, the present invention provides a recombinant cell comprising an introduced CytP450 polypeptide of the invention that metabolizes a PPO inhibiting herbicide.
Preferably, the polypeptide is produced by the cell by the expression of a CytP450 polynucleotide of the invention.
In another aspect, the present invention provides a process for preparing a CytP450 polypeptide of the invention, the process comprising cultivating a host cell of the invention encoding said polypeptide, or a vector of the invention encoding said polypeptide, under conditions which allow expression of the polynucleotide encoding the polypeptide, and recovering the expressed polypeptide.
Also provided is a CytP450 polypeptide produced using a method of the invention.
In yet another aspect, the present invention provides a composition comprising at least one polypeptide of the invention, at least one polynucleotide of the invention, a vector of the invention, a host cell of the invention, and/or a recombinant cell of the invention of the invention, and one or more acceptable carriers.
The polypeptides useful for the invention can be used as a selectable marker to detect a recombinant cell. Thus, also provided is the use of a polypeptide of the invention, or a polynucleotide encoding said polypeptide, as a selectable marker for detecting and/or selecting a recombinant cell.
In a further aspect, the present invention provides a method for detecting a recombinant cell, the method comprising
i) contacting a cell or a population of cells with a polynucleotide encoding a polypeptide of the invention under conditions which allow uptake of the polynucleotide by the cell(s), and ii) selecting a recombinant cell by exposing the cells from step i), or progeny cells thereof, to a PPO inhibiting herbicide.
Examples of a suitable cell include, but are not limited to, a plant cell, bacterial cell, fungal cell. Preferably, the cell is a plant cell.
Preferably, the PPO inhibiting herbicide is Saflufenacil.
In a further aspect, the present invention provides an extract of host cell of the invention, a recombinant cell of the invention, a transgenic plant of the invention, or a strain of the invention, comprising a polypeptide of the invention.
In another aspect, the present invention provides a product produced from a plant of the invention.
Examples of products include, but are not limited to, starch, oil, vegetables plant fibres such as cotton, malt and flour.
In a further aspect, the present invention provides a part of a plant of the invention. Examples include, but are not limited to, seeds, fruit and nuts.
In a further aspect, the present invention provides 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 a CytP450 polynucleotide encoding a polypeptide comprising a sequence selected from: i) an amino acid sequence as provided in SEQ ID NO:1 , and ii) an amino acid sequence which is at least 85% identical to i), said polypeptide metabolizing a PPO-inhibiting herbicide;
b) applying to said site an effective amount of said PPO-inhibiting herbicide herbicide.
The CytP450 polypeptides of the present invention can be mutated, and the resulting mutants screened for altered activity such as enhanced enzymatic activity. Such mutations can be site- directed or random and can be performed using any technique known in the art including, but not limited to, in vitro or in vivo mutagenesis methods, UV radiation, NTG, transposon-mediated mutagenesis and DNA shuffling.
Thus, in a further aspect the present invention provides a method of producing a polypeptide with enhanced ability to metabolize a PPO inhibiting herbicide, the method comprising
(i) altering one or more amino acids of a first CytP450 polypeptide of the invention,
(ii) determining the ability of the altered CytP450 polypeptide obtained from step (i) to metabolize a PPO inhibiting herbicide, and
(iii) selecting an altered CytP450 polypeptide with enhanced ability to metabolize a PPO
inhibiting herbicide, when compared to the first CytP450 polypeptide.
Also provided is a polypeptide produced by the method of the invention.
In yet another aspect, the present invention provides a method for screening for a microorganism capable of metabolizing a PPO inhibiting herbicide, the method comprising
i) culturing a candidate microorganism in the presence of a PPO inhibiting herbicide, and ii) determining the amount of metabolized PPO inhibiting herbicide.
In one embodiment, the microorganism is a bacterium, fungus or protozoa.
In a preferred embodiment, the microorganism is a recombinant microorganism. Furthermore, it is preferred that a population of recombinant microorganisms is screened, wherein the recombinant microorganisms comprise a plurality of different foreign DNA molecules. Examples of such foreign DNA molecules include plasmid or cosmid genomic DNA libraries.
Preferably, the PPO inhibiting herbicide is Saflufenacil.
Also provided is a microorganism isolated using a method of the invention.
As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. DESCRIPTION OF THE DRAWINGS
FIG. 1 . Influence of P450 inhibitors on S. coelicolor strain analyzed by HPLC measurements of Saflufenacil concentrations; solely medium (negative control), Saflufenacil after incubation with S. coelicolor strain and: 20 μΜ, 50 μΜ and 100 μΜ ABT, 20 μΜ, 50 μΜ and 100 μΜ tetcyclasis, 20 μΜ, 50 μΜ and 100 μΜ ΡΒΟ and medium with 300 μΜ Saflufenacil and S. coelicolor strain (positive control)
FIG. 2. Time dependent degradation pattern of Saflufenacil through S. coelicolor; shown are concentrations of Saflufenacil (line with dots), Metabolite 1 (line with asterisks), Metabolite 2 (line with crossed-out dots) and Metabolite 3 (line with triangles), measured by HPLC.
FIG. 3. Plant transformation vector map for RTP8830-3. A.) RTP8830-3 contains the
ATAHASL A122T S653N expression cassette as the selectable marker. The CYP450-657 Frdxn-656 Plast targ_Gm cassette contains the S. ceolicolor CYP450 gene and ferredoxin 656 fused together and driven by a Parsley ubiquitin promoter (p-PcUbi4-2). The S. ceolicolor CYP105D5 reductase expression is driven by the Super promoter (p-Super). Both encoded sequences are fused to the ferredoxin transit peptide from Silene pratensis for subcellular targeting to plastids (tp-SpFdx). B.) A schematic of the protein fusion of the cytochrome P450 CYP657 and the ferredoxin 656 from S. ceolicolor.
FIG. 4. Kixor treatment of wild type and transformed soybean cuttings. A.) Herbicide injury scores for cuttings of independent TO soybean events (LJ393, HT2106, HT2108, HT2145) transformed with RTP8830-3 (8830-3 in legend) and treated with 0, 6.25, 12.5, 25, 50, 100 g ai/ha Kixor + 1 % MSO. Injury scores are from 0-9, 0 being no injury and 9 being death. Injury evaluation was done 1 week after treatment. B.) Photograph of wild type (Williams 82) and RTP8830-3 event HT2145 sprayed with the indicated rates of Kixor. Photos were taken one week after treatment
KEY TO THE SEQUENCE LISTING
SEQ ID NO:1 -Amino acid sequence of Cyp450 polypeptide of the invention .
SEQ ID NO:2-Nucleotide sequence encoding Cyp450 polypeptide of the invention.
SEQ ID NO:3- Primer 1 Forward
SEQ ID NO:4: Primer 1 Reverse
SEQ ID NO.5: Primer 2 Forward
SEQ ID NO:6- Primer 2 Reverse
SEQ ID NO:7- CYP450-Frdxn-Plast targ_Gm Fusion Protein
SEQ ID NO:8- CYP450-Frdxn-Plast targ_Gm DNA
SEQ ID NO:9- Streptomyces coelicolor CYP105D5reductase Protein
SEQ ID NO:10: Streptomyces coelicolor CYP105D5reductase DNA DETAILED DESCRIPTION OF THE INVENTION General Techniques
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1 -4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), Genetic manipulation of Strep- tomyces: a laboratory manual, D. A. Hopwood et al. (2000) Norwich, The John Innes Foundation and are incorporated herein by reference.
PPO inhibitingHerbicides
Generally, 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." By "effective amount" and "effective 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 herbicide-resistant plants, plant tissues, plant cells, and host cells of the present invention. Typically, 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 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.
By 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.
Examples of PPO inhibiting herbicides which can be used according to the present invention are acifluorfen, acifluorfen-sodium, aclonifen, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxyfen, cinidon-ethyl, fluazo- late, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoro- glycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadia- zon, oxyfluorfen, pentoxazone, profluazol, pyraclonil, pyraflufen, pyraf I uf en-ethyl, saflufenacil, sulfentrazone, thidiazimin, tiafenacil, chlornitrofen, flumipropyn, fluoronitrofen, flupropacil, fu- ryloxyfen, nitrofluorfen, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo- 1 ,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100), N- ethyl-3-2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1 H-pyrazole-1 -carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1 H- pyrazole-1 -carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethyl- phenoxy)-5-methyl-1 H-pyrazole-1 -carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl-3-(2- chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1 H-pyrazole-1 -carboxamide (CAS 452100- 03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1 ,4]oxazin-6-yl]-1 ,5-dimethyl-6- thioxo-[1 ,3,5]triazinan-2,4-dione (CAS 451484-50-7), 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 (CAS 1258836-72-4), 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1 ,4]oxazin-6-yl)- 4,5,6,7-tetrahydro-isoindole-1 ,3-dione (CAS 13001 18-96-0), 1 -Methyl-6-trifluoromethyl-3-(2,2,7- trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1 ,4]oxazin-6-yl)-1 H-pyrimidine-2,4-dione, methyl (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1 H-methyl-pyrazol-3-yl]-4-fluoro- phenoxy]-3-methoxy-but-2-enoate [CAS 948893-00-3;, 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)- 1 H-benzimidazol-4-yl]-1 -methyl-6-(trifluoromethyl)-1 H-pyrimidine-2,4-dione (CAS 212754-02-4), and uracils of formula III
36
Figure imgf000011_0001
wherein
R30 and R31 independently of one another are F, CI or CN;
R32 is O or S;
R33 is H, F, CI, CH3 or OCH3; R34 is CH or N;
R35 is O or S;
R36 is H, CN, CH3, CF3, OCH3, OC2H5, SCH3, SC2H5, (CO)OC2H5 or CH2R38,
wherein R38 is F, CI, OCH3, SCH3, SC2H5, CH2F, CH2Br or CH2OH; and
R37 is (Ci-C6-alkyl)amino, (Ci-C6-dialkyl)amino, (NH)OR39, OH, OR40 or SR40
wherein R39 is CH3, C2H5 or phenyl; and
R40 is independently of one another Ci-C6-alkyl, C2-C6-alkenyl, C3-C6- alkynyl, Ci-C6-haloalkyl, Ci-C6-alkoxy-Ci-C6-alkyl, Ci-C6-alkoxy-Ci- C6-alkoxy-Ci-C6-alkyl, C2-C6-cyanoalkyl, Ci-C4-alkoxy-carbonyl-Ci-C4- alkyl, Ci-C4-alkyl-carbonyl-amino, Ci-C6-alkylsulfinyl-Ci-C6-alkyl, Ci- C6-alkyl-sulfonyl-Ci-C6-alkyl, Ci-C6-dialkoxy-Ci-C6-alkyl, Ci-C6-alkyl- carbonyloxy-Ci-C6-alkyl, phenyl-carbonyl-Ci-C6-alkyl, tri(Ci-C3-alkyl)- silyl-d-Ce-alkyl, tri(Ci-C3-alkyl)-silyl-Ci-C6-alkenyl, tri(Ci-C3-alkyl)- silyl-Ci-C6-alkynyl, tri(Ci-C3-alkyl)-silyl-Ci-C6-alkoxy-Ci-C6-alkyl, di- methylamino, tetrahydropyranyl, tetrahydrofuranyl-Ci-C3-alkyl, phe- nyl-Ci-C6-alkoxy-Ci-C6-alkyl, phenyl-Ci-C3-alkyl, pyridyl-Ci-C3-alkyl, pyridyl, phenyl,
which pyridyls and phenyls independently of one another are substituted by one to five substituents selected from the group consisting of halogen, Ci-C3-alkyl or Ci-C2-haloalkyl;
C3-C6-cycloalkyl or C3-C6-cycloalkyl-Ci-C4-alkyl,
which cycloalkyls indenpently of one another are unsubstituted or substituted by one to five substituents selected from the group consisting of halogen, Ci-C3-alkyl and Ci-C2-haloalkyl; including their agriculturally acceptable alkali metal salts or ammonium salts.
Especially preferred PPO-inhibiting herbicides are the PPO-inhibiting herbicides.1 to A.14 listed below in table A:
Table A
A.1 acifluorfen
A.2 butafenacil
A.3 benzfenidizone
A.4 carfentrazone-ethyl
A.5 cinidon-ethyl
A.6 flumioxazin
A.7 fluthiacet-methyl
A.8 fomesafen
A.9 lactofen
A.10 oxadiargyl
A.1 1 oxyfluorfen A.12 saflufenacil
A.13 sulfentrazone
A.14 ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 - 6)
A.15 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 (CAS 1258836- 72-4)
The PPO-inhibiting herbicides described above that are useful to carry out the present invention are often best applied in conjunction with one or more other herbicides to obtain control of a wider variety of undesirable vegetation. For example, PPO-inhibiting herbicides may further be used in conjunction with additional herbicides to which the crop plant is naturally tolerant, or to which it is resistant via expression of one or more additional transgenes as mentioned hereinafter. When used in conjunction with other targeting herbicides, the PPO-inhibiting herbicides, to which the plant of the present invention had been made resistant or tolerant, can be formulated with the other herbicide or herbicides, tank mixed with the other herbicide or herbicides, or applied sequentially with the other herbicide or herbicides.
Suitable components for mixtures are, for example, selected from the herbicides of class b1 ) to b15)
B) 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);
b1 1 ) cellulose biosynthesis inhibitors;
b12) decoupler herbicides;
b13) auxinic herbicides;
b14) auxin transport inhibitors; and
b15) other herbicides selected from the group consisting of bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, dalapon, dazomet, difenzoquat, dif- enzoquat-metilsulfate, dimethipin, DSMA, dymron, endothal and its salts, etoben- zanid, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flam- prop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine- ammonium, indanofan, indaziflam, maleic hydrazide, mefluidide, metam, methi- ozolin (CAS 403640-27-7), methyl azide, methyl bromide, methyl-dymron, methyl iodide, MSMA, oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoc- lamine, triaziflam, tridiphane and 6-chloro-3-(2-cyclopropyl-6-methylphenoxy)-4- pyridazinol (CAS 499223-49-3) and its salts and esters; including their agriculturally acceptable salts or derivatives.
Examples of herbicides B which can be used in combination with the PPO-inhibiting herbicides according to the present invention are: b1 ) from the group of the lipid biosynthesis inhibitors:
ACC-herbicides such as alloxydim, alloxydim-sodium, butroxydim, clethodim, clodinafop, clodinafop-propargyl, cycloxydim, cyhalofop, cyhalofop-butyl, diclofop, diclofop-methyl, fenoxa- prop, 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-ethyl, quizalofop-tefuryl, quizalo- fop-P, quizalofop-P-ethyl, quizalofop-P-tefuryl, sethoxydim, tepraloxydim, tralkoxydim,
4-(4'-Chloro-4-cyclopropyl-2'-fluoro[1 ,1 '-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran- 3(6H)-one (CAS 1312337-72-6); 4-(2,,4,-Dichloro-4-cyclopropyl[1 ,1 ,-biphenyl]-3-yl)-5-hydroxy- 2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-45-3); 4-(4'-Chloro-4-ethyl-2'-fluoro[1 ,1 '- biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1033757-93-5); 4-(2',4'- Dichloro-4-ethyl[1 ,1 '-biphenyl]-3-yl)-2,2,6,6-tetramethyl-2H-pyran-3,5(4H,6H)-dione (CAS 1312340-84-3); 5-(Acetyloxy)-4-(4,-chloro-4-cyclopropyl-2,-fluoro[1 ,1 '-biphenyl]-3-yl)-3,6- dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312337-48-6); 5-(Acetyloxy)-4-(2',4'- dichloro-4-cyclopropyl- [1 ,1 '-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one; 5- (Acetyloxy)-4-(4'-chloro-4-ethyl-2'-fluoro[1 ,1 '-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H- pyran-3-one (CAS 1312340-82-1 ); 5-(Acetyloxy)-4-(2',4'-dichloro-4-ethyl[1 ,1 '-biphenyl]-3-yl)-3,6- dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1033760-55-2); 4-(4'-Chloro-4-cyclopropyl-2'- fluoro[1 ,1 '-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1312337-51 -1 ); 4-(2',4'-Dichloro -4-cyclopropyl- [1 ,1 '-biphenyl]-3-yl)-5,6- dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester; 4-(4'-Chloro-4- ethyl-2'-fluoro[1 ,1 '-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1312340-83-2); 4-(2',4'-Dichloro-4-ethyl[1 ,1 '-biphenyl]-3-yl)-5,6-dihydro- 2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1033760-58-5); and non ACC herbicides such as benfuresate, butylate, cycloate, dalapon, dimepiperate, EPTC, esprocarb, ethofumesate, flupropanate, molinate, orbencarb, pebulate, prosulfocarb, TCA, thiobencarb, tiocarbazil, triallate and vernolate; b2) from the group of the ALS inhibitors:
sulfonylureas such as amidosulfuron, azimsulfuron, bensulfuron, bensulfuron-methyl, chlo- rimuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, flupyrsul- furon-methyl-sodium, foramsulfuron, halosulfuron, halosulfuron-methyl, imazosulfuron, iodosul- furon, iodosulfuron-methyl-sodium, iofensulfuron, iofensulfuron-sodium, mesosulfuron, met- azosulfuron, metsulfuron, metsulfuron-methyl, nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron, primisulfuron-methyl, propyrisulfuron, prosulfuron, pyrazosulfuron, pyrazosulfu- ron-ethyl, rimsulfuron, sulfometuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron, thifen- sulfuron-methyl, triasulfuron, tribenuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron, tri- flusulfuron-methyl and tritosulfuron,
imidazolinones such as imazamethabenz, imazamethabenz-methyl, imazamox, imazapic, ima- zapyr, imazaquin and imazethapyr, triazolopyrimidine herbicides and sulfonamides such as cloransulam, cloransulam-methyl, diclosulam, flumetsulam, florasulam, metosulam, penoxsu- lam, pyrimisulfan and pyroxsulam,
pyrimidinylbenzoates such as bispyribac, bispyribac-sodium, pyribenzoxim, pyriftalid, pyrimino- bac, pyriminobac-methyl, pyrithiobac, pyrithiobac-sodium, 4-[[[2-[(4,6-dimethoxy-2- pyrimidinyl)oxy]phenyl]methyl]amino]-benzoic acid-1 -methylethyl ester (CAS 420138-41 -6), 4- [[[2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]phenyl]methyl]amino]-benzoic acid propyl ester (CAS 420138-40-5), N-(4-bromophenyl)-2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzenemethanamine (CAS 420138-01 -8),
sulfonylaminocarbonyl-triazolinone herbicides such as flucarbazone, flucarbazone-sodium, propoxycarbazone, propoxycarbazone-sodium, thiencarbazone and thiencarbazone-methyl; and triafamone;
among these, a preferred embodiment of the invention relates to those compositions comprising at least one imidazolinone herbicide; b3) from the group of the photosynthesis inhibitors:
amicarbazone, inhibitors of the photosystem II, e.g. triazine herbicides, including of chlorotria- zine, triazinones, triazindiones, methylthiotriazines and pyridazinones such as ametryn, atra- zine, chloridazone, cyanazine, desmetryn, dimethametryn,hexazinone, metribuzin, prometon, prometryn, propazine, simazine, simetryn, terbumeton, terbuthylazin, terbutryn and trietazin, aryl urea such as chlorobromuron, chlorotoluron, chloroxuron, dimefuron, diuron, fluometuron, isoproturon, isouron, linuron, metamitron, methabenzthiazuron, metobenzuron, metoxuron, monolinuron, neburon, siduron, tebuthiuron and thiadiazuron, phenyl carbamates such as desmedipham, karbutilat, phenmedipham, phenmedipham-ethyl, nitrile herbicides such as bromofenoxim, bromoxynil and its salts and esters, ioxynil and its salts and esters, uraciles such as bromacil, lenacil and terbacil, and bentazon and bentazon-sodium, pyridate, pyridafol, pen- tanochlor and propanil and inhibitors of the photosystem I such as diquat, diquat-dibromide, paraquat, paraquat-dichloride and paraquat-dimetilsulfate. Among these, a preferred embodiment of the invention relates to those compositions comprising at least one aryl urea herbicide. Among these, likewise a preferred embodiment of the invention relates to those compositions comprising at least one triazine herbicide. Among these, likewise 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, cinidon-ethyl, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoroglycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, profluazol, pyraclonil, pyraflufen, pyraf I uf en-ethyl, saflufenacil, sulfentrazone, thidiazimin, tiafenacil, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100, N-ethyl-3- (2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1 /-/-pyrazole-1 -carboxamide (CAS 452098-92- 9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1 H-pyrazole-1 - carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5- methyl-1 H-pyrazole-1 -carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6- fluoro-4-trifluoromethylphenoxy)-5-methyl-1 H-pyrazole-1 -carboxamide (CAS 452100-03-7), 3-[7- fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1 ,4]oxazin-6-yl]-1 ,5-dimethyl-6-thioxo- [1 ,3,5]triazinan-2,4-dione, 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 (CAS 1258836-72-4), 2-(2,2,7- Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1 ,4]oxazin-6-yl)-4,5,6,7-tetrahydro- isoindole-1 ,3-dione, 1 -Methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro- 2H-benzo[1 ,4]oxazin-6-yl)-1 H-pyrimidine-2,4-dione (CAS 13041 13-05-0), methyl (£)-4-[2-chloro- 5-[4-chloro-5-(difluoromethoxy)-1 H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2- enoate [CAS 948893-00-3;, and 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)-1 H-benzimidazol-4-yl]-1 - methyl-6-(trifluoromethyl)-1 H-pyrimidine-2,4-dione (CAS 212754-02-4); b5) from the group of the bleacher herbicides:
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, clomazone, isoxaflutole, mesotri- one, pyrasulfotole, pyrazolynate, pyrazoxyfen, sulcotrione, tefuryltrione, tembotrione, toprame- zone and bicyclopyrone, bleacher, unknown target: aclonifen, amitrole and flumeturon; b6) from the group of the EPSP synthase inhibitors:
glyphosate, glyphosate-isopropylammonium, glyposate-potassium and glyphosate-trimesium (sulfosate); b7) from the group of the glutamine synthase inhibitors:
bilanaphos (bialaphos), bilanaphos-sodium, glufosinate, glufosinate-P and glufosinate- ammonium; b8) from the group of the DHP synthase inhibitors:
asulam; b9) from the group of the mitosis inhibitors:
compounds of group K1 : dinitroanilines such as benfluralin, butralin, dinitramine, ethalfluralin, fluchloralin, oryzalin, pendimethalin, prodiamine and trifluralin, phosphoramidates such as ami- prophos, amiprophos-methyl, and butamiphos, benzoic acid herbicides such as chlorthal, chlor- thal-dimethyl, pyridines such as dithiopyr and thiazopyr, benzamides such as propyzamide and tebutam; compounds of group K2: chlorpropham, propham and carbetamide, among these, compounds of group K1 , in particular dinitroanilines are preferred; b10) from the group of the VLCFA inhibitors: chloroacetamides such as acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, dime- thenamid-P, metazachlor, metolachlor, metolachlor-S, pethoxamid, pretilachlor, propachlor, propisochlor and thenylchlor, oxyacetanilides such as flufenacet and mefenacet, acetanilides such as diphenamid, naproanilide and napropamide, tetrazolinones such fentrazamide, and other herbicides such as anilofos, cafenstrole, fenoxasulfone, ipfencarbazone, piperophos, pyroxasulfone and isoxazoline compounds of the formulae 11.1 , II.2, II.3, II.4, II.5, II.6, II.7, II.8 and II.9
Figure imgf000017_0001
the isoxazoline compounds of the formula (l)l are known in the art, e.g. from WO 2006/024820, WO 2006/037945, WO 2007/071900 and WO 2007/096576; among the VLCFA inhibitors, preference is given to chloroacetamides and oxyacetamides; b1 1 ) from the group of the cellulose biosynthesis inhibitors:
chlorthiamid, dichlobenil, flupoxam, indaziflam, triaziflam, isoxaben and 1 -Cyclohexyl-5- pentafluorphenyloxy-14-[1 ,2,4,6]thiatriazin-3-ylamine; 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, aminocyclopy- rachlor and its salts and esters, aminopyralid and its salts such as 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, fluroxypyr, fluroxy- pyr-butometyl, fluroxypyr-meptyl, halauxifen and its salts and esters (CAS 943832-60-8); MCPA and its salts and esters, MCPA-thioethyl, MCPB and its salts and esters, mecoprop and its salts and esters, mecoprop-P and its salts and esters, picloram and its salts and esters, quinclorac, quinmerac, TBA (2,3,6) and its salts and esters and triclopyr and its salts and esters; b14) from the group of the auxin transport inhibitors: diflufenzopyr, diflufenzopyr-sodium, nap- talam and naptalam-sodium; b15) from the group of the other herbicides: bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, cyclopyrimorate (CAS 499223-49-3) and its salts and esters, dalapon, dazomet, difenzoquat, difenzoquat-metilsulfate, dimethipin, DSMA, dymron, endothal and its salts, etobenzanid, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flam- prop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, indaziflam, maleic hydrazide, mefluidide, metam, methiozolin (CAS 403640-27-7), methyl azide, methyl bromide, methyl-dymron, methyl iodide, MSMA, oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine, triaziflam and tridiphane..
The term "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. The weeds of the present invention include, for example, dicotyledonous and mono- cotyledonous 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, Alopecu- rus, and Apera. In addition, the weeds of the present invention can include, for example, crop plants that are growing in an undesired location. For example, 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. Polypeptides
By "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 or synthetically manufactured polypeptide.
The terms "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. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms "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 % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. 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.
As used herein a "biologically active" fragment is a portion of a polypeptide of the invention which maintains a defined activity of the full-length polypeptide, namely be able to metabolize a PPO inhibiting herbicide, especially Saflufenacil. Biologically active fragments can be any size as long as they maintain the defined activity. Preferably, biologically active fragments are at least 100, more preferably at least 200, and even more preferably at least 350 amino acids in length, most preferably at least 400 amino acids of SEQ ID NO:1
With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the CytP450 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%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to SEQ ID NO: 1 .
Amino acid sequence mutants of the polypeptides of the present invention can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the present invention, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.
Mutant (altered) polypeptides can be prepared using any technique known in the art. For example, a polynucleotide of the invention can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. In another example, the polynucleotides of the invention are subjected to DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they are able to metabolize a PPO inhibiting herbicide such as Saflufenacil.
In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1 ) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 .
Table 1 : Examples of conservative amino acid substitutions
Residue Conservative SubstiResidue Conservative Substitutions tutions
Ala Ser Leu lie; Val Arg Lys Lys Arg; Gin
Asn Gin; His Met Leu; lie
Asp Glu Phe Met; Leu; Tyr
Gin Asn Ser Thr; Gly
Cys Ser Thr Ser; Val
Glu Asp Trp Tyr
Gly Pro Tyr Trp; Phe
His Asn; Gin Val lie; Leu
lie Leu, Val
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 [0131]. Furthermore, if desired, unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the polypeptides of the present invention. Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4- diaminobutyric acid, [alpha]-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6- amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, [beta]-alanine, fluoro-amino acids, designer amino acids such as [beta]-methyl amino acids, C[alpha]-methyl amino acids, N[alpha]-methyl amino acids, and amino acid analogues in general.
Also included within the scope of the invention are polypeptides of the present invention which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosyla- tion, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide of the invention.
Polypeptides of the present invention can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides. In one embodiment, an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production. An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
Polynucleotides and Oligonucleotides
By 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. Preferably, 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. As the skilled addressee would be aware, an isolated polynucleotide can be an exogenous polynucleotide present in, for example, a transgenic organism which does not naturally comprise the polynucleotide. Furthermore, the term "polynucleotide" is used interchangeably herein with the term "nucleic acid".
The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Unless stated otherwise, 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. Preferably, 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. More preferably, 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.
With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that a polynucleotide of the invention comprises a 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%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO 2. As used herein, the term "gene" is to be taken in its broadest context and includes the deoxyri- bonucleotide sequences comprising the protein coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of at least about 2 kb on either end. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. The term "gene" includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above.
As used herein, the phrase "stringent conditions" refers to conditions under which a polynucleotide, probe, primer and/or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5[deg]C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1 .0 M sodium ion, typically about 0.01 to 1 .0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30[deg.] C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60[deg.] C. for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel et al. (supra), Current Protocols In Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6, as well as the Examples described herein. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6*SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65[deg.] C, followed by one or more washes in 0.2.*SSC, 0.01 % BSA at 50[deg.] C. In another embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2 under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6*SSC, 5*Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55[deg.] C, followed by one or more washes in 1 *SSC, 0.1 % SDS at 37[deg.] C. Other conditions of moderate stringency that may be used are well-known within the art, see, e.g., Ausubel et al. (supra), and Kriegler, 1990; Gene Transfer And Expression, A Laboratory Manual, Stockton Press, NY. In yet another embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NO:2, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5*SSC, 50 mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt vol) dextran sulfate at 40[deg.] C, followed by one or more washes in 2*SSC, 25 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1 % SDS at 50[deg.] C. Other conditions of low stringency that may be used are well known in the art, see, e.g., Ausubel et al. (supra) and Kriegler, 1990, Gene Transfer And Expression, A Laboratory Manual, Stockton Press, NY, as well as the Examples provided herein.
Polynucleotides of the present invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions, of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the nucleic acid).
Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. Although the terms polynucleotide and oligonucleotide have overlapping meaning, oligonucleotide are typically relatively short single stranded molecules. The minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a target nucleic acid molecule. Preferably, the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 25 nucleotides in length.
Usually, monomers of a polynucleotide or oligonucleotide are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from relatively short monomeric units, e.g., 12-18, to several hundreds of monomeric units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate.
The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, or primers to produce nucleic acid molecules. Oligonucleotides of the present invention used as a probe are typically conjugated with a detectable label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.
Probes and/or primers can be used to clone homologues of the polynucleotides of the invention from other species. Furthermore, hybridization techniques known in the art can also be used to screen genomic or cDNA libraries for such homologues.
Recombinant Vectors
One embodiment of the present invention includes a recombinant vector, which comprises at least one isolated polynucleotide molecule of the present invention, inserted into any vector capable of delivering the polynucleotide molecule into a host cell. Such a vector contains heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules of the present invention and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a trans- poson (such as described in U.S. Pat. No. 5,792,294), a virus or a plasmid.
One type of recombinant vector comprises a polynucleotide molecule of the present invention operably linked to an expression vector. The phrase operably linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, arthropod, and plant cells. Vectors of the invention can also be used to produce the polypeptide in a cell-free expression system, such systems are well known in the art.
Operably linked" as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element to a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell and/or in a cell-free expression system. Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis- acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
In particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, arthropod, nematode, plant or mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71 ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01 , metallothionein, alpha- mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Coding sequences of the polypeptides of the invention can be optimized to maximize expression is a particular host cell using known techniques.
Host Cells
Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention, or progeny cells thereof. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention. Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptides of the present invention or can be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite, nematode, arthropod, and plant cells. Examples of host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS (e.g., COS-7) cells, and Vero cells. Further examples of host cells are E. coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Particularly preferred host cells are plant cells.
Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, opera- tors, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
Transgenic Plants
The term "plant" as used herein as a noun refers to whole plants, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plant cells and the like.
Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. Examples of plant species of interest include, but are not limited to, corn or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassi- ca species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Se- cale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennise- tum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annu ), saffiower (Carthamus tinctorius), wheat (Triticum aestivum, T. turgidum ssp. durum), soybean (Glycine max), tobacco (Nicotiana taba- cum), potato (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barba- dense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers. Preferably, plants of the present invention are crop plants (for example, sunflower, Brassica sp., cotton, sugar, beet, soybean, peanut, alfalfa, saffiower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.).
The term "transgenic plant" refers to a plant that comprises a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been so altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell. In some embodiments, a "recombinant" organism is a "transgenic" organism. The term "transgenic" as used herein is not intended to encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods (e.g., crosses) or by naturally occurring events such as, e.g., self- fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacte- rial transformation, non- recombinant transposition, or spontaneous mutation.
In a preferred embodiment, the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not segregate for the desired pheno- type. The transgenic plants may also be heterozygous for the introduced transgene(s), such as, for example, in F1 progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.
A polynucleotide of the present invention may be expressed constitutively in the transgenic plants during all stages of development. Depending on the use of the plant or plant organs, the polypeptides may be expressed in a stage-specific manner. Furthermore, the polynucleotides may be expressed tissue-specifically.
Regulatory sequences which are known or are found to cause expression of a gene encoding a polypeptide of interest in plants may be used in the present invention. The choice of the regulatory sequences used depends on the target plant and/or target organ of interest. Such regulatory sequences may be obtained from plants or plant viruses, or may be chemically synthesized. Such regulatory sequences are well known to those skilled in the art.
A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmen- tally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribo- some binding site, an RNA processing signal, a transcription termination site, and/or a polyad- enylation signal.
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 ); ubi- quitin (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. 81 :581 - 588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,61 1.
For the purpose of expression in source tissues of the plant, such as the leaf, seed, root or stem, it is preferred that the promoters utilized in the present invention have relatively high expression in these specific tissues. For this purpose, one may choose from a number of promoters for genes with tissue- or cell-specific or -enhanced expression. Such 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. 1 12(3): 1331 -1341 ; Van Camp et al. (1996) Plant Physiol. 1 12(2):525-535; Canevascini et al. (1996) Plant Physiol.
1 12(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20: 181 - 196; Orozco et al. (1993) Plant Mol Biol. 23(6): 1 129-1 138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression. In one embodiment, the nucleic acids of interest are targeted to the chloroplast for expression.
The expression cassettes may additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyo- carditis 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. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991 ) Nature 353:90- 94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Job- ling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991 ) Virology 81 :382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.
The termination of transcription is accomplished by a 3' non-translated DNA sequence operably linked in the chimeric vector to the polynucleotide of interest. The 3' non-translated region of a recombinant DNA molecule contains a polyadenylation signal that functions in plants to cause the addition of adenylate nucleotides to the 3' end of the RNA. 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. (1991 ) Genes Dev. 5: 141 -149; Mogen et al. (1990) Plant Cell 2: 1261 -1272; Munroe et al. (1990) Gene 91 : 151 -158; Ballas t al. (1989) Nucleic Acids Res. 17:7891 -7903; and Joshi, CP (1987) Nucleic Acid Res. 15:9627- 9639. Where appropriate, 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 -1 1 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant- preferred genes. See, for example, U.S. Patent Nos. 5,380,831 , and 5,436,391 , and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. As it is well known to the person skilled in the art and described in the BACKGROUND section supra, most bacterial cytochrome P450 monooxygenase systems are class I systems which require a flavin adenine dinucleotide-containing NADH-dependent reductase (ferredoxin reductase) and an iron-sulfur redoxin (ferredoxin) (Sasaki et al., Appl. Environ. Microbiol. December 2005 vol. 71 no. 12 8024-8030, as well as references contained therein). Consequently, it will be easily understood by the skilled artisan that the expression cassette of the present invention additionally comprises ferredoxin- and reductase encoding nucleic acids which are operably linked to the polynucleotide of the present invention. The ferredoxin- and reductase encoding nucleic acids can be present on the same vector like the polynucleotide of the present invention, employing different promoters or can be present on two or more different vectors to be introduced into a host cell, preferably a plant cell.
The expression cassette of the present invention preferably contains the S. ceolicolor CYP450 gene comprising (i) SEQ ID NO: 2, (ii) a sequence of nucleotides encoding a polypeptide of SEQ ID NO:1 , or a polypeptide which is at least 85% identical to SEQ ID NO:1 , iii) a sequence of nucleotides which is at least 80% identical to i), iv) a sequence of nucleotides which hybridizes to i) under low stringency conditions, v) a sequence of nucleotides complementary to i) to iv) and the ferredoxin gene fused together and driven by a promoter, preferably a constitutive promoter, more preferably the Parsley ubiquitin promoter. In a particular preferred embodiment, the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 8. Additionally, the expression cassette comprises the reductase-encoding polynucleotide, preferably the S. ceol- icolor CYP105D5 reductase (SEQ ID NO. 10), the expression of which expression can be driven by the Super promoter (p-Super).
In a further preferred embodiment, both encoded sequences are fused to a transit peptide, for subcellular targeting to plastids. Such transit peptides are known in the art. With respect to plastid-targeting sequences, "operably linked" means that the nucleic acid sequence encoding a transit peptide (i.e., the plastid-targeting sequence) is linked to the CytP450 nucleic acid of the invention such that the two sequences are contiguous and in the same reading frame. See, for example, Von Heijne et al. (1991 ) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421 ; and Shah et al. (1986) Science 233:478-481. Preferably, said transit peptide is the ferredoxin transit peptide from Silene pratensis.
General methods for direct delivery of a gene into cells have been described: (1 ) chemical methods (Graham et al., 1973); (2) physical methods such as microinjection (Capecchi, 1980); electroporation (see, for example, WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO 92/09696 and WO 93/21335); and the gene gun (see, for example, U.S. Pat. No. 4,945,050 and U.S. Pat. No. 5,141 ,131 ); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediated mechanisms (Curiel et al., 1992; Wagner et al., 1992).
Acceleration methods that may be used include, for example, microprojectile bombardment and the like. One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non- biological particles (microprojectiles) that may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. A particular advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts, nor the susceptibility of Agrobacterium infection are required. An illustrative embodiment of a method for delivering DNA into Zea mays cells by acceleration is a biolistics [alpha]-particle delivery system that can be used to propel particles coated with DNA through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with corn cells cultured in suspension. A particle delivery system suitable for use with the present invention is the helium acceleration PDS-1000/He gun is available from Bio-Rad Laboratories.
For the bombardment, cells in suspension may be concentrated on filters. Filters containing the cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the gun and the cells to be bombarded.
In bombardment transformation, one may optimize the pre-bombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids.
In another alternative embodiment, plastids can be stably transformed. Methods disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (U.S. Pat. No. 5,451 ,513, U.S. Pat. No. 5,545,818, U.S. Pat. No. 5,877,402, U.S. Pat. No. 5,932,479, and WO 99/05265).
Accordingly, it is contemplated that one may wish to adjust various aspects of the bombardment parameters in small scale studies to fully optimize the conditions. The execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.
Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium- mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the 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-medlated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP
1 198985 A1 , Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491 -506, 1993), Hiei et al. (Plant J 6 (2): 271 -282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1 ): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991 ) 205-225. The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 871 1 ). 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. 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, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
A transgenic plant formed using Agrobacterium transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene. More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants for the gene of interest.
In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic (Feldman, KA and Marks MD (1987). Mol Gen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289). Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension (Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1 194-1 199), while in the case of the "floral dip" method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension (Clough, SJ and Bent AF (1998) The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non- transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 (Nature Biotechnology 22 (2), 225-229). Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001 ) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21 ; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21 , 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229). The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. Application of these systems to different plant varieties depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).
Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos.
The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif., (1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign, exogenous gene is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed- grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired exogenous nucleic acid is cultivated using methods well known to one skilled in the art.
Methods for transformation of cereal plants such as wheat and barley for introducing genetic variation into the plant by introduction of an exogenous nucleic acid and for regeneration of plants from protoplasts or immature plant embryos are well known in the art, see for example, Canadian Patent Application No. 2,092,588, Australian Patent Application No 61781/94, Australian Patent No 667939, U.S. Pat. No. 6,100,447, International Patent Application
PCT/US97/10621 , U.S. Pat. No. 5,589,617, U.S. Pat. No. 6,541 ,257, and other methods are set out in Patent specification W099/14314. Preferably, transgenic wheat or barley plants are produced by Agro bacterium tumefaciens mediated transformation procedures. Vectors carrying the desired nucleic acid construct may be introduced into regenerable wheat cells of tissue cultured plants or explants, or suitable plant systems such as protoplasts.
To confirm the presence of the transgenes in transgenic cells and plants, a polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. One particularly useful way to quantitate protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.
In other embodiments, PPO inhibitor-tolerant, preferably PPO inhibitor-tolerant characteristics/traits of the present invention can be stacked with any combination of plant characteristics/trails) of interest to provide plants with a desired combination of characteristics/traits.
Thus, transgenic plants of the present invention include those plants which, in addition to being PPO inhibitor-tolerant, 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; protoporphyrinogen-IX oxidase (PPO) inhibitors to which the plant have been rendered tolerant by mechanisms other than inactivation or metabolization, e.g. overexpression of a mutated PPO which confers tolerance/resistance to said inhibitor; lipid biosynthesis inhibitors such as acetyl CoA carboxylase (ACCase) inhibitors; or oxynil {i.e. bromoxynil or ioxynil) herbicides as a result of conventional methods of breeding or genetic engineering, Thus, PPO inhibitor-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. These herbicide resistance technologies are, for example, described in Pest Management Science (at volume, year, page): 61 , 2005, 246; 61 , 2005, 258; 61 , 2005, 277; 61 , 2005, 269; 61 , 2005, 286; 64, 2008, 326; 64, 2008, 332; Weed Science 57, 2009, 108; Australian Journal of Agricultural Research 58, 2007, 708; Science 316, 2007, 1 185; and references quoted therein. For example, PPO inhibitor-tolerant plants of the invention, in some embodiments, may be tolerant to ACCase inhibitors, such as "dims" {e.g., cycloxydim, sethoxydim, clethodim, or tepraloxydim), "fops" {e.g. , clodinafop, diclofop, fluazifop, haloxyfop, or quizalo- fop), 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.
In addition to these classes of inhibitors, PPO inhibitor-tolerant plants of the invention may also be tolerant to herbicides having other modes of action, for example, chlorophyll/carotenoid pigment inhibitors, cell membrane disrupters, photosynthesis inhibitors, cell division inhibitors, root inhibitors, shoot inhibitors, and combinations thereof Such tolerance traits may be expressed, e.g. : as mutant AHASL proteins, mutant ACCase proteins, mutant EPSPS proteins, or mutant glutamine synthetase proteins; or as mutant native, inbred, or transgenic aryloxyalka- noate dioxygenase (AAD or DHT), haloarylnitrilase (BXN), 2,2-dichloropropionic acid dehalo- genase (DEH), glyphosate-N- acetyltransf erase (GAT), glyphosate decarboxylase (GDC), glyphosate oxidoreductase (GOX), glutathione-S-transferase (GST), phosphinothricin acetyl- transferase (PAT or bar), or CYP450 - other than the CYP450 of the present invention - having an herbicide-degrading or -metabolizing activity. PPO inhibitor- 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.
As described above, the present invention teaches compositions and methods for increasing the PPO-inhibiting tolerance of a crop plant or seed comprising a polynucleotide encoding a CYP450 polypeptide according to the present invention as compared to an untransformed wild- type variety of the plant or seed. In a preferred embodiment, the PPO-inhibiting tolerance of a crop plant or seed is increased such that the plant or seed can withstand a PPO-inhibiting 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. As used herein, to "withstand" a PPO-inhibiting 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). Furthermore, the present invention provides methods that involve the use of at least one PPO- inhibiting herbicide, optionally in combination with one or more herbicidal compounds B, as described in detail supra.
In these methods, the PPO-inhibiting herbicide can be applied by any method known in the art including, but not limited to, seed treatment, soil treatment, and foliar treatment. Prior to application, the PPO-inhibiting herbicide 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.
By providing plants having increased tolerance to PPO-inhibiting 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-inhibiting 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-inhibiting herbicide formulation can be used that contains other additives. The PPO-inhibiting herbicide can also be used as a seed treatment. Additives found in a PPO-inhibiting herbicide formulation include other herbicides, detergents, adjuvants, spreading agents, sticking agents, stabilizing agents, or the like. The PPO-inhibiting 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-inhibiting herbicide and herbicide formulations can be applied in accordance with conventional methods, for example, by spraying, irrigation, dusting, or the like.
Suitable formulations are described in detail in PCT/EP2009/063387 and PCT/EP2009/063386, which are incorporated herein by reference.
Compositions
A polypeptide of the present invention can be provided in a composition which enhances the rate and/or degree of metabolization of a PPO inhibiting herbicide, or increases the stability of the polypeptide. For example, the polypeptide can be immobilized on a polyurethane matrix (Gordon et al., 1999), or encapsulated in appropriate liposomes (Petrikovics et al. 2000a and b). One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a polypeptide of the present invention into a plant, plant material, or the environment (including soil and water samples).
As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, micro- particles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres. Preferred controlled release formulations are biodegradable (i.e., bioerodible).
It should be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.
EXAMPLES
Example 1 : Screening for PPO inhibitor-metabolizing microorganisms
To find out which microorgamisms are able to metabolize PPO-inhibiting herbicides such as Saflufenacil, a screening was performed in which several microorganisms, including different actinobacteria, were cultivated with 200 μΜ Saflufenacil. Supernatants of these cultures were added to Arabidopsis thaliana plants cultivated in 96 MTPs. If growth of the plants was visible it was concluded that the microorganism of which the supernatant was taken, was able to metabolize Saflufenacil to non-herbicidal metabolites. In this screening approach, S. coelicolor was identified as a potential Saflufenacil metabolizing strain.
Example 2: CytP450 monooxygenase inhibitors indicate CytP450 monooxygenase to be involved in Saflufenacil metabolization
CytP450 enzymes are known for their detoxifying potential (Thies et al., 1996; De Schrijver et al., 1999). To sustain the assumption, that a CytP450 demethylates Saflufenacil and thereby turns it into a non herbicidal derivative, several CytP450 monooxygenase inhibitors were tested. Five different inhibitors of CytP450 enzymes have been described (Thies et al., 1996) which are able to suppress the enzymatic activity of CytP450 monooxygenases. These substances were tested in a Streptomyces Saflufenacil degradation assay. Therefore a 3 days old preculture (2xYT broth: Tryptone 16 g/L; yeast extract 10 g/L; NaCI 5 g/L) of a S. coelicolor strain was transferred to M12 minimal medium (NH42*S04 1 g/L; NaCI 0.1 g/L; MgS0 *7H20 0.2 g/L;
CaCl2*H20 0.02 g/L; yeast extract 0.05 g/L; trace element solution 1 mL/L sugar mix 4 g/L; vitamin solution 10 mL/L; KhbPC pH 6.7) containing 300 μΜ Saflufenacil and different concentrations of the inhibiting substances tetcyclasis, aminobenzotriazole, cinnamonic acid and piper- onylbutoxide. After an incubation period of 7 d at 30 °C and 290 rpm the supernatants were analyzed by UPLC analysis. The results clearly indicate an inhibition of the Saflufenacil metabolizing activity by all CytP450 inhibitors, with tetcyclasis exhibiting the most prominent inhibitory activity. No demethylation or any other Saflufenacil turnover could be detected. Figure 1 shows the results of this assay, including a negative control (solely medium), a positive control (a S. coelicolor strain with Saflufenacil) and the samples.
Example 3: Time-dependent metabolization of Saflufenacil by a S. coelicolor strain
It was determined if Saflufenacil metabolization is time-dependent. The results of the measurements of Saflufenacil, metabolite 1 , metabolite 2, and metabolite 3 are shown in Figure 2. As can be seen, demethylation of Saflufenacil starts 24-26 h after incubation of the main culture. Within less than 24 h more than 90 % of Saflufenacil are demethylated. Example 4: Identification of a CytP450 monooxygenase candidate gene locus on the
S. coelicolor genome
To check which S. coelicolor CytP450 enzyme is responsible for Saflufenacil meabilzation several candidate genes were knocked out via the homologous insertion of a hygromycinre- sistance gene. The knockout of one gene lead to abolished Saflufenacil metabolization.
To proof the function of CytP105D5 (SEQ ID NO:1 as decribed by Chun et al.) in Saflufenacil demethylation, the gene was knocked out. The primers used for knock-out experiments are depicted in SEQ ID NOs: 3, 4, 5, and 6.The knockout plasmid was constructed using two PCR products homologous to the Streptomyces genome, covering the locus of the CytP450 gene. The two PCR fragments with 1510 bp and 1557 bp, respectively, were cloned into the shuttle plasmid pKC1 132, flanking the hygromycin resistance conferring gene hph. pKC1 132 is a plasmid not able to replicate in Streptomyces. In the first step the resistance gene was cloned to the plasmid backbone, using BamHI and EcoRV restriction sites, followed by the homologous fragment covering the upstream region and the 5' end of SEQ ID NO: 2, utilizing Xbal and BamHI restriction sites. In the next step the homologous fragment covering the 3' end, as well as a downstream region of SEQ ID NO: 2, was cloned to the plasmid using EcoRV restriction sites. The resulting plasmid was named p62. Correct cloning was verified by restriction analysis and sequencing. The verified plasmid was introduced into E. coli ET12567 cells containing the plasmid pUZ8002, cultivated in LB medium (NaCI 5 g/L, yeast extract 5 g/L, Tryptone 10 g/L), conferring the ability to conjugate / mate with other bacterial cells, i. e. actinomycetes. By means of conjugation p62 was introduced into the respective S. coelicolor strain. Exconjugands were plated out on MS plates (mannitol 20 g/L, soy flour 20 g/L) containing 100 μg/μL hygromycin, for selection for crossover events. Colonies resistant to hygromycin were transferred onto plates containing apramycin (for selection on the plasmid p62) and plates containing hygromycin. Such colonies that solely grew on agar containing only the latter antibiotic indicated a double-crossover event. Those colonies were chosen for further experiments.
Example 5: Characterization of S. coelicolor SEQ ID NO: 2 knockout
After the CytP450 encoding gene comprising SEQ ID NO: 2 had been knocked out, the knockout strain was cultivated and checked in the metabolization assay, as described supra. The HPLC based assay clearly showed, that the mutant had nearly completely lost the ability to metabolize Saflufenacil to metabolite 1 , metabolite 2 and metabolite 3. Results from the corresponding HPLC analysis are shown in Table 2.
Table 2: Data from HPLC; S. coelicolor wildtype (WT) cultivated for 5 d with -200 μΜ
Saflufenacil, culture broth with -200 μΜ Saflufenacil and S. coelicolor SEQ ID NO: 2 knockout cultivated for 5 d with -200 μΜ Saflufenacil
Sample metabolite 1/μΜ metabolite 2/μΜ metabolite 3/μΜ Saflufenacil/μΜ
S.c. WT 36,03 24,98 - -
Broth - - - 200,25
S.c. k.o. 3,94 - - 177,63 To verify the knockout event by molecular biology methods a PCR was performed with genomic DNA from S. coelicolor as a template. The size of the PCR products showed that the knockout construct had inserted in the right locus. In a second step, the chromosomal region of the knockout was sequenced, also verifying the insertion of the knockout construct at the right position on the chromosome.
Example 6: Material and Methods
Enzymes
Restriction enzymes, Roche, according to manufacturer's conditions
Polymerase "Phusion", Finnzme, according to manufacturer's conditions
T4-Ligase from Roche, according to manufacturer's conditions
HPLC analytics
Method used for 96 dwps: B800FAST
Precolumn C18 0DS
Column Luna C8(2), 50*3.0 mm (Phenomenex)
Temperature 40 °C
Flowrate 2 mL/min
Volume of injection 5 μΙ_
Detection UV 271 nm
Stop 1.8 min
Max. pressure 300 bar
eluentA 10 mM KH2PO4, pH 2.5
eluentB acetonitril
method of elution isocratic (50 %:50 %)
pretreatment filtration 0.22 μηη pores
Example 7: Plant transformation with Streptomyces coelicolor CYP105D5 cytochromo P450 system results in Kixor tolerance.
(a) Plant transformation vector construction:
Using standard cloning practices, two expression cassettes were cloned into the T-DNA portion of a base plant transformation vector containing imidazolinone selectability (AtAHASL A122T S653N cassette) to make RTP8830-3 (Figure 3A). The first cassette contains the Parsley ubiq- uitin promoter driving the expression of a fusion of sequences (named CYP450-657 Frdxn-656 Plast targ_Gm, see SEQ ID NO: 8) (Figure 3B) that encode the plastid targeting transit peptide from Silene pratensis ferredoxin (51 amino acids), the open reading frame of the Streptomyces coelicolor CYP105D5 (410 amino acids), a linker sequence from Methylococcus capsulatus MCCYP51 FX (21 amino acids) (see Jackson et al., J. Biol. Chem. 2002:277(49):46959-46965), and the open reading frame of a S. coelicolor ferredoxin (66 amino acids). The second cassette contains the SUPER promoter (originating from Agrobacterium tumefaciens and contains 3 repeats of OCS enhancers upstream of mas2 promoter) driving the expression of the S. coelicolor 105D5 reductase (see SEQ ID NO: 10). The coding sequences for each cassette were codon optimized for soybean based on frequency of codon usage and made via de novo DNA synthesis.
(b) Soybean transformation and Kixor tolerance testing:
Soybean cv Williams 82 was transformed as previously described (see Hong HP, et al., In Vitro Cell. Dev. Biol. -Plant 2007: 43: 558-568). After regeneration, transformants were transplanted to soil in small pots, placed in growth chambers (18 hr day/ 6 hr night; 26°C constant; 65% relative humidity; 130-150 DE nr2 s_1) and subsequently tested for the presence of the T-DNA via Taq- man analysis. After a few weeks, healthy, transgenic positive, 1 -2 copy events were transplanted to larger pots and allowed to grow in the growth chamber for subsequent generation of clones via shoot cutting. An optimal shoot for cutting was about 3-4" tall, with at least two nodes present. Each cutting was taken from the original transformant (mother plant) and dipped into rooting hormone powder (indole-3-butyric acid, IBA). The cutting was then placed in oasis wedges inside a bio-dome. The mother plant was taken to maturity in the greenhouse and harvested for seed. Wild type cuttings were also taken simultaneously to serve as negative controls. The cuttings were kept in the bio-dome for 5-7 days and then transplanted to 3" pots and then acclimated in the growth chamber for two more days. Subsequently, the cuttings were transferred to the greenhouse, acclimated for approximately 4 days, and then subjected to spray tests containing 0, 6.25, 12.5, 25, and 50, 100 g ai/ha Saflufenacil (Kixor® or Sharpen®) plus 1 % MSO. Herbicide injury evaluations were taken at 7 days after treatment.
(c) Soybean transformation and Kixor tolerance testing:
A plant transformation vector, RTP8830-3, (Figure 3) harboring two transgene expression cassettes in addition to the selectable marker was tested for the ability to confer tolerance to Saflufenacil in TO soybean cuttings. Figures 4 and 5 indicate the level of injury of those plants treated with 0, 6.25, 12.5 25, 50, and 100 g ai/ha Saflufenacil + 1 % MSO. 1 out of 4 events showed increased tolerance to Saflufenacil at all spray rates tested. Table 3 describes the health of the plant an type of injury observed on the 10 point injury rating scale indicated in Figure 4.
Table 3: Description of the 10 point injury rating scale.
Score Description of injury
0 No Injury
1 Minimal injury, only a few patches of leaf injury or chlorosis.
Minimal injury with slightly stronger chlorosis. Overall growth points
2 remain undamaged.
Slightly stronger injury on secondary leaf tissue, but primary leaf and
3 growth points are still undamaged.
Overall plant morphology is slightly different, some chlorosis and ne¬
4 crosis in secondary growth points and leaf tissue. Stems are intact.
Regrowth is highly probable within 1 week.
Overall plant morphology is clearly different, some chlorosis and necro¬
5 sis on a few leaves and growth points, but primary growth point is
intact. Stem tissue is still green. Regrowth is highly probably within 1 week.
Strong injury can be seen on the new leaflet growth. Plant has a high
6 probability to survive only through regrowth at different growth points.
Most of the leaves are chlorotic/ necrotic but stem tissue is still green. May have regrowth but with noticeable injured appearance.
Most of the active growth points are necrotic. There may be a single
7 growth point that could survive and may be partially chlorotic or green and partially necrotic. Two leaves may still be chlorotic with some green; the rest of the plant including stem is necrotic.
8 Plant will likely die, and all growth points are necrotic. One leaf may still be chlorotic with some green. The remainder of the plant is necrotic.
9 Plant is dead.
* Plant is missing

Claims

Claims:
1. A method for metabolizing a PPO inhibiting herbicide, the method comprising contacting the PPO inhibiting herbicide with a CytP450 polypeptide comprising a sequence selected from: i) an amino acid sequence as provided in SEQ ID NO:1 , and ii) an amino acid sequence which is at least 85% identical to i).
2. A transgenic plant comprising an exogenous CytP450 polynucleotide, the polynucleotide encoding at least one polypeptide according as defined in claim 1.
3. A method of producing plants with enhanced resistance to a PPO inhibiting herbicide comprising the steps of: a) inserting into the genome of a plant cell a CytP450 polynucleotide comprising: a promoter that functions in plant cells to cause the production of a RNA sequence, operably linked to; a structural DNA sequence that causes the production of a RNA sequence that encodes a CytP450 polypeptide as defined in claim 1 , operably linked to; a 3' non-translated region that functions in plant cells to cause the addition of polyadenyl nucleotides at the 3' end of the RNA sequence; where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the polypeptide to enhance resistance to an PPO inhibiting herbicide of a plant cell transformed with the DNA molecule; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which has increased resistance to a PPO inhibiting herbicide, wherein the CytP450 polynucleotide comprising nucleotides having a sequence selected from: i) SEQ ID NO:2, ii) a sequence of nucleotides encoding a polypeptide of SEQ ID NO:1 , or a polypeptide which is at least 85% identical to SEQ ID NO:1 , iii) a sequence of nucleotides which is at least 80% identical to i), iv) a sequence of nucleotides which hybridizes to i) under low stringency conditions, v) a sequence of nucleotides complementary to i) to iv).
4. The method according to claim 3, wherein the polynucleotide encodes a polypeptide that metabolizes a PPO inhibiting herbicide.
5. A transgenic-plant produced using a method of claim 3 or 4.
6. A product produced from a plant according to claim 5.
7. A part of a plant according to claim 5.
8. 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 a CytP450 polynucleotide encoding a polypeptide comprising a sequence selected from: i) an amino acid sequence as provided in SEQ ID NO: 1 , and ii) an amino acid sequence which is at least 85% identical to i), said polypeptide metabolizing a PPO-inhibiting herbicide; applying to said site an effective amount of said PPO-inhibiting herbicide.
9. An expression cassette comprising at least
a. a CytP450 polynucleotide comprising nucleotides having a sequence selected from: i) SEQ ID NO:2, ii) a sequence of nucleotides encoding a polypeptide of SEQ ID NO:1 , or a polypeptide which is at least 85% identical to SEQ ID NO:1 , iii) a sequence of nucleotides which is at least 80% identical to i), iv) a sequence of nucleotides which hybridizes to i) under low stringency conditions, v) a sequence of nucleotides complementary to i) to iv), operably linked to
b. a ferredoxin-encoding polynucleotide,
c. a ferredoxin-reductase-encoding polynucleotide, and
d. a regulatory sequence which causes the expression of (a) in plants.
The expression cassette of claim 8, comprising the sequence of SEQ ID NO:8 and SEQ ID NO: 10.
1 1 . A method of producing a polypeptide with enhanced ability to metabolize an PPO inhibiting herbicide, the method comprising (i) altering one or more amino acids of a first polypeptide as defined in claim 1 , (ii) determining the ability of the altered polypeptide obtained from step (i) to metabolize a PPO inhibiting herbicide, and (iii) selecting an altered polypeptide with enhanced ability to metabolize an PPO inhibiting herbicide, when compared to the first polypeptide.
12. A method for screening for a microorganism capable of metabolizing an PPO inhibiting herbicide, the method comprising i) culturing a candidate microorganism in the presence of an PPO inhibiting herbicide, and ii) determining the amount of metabolized PPO inhibiting herbicide.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10308953B2 (en) 2013-12-18 2019-06-04 BASF Agro B.V. Plants having increased tolerance to herbicides
US11827610B2 (en) 2021-09-15 2023-11-28 Enko Chem, Inc. Protoporphyrinogen oxidase inhibitors
US11889833B2 (en) 2022-01-14 2024-02-06 Enko Chem, Inc. Protoporphyrinogen oxidase inhibitors

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101182521A (en) * 2007-11-02 2008-05-21 浙江大学 Applications of corn cytochrome P450 gene
CN101998988A (en) * 2007-05-30 2011-03-30 先正达参股股份有限公司 Cytochrome P450 genes conferring herbicide resistance
WO2011085221A2 (en) * 2010-01-07 2011-07-14 Basf Agro B.V., Arnhem (Nl), Zweigniederlassung Wadenswil Herbicide-tolerant plants

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101998988A (en) * 2007-05-30 2011-03-30 先正达参股股份有限公司 Cytochrome P450 genes conferring herbicide resistance
CN101182521A (en) * 2007-11-02 2008-05-21 浙江大学 Applications of corn cytochrome P450 gene
WO2011085221A2 (en) * 2010-01-07 2011-07-14 Basf Agro B.V., Arnhem (Nl), Zweigniederlassung Wadenswil Herbicide-tolerant plants

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 19 January 2012 (2012-01-19), BENTLEY, S.D.: "Cytochrome P450 [Streptomyces coelicolorA3(2)].", accession no. 25076 *
WU, FAQIANG ET AL.: "Research progress on herbicide resistant transgenic rice and its safety issues.", MOLECULAR PLANT BREEDING, vol. 4, no. 6, 28 November 2006 (2006-11-28), pages 846 - 852 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10308953B2 (en) 2013-12-18 2019-06-04 BASF Agro B.V. Plants having increased tolerance to herbicides
US11306322B2 (en) 2013-12-18 2022-04-19 BASF Agro B.V. Plants having increased tolerance to herbicides
US11827610B2 (en) 2021-09-15 2023-11-28 Enko Chem, Inc. Protoporphyrinogen oxidase inhibitors
US11889833B2 (en) 2022-01-14 2024-02-06 Enko Chem, Inc. Protoporphyrinogen oxidase inhibitors

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