WO2010029311A2 - Herbicide tolerant plants - Google Patents

Herbicide tolerant plants Download PDF

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Publication number
WO2010029311A2
WO2010029311A2 PCT/GB2009/002188 GB2009002188W WO2010029311A2 WO 2010029311 A2 WO2010029311 A2 WO 2010029311A2 GB 2009002188 W GB2009002188 W GB 2009002188W WO 2010029311 A2 WO2010029311 A2 WO 2010029311A2
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WO
WIPO (PCT)
Prior art keywords
hst
hppd
herbicide
region
enzyme
Prior art date
Application number
PCT/GB2009/002188
Other languages
French (fr)
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WO2010029311A3 (en
Inventor
Timothy Robert Hawkes
Paul Richard Drayton
Richard Dale
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Syngenta Limited
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Filing date
Publication date
Priority to CN2009801358622A priority Critical patent/CN102159718A/en
Priority to EP09785101.8A priority patent/EP2326722B1/en
Priority to BRPI0918730-8A priority patent/BRPI0918730A2/en
Priority to EA201100481A priority patent/EA201100481A1/en
Priority to CA2735476A priority patent/CA2735476A1/en
Priority to UAA201104310A priority patent/UA107184C2/en
Priority to US13/119,123 priority patent/US20110173718A1/en
Priority to ES09785101.8T priority patent/ES2509897T3/en
Priority to JP2011526554A priority patent/JP5770631B2/en
Priority to AU2009290688A priority patent/AU2009290688B2/en
Application filed by Syngenta Limited filed Critical Syngenta Limited
Publication of WO2010029311A2 publication Critical patent/WO2010029311A2/en
Publication of WO2010029311A3 publication Critical patent/WO2010029311A3/en
Priority to ZA2011/01559A priority patent/ZA201101559B/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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
    • A01N43/58—1,2-Diazines; Hydrogenated 1,2-diazines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)

Definitions

  • the present invention relates to methods for selectively controlling weeds at a locus.
  • the invention further relates to recombinant DNA technology, and in particular to the production of transgenic plants which exhibit substantial resistance or substantial tolerance to herbicides when compared with non transgenic like plants.
  • Plants which are substantially "tolerant” to a herbicide when they are subjected to it provide a dose/response curve which is shifted to the right when compared with that provided by similarly subjected non tolerant like plants.
  • Such dose/response curves have "dose” plotted on the x-axis and “percentage kill", "herbicidal effect” etc. plotted on the y-axis.
  • Tolerant plants will typically require at least twice as much herbicide as non tolerant like plants in order to produce a given herbicidal effect. Plants which are substantially "resistant" to the herbicide exhibit few, if any, necrotic, lytic, chlorotic or other lesions when subjected to the herbicide at concentrations and rates which are typically employed by the agricultural community to kill weeds in the field.
  • the present invention relates to the production of plants that are resistant to herbicides that inhibit hydroxyphenyl pyruvate dioxygenase (HPPD) and/ or herbicides that inhibit the subsequent, homogentisate solanesyl transferase (HST) step in the pathway to plastoquinone.
  • HPPD hydroxyphenyl pyruvate dioxygenase
  • HST homogentisate solanesyl transferase
  • HPPD-inhibiting herbicides are phloem-mobile bleachers which cause the light-exposed new meristems and leaves to emerge white where, in the absence of carotenoids, chlorophyll is photo-destroyed and becomes itself an agent of photo-destruction via the photo-generation of singlet oxygen.
  • HST The enzyme catalysing the following step from HPPD in the plastoquinone biosynthesis pathway.
  • the HST enzyme is a prenyl tranferase that both decarboxylates homogentisate and also transfers to it the solanesyl group from solanesyl diphosphate and thus forms 2-methyl-6-solanesyl-l,4-benzoquinol (MSBQ), an intermediate along the biosynthetic pathway to plastoquinone.
  • MSBQ 2-methyl-6-solanesyl-l,4-benzoquinol
  • HST enzymes are membrane bound and the genes that encode them include a plastid targeting sequence. Methods for assaying HST have recently been disclosed.
  • HST Over expression of HST in transgenic plants has been reported - and said plants are said to exhibit slightly higher concentrations of ⁇ -tocopherol.
  • HST is the target site for certain classes of herbicidal compounds - which act wholly or in part by inhibiting HST.
  • over expression of HST in a transgenic plant provides tolerance to HST-inhibiting and/or HPPD-inhibiting herbicides.
  • a method of selectively controlling weeds at a locus comprising crop' plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide and/or hydroxyphenyl pyruvate dioxygenase (HPPD) inhibiting herbicide, wherein the crop plants comprise at least one heterologous polynucleotide which comprises a region which encodes an HST.
  • the crop plants further comprise an additional heterologous polynucleotide which comprises a region which encodes a hydroxyphenyl pyruvate dioxygenase (HPPD).
  • the invention still further provides a method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an HST-inhibiting herbicide, wherein the crop plants comprise at least one heterologous polynucleotide which comprises a region which encodes a HPPD enzyme.
  • an HST inhibiting herbicide is one which itself, or as a procide generates a molecule that inhibits Arabidopsis HST exhibits an IC50 less than 150 ppm, preferably less than 60 ppm using the "total extract” assay method as set out herein.
  • the HST inhibiting herbicides may also act as a HPPD inhibitors (possible to identify using, for example, HPPD enzyme assays and/or the differential responses of HPPD or HST over expressing transgenic plant lines) and, therefore, as shown below, self-synergise the effect of their inhibition of HST.
  • the HST inhibiting herbicide is selected from the group consisting of a compound of formula (Ha)
  • R 1 , R 2 , R 3 and R 4 are independently hydrogen or halogen; provided that at least three of R 1 , R 2 , R 3 and R 4 are halogen; or salts thereof;
  • R 1 and R 2 are independently hydrogen, Ci-C 4 alkyl, Ci-C 4 haloalkyl, halo, cyano, hydroxy, Ci-C 4 alkoxy, Ci-C 4 alkylthio, aryl or aryl substituted by one to five R 6 , which may be the same or different, or heteroaryl or heteroaryl substituted by one to five R 6 , which may be the same or different;
  • R 3 is hydrogen, Ci-Ci O alkyl, C 2 -Ci O alkenyl, C 2 -Cioalkynyl, C 3 -Ci 0 cycloalkyl, C 3 - Ciocycloalkyl-Ci-C 6 alkyl-, d-Cioalkoxy-Ci-Qalkyl-, Ci-Ciocyanoalkyl-, C 1 - Cioalkoxycarbonyl-Ci-Cealkyl-, N-Ci-C 3 alkyl-aminocarbonyl-Ci-C 6 alkyl-, N,N-di- (Ci-C 3 alkyl)-aminocarbonyl-Ci-C 6 alkyl-, aryl-Ci-C 6 alkyl- or aryl-Ci-C 6 alkyl- wherein the aryl moiety is substituted by one to three R 7 , which may be the same or different, or heterocyclyl-C
  • R 4 is aryl or aryl substituted by one to five R 8 , which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R 8 , which may be the same or different;
  • R 5 is hydroxy, R 9 -oxy-, R 10 -carbonyloxy-, tri-R ⁇ -silyloxy- or R 12 -sulfonyloxy-
  • each R 6 , R 7 and R 8 is independently halo, cyano, nitro, Ci-Cioalkyl, Ci-C 4 haloalkyl, C 2 -Cioalkenyl, C 2 -Cioalkynyl, hydroxy, CpCioalkoxy, Ci-C 4 haloalkoxy, Ci- Cioalkoxy-Ci-C 4 alkyl-, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloalkoxy, C 3 -C 7 cycloalkyl-Ci- C 4 alkyl-, C 3 -C 7 cycloalkyl-Ci-C 4 alkoxy-, Ci-C ⁇ alkylcarbonyl-, formyl, Ci-C 4 alk
  • R 9 is d-Cioalkyl, C 2 -Ci 0 alkenyl, C 2 -Ci 0 alkynyl or aryl-Ci-C4alkyl- or aryl-d- C 4 alkyl- wherein the aryl moiety is substituted by one to five substituents independently selected from halo, cyano, nitro, C)-C 6 alkyl, Ci-C 6 haloalkyl or C 1 - C 6 alkoxy;
  • R 10 is Ci-Cioalkyl, C 3 -Ci 0 cycloalkyl, C 3 -Ci 0 cycloalkyl-C r C 10 alkyl-, Ci-Ciohaloalkyl, C 2 -Ci O alkenyl, C 2 -Ci 0 alkynyl, C 1 -C 4 alkoxy-C 1 -Ci 0 alkyl-, C r C 4 alkylthio-C 1 -C 4 alkyl-, C]-Ci 0 alkoxy, C 2 -Cioalkenyloxy, C 2 -Cioalkynyloxy, Ci-Cioalkylthio-, TV-d-C t alkyl- amino-, jV,iV-di-(Ci-C 4 alkyl)-ammo-, aryl or aryl substituted by one to three R 14 , which may be the same or different, heteroaryl or hetero
  • R 1 and R 2 are independently hydrogen, Ci-C 4 alkyl, Ci-C 4 haloalkyl, halo, cyano, hydroxy, Ci-C 4 alkoxy, Ci-C 4 alkylthio, aryl or aryl substituted by one to five R 6 , which may be the same or different, or heteroaryl or heteroaryl substituted by one to five R 6 , which may be the same or different;
  • R 3 is Ci-C 4 haloalkyl, C 2 -C 4 haloalkenyl or C 2 -C 4 haloalkynyl;
  • R is aryl or aryl substituted by one to five R , which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R 8 , which may be the same or different;
  • R 5 is hydroxy or a group which can be metabolised to the hydroxy group; each R and R is independently halo, cyano, nitro, Ci-Cioalkyl, Ci-C 4 haloalkyl, C 2 - Ci oalkenyl, C 2 -C i oalkynyl, hydroxy, Ci-Ci oalkoxy, C i -Qhaloalkoxy, CpC i O alkoxy- Ci-C 4 alkyl-, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloalkoxy, C 3 -C 7 cycloalkyl-Ci-C 4 alkyl-, C 3 - C 7 cycloalkyl-Ci-C 4 alkoxy-, Ci-C ⁇ alkylcarbonyl-, formyl, d-Qalkoxycarbonyl-, Ci- C 4 alkylcarbonyloxy-, Ci-Cioalkylthio-
  • R and R are independently hydrogen, Ci-C 4 alkyl, Ci-C 4 haloalkyl, halo, cyano, hydroxy, Ci-C 4 alkoxy, Ci-C 4 alkylthio, aryl or aryl substituted by one to five R 6 , which may be the same or different, or heteroaryl or heteroaryl substituted by one to five R 6 , which may be the same or different;
  • R 3 is hydrogen, Ci-Ci O alkyl, Ci-C 4 haloalkyl, C 2 -Ci 0 alkenyl, C 2 -C 4 haloalkenyl, C 2 -Ci 0 alkynyl, C 2 -C 4 haloalkynyl, C 3 -Ciocycloalkyl, C 3 -Ciocycloalkyl-C]-C 6 alkyl-, Ci-Cioalkoxy-Ci-Qalkyl-, Ci-Ciocyanoalkyl-, Ci-Ci 0 alkoxycarbonyl-Ci-C 6 alkyl-, TV- C i -Qalkyl-aminocarbonyl-C i -C 6 alkyl-, 7V,iV-di-(C i -Csalkyty-aminocarbonyl-C i - C 6 alkyl-, aryl-Ci-C 6 alkyl
  • R 4 is aryl or aryl substituted by one to five R 8 , which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R 8 , which may be the same or different;
  • R 5 is hydroxy or a group which can be metabolised to the hydroxy group;
  • each R 6 , R 7 and R 8 is independently halo, cyano, nitro, C 1 -C 1O aIlCyI, Ci-C 4 haloalkyl, C 2 -C
  • a 1 , A A 2 , A / and A are independently C-R or N, provided at least one of A , A , A and A 4 is N, and provided that if A 1 and A 4 are both N, A 2 and A 3 are not both C-R 1 ; each R 1 is independently hydrogen, CrQalkyl, Ci-C 4 haloalkyl, halo, cyano, hydroxy, Ci-C 4 alkoxy, C[-C 4 alkylthio, aryl or aryl substituted by one to five R 6 , which maybe the same or different, or heteroaryl or heteroaryl substituted by one to five R 6 , which may be the same or different; R 3 is hydrogen, Ci-C 10 alkyl, d-C 4 haloalkyl, C 2 -Ci 0 alkenyl, C 2 -C 4 haloalkenyl, C 2 -Ci O alkynyl, C 2 -C 4 haloalkynyl, Q-
  • R is aryl or aryl substituted by one to five R 8 , which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R 8 , which may be the same or different;
  • R 5 is hydroxy or a group which can be metabolised to a hydroxy group; each R , R and R is independently halo, cyano, nitro, Ci-Ci O alkyl, Ci-C 4 haloalkyl, C 2 -C
  • R 1 is Ci-C 6 alkyl or Ci-C 6 alkyloxy-C r C 6 alkyl
  • R 2 is hydrogen or Ci-C 6 alkyl
  • R 3 is Ci-C 6 alkyl, C 3 -C 8 cycloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 6 -C ]O aryl, C 6 - Ci 0 aryl-Ci-C 6 alkyl-, Ci-C 6 alkyloxy, C 3 -C 8 cycloalkyloxy, C 2 -C 6 alkenyloxy, C 2 - C 6 alkynyloxy, C 6 -Cioaryloxy, Q-Cioaryl-Ci-C ⁇ alkyloxy-, amino, C r C 6 alkylamino, C 2 -C 6 alkenylamino, C 6 -Ci 0 arylamino, di(Ci-C 6 alkyl)amino, di(C 2 -C 6 alkenyl)amino, (Ci-C ⁇ alkyl)(C 6 -Cioaryl)a
  • R 4 is Ci-C 6 alkyl, C 6 -Ci 0 aryl, Ci-C 6 alkylamino group or di(Ci-C 6 alkyl)amino; and R 5 and R 6 maybe same or different and are independently Ci-C ⁇ alkyl, C 3 - 8 cycloalkyl, C 2 -C 6 alkenyl, C 6 -Ci 0 aryl, Ci-C 6 alkyloxy, C 3 -C 8 cycloalkyloxy, C 6 -Ci 0 aryloxy, C 6 - Cioaryl-Ci-C 6 alkyloxy, Ci-C 6 alkylthio, Ci-C 6 alkylamino or di(Ci-C 6 alkyl)amino, whereby any R 3 , R 4 , R 5 and R 6 group may be substituted with halogen, C 3 - Cscycloalkyl, C 6 -Cioaryl, C 6 -C ⁇ 0 ary
  • the HST inhibitors of formula (Ha) are known, for example haloxydine and pyriclor.
  • the HST inhibitors of formula (lib) are known from, for example WO 2008/009908.
  • the HST inhibitors of formula (lie) are known from, for example WO 2008/071918.
  • the HST inhibitors of formula (Hd) are known from, for example WO 2009/063180.
  • the HST inhibitors of formula (He) are known from, for example WO2009/090401 and WO2009/090402.
  • the HST inhibitors of formula (Hf) are known from, for example WO 2007/119434.
  • R 1 , R 2 , R 3 and R 4 are independently hydrogen, bromo, chloro or fiuoro; provided that at least three of R 1 , R 2 , R 3 and R 4 are either bromo, chloro or fiuoro, most preferred is the compound of formula (Ha) wherein R 1 and R 4 are fiuoro and R 2 and R 3 are chloro (haloxydine) or wherein R 1 , R 2 and R 3 are chloro and R 4 is hydrogen (pyriclor).
  • HPPD inhibiting herbicide refers to herbicides that act either directly or as procides to inhibit HPPD and that, in their active form, exhibit a Ki value of less than 5 iiM, preferably 1 iiM versus Arabidopsis HPPD when assayed using the on and off rate methods described in WO 02/46387.
  • hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase HPPD
  • p-HPPD p-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase
  • the HPPD-inhibiting herbicide is selected from the group consisting of
  • R 1 and R 2 are hydrogen or together form an ethylene bridge
  • R 3 is hydroxy or phenylthio-;
  • R 4 is halogen, nitro, Ci-C 4 alkyl, Ci-C 4 alkoxy-Ci- C 4 alkyl-, C i -C 4 alkoxy-C i -C 4 alkoxy-C i -C 4 alkyl-;
  • X is methine, nitrogen, or C-R 5 wherein R 5 is hydrogen, Ci-C 4 haloalkoxy-Ci-C 4 alkyl-, or a group
  • R 6 is Ci-Qalkylsulfonyl- or Ci-C 4 haloalkyl
  • R 1 and R 2 are independently Ci -C 4 alkyl; and the free acids thereof; a compound of formula (Ic)
  • R 1 is hydroxy, phenylcarbonyl-Ci-Qalkoxy- or phenylcarbonyl-Ci-
  • R 2 is Ci-C 4 alkyl
  • R 3 is hydrogen or Ci-C 4 alkyl;R 4 and R 6 are independently halogen, Ci-C 4 alkyl, Ci-
  • R 5 is hydrogen, Ci-C 4 alkyl, Ci-C 4 alkoxy-Ci-C 4 alkoxy-, or a group
  • R 1 is hydroxy
  • R 2 is C r C 4 alkyl
  • R is hydrogen; andR >4 , r R,5 and R are independently Ci-C 4 alkyl;
  • R 1 is cyclopropyl
  • R 2 and R 4 are independently halogen, Ci-C 4 haloalkyl, or Ci-C 4 alkylsulfonyl-
  • R 3 is hydrogen
  • R 1 is cyclopropyl
  • R 2 and R 4 are independently halogen, Ci-C 4 haloalkyl, or Ci-C 4 alkylsulfonyl-;
  • R 3 is hydrogen
  • Example HPPD-inhibitors are also disclosed in WO2009/016841.
  • the HPPD inhibitor is selected from the group consisting of benzobicyclon, mesotrione, sulcotrione, tefuryltrione, tembotrione, 4-hydroxy-3-[[2- (2-methoxyethoxy)methyl] -6-(trifluoromethyl)-3 -pyridinyl] carbonyl] -bicyclo [3.2.1]- oct-3-en-2-one (bicyclopyrone), ketospiradox or the free acid thereof, benzofenap, pyrasulfotole, pyrazolynate, pyrazoxyfen, topramezone, [2-chloro-3-(2- methoxyethoxy)-4-(methylsulfonyl)phenyl](l -ethyl-5-hydroxy- l/i-pyrazol-4-yl)- methanone, (2,3-dihydro-3,
  • HPPD inhibitors are known and have the following Chemical Abstracts registration numbers: benzobicyclon (CAS RN 156963-66-5), mesotrione (CAS RN 104206-82-8), sulcotrione (CAS RN 99105-77-8), tefuryltrione (CAS RN 473278-76- 1), tembotrione (CAS RN 335104-84-2), 4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]- 6-(trifluoromethyl)-3-pyi-idinyl]carbonyl]-bicyclo[3.2.
  • Alkyl moiety (either alone or as part of a larger group, such as alkoxy, alkoxy- carbonyl, alkylcarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl) is a straight or branched chain and is, for example, methyl, ethyl, ⁇ -propyl, /2-butyl, r ⁇ -pentyl, n- hexyl, wO-propyl, /2-butyl, sec-butyl, wo-butyl, tert-huty ⁇ or r ⁇ e ⁇ -pentyl.
  • the alkyl giOups are preferably Ci to C 6 alkyl groups, more preferably Cj-C 4 and most preferably methyl groups.
  • Alkenyl and alkynyl moieties can be in the form of straight or branched chains, and the alkenyl moieties, where appropriate, can be of either the (E)- or ⁇ -configuration. Examples are vinyl, allyl and propargyl.
  • the alkenyl and alkynyl groups are preferably C 2 to C 6 alkenyl or alkynyl groups, more preferably C 2 -C 4 and most preferably C 2 -C 3 alkenyl or alkynyl groups.
  • Alkoxyalkyl groups preferably have a chain length of from 2 to 8 carbon or oxygen atoms.
  • An example of an alkoxyalkyl group is 2-methoxy-ethyl-.
  • Halogen is generally fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine. The same is true of halogen in conjunction with other meanings, such as haloalkyl.
  • Haloalkyl groups preferably have a chain length of from 1 to 4 carbon atoms.
  • Haloalkyl is, for example, fluoromethyl, difluoromethyl, ti ⁇ fluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2-fluoroethyl, 2- chloroethyl, pentafluoroethyl, 1 , 1 -difluoro-2,2,2-trichloroethyl, 2,2,3 ,3 - tetrafluoroethyl or 2,2,2-trichloroethyl; preferably trichloromethyl, difluorochloromethyl, difluoromethyl, trifluoromethyl or dichlorofluoromethyl.
  • Haloalkoxyalkyl groups preferably have a chain length of from 2 to 8 carbon or oxygen atoms.
  • An example of an alkoxyalkyl group is 2,2,2-trifiuoroethoxymethyl-
  • Alkoxyalkoxy groups preferably have a chain length of from 2 to 8 carbon or oxygen atoms.
  • alkoxyalkoxy are: methoxymethoxy, 2-methoxy-ethoxy, methoxypropoxy, ethoxymethoxy, ethoxyethoxy, propoxymethoxy and butoxybutoxy.
  • Alkoxyalkyl groups have a chain length of preferably from 1 to 6 carbon atoms.
  • Alkoxyalkyl is, for example, methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl, n-propoxymethyl, n-propoxyethyl, isopropoxymethyl or isopropoxyethyl.
  • Alkoxyalkoxyalkyl groups preferably have a chain length of from 3 to 8 carbon or oxygen atoms.
  • alkoxy-alkoxy-alkyl are: methoxymethoxymethyl, methoxyethoxymethyl, ethoxymethoxymethyl and methoxyethoxy ethyl .
  • Cyanoalkyl giOups are alkyl groups which are substituted with one or more cyano groups, for example, cyanomethyl or 1,3-dicyanopropyl.
  • Cycloalkyl groups can be in mono- or bi-cyclic form and may optionally be substituted by one or more methyl groups.
  • the cycloalkyl groups preferably contain 3 to 8 carbon atoms, more preferably 3 to 6 carbon atoms.
  • Examples of monocyclic cycloalkyl groups are cyclopropyl, 1 -methyl cyclopropyl, 2-methylcyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • aryl refers to a ring system which may be mono-, bi- or tricyclic. Examples of such rings include phenyl, naplithalenyl, anthracenyl, indenyl or phenanthrenyl. A preferred aryl group is phenyl.
  • heteroaryl refers to an aromatic ring system containing at least one heteroatom and consisting either of a single ring or of two or more fused rings.
  • single rings will contain up to three and bicyclic systems up to four heteroatoms which will preferably be chosen from nitrogen, oxygen and sulfur.
  • Examples of such groups include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl and tetrazolyl.
  • a preferred heteroaryl group is pyridine.
  • bicyclic groups are benzothiophenyl, benzimidazolyl, benzothiadiazolyl, quinolinyl, cinnolinyl, quinoxalinyl and pyrazolo[ 1 ,5-a]pyrimidinyl.
  • heterocyclyl is defined to include heteroaryl and in addition their unsaturated or partially unsaturated analogues such as 4,5,6,7-tetrahydro- benzothiophenyl, chromen-4-onyl, 9H-fluorenyl, 3,4-dihydro-2H-benzo-l,4- dioxepinyl, 2,3-dihydro-benzofuranyl, piperidinyl, 1,3-dioxolanyl, 1,3-dioxanyl, 4,5- dihydro-isoxazolyl, tetrahydro furanyl and morpholinyl.
  • analogues such as 4,5,6,7-tetrahydro- benzothiophenyl, chromen-4-onyl, 9H-fluorenyl, 3,4-dihydro-2H-benzo-l,4- dioxepinyl, 2,3-dihydro-benzofuranyl, piperidinyl, 1,
  • the herbicide composition may be applied to the locus pre-emergence of the crop and/or post- emergence of the crop.
  • the herbicide composition is applied post-emergence of the crop - a so-called “over-the-top” application.
  • Single or indeed multiple applications may be applied as necessary to obtain the desired weed control.
  • the term "weeds" relates to any unwanted vegetation and includes, for example, carry-over or “rogue” or “volunteer” crop plants in a field of soybean crop plants.
  • the heterologous polynucleotide will comprise (i) a plant operable promoter operably linked to (ii) the region encoding the HST enzyme and (iii) a transcription terminator.
  • the heterologous polynucleotide will further comprise a region which encodes a polypeptide capable of targeting the HST enzyme to subcellular organelles such as the chloroplast or mitochondria — preferably the chloroplast.
  • the heterologous polynucleotide may further comprise, for example, transcriptional enhancers.
  • the region encoding the HST enzyme can be "codon-optimised" depending on plant host in which expression of the HST enzyme is desired. The skilled person is well aware of plant operable promoters, transcriptional terminators, chloroplast transit peptides, enhancers etc that have utility with the context of the present invention.
  • the HST may be a "wild type" enzyme or it may be one which has been modified in order to afford preferential kinetic properties with regard to provision of herbicide tolerant plants.
  • the HST is characterised in that it comprises one or more of the following polypeptide motifs:- W-(RZK)-F-L-R-P-H-T-I-R-G-T; and/or N-G-(YZF)-I-V-G-I-N-Q-I-(YZF)-D; and/or I-A-I-T-K-D-L-P; andZor Y-(RZQ)-(FZW)-(IZV)-W-N-L-F-Y.
  • Suitable HSTs are derived from Arabidopsis thaliana, Glycine max, Oryza sativa or Chlamydomonas reinhardtii.
  • the HST is selected from the group consisting of SEQ ID NO:1 to SEQ ID NO. 10.
  • amino acid sequences provided in SEQ ID NOS :1 to 10 are examples of HST amino acid sequences that include a region encoding a chloroplast transit peptide.
  • SEQ ID NOS 11-20 correspond to DNA sequences encoding the HSTs depicted as SEQ ID NO. 1-10 while SEQ ID NOS 21-24 are examples of DNA sequences encoding truncated mature HST sequences without the transit peptide region.
  • Amino acid sequences provided in SEQ ID NOS 25-28 are examples of HPPD amino acid sequences and SEQ ID NOS 29-32 are examples of DNA sequences encoding them.
  • HPPDs suitable for providing tolerance to HPPD-inhibiting herbicides are well known to the skilled person- e.g WO 02/46387.
  • SEQ ID No 33 provides the DNA sequence of the TMV translational enhancer and SEQ ID No 34 provides the DNA sequence of the TMV translational enhancer fused 5' to the DNA sequence encoding Arabidopsis HST.
  • the crop plant used in said method may further comprise a further heterologous polynucleotide encoding a further herbicide tolerance enzyme.
  • further herbicide tolerance enzymes include, for example, herbicide tolerance enzymes selected from the group consisting of, 5- enolpymvylshikimate-3 -phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPGO), Phytoene desaturase (PD), dicamba degrading enzymes (e.g WO 02/068607), and aryloxy herbicide degrading enzymes as taught in WO2007/053482 & WO2005/107437.
  • EPSPS 5- enolpymvylshikimate-3 -phosphate synthase
  • the pesticide composition used in the aforementioned methods may further comprise one or more additional pesticides - in particular 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 herein.
  • the one or more additional herbicides are selected from the group consisting of glyphosate (including agrochemically acceptable salts thereof); glufosinate (including agrochemically acceptable salts thereof); chloroacetanilides e.g alachlor, acetochlor, metolachlor, S- metholachlor; photo system II inhibitors e.g triazines such as ametryn, atrazine, cyanazine and terbuthylazine, triazinones such as hexazinone and metribuzin, ureas such as chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; ALS -inhibitors e.
  • the present invention further provides a recombinant polynucleotide which comprises a region which encodes an HST-enzyme operably linked to a plant operable promoter, wherein the region which encodes the HST-enzyme does not include the polynucleotide sequence depicted in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 14 or SEQ ID NO. 15.
  • the HST-enzyme is selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.
  • the present invention still further provides a recombinant polynucleotide comprising (i) a region which encodes a HST enzyme operably linked to a plant operable promoter and (ii) at least one additional heterologous polynucleotide, which comprises a region which encodes an additional herbicide tolerance enzyme, operably linked to a plant operable promoter.
  • the additional herbicide tolerance enzyme is, for example, selected from the group consisting of hydroxyphenyl pyruvate dioxygenase (HPPD), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPGO), Phytoene desaturase (PD) and dicamba degrading enzymes as taught in WO 02/068607.
  • HPPD hydroxyphenyl pyruvate dioxygenase
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • GAT Glyphosate acetyl transferase
  • Cytochrome P450 phosphinothricin acetyltransfera
  • the recombinant polynucleotide comprises (i) a region which encodes a HST operably linked to a plant operable promoter and (ii) a region which encodes an HPPD operably linked to a plant operable promoter. It is also possible for the recombinant polynucleotide to comprise at least two, three, or more additional regions each encoding a herbicide tolerance enzyme for example as defined previously. Thus, in another preferred embodiment the recombinant polynucleotide comprises (i) a region which encodes a HST enzyme, (ii) a region which encodes a HPPD enzyme and (iii) a region which encodes a glyphosate tolerance enzyme.
  • the present invention further provides a vector comprising a recombinant polynucleotide according to the present invention.
  • the present invention further relates to transformed plants over expressing an HST enzyme which exhibit substantial resistance or substantial tolerance to HST- inhibiting herbicides and/or HPPD-inhibiting herbicides when compared with non transgenic like plants. It should also be appreciated that the transformed plants of the present invention typically exhibit enhanced stress tolerance including heat and drought tolerance.
  • the present invention further provides a plant cell which exhibits substantial resistance or substantial tolerance to HST-inhibiting herbicides and/or
  • HPPD-inhibiting herbicides when compared with non transgenic like plant cell - said plant cell comprising the recombinant polynucleotide of the present invention as herein described. It should be appreciated that the region encoding the HST and any region encoding one or more additional herbicide tolerance enzymes may be provided on the same ("linked") or indeed separate transforming recombinant polynucleotide molecules.
  • the plant cell may further comprise further transgenic traits, for example heterologous polynucleotides providing resistance to insects, fungi and/or nematodes.
  • further transgenic traits for example heterologous polynucleotides providing resistance to insects, fungi and/or nematodes.
  • the present invention further provides morphologically normal fertile HST- inhibitor tolerant plants, plant cells, tissues and seeds which comprise a plant cell according to the present invention.
  • Plants or plant cells transformed include but are not limited to, field crops, fruits and vegetables such as canola, sunflower, tobacco, sugar beet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, ⁇ worzel, potato, carrot, lettuce, cabbage, onion, etc.
  • Particularly preferred genetically modified plants are soya spp, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses, flax and oilseed rape, and nut producing plants insofar as they are not already specifically mentioned .
  • the said plant is a dicot, preferably selected from the group consisting of canola, sunflower, tobacco, sugar beet, soybean, cotton, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, and is particularly preferably soybean.
  • the said plant is maize or rice.
  • the plant of the invention is soybean, rice or maize.
  • the invention also includes the progeny of the plant of the preceding sentence, and the seeds or other propagating material of such plants and progeny.
  • the recombinant polynucleotide of the present invention is used to protect soybean crops from the herbicidal injury of HPPD inhibitor herbicides of the classes of HPPD chemistry selected from the group consisting of the compounds of formula Ia or Ig.
  • HPPD inhibitor herbicide is selected from sulcotrione, mesotrione, tembotrione and compounds of formula Ia where X is nitrogen and R 4 is CF 3 , CF 2 H or CFH 2 and/or where Ri and R 2 together form an ethylene bridge.
  • the present invention still further provides a method of providing a transgenic plant which is tolerant to HST-inhibiting and/or HPPD-inhibiting herbicides which comprises transformation of plant material with a recombinant polynucleotide(s) which comprises a region which encodes an HST enzyme, selection of the transformed plant material using an HST-inhibiting herbicide and/or HPPD-inhibiting herbicide, and regeneration of that material into a morphological normal fertile plant.
  • the transformed plant material is selected using a HST- inhibiting herbicide alone or in combination with a HPPD-inhibiting herbicide.
  • the present invention further relates to the use of polynucleotide which comprises a region which encodes an HST enzyme as a selectable marker in plant transformation and to the use of a polynucleotide comprising a region which encodes an HST enzyme in the production of plants which are tolerant to herbicides which act wholly or in part by inhibiting HST.
  • the present invention still further relates to the use of HST inhibitors as selection agents in plant transformation and to the use of a recombinant HST enzyme in in vitro screening of potential herbicides.
  • the present invention still further provides a herbicidal composition, preferably a synergistic herbicide composition, comprising an HPPD-inhibiting herbicide (as defined herein) and a HST-inhibiting herbicide (as defined herein).
  • a herbicidal composition preferably a synergistic herbicide composition, comprising an HPPD-inhibiting herbicide (as defined herein) and a HST-inhibiting herbicide (as defined herein).
  • the ratio of the HPPD-inhibiting herbicide to the HST-inhibiting herbicide in the composition is any suitable ratio - typically from 100:1 to 1:100, preferably from 1:10 to 1 :100, even more preferably from 1 :1 to 1 :20.
  • the skilled person will recognise that the optimal ratio will depend on the relative potencies and spectrum of the two herbicides which can be derived as a matter of routine experimental optimisation.
  • the herbicidal composition may further comprise one or more additional pesticidal ingredient(s).
  • the additional pesticides may include, for example, herbicides, fungicides or insecticides (such as thiomethoxam) - however herbicides are preferred.
  • the additional herbicide is preferably selected from the group consisting of glyphosate (including agrochemically acceptable salts thereof); glufosinate (including agrochemically acceptable salts thereof); chloroacetanilides e.g alachlor, acetochlor, metolachlor, S-metholachlor; photo system II (PS-II) inhibitors e.g triazines such as ametryn, atrazine, cyanazine and terbuthylazine, triazinones such as hexazinone and metribuzin, and ureas such as chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; ALS -inhibitors e.g sulfonyl ureas such as amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, primisulfuron, prosulfuron, rim
  • the present invention still further provides a method of selectively controlling weeds at a locus comprising crop plants and weeds comprising applying to the locus a weed controlling amount of a synergistic herbicidal composition as previously defined.
  • HPPD and HST inhibiting herbicides are sprayed sequentially rather than at the same time as a mixture.
  • the HST herbicide can be advantageously applied over a crop locus to which an HPPD herbicide has already been previously applied. Normally this would be in the same season but, especially in the case of more persistent HPPD herbicides, there would also be an advantage in using the HST herbicide in the following season.
  • HST inhibiting herbicides are advantageously used as part of a programme of weed control wherein an HPPD inhibiting herbicide is applied earlier in the season or even in the preceding season.
  • the HPPD-inhibiting herbicide may be applied to the locus at any suitable rate — for example from 1 to 1000 g/ha, more preferably from 2 to 200 g/ha.
  • the HST-inhibiting herbicide may be applied at any suitable rate — for example from 10 to 2000 g/ha, more preferably from 50 to 400 g/ha.
  • the HPPD-inhibiting herbicide is applied to the locus at a rate which is sub-lethal to the weeds were the HPPD-inhibiting herbicide to be applied in the absence of other herbicides.
  • the actual sub-lethal rate will depend on weed species present and the actual HPPD inhibitor - but will typically be less than 50 g/ha - more preferably less than 10 g/ha.
  • the present invention further provides the use of a sub-lethal application of an HPPD-mhibiting herbicide to increase the weed controlling efficacy of an HST-inhibiting herbicide.
  • the invention will be further apparent from the following non-limiting examples and sequence listings. SEQUENCE LISTING
  • SEQ ID NO. 28 HPPD a/a sequence from Shewanella collwelliana
  • SEQ ID NO. 33 TMV translational enhancer nucleotide sequence tatttttacaacaattaccaacaacaacaaacaacaacaacaacattacaattactatt tacaattacac
  • GR50 values derived from dose/response curves having "dose” plotted on the x-axis and “percentage kill", "herbicidal effect”, “numbers of emerging green plants” etc. plotted on the y-axis where increased GR50 values may, for example, correspond to increased levels of inherent inhibitor-tolerance (e.g increased Ki x kcat./ Kni H PP value) and/or level of expression of the expressed HPPD and/or HST.
  • EXAMPLE 1 Cloning and expressing plant HST enzymes in insect cells and in E.coli
  • HST coding sequences (minus the ATG start codon) are amplified, with flanking EcoRI sites, from Arabidopsis (SEQ ID 11) and Rice (SEQ ID 12 or SEQ ID 13) from cDNA libraries or made synthetically. Both these full length and also the truncated coding sequences (encoding the mature sequences starting from ARG 64) Arabidopsis SEQ ID 21, rice SEQ ID 22 and rice SEQ ID 23) were cloned into the EcoRI site of the pAcG3X vector (BD Biosciences Cat. No.
  • Arabidopsis HST is cloned into the EcoRI site of pAcG3X and expressed in SfP cells as a N-terminal GST fusion protein.
  • the Arabidopsis HST SWISSPROT accession number (protein) is Ql ACB3 and the Arabidopsis HST EMBL accession number (DNA): DQ231060.
  • HST coding sequences are cloned as GST N-terminal fusion enzymes and expressed in E.coli.
  • EXAMPLE 2 Growth of Cells and Preparation of HST Enzyme Extracts.
  • BL21A1 cells expressing mature Arabidopsis or Chlamydomonas HST as a GST N-terminal fusion proteins are grown, harvested, broken and membrane fractions expressing HST produced.
  • Ing of recombinant DNA is used to transform BL21DE3 cells to obtain a plateful of individual colonies.
  • One of these colonies is picked and used to inoculate an overnight culture of 100ml of Luria Broth (LB) supplemented with 50ug/ml of kanamycin at final concentration, grown at 37° C with shaking at 220rpm.
  • LB Luria Broth
  • lOmls of the overnight culture is used to inoculate 11 of fresh sterile LB supplemented with 50ug/ml of kanamycin at final concentration, grown at 37° C with shaking at 220rpm until the OD reached 0.6 at 600nm, induced with the addition of 0. ImM IPTG and left to induce at 15° C overnight.
  • the cells are harvested by centrifugation at 4600 rpm for 10 min at 4° C and the pellet stored at -80°C.
  • E.coli cell pellet is then resuspended in 25 ml of 5OmM Tris, pH 7.5 supplemented with Roche EDTA- free protease inhibitor tablet (one tablet in 20OmIs of buffer). 10 ml of cells are lysed by sonication on ice. The resultant lysed cells are centrifuged at 300Og for lOmin to pellet the cell nuclei/debris etc. lOmls of supernatant is aspirated and centrifuged at 150,00Og for 60 min at 4° C. The pellet containing the membranes is resuspended in 2 ml of the above buffer. These samples are stored as lOOul aliquots at -80° C, after being diluted with addition of glycerol to 50% v/v.
  • the HST expression pAcG3X -derived transfer vectors (described above) are independently co-transfo ⁇ ned into Sf9 suspension cells with FlashBac (Oxford Expression Technologies) parental baculovirus vector.
  • Baculovirus amplification and HST protein expression is performed in accordance with the manufacturer's instructions Sf9 Suspension cell cultures are subcultured at a density of 1.0 EXP 6 cells / ml in 140 ml SfPOOII medium (Invitrogen Cat No.10902) in 500 ml Erlenmeyer flasks. After 24 hours culture at 27 0 C shaking at 120rpm the cell density is measured and readjusted to 2.0 EXP 6 cells / ml in 140 ml.
  • volumes of amplified virus stock of known titre are added to prepared suspension flasks to give a multiplicity of infection of 10. Flasks are sealed and incubated at 27 0 C shaking at 125rpm for 72 hours to allow adequate protein expression without cell lysis. Cells are harvested by dividing flask contents evenly between three 50 ml Falcon tubes and centrifuging at 900rpm for 4 minutes. Medium is discarded leaving a 3 ml cell pellet which is snap frozen in liquid nitrogen and maintained at -80 0 C
  • the pellet from 25mls of Sf9 cells (after induction of expression for 4 days) is resuspended in lOmls of 5OmM Tris, pH 7.5 supplemented with Roche-EDTA free protease inhibitor tablet (1 tablet in 20OmIs of buffer) and homogenised using a hand held homogeniser.
  • the resultant lysed cells are centrifuged at 3000g for lOmin to pellet the cell nuclei/debris etc. 1 OmIs of supernatant is aspirated and centrifuged at 15O 5 OOOg for 60 min at 4° C.
  • the pellet containing the membranes is resuspended in ImI of above buffer and samples are stored as lOOul aliquots at -80° C, after first being diluted with addition of glycerol to 50% v/v.
  • HST enzyme preparations for assay are also prepared directly from fresh plant material.
  • HST enzyme preparations are from spinach.
  • intact spinach chloroplasts are prepared from two lots of 500 g of fresh baby spinach leaves (e.g from the salad section of the local supermarket). Prepacked spinach is usually already washed, but if buying loose leaves these must be rinsed in water before proceeding. Stalks, large leaves and mid-ribs are removed. Each 500g lot of leaves is added to 1.5 1 of 'Grinding medium' in a 2L plastic beaker.
  • Grinding medium is cold (4 0 C) 50 mM Tricine/ NaOH buffer at pH 7.1 containing 330 mM glucose, 2 mM sodium isoascorbate, 5 mM MgC12 and 0.1% bovine serum albumen
  • the beaker kept at 4 0 C, is placed under a Polytron 6000 blender, fitted with a 1.5"cutting probe and the mixture blended in short bursts of 5-8sec up to 8- 1OK rpm until all the leaves are macerated.
  • the homogenate is filtered into a 5L beaker (embedded in an ice bucket) through four layers of muslin, and two layers of 50 ⁇ mesh nylon cloth.
  • the filtrate is transferred to 250ml buckets of a Beckman GS-6 centrifuge and spun at 200 x g (3020rpm) for 2 min at 4° C. The supernatant is drained away and discarded to leave a sediment of chloroplasts.
  • Chloroplasts are resuspended in a few ml of cold resuspension medium by gentle swirling and gentle use of a quill brush soaked in resuspension medium.
  • Resuspension medium is 50 mM Hepes/ KOH pH 7.8 containing 330 mM sorbitol, 2 mM EDTA, 5mM KH 2 PO 4 , 2 mM MgC12 and 0.1% bovine serum albumen at 4 0 C.
  • the chloroplasts are resuspended in 5-10 ml of resuspension buffer, recentrifuged down and resuspended again in order to wash them.
  • the chloroplasts are then once again centrifuged down and then broken by resuspension in about 5 ml of 50 mM Tricine-NaOH pH 7.8 to a protein concentration of about 40 mg/ ml.
  • the solution is stored frozen at -8O 0 C in aliquots. This resuspension is defrosted and used directly in HST activity assays.
  • chloroplasts are prepared resuspended in 50 mM Tris/ HCl buffer at pH 7.8 containing 330 mM sorbitol (alternative resuspension buffer) and layered on top of a percol gradient (comprising the same buffer containing 45% percol), spun down, the intact chloroplast fraction taken and washed 2 or 3 times in the alternative resuspension buffer and then spun down again, resuspended in breaking buffer (without sorbitol), flash frozen and stored in aliquots at - 80 0 C.
  • a percol gradient comprising the same buffer containing 45% percol
  • Prenyltransferase activities are measured by determining the prenylation rates of [U- I4 C]homogentisate using farnesyl diphosphate (FDP) as prenyl donor.
  • 14 C homogentisate is prepared from 14 C tyrosine using L amino acid oxidase and HPPD.
  • compounds are dissolved in dimethylsulfoxide (DMSO). DMSO added at up to 2% v/v has no effect on assays. Control assays contain DMSO at the same concentration as in inhibitor containing assays.
  • Assays using spinach chloroplast extracts contain up to 2 mg of chloroplast protein, 50 mM Tricine-NaOH pH 8.5, 50 mM MgCl 2 , 200 ⁇ M farnesyl diphosphate (FDP) and 26 ⁇ M 14 C-homogentisate (167 dpm/pmol). Assays are run for about an hour at 28 0 C. For inhibitor studies, haloxydine at a final concentration of 500 ppm is found to completely inhibit the reaction. Alternatively, stopping the reaction and carrying out solvent extraction at zero time also provides a 100% inhibition baseline reference. Lipophilic reactions products are extracted and analyzed essentially as described in the literature.
  • Chlamydomonas HST expressed in E.coli membranes is assayed in standard reaction mixtures with 200 ⁇ M FDP and 100 ⁇ M 14 C-homogentisate
  • Recombinant Arabidopsis and Rice HSTs are expressed in insect cells. Assays are run as for Chlamydomonas HST except that assay temperature is 27 0 C. Assays are stopped with 300ul of solvent mix (1:2, Chloroform: Methanol) and lOOul of 0.5% NaCl, agitated / mixed and spun at 13,000rpm in abenchtop eppendorf centrifuge for 5 minutes. 80ul of of the lower phase extract is loaded onto a TLC plate (silica Gel 60, 20cm x 20cm) FLA3000 system and run for 35 minutes in dichloromethane. The radioactivity is quantified using a Fuji Phosphoimager and band intensity integrated as quantitative measure of product amount.
  • the bands corresponding to oxidised and reduced 2 ⁇ methyl-6-farnesyl-l,4-benzoquinol (MFBQ) are identified and the total of the two (oxidised and reduced) band intensities is calculated in order to estimate the total amount of MFBQ product formation.
  • Specific activities of 8 pmol MFBQ min "1 mg '1 protein (23 pmol) and 7 pmol MFBQ min "1 mg "1 protein (14 pmol) are , for example, estimated for the GST-fusion truncated Arabidopsis HST gene (SEQ ID # 3) expressed in membranes from insect cells 4 days and 5 days after transfection respectively. Similar results are noted from past literature on E. coli expressed Arabidopsis HST.
  • inhibitors such as haloxydine inhibit the formation of these other products in a way that, as dose is varied, is apparently co-linear with inhibition of the formation of MFBQ.
  • 500 ppra haloxydine about completely inhibits the HST enzyme reaction and neither MFBQ nor any of the other products are formed.
  • the TLC step is dispensed with, treatment with 500 ppm haloxydine (or other inhibitor at a suitable concentration) is used as the 100% inhibition 'control' and a portion of the chloroform/ methanol extract is taken directly into a scintillation vial and counted.
  • Arabidopsis HST SEQ ID # 11 is cloned behind a double 35s CMV promoter sequence and a TMV translational enhancer sequence and in front of the 3' terminator from the nos gene.
  • This expression cassette is ligated into pMJBl (described in WO98/20144) and then into pBIN19 and then transformed into Agrobacterium titmefaciens strains LBA4404 prior to plant transformation.
  • the full length Arabidopsis HST seq ID # 11 is fused to the TMV translational enhancer sequence (SEQ ID #33) by overlapping PCR and, at the same time, 5' Xhol site and a 3'KpnI site are added by PCR. Site-directed mutagenesis is performed to remove an internal Xhol site.
  • the TMV/HPPD fusion is removed from the pBIN19 by digestion with Xhol/Kpnl and is replaced by the TMV/HST fusion (SEQ ID #34).
  • the TMV/HST fusion is now cloned behind a double 35s promoter and in front of the 3' terminator from the nos gene. Again the modified pBIN19 vector ( ⁇ pBinAT HST') is then transformed into Agrobacterium tumefaciens strain LBA4404.
  • vectors for plant transformation are constructed to comprise DNA sequences any HST and, for example, SEQ Ids nos. 12-20.
  • vectors comprise DNA encoding HSTs from photosynthetic protozoans, higher and lower plants the sequences of which are derived from cDNA libraries using methods known to the skilled man.
  • total RNA is prepared from 5- 20 -day-old plant seedlings using the method of Tri-Zol extraction (Life Technologies Inc.
  • mRNA is obtained, for example, from Avena sativa using the Oligotex mRNA purification system (Qiagen).
  • the 5' end of, for example, the A. sativa HST gene is identified using 5' RACE, performed using the Gene Racer kit (Invitrogen) with internal HST gene specific primers (based on HST consensus regions e.g SEQ No. 35, 36, 37 and 38).
  • the 3' end of the gene is identified by 3' RACE, performed using Themoscript RT (Life Technologies) with appropriate oligo dT primer and an appropriate internal HST gene primer, followed by PCR All methodologies are performed according to protocols provided by the various stated manufacturers.
  • Products obtained from the 5' and 3' RACE reactions are cloned into pCR 2.1 TOPO (invitrogen) and the cloned products sequenced using universal M 13 forward and reverse primers with an automated ABI377 DNA sequencer. Primers are then designed to the translation initiation and termination codons of the HST gene respectively. Both primers are used in conjunction with the One-step RTPCR kit (Qiagen or Invitrogen) to obtain full length coding sequences. Products obtained are cloned into pCR 2.1 TOPO, sequenced, and identified as HST by comparison with sequences known in the art (and for example the HST sequences herein).
  • a master plate of Agrobacterium tumefaciens containing the binary vector pBinAT HST (described above) or analogous binary vector comprising a different HST is used to inoculate 10 ml LB containing 100 mg / 1 Rifampicin plus 50 mg / 1 Kanamycin using a single bacterial colony. This is incubated overnight at 28 0 C shaking at 200 rpm. This entire overnight culture is used to inoculate a 50 ml volume of LA (plus antibiotics). Again this is cultured overnight at 28 0 C shaking at 200 rpm.
  • Explants are then transferred to NBM medium containing 100 mg / 1 Kanamycin plus antibiotics to prevent further growth of Agrobacterium (200 mg / 1 timentin with 250 mg / 1 carbenicillin). Further subculture on to this same medium is then performed every 2 weeks.
  • Rooted transgenic TO plantlets are transferred from agar and potted into 50% peat, 50% John Innes soil no 3 or, for example, MetroMix® 380 soil (Sun Gro Horticulture, Bellevue, WA) with slow-release fertilizer in 3 inch round or 4 inch square pots and left regularly watered to establish for 8-12d in the glass house.
  • Glass house conditions are about 24-27 0 C day; 18-21 0 C night and approximately a 14h (or longer in UK summer) photoperiod.
  • Humidity is ⁇ 65% and light levels up to 2000 ⁇ mol/ m 2 at bench level.
  • test chemicals dissolved in water with 0.2-0.25% X-77 surfactant and sprayed from a boom on a suitable track sprayer moving at 2 mph in a DeVries spray chamber with the nozzle about 2 inches from the plant tops.
  • Spray volume is suitably 25 gallons per acre or, for example, 2001/ ha.
  • Test chemicals are, for example, compound 2.3 at 500 g/ha.
  • transgenic plants are sprayed so too are w/t Samsun tobacco plants grown from seed as well as non-transgenic plants regenerated from tissue culture and non-transgenic tissue culture escapes. Damage is assessed versus unsprayed control plants of like size and development.
  • Arabidopsis HST transgenic tobacco plants assessed 11 DAT with compound 2.3 (designated compound 3 in the table). Compared to untreated controls all the plants are affected by treatment at 50Og/ ha and are smaller and growth is set back. However, unlike controls that show white meristems and that are essentially dead many of the HST transgenics show green meristems and are recovering and some show essentially no bleaching. Plants are scored on a scale from 0 to 10 with 0 meaning plant substantially bleached / burnt and meristem dead/ white and 10 meaning that the entire plant looks green and undamaged.
  • the plants are assessed at various times after treatment up to 28 DAT. Those events (e.g C8, G9, E9) showing the least damage from HST herbicides are grown on to flowering, bagged and allowed to self. The seed from selected events are collected sown on again in pots and tested again for herbicide resistance in a spray test for herbicide resistance. Single copy events amongst the Tl plant lines are identified by their 3 : 1 segregation ratio (for example, dependent on the construct, by both kanamycin selection and wrt herbicide resistance phenotype) and by quantitative RT- PCR.
  • 3 : 1 segregation ratio for example, dependent on the construct, by both kanamycin selection and wrt herbicide resistance phenotype
  • EXAMPLE 5 Production and further testing of Tl and T2 transgenic plants transformed to express Arabidopsis HST , Avena HPPD or Pseudomonas HPPD TO transgenic tobacco plant lines B 8 and G9 described above in the foregoing examples are selfed. About 50 of the resultant seed from each selfing each line are planted out into a soil/ peat mixture in 3 inch pots, grown in the glass house for 7-10 d and sprayed with 500g/ ha of compound 2.3 (all as described in the foregoing examples). For each line about three quarters of the plants display visible resistance to the herbicide and of these a few plants (possible homozygotes at a single insertion event) appear the most resistant. A few of these more highly tolerant Tl plants are selfed again to produce batches of T2 seed.
  • Seed from w/t Samsun tobacco and 8 Tl seed from events B8 and G9 are also planted out in 3 inch pots , grown on for 7 to 12 d and then, as described in the foregoing examples, the plantlets spray tested for resistance to various chemicals and assessed at 14 DAT. Chemicals are formulated in 0.2% X77 and sprayed at a spray volume of 200 1/lia. Results are depicted in Table 4 below. The results clearly demonstrate the heritability of the herbicide resistance phenotype.
  • the transgenic Arabidopsis HST Tobacco Tl plants display resistance to HST herbicides as exemplified using compounds 2.15 and 2.30 but that the phenotype is specific and the plants are not significantly tolerant to the other two herbicides tested, norflurazon and atrazine.
  • Table 4 Assessment of herbicide % damage to Tl progeny plants of Arabidopsis HST lines B8 and G9 at 14 DAT with various herbicides.
  • a TO event exhibiting tolerance to mesotrione was selfed to produce a single insertion Tl line (exhibiting 3:1 segregation of the herbicide tolerance and kanamycin selection phenotypes) which was again further selfed to provide the T2 line designated C2.
  • Arabidopsis HST 5 lines of tobacco transformed with Arabidopsis HST were less damaged 10 DAT with 10 g/ha mesotrione than like-treated wild-type lines. Expression of Arabidopsis HST confers a degree of tolerance to the HPPD herbicide, mesotrione.
  • EXAMPLE 6 Resistance of plants expressing Avena HPPD to HST herbicides.
  • Tl lines of tobacco expressing the wild-type HPPD gene of Avena sativa under operable control of the double enhanced 35S CMV promoter region , Nos3' terminator and TMV translational enhancer were provided as described in WO0246387.
  • About 30-40 Tl seed derived from selfing a mesotrione-tolerant TO event were grown up to 7-10 d old plantlets sprayed and assessed as described above and the results (at 6 DAT) are depicted in the Table 7 below. Under the conditions of the experiment the Avena HPPD appearsto offer a degree tolerance to both HST inhibitors 2.30 and 3.6.
  • a master plate of Agrobacterhim tiimefaciens containing the binary vector pBinAT HST (described in example 6) is used to inoculate 10 ml LB containing 100 mg / 1 Rifampicin plus 50 mg / 1 Kanamycin using a single bacterial colony. This is incubated overnight at 28 0 C shaking at 200 rpm. This entire overnight culture is used to inoculate a 50 ml volume of LA (plus antibiotics). Again this is cultured overnight at 28 0 C shaking at 200 rpm.
  • Explants are then transferred to NBM medium containing 0.5 mg / 1 Haloxydine plus antibiotics to prevent further growth of Agrobacterium (200 mg / 1 timentin with 250 mg / 1 carbenicillin). Further subculture on to this same medium is then performed every 2 weeks.
  • HST gene in combination with a HST-inhibitor herbicide provides a means for the selection of transgenic plant tissue EXAMPLE 8.
  • Preparation and testing of stable transgenic plants lines expressing a heterologous HPPD enzyme Transgenic lines of tobacco, soyabean and corn etc. can be engineered to express various heterologous HPPDs derived from, for example Avena (SEQ ID #26), Wheat (SEQ ID #27), Pseudomonas fluorescein (SEQ ID # 25) and Shewanella colwelliana (SEQ ID #28) as, for example, described in WO 02/46387.
  • the seed from selected events are collected sown on again in pots and tested again for herbicide resistance in a spray test for resistance to HPPD herbicide (for example mesotrione).
  • Single copy events amongst the Tl plant lines are identified by their 3:1 segregation ratio (wrt kanamycin and or herbicide) and by quantitative RT-PCR.
  • Seed from the thus selected Tl tobacco (var Samsun) lines are sown in 3 inch diameter pots containing 50% peat, 50% John Innes soil no 3. After growth to the 3 leaf stage, plants are sprayed, as described above, in order to test for herbicide tolerance relative to like- treated non-transgenic tobacco plants.
  • Control tobacco plants and transgenic Tl plants expressing either the Pseudomonas or the wheat HPPD gene are sprayed at 37, 111, 333 and 1000 g/lia rates of HST inhibitors and, for example, compound 2.3.
  • Plants are assessed and scored for % damage at 16 DAT.
  • EXAMPLE 9 Preparation of transgenic plants lines expressing different heterologous HST and HPPD enzymes and stacked combinations thereof. Glasshouse testing for herbicide tolerance
  • the full length Arabidopsis (plus start codon) HST seq ID 11 is cloned behind a double 35s CMV promoter sequence and a TMV translational enhancer sequence and in front of the 3' terminator from the nos gene as described previously.
  • this expression construct is cloned into a binary vector (pBIN 35S Arabidopsis HST) that is transformed into tobacco to produce populations of 30-50 transgenic events which are subdivided at the callus stage to produce 2-4 clonal plants from each transgenic 'event' which are then regenerated and transferred into soil before transfer to the glass hosue and testing.
  • Chlamydomonas HST gene sequence (AM285678) is codon-optimised for tobacco and cloned behind the double Cauliflower mosaic virus 35S promoter and Tobacco mosaic virus enhancer sequences and in front of a nos gene terminator, cloned into a binary vector and transformed into tobacco to produce a population of TO plants.
  • the wheat HPPD gene sequence (Embl DD064495) is cloned behind an Arabidopsis Rubisco small subunit (SSU) promoter and in front of a nos gene terminator to produce an ' SSU Wheat HPPD nos expression cassette' which is cloned into a binary vector and transformed into tobacco to produce a population of 30-50 transgenic events.
  • SSU Arabidopsis Rubisco small subunit
  • a "pBin Arabidopsis HST/Wheat HPPD” vector is also built in order to provide a population of plants that co-express the HST and HPPD enzymes.
  • the SSU Wheat HPPD nos cassette (described above) is cloned into the EcoRI site of the pBin 35S Arabidopsis HST vector (described above) to generate the HST/HPPD expression construct and binary vector. Again this is transformed into tobacco to produce a population of primary transformants.
  • transgenic plants expressing both HPPD and HST are produced by first transforming to express either a heterologous HST or HPPD and then the progeny tissue are subsequently transformed with a construct designed to express the other enzyme.
  • tobacco plants are transformed to express wheat, Avena or Pseudomonas HPPD under expression control of the
  • Leaves from these shoot cultures are subject to transformation using constructs and selection methods described previously.
  • To initially evaluate whether co-expression of HPPD and HST results in elevated levels of plant resistance to mesotrione compared to expression of HPPD alone shoot culture derived leaf explants from HPPD only and HPPD plus HST transformants are plated onto NBM medium containing a range of mesotrione concentrations between 0.1 to 5 mg / 1. Explants from transgenics combining HPPD and HST may exhibit green callus and more limited bleaching of regenerating shoots at higher mesotrione concentrations than the HPPD only 'background' explants from the clonal single plant, HPPD event derived material.
  • 'ControPplantlets are regenerated from the untransformed (with HST) HPPD- expressing clonal background material derived from a single plant of a single event.
  • TO HST transgenic plantlets that are additionally transformed with HST are selected against this background as described in the previous example on haloxydine, and are also regenerated. Plantlets are micropropagated into further clones, rooted and grown on in pots in the glass house as described in the previous examples.
  • EXAMPLE 10 Construction of soybean transformation vectors.
  • a binary vector (17107) for dicot (soybean) transformation is, for example, constructed, with the Arabidopsis UBQ3 promoter driving expression of the
  • Chlamydomonas HST coding sequence (SEQ ID # 15), followed by Nos gene 3' terminator.
  • the gene is codon optimized for soybean expression based upon the predicted amino acid sequence of the HST gene coding region.
  • the amino acid sequence of the protein encoded by Chlamydomonas HST gene is provided in SEQ ID # 5.
  • the transformation vector also contains two PAT gene cassettes (one with the 35S promoter and one with the CMP promoter, and both PAT genes are followed by the nos terminator) for glufosinate based selection during the transformation process.
  • a similar binary vector (17108) is similarly constructed but also comprising an expression cassette expressing the soyabean codon-optimized Avena HPPD gene. In this case there is no PAT gene and selection is earned out using a HPPD herbicide or, as described herein, a HST herbicide.
  • Soybean transformation is achieved using methods well known in the art. TO plants were taken from tissue culture to the greenhouse where they are transplanted into saturated soil (Redi-Earth® Plug and Seedling Mix, Sun Gro Horticulture, Bellevue, WA) mixed with 1% granular Marathon® (Olympic Horticultural Products, Co., venue, PA) at 5-10 g/gal Redi-Earth® Mix in 2" square pots. The plants are covered with humidity domes and placed in a Conviron chamber (Pembina, ND) with the following environmental conditions: 24 0 C day; 18 0 C night; 23 hr photoperiod; 80% relative humidity.
  • Conviron chamber Pembina, ND
  • plants are sampled and tested for the presence of desired transgene by TaqmanTM analysis using appropriate probes for the HST and/or HPPD genes, or promoters (for example prCMP and prUBq3). All positive plants and several negative plants are transplanted into 4" square pots containing MetroMix® 380 soil (Sun Gro Horticulture, Bellevue, WA). Sierra 17-6-12 slow release fertilizer is incorporated into the soil at the recommended rate. The negative plants serve as controls for the spray experiment. The plants are then relocated into a standard greenhouse to acclimatize ( ⁇ 1 week). The environmental conditions are: 27 0 C day; 21 0 C night; 12 hr photoperiod (with ambient light); ambient humidity. After acclimatizing ( ⁇ 1 week), the plants are ready to be sprayed with the desired herbicides.
  • Example 12 HPPD/ HST herbicide mixtures. Effect of adding small amounts of HPPD inhibitor on the herbicidal activity of HST herbicides
  • Tobacco var Samsun plantlets germinated aseptically in agar made up in 1/3 strength Murashige and Skoog salts medium along with various doses of herbicide. Bleaching damage to emerging plantlets is assessed 7 DAT. The plantlets are kept covered under clear perspex and grown at 18 0 C (night) and 24° C (day) under a 16h day ( ⁇ 500-900 umol/ m 2 ) , 8 h darkness regime. Herbicide affected plantlets are bleached white and grow less. Synergistic / antagonistic responses are calculated using the Colby formula (Colby, S. R. (Calculating synergistic and antagonistic responses of herbicide Combinations", Weeds, 15, p. 20-22, 1967).
  • Haloxydine / Haloxydine (0.004ppm) (0.004ppm) ppm only (OBSERVED) (EXPECTED) (O-E)
  • HST + HPPD herbicide effects on tobacco seedlings in agar The % bleaching observed 7 DAT of germinating tobacco seeds in agar is assessed with various doses of haloxydine and 0.75% v/v DMSO in the presence or absence of 0.004 ppm mesotrione. At this dose the mesotrione by itself consistently gives 35% bleaching damage and the expected values for the damage in mixture with the various doses of haloxydine are therefore calculated accordingly as described by Colby (1967).
  • HST + HPPD herbicide effects on tobacco seedlings growing on liquid The % bleaching observed 20 DAT of tobacco seedlings on liquid culture medium is assessed versus the presence of various concentrations of the HST herbicide, compound 2.13 with 0.75% v/v DMSO in the presence or absence of 0.001 or 0.0005 ppm of mesotrione. At these doses the mesotrione by itself produced either minimal, 20% , or zero bleaching damage.
  • Weed seeds are planted out in trays containing suitable soil (for example 50% peat, 50% John Innes soil no 3) and grown in the glass house conditions under 24-27 0 C day; 18- 21 0 C night and approximately a 14h (or longer in UK summer) photoperiod.
  • suitable soil for example 50% peat, 50% John Innes soil no 3
  • Humidity is ⁇ 65% and light levels up to 2000 ⁇ mol/ m 2 at bench level. Trays are sprayed with test chemicals dissolved in water with 0.2-0.25% X-77 surfactant and sprayed from a boom on a suitable track sprayer moving at about 2 mph in a suitable track sprayer (for example a DeVries spray chamber with the nozzle about 2 inches from the plant tops). Spray volume is suitably 500 - 1000 1/ha. Sprays are carried out both pre-emergence and over small plants at about 7-12 d post-emergence
  • Mesotrione is applied at a very low rate of lg/ha at which it causes essentially no ( ⁇ 10% damage).
  • weed seeds are planted out in trays containing 50% peat/ 50% John Iniies no. 3 soil and grown in the glass house at 24- 27 C day; 18-21 C night and approximately a 15h photoperiod.
  • Humidity is ⁇ 65% and light levels at bench level are up to 2mmol/ m 2 .
  • All spray chemicals are dissolved in 0.2% X77 surfactant and sprayed from a boom on a track sprayer moving at 2 mph with the nozzle set about 2 inches above the plant tops.
  • the spray volume is 5001/ ha. Sprays are carried out both pre-emergence and post- emergence over small plants at about 7-12d post-emergence.
  • HPPD inhibiting herbicide is compound A22 (4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]-6- (trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1 ]oct-3-en-2-one) which is sprayed at 2g/ ha both alone and in mixture with various HST herbicides. Results and spray rates are depicted in Tables 18 and 19. Again the Colby formula has been used to calculate synergy scores observed following treatment with the mixture based on the results obtained with the single components alone. Positive synergy is observed between the HPPD herbicide and a wide variety of HST inhibitor herbicides applied both pre and postemergence across a variety of weeds.
  • 625 is -225 2875

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Abstract

The present invention relates, inter alia, a method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide and/or hydroxyphenyl pyruvate dioxygenase (HPPD) inhibiting herbicide, wherein the crop plants comprise at least one recombinant polynucleotide which comprises a region which encodes an HST; to a method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide, wherein the crop plants comprise at least one recombinant polynucleotide which comprises a region which encodes a HPPD enzyme and to recombinant polynucleotides and vectors for utilised in the methods. The present invention further relates to a herbicidal composition comprising a HPPD-inhibiting herbicide and a HST-inhibiting herbicide.

Description

HERBICIDE TOLERANT PLANTS
The present invention relates to methods for selectively controlling weeds at a locus. The invention further relates to recombinant DNA technology, and in particular to the production of transgenic plants which exhibit substantial resistance or substantial tolerance to herbicides when compared with non transgenic like plants. Plants which are substantially "tolerant" to a herbicide when they are subjected to it provide a dose/response curve which is shifted to the right when compared with that provided by similarly subjected non tolerant like plants. Such dose/response curves have "dose" plotted on the x-axis and "percentage kill", "herbicidal effect" etc. plotted on the y-axis. Tolerant plants will typically require at least twice as much herbicide as non tolerant like plants in order to produce a given herbicidal effect. Plants which are substantially "resistant" to the herbicide exhibit few, if any, necrotic, lytic, chlorotic or other lesions when subjected to the herbicide at concentrations and rates which are typically employed by the agricultural community to kill weeds in the field.
More particularly, the present invention relates to the production of plants that are resistant to herbicides that inhibit hydroxyphenyl pyruvate dioxygenase (HPPD) and/ or herbicides that inhibit the subsequent, homogentisate solanesyl transferase (HST) step in the pathway to plastoquinone.
Herbicides that act by inhibiting HPPD are well known in the art. Inhibition of HPPD blocks the biosynthesis of plastoquinone (PQ) from tyrosine. PQ is an essential cofactor in the biosynthesis of carotenoid pigments which are essential for photopro tecti on of the photosynthetic centres. HPPD-inhibiting herbicides are phloem-mobile bleachers which cause the light-exposed new meristems and leaves to emerge white where, in the absence of carotenoids, chlorophyll is photo-destroyed and becomes itself an agent of photo-destruction via the photo-generation of singlet oxygen. Methods for production of transgenic plants which exhibit substantial resistance or substantial tolerance to HPPD-inhibiting herbicides have been reported - for example WO02/46387. The enzyme catalysing the following step from HPPD in the plastoquinone biosynthesis pathway is HST. The HST enzyme is a prenyl tranferase that both decarboxylates homogentisate and also transfers to it the solanesyl group from solanesyl diphosphate and thus forms 2-methyl-6-solanesyl-l,4-benzoquinol (MSBQ), an intermediate along the biosynthetic pathway to plastoquinone. HST enzymes are membrane bound and the genes that encode them include a plastid targeting sequence. Methods for assaying HST have recently been disclosed.
Over expression of HST in transgenic plants has been reported - and said plants are said to exhibit slightly higher concentrations of α-tocopherol. However, it has not hitherto been recognised that HST is the target site for certain classes of herbicidal compounds - which act wholly or in part by inhibiting HST. Furthermore, it has now been found, inter alia, that over expression of HST in a transgenic plant provides tolerance to HST-inhibiting and/or HPPD-inhibiting herbicides.
Thus, according to the present invention there is provided a method of selectively controlling weeds at a locus comprising crop' plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide and/or hydroxyphenyl pyruvate dioxygenase (HPPD) inhibiting herbicide, wherein the crop plants comprise at least one heterologous polynucleotide which comprises a region which encodes an HST. In a preferred embodiment of the method the crop plants further comprise an additional heterologous polynucleotide which comprises a region which encodes a hydroxyphenyl pyruvate dioxygenase (HPPD).
The invention still further provides a method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an HST-inhibiting herbicide, wherein the crop plants comprise at least one heterologous polynucleotide which comprises a region which encodes a HPPD enzyme.
In a preferred embodiment the pesticide composition referred to in the aforementioned methods comprises both an HST-inhibiting herbicide and an HPPD- inhibiting herbicide
For the purposes of the present invention - an HST inhibiting herbicide is one which itself, or as a procide generates a molecule that inhibits Arabidopsis HST exhibits an IC50 less than 150 ppm, preferably less than 60 ppm using the "total extract" assay method as set out herein. It should be appreciated that the HST inhibiting herbicides may also act as a HPPD inhibitors (possible to identify using, for example, HPPD enzyme assays and/or the differential responses of HPPD or HST over expressing transgenic plant lines) and, therefore, as shown below, self-synergise the effect of their inhibition of HST. Preferably the HST inhibiting herbicide is selected from the group consisting of a compound of formula (Ha)
Figure imgf000004_0001
wherein
R1, R2, R3 and R4 are independently hydrogen or halogen; provided that at least three of R1, R2, R3 and R4 are halogen; or salts thereof;
a compound of formula (lib)
Figure imgf000004_0002
wherein
R1 and R2 are independently hydrogen, Ci-C4alkyl, Ci-C4haloalkyl, halo, cyano, hydroxy, Ci-C4alkoxy, Ci-C4alkylthio, aryl or aryl substituted by one to five R6, which may be the same or different, or heteroaryl or heteroaryl substituted by one to five R6, which may be the same or different;
R3 is hydrogen, Ci-CiOalkyl, C2-CiOalkenyl, C2-Cioalkynyl, C3-Ci0cycloalkyl, C3- Ciocycloalkyl-Ci-C6alkyl-, d-Cioalkoxy-Ci-Qalkyl-, Ci-Ciocyanoalkyl-, C1- Cioalkoxycarbonyl-Ci-Cealkyl-, N-Ci-C3alkyl-aminocarbonyl-Ci-C6alkyl-, N,N-di- (Ci-C3alkyl)-aminocarbonyl-Ci-C6alkyl-, aryl-Ci-C6alkyl- or aryl-Ci-C6alkyl- wherein the aryl moiety is substituted by one to three R7, which may be the same or different, or heterocyclyl-Ci-Cόalkyl- or heterocyclyl-Cj-Cόalkyl- wherein the heterocyclyl moiety is substituted by one to three R7, which may be the same or different;
R4 is aryl or aryl substituted by one to five R8, which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R8, which may be the same or different;
R5 is hydroxy, R9-oxy-, R10-carbonyloxy-, tri-Rπ-silyloxy- or R12-sulfonyloxy-, each R6, R7 and R8 is independently halo, cyano, nitro, Ci-Cioalkyl, Ci-C4haloalkyl, C2-Cioalkenyl, C2-Cioalkynyl, hydroxy, CpCioalkoxy, Ci-C4haloalkoxy, Ci- Cioalkoxy-Ci-C4alkyl-, C3-C7cycloalkyl, C3-C7cycloalkoxy, C3-C7cycloalkyl-Ci- C4alkyl-, C3-C7cycloalkyl-Ci-C4alkoxy-, Ci-Cόalkylcarbonyl-, formyl, Ci-C4alkoxy- carbonyl-, Ci-C4alkylcarbonyloxy-, Ci-Ci0alkylthio-, Ci-C4haloalkylthio-, Ci- Cioalkylsulfinyl-, Ci-Qhaloalkylsulfmyl-, Ci-Cioalkylsulfonyl-, Ci -
C^ialoalkylsulfonyl-, amino, Ci-Cioalkylamino-, di-Ci-Cioalkylamino-, Ci- Cioalkylcarbonylamino-, aryl or aryl substituted by one to three R13, which may be the same or different, heteroaryl or heteroaryl substituted by one to three R13, which may be the same or different, aryl-Ci-C4alkyl- or aryl-Ci-C4alkyl- wherein the aryl moiety is substituted by one to three R13, which may be the same or different, heteroaryl-Ci- C4alkyl- or heteroaryl-Ci-C4alkyl- wherein the heteroaryl moiety is substituted by one to three R13, which may be the same or different, aryloxy- or aryloxy- substituted by one to three R13, which may be the same or different, heteroaryloxy- or heteroaryloxy- substituted by one to three R13, which may be the same or different, arylthio- or arylthio- substituted by one to three R13, which may be the same or different, or heteroarylthio- or heteroarylthio- substituted by one to three R13, which may be the same or different;
R9 is d-Cioalkyl, C2-Ci0alkenyl, C2-Ci0alkynyl or aryl-Ci-C4alkyl- or aryl-d- C4alkyl- wherein the aryl moiety is substituted by one to five substituents independently selected from halo, cyano, nitro, C)-C6alkyl, Ci-C6haloalkyl or C1- C6alkoxy;
R10 is Ci-Cioalkyl, C3-Ci0cycloalkyl, C3-Ci0cycloalkyl-CrC10alkyl-, Ci-Ciohaloalkyl, C2-CiOalkenyl, C2-Ci0alkynyl, C1-C4alkoxy-C1-Ci0alkyl-, CrC4alkylthio-C1-C4alkyl-, C]-Ci0alkoxy, C2-Cioalkenyloxy, C2-Cioalkynyloxy, Ci-Cioalkylthio-, TV-d-Ctalkyl- amino-, jV,iV-di-(Ci-C4alkyl)-ammo-, aryl or aryl substituted by one to three R14, which may be the same or different, heteroaryl or heteroaryl substituted by one to three R14, which may be the same or different, aryl-Ci-C4alkyl- or aryl-Ci-C4alkyl- wherein the aryl moiety is substituted by one to three R14, which may be the same or different, heteroaryl-Ci-C4alkyl- or heteroaryl-Ci-C4alkyl- wherein the heteroaryl moiety is substituted by one to three R14, which may be the same or different, aryloxy- or aryloxy- substituted by one to three R14, which may be the same or different, heteroaryloxy- or heteroaryloxy- substituted by one to three R14, which may be the same or different, arylthio- or arylthio- substituted by one to three R14, which may be the same or different, or heteroarylthio- or heteroarylthio- substituted by one to three R14, which may be the same or different; each R1 ' is independently Ci-Cioalkyl or phenyl or phenyl substituted by one to five substituents independently selected from halo, cyano, nitro, Ci-C6alkyl, C1- C6haloalkyl or Ci-C6alkoxy; R12 is Ci-CiQalkyl or phenyl or phenyl substituted by one to five substituents independently selected from halo, cyano, nitro, Ci-Cδalkyl, Ci-C6haloalkyl or Cr C6alkoxy; each R13 is independently halo, cyano, nitro, Ci-C6alkyl, Ci-C6haloalkyl or d- C6alkoxy; and each R14 is independently halo, cyano, nitro, Ci-CiOalkyl, Ci-C4haloalkyl, d- Cioalkoxy, d-Qalkoxycarbonyl-, Ci-C4haloalkoxy, d-doalkylthio-, d-
C4haloalkylthio-, Ci-CiQalkylsulfmyl-,
Figure imgf000006_0001
Ci-Ci0alkylsulfonyl-, Ci-Qhaloalkylsulfonyl-, aryl or aryl substituted by one to five substituents independently selected from halo, cyano, nitro, Ci-C6alkyl, Ci-C6haloalkyl or Ci- C6alkoxy, or lieteroaryl or heteroaryl substituted by one to four substituents independently selected from halo, cyano, nitro, Ci-C6alkyl, Ci-Cόhaloalkyl or Ci- C6alkoxy; or salts or JV-oxides thereof;
a compound of formula (lie)
Figure imgf000007_0001
wherein R1 and R2 are independently hydrogen, Ci-C4alkyl, Ci-C4haloalkyl, halo, cyano, hydroxy, Ci-C4alkoxy, Ci-C4alkylthio, aryl or aryl substituted by one to five R6, which may be the same or different, or heteroaryl or heteroaryl substituted by one to five R6, which may be the same or different; R3 is Ci-C4haloalkyl, C2-C4haloalkenyl or C2-C4haloalkynyl; R is aryl or aryl substituted by one to five R , which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R8, which may be the same or different;
R5 is hydroxy or a group which can be metabolised to the hydroxy group; each R and R is independently halo, cyano, nitro, Ci-Cioalkyl, Ci-C4haloalkyl, C2- Ci oalkenyl, C2-C i oalkynyl, hydroxy, Ci-Ci oalkoxy, C i -Qhaloalkoxy, CpC i Oalkoxy- Ci-C4alkyl-, C3-C7cycloalkyl, C3-C7cycloalkoxy, C3-C7cycloalkyl-Ci-C4alkyl-, C3- C7cycloalkyl-Ci-C4alkoxy-, Ci-Cόalkylcarbonyl-, formyl, d-Qalkoxycarbonyl-, Ci- C4alkylcarbonyloxy-, Ci-Cioalkylthio-, Ci-Qhaloalkylthio-, Ci-Cioalkylsulfmyl-, Ci- C4haloalkylsulfinyl-, Ci-Cioalkylsulfonyl-,
Figure imgf000007_0002
amino, Cr Cioalkylamino-, di-Ci-Cioalkylamino-, Ci-Cioalkylcarbonylamino-, aryl or aryl substituted by one to three R13, which may be the same or different, heteroaryl or heteroaryl substituted by one to three R13, which may be the same or different, aryl- Ci-C4alkyl- or aryl-Ci-C4alkyl- wherein the aryl moiety is substituted by one to three R13, which may be the same or different, heteroaryl-Ci-C4alkyl- or heteroaryl-Cr C4alkyl- wherein the heteroaryl moiety is substituted by one to three R13, which may be the same or different, aryloxy- or aryloxy- substituted by one to three R13, which may be the same or different, heteroaryloxy- or heteroaryloxy- substituted by one to three R13, which may be the same or different, arylthio- or arylthio- substituted by one to three R13, which may be the same or different, or heteroarylthio- or heteroarylthio- substituted by one to three R13, which may be the same or different; and each R13 is independently halo, cyano, nitro, Ci-C6alkyl, C]-C6haloalkyl or C1- C6alkoxy; or a salt or iV-oxide thereof;
a compound of formula (Hd)
Figure imgf000008_0001
wherein
R and R are independently hydrogen, Ci-C4alkyl, Ci-C4haloalkyl, halo, cyano, hydroxy, Ci-C4alkoxy, Ci-C4alkylthio, aryl or aryl substituted by one to five R6, which may be the same or different, or heteroaryl or heteroaryl substituted by one to five R6, which may be the same or different;
R3 is hydrogen, Ci-CiOalkyl, Ci-C4haloalkyl, C2-Ci0alkenyl, C2-C4haloalkenyl, C2-Ci0alkynyl, C2-C4haloalkynyl, C3-Ciocycloalkyl, C3-Ciocycloalkyl-C]-C6alkyl-, Ci-Cioalkoxy-Ci-Qalkyl-, Ci-Ciocyanoalkyl-, Ci-Ci0alkoxycarbonyl-Ci-C6alkyl-, TV- C i -Qalkyl-aminocarbonyl-C i -C6alkyl-, 7V,iV-di-(C i -Csalkyty-aminocarbonyl-C i - C6alkyl-, aryl-Ci-C6alkyl- or aryl-Ci-Cδalkyl- wherein the aryl moiety is substituted by one to three R7, which may be the same or different, or heterocyclyl-Ci-Cόalkyl- or heterocyclyl-Ci-Qalkyl- wherein the heterocyclyl moiety is substituted by one to three R7, which may be the same or different;
R4 is aryl or aryl substituted by one to five R8, which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R8, which may be the same or different; R5 is hydroxy or a group which can be metabolised to the hydroxy group; each R6, R7 and R8 is independently halo, cyano, nitro, C1-C1OaIlCyI, Ci-C4haloalkyl, C2-C|Oalkenyl, C2-Cioalkynyl, hydroxy, Ci-Ci0alkoxy, Ci-C4haloalkoxy, Cr Cioalkoxy-Ci-C4alkyl-, C3-C7cycloalkyl, C3-C7cycloalkoxy, C3-C7cycloalkyl-Ci- Qalkyl-, C3-C7cycloalkyl-Ci-C4alkoxy-, CrC6alkylcarbonyl-, formyl, Ci-Qalkoxy- carbonyl-, Ci-C4alkylcarbonyloxy-, Ci-Cioalkylthio-, Ci-Qhaloalkylthio-, C1- Cioalkylsulfmyl-, Ci-C4haloalkylsulfinyl-, Ci-Ci0alkylsulfonyl-, Ci- C4haloalkylsulfonyl-, amino, Ci-Cioalkylamino-, di-Q-Cioalkylamino-, C1- Cioalkylcarbonylamino-, aryl or aryl substituted by one to three R13, which may be the same or different, heteroaryl or heteroaryl substituted by one to three R13, which may be the same or different, aryl-Q -Qalkyl- or aryl-Ci-C4alkyl- wherein the aryl moiety is substituted by one to three R13, which may be the same or different, heteroaryl-Cp C4alkyl- or heteroaryl-Ci -Qalkyl- wherein the heteroaryl moiety is substituted by one to three R13, which may be the same or different, aryloxy- or aryloxy- substituted by one to three R13, which may be the same or different, heteroaryloxy- or heteroaryloxy- substituted by one to three R13, which may be the same or different, arylthio- or arylthio- substituted by one to three R13, which may be the same or different, or heteroarylthio- or heteroarylthio- substituted by one to three R13, which may be the same or different; and each R13 is independently halo, cyano, nitro, Ci-C6alkyl, Ci-Cδhaloalkyl or Cr C6alkoxy; or a salt or iV-oxide thereof;
lie) a compound of formula (He)
Figure imgf000009_0001
wherein
A 1 , A A 2 , A / and A are independently C-R or N, provided at least one of A , A , A and A4 is N, and provided that if A1 and A4 are both N, A2 and A3 are not both C-R1; each R1 is independently hydrogen, CrQalkyl, Ci-C4haloalkyl, halo, cyano, hydroxy, Ci-C4alkoxy, C[-C4alkylthio, aryl or aryl substituted by one to five R6, which maybe the same or different, or heteroaryl or heteroaryl substituted by one to five R6, which may be the same or different; R3 is hydrogen, Ci-C10alkyl, d-C4haloalkyl, C2-Ci0alkenyl, C2-C4haloalkenyl, C2-CiOalkynyl, C2-C4haloalkynyl, Q-Ciocycloalkyl, C3-Ciocycloalkyl-Ci-C6alkyl-, CrCioalkoxy-Cj-Qalkyl-, Cj-Ciocyanoalkyl-, Ci-Ci0alkoxycarbonyl-Ci-C6alkyl-, N- CrCsalkyl-aminocarbonyl-Ci-Cealkyl-, N,N-di-(Ci-C3alkyl)-aminocarbonyl-C|- C6alkyl-, aryl-CrC6alkyl- or aryl-Ci-C6alkyl- wherein the aryl moiety is substituted by one to three R7, which may be the same or different, or heterocyclyl-Ci-Cealkyl- or heterocyclyl-Ci-Cealkyl- wherein the heterocyclyl moiety is substituted by one to three R7, which may be the same or different;
R is aryl or aryl substituted by one to five R8, which may be the same or different, or heteroaryl or heteroaryl substituted by one to four R8, which may be the same or different;
R5 is hydroxy or a group which can be metabolised to a hydroxy group; each R , R and R is independently halo, cyano, nitro, Ci-CiOalkyl, Ci-C4haloalkyl, C2-C|Oalkenyl, C2-Cioalkynyl, hydroxy, CpCioalkoxy, C[-C4haloalkoxy, Ci- Cioalkoxy-Ci-C4alkyl-, C3-C7cycloalkyl, C3-C7cycloalkoxy, C3-C7cycloalkyl-Ci- C4alkyl-, C3-C7cycloalkyl-Ci-C4alkoxy-, Ci-Qalkylcarbonyl-, formyl, Ci-C4alkoxy- carbonyl-, Ci-C4alkylcarbonyloxy-, Ci-Cioalkylthio-, Ci-C4haloalkylthio-, Ci- Cioalkylsulfinyl-, Ci-C4haloalkylsulfmyl-, Ci-Ci0alkylsulfonyl-, Ci- C4haloalkylsulfonyl-, amino, Ci-Cioalkylamino-, di-Ci-Cioalkylamino-, C\- Cioalkylcarbonylamino-, aryl or aryl substituted by one to three R13, which may be the same or different, heteroaryl or heteroaryl substituted by one to three R13, which may be the same or different, aryl-Ci-C4alkyl- or aryl-C[-C4alkyl- wherein the aryl moiety is substituted by one to three R13, which may be the same or different, heteroaryl-Ci- C4alkyl- or heteroaryl-Ci-C4alkyl- wherein the heteroaryl moiety is substituted by one to three R13, which may be the same or different, aryloxy- or aryloxy- substituted by one to three R13, which may be the same or different, heteroaryloxy- or heteroaryloxy- substituted by one to three R13, which may be the same or different, arylthio- or arylthio- substituted by one to three R13, which may be the same or different, or heteroarylthio- or hetero arylthio- substituted by one to three R13, which may be the same or different; and each R13 is independently halo, cyano, nitro, Ci-C6alkyl, Ci-C6haloalkyl or C1- Qalkoxy; or a salt or JV-oxide thereof; and
a compound of formula (Hf)
Figure imgf000011_0001
wherein
R1 is Ci-C6 alkyl or Ci-C6alkyloxy-CrC6alkyl; R2 is hydrogen or Ci-C6alkyl;
G is a hydrogen, -(C=L)R3, -(SO2)R4, or -(P=L)R5R6, wherein L is oxygen or sulfur;
R3 is Ci-C6alkyl, C3-C8cycloalkyl, C2-C6alkenyl, C2-C6alkynyl, C6-C]Oaryl, C6- Ci0aryl-Ci-C6alkyl-, Ci-C6alkyloxy, C3-C8cycloalkyloxy, C2-C6alkenyloxy, C2- C6alkynyloxy, C6-Cioaryloxy, Q-Cioaryl-Ci-Cόalkyloxy-, amino, CrC6alkylamino, C2-C6alkenylamino, C6-Ci0arylamino, di(Ci-C6alkyl)amino, di(C2-C6alkenyl)amino, (Ci-Cόalkyl)(C6-Cioaryl)amino or a three- to eight-membered nitrogen containing heterocyclic ring,
R4 is Ci-C6alkyl, C6-Ci0aryl, Ci-C6alkylamino group or di(Ci-C6 alkyl)amino; and R5 and R6 maybe same or different and are independently Ci-Cόalkyl, C3-8cycloalkyl, C2-C6alkenyl, C6-Ci0aryl, Ci-C6alkyloxy, C3-C8cycloalkyloxy, C6-Ci0aryloxy, C6- Cioaryl-Ci-C6alkyloxy, Ci-C6alkylthio, Ci-C6alkylamino or di(Ci-C6alkyl)amino, whereby any R3, R4, R5 and R6 group may be substituted with halogen, C3- Cscycloalkyl, C6-Cioaryl, C6-Cι0aryl-Ci-C6alkyl-, C3-Cscycloalkyloxy, C6-Ci0aryloxy, Cβ-Cioaryl-d-Cβalkyloxy-, C6-Ci0arylamino, (Ci-C6 alkyl)(C6-Ci0aryl)amino and a three- to eight-membered nitrogen containing heterocyclic ring which may be substituted with at least one Ci-C6alkyl; Z1 is Ci-C6alkyl; Z2 is Ci-C6alkyl; n is O, 1, 2, 3 or 4; and each of Z2 may be same or different when n represents an integer of 2 or more, and a sum of the number of carbon atoms in the group represented by Z1 and that in the group represented by Z2 is equal to 2 or more. The HST inhibitors of formula (Ha) are known, for example haloxydine and pyriclor. The HST inhibitors of formula (lib) are known from, for example WO 2008/009908. The HST inhibitors of formula (lie) are known from, for example WO 2008/071918. The HST inhibitors of formula (Hd) are known from, for example WO 2009/063180. The HST inhibitors of formula (He) are known from, for example WO2009/090401 and WO2009/090402. The HST inhibitors of formula (Hf) are known from, for example WO 2007/119434.
Preferred are the compounds of formula (Ha)
Figure imgf000012_0001
wherein
R1, R2, R3 and R4 are independently hydrogen, bromo, chloro or fiuoro; provided that at least three of R1, R2, R3 and R4 are either bromo, chloro or fiuoro, most preferred is the compound of formula (Ha) wherein R1 and R4 are fiuoro and R2 and R3 are chloro (haloxydine) or wherein R1, R2 and R3 are chloro and R4 is hydrogen (pyriclor).
The term "HPPD inhibiting herbicide" refers to herbicides that act either directly or as procides to inhibit HPPD and that, in their active form, exhibit a Ki value of less than 5 iiM, preferably 1 iiM versus Arabidopsis HPPD when assayed using the on and off rate methods described in WO 02/46387. Within the context of the present invention the terms hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (HPPD), 4-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (4- HPPD) and p-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (p-HPPD) are synonymous.
Preferably, the HPPD-inhibiting herbicide is selected from the group consisting of
a compound of formula (Ia)
Figure imgf000013_0001
wherein R1 and R2 are hydrogen or together form an ethylene bridge;
R3 is hydroxy or phenylthio-;R4 is halogen, nitro, Ci-C4alkyl, Ci-C4alkoxy-Ci- C4alkyl-, C i -C4alkoxy-C i -C4alkoxy-C i -C4alkyl-;
X is methine, nitrogen, or C-R5 wherein R5 is hydrogen, Ci-C4haloalkoxy-Ci-C4alkyl-, or a group
Figure imgf000013_0002
R6 is Ci-Qalkylsulfonyl- or Ci-C4haloalkyl;
a compound of formula (Ib)
Figure imgf000013_0003
R1 and R2 are independently Ci -C4alkyl; and the free acids thereof; a compound of formula (Ic)
Figure imgf000014_0001
wherein R1 is hydroxy, phenylcarbonyl-Ci-Qalkoxy- or phenylcarbonyl-Ci-
C4alkoxy- wherein the phenyl moiety is substituted in para-position by halogen or Ci-
C4alkyl, or phenylsulfonyloxy- or phenylsulfonyloxy- wherein the phenyl moiety is substituted in para-position by halogen or Ci-C4alkyl;
R2 is Ci-C4alkyl;
R3 is hydrogen or Ci-C4alkyl;R4 and R6 are independently halogen, Ci-C4alkyl, Ci-
C4haloalkyl, or Ci-C4alkylsulfonyl-; and
R5 is hydrogen, Ci-C4alkyl, Ci-C4alkoxy-Ci-C4alkoxy-, or a group
Figure imgf000014_0002
a compound of formula (Id)
Figure imgf000014_0003
wherein R1 is hydroxy; R2 is CrC4alkyl;
R is hydrogen; andR >4 , r R,5 and R are independently Ci-C4alkyl;
a compound of formula (Ie)
Figure imgf000014_0004
wherein R1 is cyclopropyl; R2 and R4 are independently halogen, Ci-C4haloalkyl, or Ci-C4alkylsulfonyl-; and R3 is hydrogen; and
a compound of formula (If)
Figure imgf000015_0001
wherein R1 is cyclopropyl;
R2 and R4 are independently halogen, Ci-C4haloalkyl, or Ci-C4alkylsulfonyl-; and
R3 is hydrogen.
Example HPPD-inhibitors are also disclosed in WO2009/016841. In a preferred embodiment the HPPD inhibitor is selected from the group consisting of benzobicyclon, mesotrione, sulcotrione, tefuryltrione, tembotrione, 4-hydroxy-3-[[2- (2-methoxyethoxy)methyl] -6-(trifluoromethyl)-3 -pyridinyl] carbonyl] -bicyclo [3.2.1]- oct-3-en-2-one (bicyclopyrone), ketospiradox or the free acid thereof, benzofenap, pyrasulfotole, pyrazolynate, pyrazoxyfen, topramezone, [2-chloro-3-(2- methoxyethoxy)-4-(methylsulfonyl)phenyl](l -ethyl-5-hydroxy- l/i-pyrazol-4-yl)- methanone, (2,3-dihydro-3,3,4-trimethyl-l,l-dioxidobenzo[b]thien-5-yl)(5-hydroxy- 1 -methyl- lZ7-pyrazol-4-yl)-methanone, isoxachlortole, isoxaflutole, α- (cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-chloro-benzenepropanenitrile, and α-(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-(trifluoromethyl)- benzenepropanenitrile.
These HPPD inhibitors are known and have the following Chemical Abstracts registration numbers: benzobicyclon (CAS RN 156963-66-5), mesotrione (CAS RN 104206-82-8), sulcotrione (CAS RN 99105-77-8), tefuryltrione (CAS RN 473278-76- 1), tembotrione (CAS RN 335104-84-2), 4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]- 6-(trifluoromethyl)-3-pyi-idinyl]carbonyl]-bicyclo[3.2. l]oct-3-en-2-one (CAS RN 352010-68-5), ketospiradox (CAS RN 192708-91-1) or its free acid (CAS RN 187270-87-7), benzofenap (CAS RN 82692-44-2), pyrasulfotole (CAS RN 365400- 11-9), pyrazolynate (CAS RN 58011-68-0), pyrazoxyfen (CAS RN 71561-11-0), topramezone (CAS RN 210631-68-8), [2-chloro-3-(2-methoxyethoxy)-4- (methylsulfonyl)phenyl](l-ethyl-5-hydroxy-l/J-pyrazol-4-yl)-methanone (CAS RN 128133-27-7), (2,3-dihydro-3,3,4-trimethyl-l, l-dioxidobenzo[b]thien-5-yl)(5- hydroxy-1 -methyl- l/J-pyrazol-4-yl)-methanone (CAS RN 345363-97-5), isoxachlortole (CAS RN 141112-06-3), isoxaflutole (CAS RN 141112-29-0), α- (cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-chloro-benzenepropanenitrile (CAS RN 143701-66-0), and ^(cyclopropylcarbonyO^-OiiethylsulfonyO-β-oxo^- (trifluoromethyl)-benzenepropanenitrile (CAS RN 143701-75-1).
The following definitions apply to those terms used in respect of Formula I and Formula II.
Alkyl moiety (either alone or as part of a larger group, such as alkoxy, alkoxy- carbonyl, alkylcarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl) is a straight or branched chain and is, for example, methyl, ethyl, ^-propyl, /2-butyl, rø-pentyl, n- hexyl, wO-propyl, /2-butyl, sec-butyl, wo-butyl, tert-huty\ or røeσ-pentyl. The alkyl giOups are preferably Ci to C6 alkyl groups, more preferably Cj-C4 and most preferably methyl groups.
Alkenyl and alkynyl moieties (either alone or as part of a larger group, such as alkenyloxy or alkynyloxy) can be in the form of straight or branched chains, and the alkenyl moieties, where appropriate, can be of either the (E)- or ©-configuration. Examples are vinyl, allyl and propargyl. The alkenyl and alkynyl groups are preferably C2 to C6 alkenyl or alkynyl groups, more preferably C2-C4 and most preferably C2-C3 alkenyl or alkynyl groups.
Alkoxyalkyl groups preferably have a chain length of from 2 to 8 carbon or oxygen atoms. An example of an alkoxyalkyl group is 2-methoxy-ethyl-.
Halogen is generally fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine. The same is true of halogen in conjunction with other meanings, such as haloalkyl. Haloalkyl groups preferably have a chain length of from 1 to 4 carbon atoms. Haloalkyl is, for example, fluoromethyl, difluoromethyl, tiϊfluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2-fluoroethyl, 2- chloroethyl, pentafluoroethyl, 1 , 1 -difluoro-2,2,2-trichloroethyl, 2,2,3 ,3 - tetrafluoroethyl or 2,2,2-trichloroethyl; preferably trichloromethyl, difluorochloromethyl, difluoromethyl, trifluoromethyl or dichlorofluoromethyl.
Haloalkoxyalkyl groups preferably have a chain length of from 2 to 8 carbon or oxygen atoms. An example of an alkoxyalkyl group is 2,2,2-trifiuoroethoxymethyl-
Alkoxyalkoxy groups preferably have a chain length of from 2 to 8 carbon or oxygen atoms. Examples of alkoxyalkoxy are: methoxymethoxy, 2-methoxy-ethoxy, methoxypropoxy, ethoxymethoxy, ethoxyethoxy, propoxymethoxy and butoxybutoxy. Alkoxyalkyl groups have a chain length of preferably from 1 to 6 carbon atoms. Alkoxyalkyl is, for example, methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl, n-propoxymethyl, n-propoxyethyl, isopropoxymethyl or isopropoxyethyl.
Alkoxyalkoxyalkyl groups preferably have a chain length of from 3 to 8 carbon or oxygen atoms. Examples of alkoxy-alkoxy-alkyl are: methoxymethoxymethyl, methoxyethoxymethyl, ethoxymethoxymethyl and methoxyethoxy ethyl .
Cyanoalkyl giOups are alkyl groups which are substituted with one or more cyano groups, for example, cyanomethyl or 1,3-dicyanopropyl.
Cycloalkyl groups can be in mono- or bi-cyclic form and may optionally be substituted by one or more methyl groups. The cycloalkyl groups preferably contain 3 to 8 carbon atoms, more preferably 3 to 6 carbon atoms. Examples of monocyclic cycloalkyl groups are cyclopropyl, 1 -methyl cyclopropyl, 2-methylcyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
In the context of the present specification the term "aryl" refers to a ring system which may be mono-, bi- or tricyclic. Examples of such rings include phenyl, naplithalenyl, anthracenyl, indenyl or phenanthrenyl. A preferred aryl group is phenyl.
The term "heteroaryl" refers to an aromatic ring system containing at least one heteroatom and consisting either of a single ring or of two or more fused rings. Preferably, single rings will contain up to three and bicyclic systems up to four heteroatoms which will preferably be chosen from nitrogen, oxygen and sulfur. Examples of such groups include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl and tetrazolyl. A preferred heteroaryl group is pyridine. Examples of bicyclic groups are benzothiophenyl, benzimidazolyl, benzothiadiazolyl, quinolinyl, cinnolinyl, quinoxalinyl and pyrazolo[ 1 ,5-a]pyrimidinyl.
The term "heterocyclyl" is defined to include heteroaryl and in addition their unsaturated or partially unsaturated analogues such as 4,5,6,7-tetrahydro- benzothiophenyl, chromen-4-onyl, 9H-fluorenyl, 3,4-dihydro-2H-benzo-l,4- dioxepinyl, 2,3-dihydro-benzofuranyl, piperidinyl, 1,3-dioxolanyl, 1,3-dioxanyl, 4,5- dihydro-isoxazolyl, tetrahydro furanyl and morpholinyl.
It should be understood that in the aforementioned methods the herbicide composition may be applied to the locus pre-emergence of the crop and/or post- emergence of the crop. In a preferred embodiment the herbicide composition is applied post-emergence of the crop - a so-called "over-the-top" application. Single or indeed multiple applications may be applied as necessary to obtain the desired weed control. The term "weeds" relates to any unwanted vegetation and includes, for example, carry-over or "rogue" or "volunteer" crop plants in a field of soybean crop plants.
Typically, the heterologous polynucleotide will comprise (i) a plant operable promoter operably linked to (ii) the region encoding the HST enzyme and (iii) a transcription terminator. Typically, the heterologous polynucleotide will further comprise a region which encodes a polypeptide capable of targeting the HST enzyme to subcellular organelles such as the chloroplast or mitochondria — preferably the chloroplast. The heterologous polynucleotide may further comprise, for example, transcriptional enhancers. Furthermore, the region encoding the HST enzyme can be "codon-optimised" depending on plant host in which expression of the HST enzyme is desired. The skilled person is well aware of plant operable promoters, transcriptional terminators, chloroplast transit peptides, enhancers etc that have utility with the context of the present invention.
The HST may be a "wild type" enzyme or it may be one which has been modified in order to afford preferential kinetic properties with regard to provision of herbicide tolerant plants. In a preferred embodiment the HST is characterised in that it comprises one or more of the following polypeptide motifs:- W-(RZK)-F-L-R-P-H-T-I-R-G-T; and/or N-G-(YZF)-I-V-G-I-N-Q-I-(YZF)-D; and/or I-A-I-T-K-D-L-P; andZor Y-(RZQ)-(FZW)-(IZV)-W-N-L-F-Y.
Suitable HSTs are derived from Arabidopsis thaliana, Glycine max, Oryza sativa or Chlamydomonas reinhardtii. In an even more preferred embodiment the HST is selected from the group consisting of SEQ ID NO:1 to SEQ ID NO. 10. It should be noted that amino acid sequences provided in SEQ ID NOS :1 to 10 are examples of HST amino acid sequences that include a region encoding a chloroplast transit peptide. SEQ ID NOS 11-20 correspond to DNA sequences encoding the HSTs depicted as SEQ ID NO. 1-10 while SEQ ID NOS 21-24 are examples of DNA sequences encoding truncated mature HST sequences without the transit peptide region.
Amino acid sequences provided in SEQ ID NOS 25-28 are examples of HPPD amino acid sequences and SEQ ID NOS 29-32 are examples of DNA sequences encoding them. HPPDs suitable for providing tolerance to HPPD-inhibiting herbicides are well known to the skilled person- e.g WO 02/46387. SEQ ID No 33 provides the DNA sequence of the TMV translational enhancer and SEQ ID No 34 provides the DNA sequence of the TMV translational enhancer fused 5' to the DNA sequence encoding Arabidopsis HST.
It should be further understood that the crop plant used in said method may further comprise a further heterologous polynucleotide encoding a further herbicide tolerance enzyme. Examples of further herbicide tolerance enzymes include, for example, herbicide tolerance enzymes selected from the group consisting of, 5- enolpymvylshikimate-3 -phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPGO), Phytoene desaturase (PD), dicamba degrading enzymes (e.g WO 02/068607), and aryloxy herbicide degrading enzymes as taught in WO2007/053482 & WO2005/107437.
The pesticide composition used in the aforementioned methods may further comprise one or more additional pesticides - in particular 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 herein. In a preferred embodiment the one or more additional herbicides are selected from the group consisting of glyphosate (including agrochemically acceptable salts thereof); glufosinate (including agrochemically acceptable salts thereof); chloroacetanilides e.g alachlor, acetochlor, metolachlor, S- metholachlor; photo system II inhibitors e.g triazines such as ametryn, atrazine, cyanazine and terbuthylazine, triazinones such as hexazinone and metribuzin, ureas such as chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; ALS -inhibitors e.g sulfonyl ureas such as amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosul&ron, primisulfuron, prosulfuron, rimsulfuron, triasulfuron, trifloxysulfuron and tritosulfuron; diphenyl ethers e.g aciflurofen and fomesafen.
The present invention further provides a recombinant polynucleotide which comprises a region which encodes an HST-enzyme operably linked to a plant operable promoter, wherein the region which encodes the HST-enzyme does not include the polynucleotide sequence depicted in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 14 or SEQ ID NO. 15. In a preferred embodiment the HST-enzyme is selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.
The present invention still further provides a recombinant polynucleotide comprising (i) a region which encodes a HST enzyme operably linked to a plant operable promoter and (ii) at least one additional heterologous polynucleotide, which comprises a region which encodes an additional herbicide tolerance enzyme, operably linked to a plant operable promoter. The additional herbicide tolerance enzyme is, for example, selected from the group consisting of hydroxyphenyl pyruvate dioxygenase (HPPD), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPGO), Phytoene desaturase (PD) and dicamba degrading enzymes as taught in WO 02/068607.
Preferably the recombinant polynucleotide comprises (i) a region which encodes a HST operably linked to a plant operable promoter and (ii) a region which encodes an HPPD operably linked to a plant operable promoter. It is also possible for the recombinant polynucleotide to comprise at least two, three, or more additional regions each encoding a herbicide tolerance enzyme for example as defined previously. Thus, in another preferred embodiment the recombinant polynucleotide comprises (i) a region which encodes a HST enzyme, (ii) a region which encodes a HPPD enzyme and (iii) a region which encodes a glyphosate tolerance enzyme.
The present invention further provides a vector comprising a recombinant polynucleotide according to the present invention.
The present invention further relates to transformed plants over expressing an HST enzyme which exhibit substantial resistance or substantial tolerance to HST- inhibiting herbicides and/or HPPD-inhibiting herbicides when compared with non transgenic like plants. It should also be appreciated that the transformed plants of the present invention typically exhibit enhanced stress tolerance including heat and drought tolerance.
Thus, the present invention further provides a plant cell which exhibits substantial resistance or substantial tolerance to HST-inhibiting herbicides and/or
HPPD-inhibiting herbicides when compared with non transgenic like plant cell - said plant cell comprising the recombinant polynucleotide of the present invention as herein described. It should be appreciated that the region encoding the HST and any region encoding one or more additional herbicide tolerance enzymes may be provided on the same ("linked") or indeed separate transforming recombinant polynucleotide molecules.
The plant cell may further comprise further transgenic traits, for example heterologous polynucleotides providing resistance to insects, fungi and/or nematodes.
The present invention further provides morphologically normal fertile HST- inhibitor tolerant plants, plant cells, tissues and seeds which comprise a plant cell according to the present invention.
Plants or plant cells transformed include but are not limited to, field crops, fruits and vegetables such as canola, sunflower, tobacco, sugar beet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, mangelworzel, potato, carrot, lettuce, cabbage, onion, etc. Particularly preferred genetically modified plants are soya spp, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses, flax and oilseed rape, and nut producing plants insofar as they are not already specifically mentioned . In a particularly preferred embodiment of the method the said plant is a dicot, preferably selected from the group consisting of canola, sunflower, tobacco, sugar beet, soybean, cotton, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, and is particularly preferably soybean. In further preferred embodiments the said plant is maize or rice. Preferably the plant of the invention is soybean, rice or maize. The invention also includes the progeny of the plant of the preceding sentence, and the seeds or other propagating material of such plants and progeny.
In a particularly preferred aspect, the recombinant polynucleotide of the present invention is used to protect soybean crops from the herbicidal injury of HPPD inhibitor herbicides of the classes of HPPD chemistry selected from the group consisting of the compounds of formula Ia or Ig. In a further embodiment the HPPD inhibitor herbicide is selected from sulcotrione, mesotrione, tembotrione and compounds of formula Ia where X is nitrogen and R4 is CF3, CF2H or CFH2 and/or where Ri and R2 together form an ethylene bridge.
The present invention still further provides a method of providing a transgenic plant which is tolerant to HST-inhibiting and/or HPPD-inhibiting herbicides which comprises transformation of plant material with a recombinant polynucleotide(s) which comprises a region which encodes an HST enzyme, selection of the transformed plant material using an HST-inhibiting herbicide and/or HPPD-inhibiting herbicide, and regeneration of that material into a morphological normal fertile plant. In a preferred embodiment the transformed plant material is selected using a HST- inhibiting herbicide alone or in combination with a HPPD-inhibiting herbicide. The present invention further relates to the use of polynucleotide which comprises a region which encodes an HST enzyme as a selectable marker in plant transformation and to the use of a polynucleotide comprising a region which encodes an HST enzyme in the production of plants which are tolerant to herbicides which act wholly or in part by inhibiting HST.
The present invention still further relates to the use of HST inhibitors as selection agents in plant transformation and to the use of a recombinant HST enzyme in in vitro screening of potential herbicides.
The present invention still further provides a herbicidal composition, preferably a synergistic herbicide composition, comprising an HPPD-inhibiting herbicide (as defined herein) and a HST-inhibiting herbicide (as defined herein). The ratio of the HPPD-inhibiting herbicide to the HST-inhibiting herbicide in the composition is any suitable ratio - typically from 100:1 to 1:100, preferably from 1:10 to 1 :100, even more preferably from 1 :1 to 1 :20. The skilled person will recognise that the optimal ratio will depend on the relative potencies and spectrum of the two herbicides which can be derived as a matter of routine experimental optimisation.
The herbicidal composition may further comprise one or more additional pesticidal ingredient(s). The additional pesticides may include, for example, herbicides, fungicides or insecticides (such as thiomethoxam) - however herbicides are preferred. Thus, the additional herbicide is preferably selected from the group consisting of glyphosate (including agrochemically acceptable salts thereof); glufosinate (including agrochemically acceptable salts thereof); chloroacetanilides e.g alachlor, acetochlor, metolachlor, S-metholachlor; photo system II (PS-II) inhibitors e.g triazines such as ametryn, atrazine, cyanazine and terbuthylazine, triazinones such as hexazinone and metribuzin, and ureas such as chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; ALS -inhibitors e.g sulfonyl ureas such as amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, primisulfuron, prosulfuron, rimsulfuron, triasulfuron, trifloxysulfuiOn and tritosulfuron; diphenyl ethers e.g aciflurofen and fomesafen. PS-II herbicides are a particularly preferred as such mixtures exhibit particularly good efficacy.
Thus, the present invention still further provides a method of selectively controlling weeds at a locus comprising crop plants and weeds comprising applying to the locus a weed controlling amount of a synergistic herbicidal composition as previously defined. In a further embodiment of the current invention HPPD and HST inhibiting herbicides are sprayed sequentially rather than at the same time as a mixture. Thus for example, in a programme of weed control of the current invention, the HST herbicide can be advantageously applied over a crop locus to which an HPPD herbicide has already been previously applied. Normally this would be in the same season but, especially in the case of more persistent HPPD herbicides, there would also be an advantage in using the HST herbicide in the following season. In addition HST inhibiting herbicides are advantageously used as part of a programme of weed control wherein an HPPD inhibiting herbicide is applied earlier in the season or even in the preceding season.
The HPPD-inhibiting herbicide may be applied to the locus at any suitable rate — for example from 1 to 1000 g/ha, more preferably from 2 to 200 g/ha. Likewise, the HST-inhibiting herbicide may be applied at any suitable rate — for example from 10 to 2000 g/ha, more preferably from 50 to 400 g/ha.
In another embodiment, the HPPD-inhibiting herbicide is applied to the locus at a rate which is sub-lethal to the weeds were the HPPD-inhibiting herbicide to be applied in the absence of other herbicides. The actual sub-lethal rate will depend on weed species present and the actual HPPD inhibitor - but will typically be less than 50 g/ha - more preferably less than 10 g/ha. Thus, the present invention further provides the use of a sub-lethal application of an HPPD-mhibiting herbicide to increase the weed controlling efficacy of an HST-inhibiting herbicide. The invention will be further apparent from the following non-limiting examples and sequence listings. SEQUENCE LISTING
SEQ ID NO. 1 ARABIDOPSIS HST AMINO ACID SEQUENCE melsisqsprvrfsslaprflaashhhrpsvhlagkfislprdvrftslstsrmrsk fvstnyrkisiracsqvgaaesddpvldriarfqnacwrflrphtirgtalgstalv tralienthlikwslvlkalsgllalicgngyivginqiydigidkvnkpylpiaag dlsvqsawllviffaiagllvvgfnfgpfitslyslglflgtiysvpplrmkrfpva afliiatvrgfllnfgvyhatraalglpfqwsapvafitsfvtlfalviaitkdlpd vegdrkfqistlatklgvrniaflgsglllvnyvsaislafympqvfrgslmipahv ilasglifqtwvlekanytkeaisgyyrfiwnlfyaeyllfpf1
SEQ ID NO.2 RICE HST AMINO ACID SEQUENCE maslaspplpcraaatasrsgrpaprllgpppppaspllssasarfprapcnaarws rrdavrvcsqagaagpaplsktlsdlkdscwrfIrphtirgtalgsmslvaralien pqlinwwlvfkafyglvalicgngyivginqiydiridkvnkpylpiaagdlsvqta wllvvlfaaagfsivvtnfgpfitslyclglflgtiysvppfrlkrypvaafliiat vrgfllnfgvyyatraalgltfqwsspvafitcfvtlfalviaitkdlpdvegdrky qistlatklgvrniaflgsgllianyvaaiavaflmpqafrrtvmvpvhaalavgii fqtwvleqakytkdaisqyyrfiwnlfyaeyiffpli SEQ ID NO.3 RICE HST VARIANT AMINO ACID SEQUENCE maslaspplpcraaatasrsgrpaprllgpppppaspllssasarfprapcnaarws rrdavrvcsqagaagpaplsktlsdlkdscwrflrphtirgtalgsialvaralien pqlinwwlvfkafyglvalicgngyivginqiydiridkvnkpylpiaagdlsvqta wllvvlfaaagfsivvtnfgpfitslyclglflgtiysvppfrlkrypvaafliiat vrgfllnfgvyyatraalgltfqwsspvafitcfvtlfalviaitkdlpdvegdrky qistlatklgvrniaflgsgllianyvaaiavaflmpqafrrtvmvpvhaalavgii fqtwvleqakytkdaisqyyrfiwnlfyaeyiffpli
SEQ ID NO.4 SOYA HST AMINO ACID SEQUENCE melslsptshrvpstiptlnsaklsstkatksqqplflgfskhfnsiglhhhsyrcc snavperpqrpssiractgvgasgsdrplaerlldlkdacwrflrphtirgtalgsf alvaralientnlikwslffkafcglfalicgngyivginqiydisidkvnkpylpi aagdlsvqsawflviffaaaglsiaglnfgpfifslytlglflgtiysvpplrmkrf pvaafliiatvrgfllnfgvyyatraslglafewsspvvfittfvtffalviaitkd lpdvegdrkyqistfatklgvrniaflgsgillvnyivsvlaaiympqafrrwllip ahtifaisliyqarileqanytkdaisgfyrfiwnlfyaeyaifpfi
SEQ ID NO. 5 CHLAMYDOMONAS HST AMINO ACID SEQUENCE mdlcsstgrgaclspastsrpcpapvhlrgrrlafspaqpagrrhlpvlssaavpap lpnggndesfaqklanfpnafwkflrphtirgtilgttavtakvlmenpgcidwall pkallglvallcgngyivginqiydvdidvvnkpflpvasgelspalawglclslaa agagivaanfgnlitslytfglflgtvysvpplrlkqyavpafmiiatvrgfllnfg vysatraalglpfewspavsfitvfvtlfatviaitkdlpdvegdqannistfatrm gvrnvallaigllmanylgaialaltystafnvplmagahailaatlalrtlklhaa sysreavasfyrwiwnlfyaeyallpf1
SEQ ID NO.6 NICOTINIA HST AMINO ACID SEQUENCE melacsscsslrfssvlthqdtaasryrklpptspsckaanfvlkssknlsssaglh igytnfsktvsyrkyrhisiracsqvgtagsepvldklsqfkdafwrflrphtirgt algslslvtralienpnlirwslamkafsglialicgngyivginqiydigidkvnk pylpiaagdlsvqsawflvllfamagllivginfgpfitslyclglflgtiysvppf rmkrfavvafliiatvrgfllnygvyyattaalglsfqwsspvafittfvtlfalvi aitkdlpdvegdrkfqistlatklgvrniaflgsglllanyigavvaaiympqafrs slmipvhailalclvfqawllekanytkeaisayyqfiwnffyaeylifpfi
SEQ ID NO.7 AQUILEGIA HST AMINO ACID SEQUENCE lcfsspsisipphcsttthyrkipinstfkstnflskasnnlttfgfsrnkkysrsi 1srksrhfsiwassqvgaagsddpllkkipdfkdavwrflrphtirgtalgsialvs ralienthlikwsllfkaicgvfalmcgngyivginqiydigidkvnkpylpiaagd IsvqsawslvtffavagvcivafnfgpfitslyclglfIgtiysvpplrmkrypvaa fliiatvrgfllnfgvyhatraalgltfewsypvafittfvtmfalviaitkdlpdv egdrkfqistlatklgvrniallgtglllanyigaivaaiylpqafrrnlmipahti lalglvfqawaleqakyskeaildfyrfvwnlfyseyflfpfi
SEQ ID NO.8 BRASSICA NAPUS HST AMINO ACID SEQUENCE melsishspclrfssssprflaasshhyrpsvhlagkllsrskdadltslssscmrs kfvstnyrkisirassqvgaagsdpvldrlarfqnacwrflrphtirgtalgstalv tralienthlikwslvlkalsgllalicgngyivginqiydigidkvnkpylpiaag dlsvqsawllviffaiagltvvgfnfgpfitclyslglflgtiysvppfrmkrfpva afliiatvrgfllnfgvyhatraalglsfqwsapvafitsfvtlfalviaitkdlpd vegdrkfqistlatklgvrniafIgsglllvnyisaislafympqvfrgslmipahm ilasclvfqtwvlekanytkeaiagyyrfiwnlfyaeyllfpff
SEQ ID NO.9 VITIS VINIFERA HST AMINO ACID SEQUENCE mkvdavqastqvgaagsdpplnkfsvfkdacwrflrphtirgtalgstalvaralie npnlikwsllfkafsgllalicgngyivginqiydisidkvnkpylpiaagdlsvqs awflvlffavagvlivgsnfgsfitslyclglvlgtiysvppfrmkrfpvaafliia tvrgfllnfgvyyatraalglpfmwsapvvfittfvtIfalviaitkdlpdvegdrk yqistlatklgvrniaflgsglllvnyigsilaaiympqafrlslmipahailaagl ifqarvleqanytkeaisdfyrfiwnlfyveyiifpfi
SEQ ID NO.10 PHYSCOMITRELLA PATENS HST SEQUENCE mgltaivvdvaqassssvalsqgrgatrrlpgglalgdafkglrkreyaqglqcrvr reggcasearvwkvrcssdsagslggdlpasqpqqsevsgirdpaaasaasfaplpq rialfydafwrflrphtirgtflgtsalvtrallenptlinwallpkalrgllallc gngfivginqifdsgidkvnkpfIpiaagdlsvpaawalvgglaalgvglvatnfgp littlytfglflgtiysvpplrlkqypvpafmiiatvrgfllnfgvyyatraalgls yewspsvmfitifvtIfatviaitkdlpdiegdkkfnistfatnlgvrkisflgagl llvnyigaivaafylpqafktkirαvtghavlglsliyqtwlldtakyskeaisnfyr fiwnlfyseyalfpfi SEQ ID NO.11 ARABIDOPSIS HST (DNA)
atggagctctcgatctcacaatcaccgcgtgttcggttctcgtctctggcgcctcgt ttcttagcagcttctcatcatcatcgtccttctgtgcatttagctgggaagtttata agcctccctcgagatgttcgcttcacgagcttatcaacttcaagaatgcggtccaaa tttgtttcaaccaattatagaaaaatctcaatccgggcatgttctcaggttggtgct gctgagtctgatgatccagtgctggatagaattgcccggttccaaaatgcttgctgg agatttcttagaccccatacaatccgcggaacagctttaggatccactgccttggtg acaagagctttgatagagaacactcatttgatcaaatggagtcttgtactaaaggca ctttcaggtcttcttgctcttatttgtgggaatggttatatagtcggcatcaatcag atctacgacattggaatcgacaaagtgaacaaaccatacttgccaatagcagcagga gatctatcagtgcagtctgcttggttgttagtgatattttttgcgatagcagggctt ttagttgtcggatttaactttggtccattcattacaagcctatactctcttggcctt tttctgggaaccatctattctgttccacccctcagaatgaaaagattcccagttgca gcatttcttattattgccacggtacgaggtttccttcttaactttggtgtgtaccat gctacaagagctgctcttggacttccatttcagtggagtgcacctgtggcgttcatc acatcttttgtgacactgtttgcactggtcattgctattacaaaggaccttcctgat gttgaaggagatcgaaagttccaaatatcaaccctggcaacaaaacttggagtgaga aacattgcattcctcggttctggacttctgctagtaaattatgtttcagccatatca ctagctttctacatgcctcaggtttttagaggtagcttgatgattcctgcacatgtg atcttggcttcaggcttaattttccagacatgggtactagaaaaagcaaactacacc aaggaagctatctcaggatattatcggtttatatggaatctcttctacgcagagtat ctgttattccccttcctctag
SEQ ID NO.12 RICE HST (DNA) atggcttccctcgcctcccctcctctcccctgccgcgccgccgccaccgccagccgc agcgggcgtcctgctccgcgcctcctcggccctccgccgccgcccgcttcccctctc ctctcctccgcttcggcgcgcttcccgcgtgccccctgcaacgccgcacgctggagc cggcgcgacgccgtgcgggtttgctctcaagctggtgcagctggaccagccccatta tcgaagacattgtcagacctcaaggattcctgctggagatttttacggccacataca attcgaggaactgccttgggatccatgtcattagttgctagagctttgatagagaac ccccaactgataaattggtggttggtattcaaagcgttctatgggctcgtggcgtta atctgtggcaatggttacatcgttgggatcaatcagatctatgacattagaatcgat aaggtaaacaagccatatttaccaattgctgccggtgatctctcagttcagacagca tggttattggtggtattatttgcagctgcgggattttcaattgttgtgacaaacttt ggacctttcattacctctctatattgccttggtctatttcttggcaccatatactct gttcctccattcagacttaagagatatcctgttgctgcttttcttatcattgcaacg gtccgtggttttcttctcaactttggtgtgtactatgctactagagcagcactgggt cttacattccaatggagctcgcctgttgctttcattacatgcttcgtgactttattt gctttggtcattgctataaccaaagatctcccagatgttgaaggggatcggaagtat caaatatcaactttggcgacaaagctcggtgtcagaaacattgcatttcttggctct ggtttattgatagcaaattatgttgctgctattgctgtagcttttctcatgcctcag gctttcaggcgcactgtaatggtgcctgtgcatgctgcccttgccgttggtataatt ttccagacatgggttctggagcaagcaaaatatactaaggatgctatttcacagtac taccggttcatttggaatctcttctatgctgaatacatcttcttcccgttgata
SEQ ID NO.13 RICE VARIANT HST (DNA) atggcttccctcgcctcccctcctctcccctgccgcgccgccgccaccgccagccgc agcgggcgtcctgctccgcgcctcctcggccctccgccgccgcccgcttcccctctc ctctcctccgcttcggcgcgcttcccgcgtgccccctgcaacgccgcacgctggagc cggcgcgacgccgtgcgggtttgctctcaagctggtgcagctggaccagccccatta. tcgaagacattgtcagacctcaaggattcctgctggagatttttacggccacataca attcgaggaactgccttgggatccatagcattagttgctagagctttgatagagaac ccccaactgataaattggtggttggtattcaaagcgttctatgggctcgtggcgtta atctgtggcaatggttacatcgttgggatcaatcagatctatgacattagaatcgat aaggtaaacaagccatatttaccaattgctgccggtgatctctcagttcagacagca tggttattggtggtattatttgcagctgcgggattttcaattgttgtgacaaacttt ggacctttcattacctctctatattgccttggtctatttcttggcaccatatactct gttcctccattcagacttaagagatatcctgttgctgcttttcttatcattgcaacg gtccgtggttttcttctcaactttggtgtgtactatgctactagagcagcactgggt cttacattccaatggagctcgcctgttgctttcattacatgcttcgtgactttattt gctttggtcattgctataaccaaagatctcccagatgttgaaggggatcggaagtat caaatatcaactttggcgacaaagctcggtgtcagaaacattgcatttcttggctct ggtttattgatagcaaattatgttgctgctattgctgtagcttttctcatgcctcag gctttcaggcgcactgtaatggtgcctgtgcatgctgcccttgccgttggtataatt ttccagacatgggttctggagcaagcaaaatatactaaggatgctatttcacagtac taccggttcatttggaatctcttctatgctgaatacatcttcttcccgttgatatag SEQ ID NO.14 SOYA HST (DNA) tctgctaaattatcttctactaaagctactaaatctcaacaacctttatttttagga ttttctaaacattttaattctattggattacatcatcattcttatagatgttgttct aatgctgtacctgaaagacctcaaagaccttcttctattagagcttgtactggagta ggagcttctggatctgatagacctttagctgaaagattattagatttaaaagatgct tgttggagatttttaagacctcatactattagaggaactgctttaggatcttttgct ttagtagctagagctttaattgaaaatactaatttaattaaatggtctttatttttt aaagctttttgtggattatttgctttaatttgtggaaatggatatattgtaggaatt aatcaaatttatgatatttctattgataaagtaaataaaccttatttacctattgct gctggagatttatctgtacaatctgcttggtttttagtaattttttttgctgctgct ggattatctattgctggattaaattttggaccttttattttttctttatatacttta ggattatttttaggaactatttattctgtacctcctttaagaatgaaaagatttcct gtagctgcttttttaattattgctactgtaagaggatttttattaaattttggagta tattatgctactagagcttctttaggattagcttttgaatggtcttctcctgtagta tttattactacttttgtaactttttttgctttagtaattgctattactaaagattta cctgatgtagaaggagatagaaaatatcaaatttctacttttgctactaaattagga gtaagaaatattgcttttttaggatctggaattttattagtaaattatattgtatct gtattagctgctatttatatgcctcaagcttttagaagatggttattaattcctgct catactatttttgctatttctttaatttatcaagctagaattttagaacaagctaat tatactaaagatgctatttctggattttatagatttatttggaatttattttatgct gaatatgctatttttccttttatt
SEQ ID NO.15 CHLAMYDOMONAS HST (DNA) atggacctttgcagctcaactggaagaggagcatgcctttcgccggcatccacgtcg cggccgtgcccagcaccagtgcatttgcgcggccgacgcctggctttctctccggct cagcctgctggacggcgccacttgccggtgctctcatctgcagcggtccccgctccc ctcccaaatggtggaaacgacgagagcttcgcacaaaaactggctaactttccaaac gccttctggaagttcctgcggccacacaccatccgggggactatcctgggcaccaca gctgtgaccgccaaggtccttatggagaaccccggctgcatagactgggcactgctg ccgaaggcgctgctcggcctggtggcgctgctgtgcggcaacggctacattgtgggc atcaaccaaatctacgacgtcgacattgacgtggtcaacaagccattcctccccgtg gcgtcgggcgagctgtcgccggcgctggcgtggggcctgtgtctgtcgctggcggct gcgggcgcgggcatcgtagccgccaacttcggcaacctcatcaccagcctctacacc tttggcctcttcctgggcaccgtgtacagtgtgcctcccctgcgcctgaagcagtac gcggtgccggccttcatgatcatcgccacggtgcgcggcttcctgetcaacttcggc gtgtacagcgccacgcgggcggcactgggactgcccttcgagtggagcccggccgtc agcttcatcacggtgtttgtgacgctgtttgccactgtgatcgccatcaccaaggac ctgccggacgtggagggcgaccaggccaacaacatctccaccttcgccacgcgcatg ggcgtgcgcaacgtggcactgctggccatcggccttctcatggccaactacctgggt gccatcgcgctggcactcacctactccaccgccttcaacgtgccgctcatggcgggc gcgcacgccatcctggccgccacgctggcgctgcgcacgctcaagctgcacgccgcc agctacagccgggaggcggtggcgtccttctaccgctggatctggaacctgttctac gccgagtacgcgctgctgccgttcctgtag
SEQ ID NO.16 NICOTINIA HST DNA
atggaattagcttgttcttcttgttcttctttaagattttcttctgtattaactcat caagatactgctgcttctagatatagaaaattacctcctacttctccttcttgtaaa gctgctaattttgtattaaaatcttctaaaaatttatcttcttctgctggattacat attggatatactaatttttctaaaactgtatcttatagaaaatatagacatatttct attagagcttgttctcaagtaggaactgctggatctgaacctgtattagataaatta tctcaatttaaagatgctttttggagatttttaagacctcatactattagaggaact gctttaggatctttatctttagtaactagagctttaattgaaaatcctaatttaatt agatggtctttagctatgaaagctttttctggattaattgctttaatttgtggaaat ggatatattgtaggaattaatcaaatttatgatattggaattgataaagtaaataaa ccttatttacctattgctgctggagatttatctgtacaatctgcttggtttttagta ttattatttgctatggctggattattaattgtaggaattaattttggaccttttatt acttctttatattgtttaggattatttttaggaactatttattctgtacctcctttt agaatgaaaagatttgctgtagtagcttttttaattattgctactgtaagaggattt ttattaaattatggagtatattatgctactactgctgctttaggattatcttttcaa tggtcttctcctgtagcttttattactacttttgtaactttatttgctttagtaatt gctattactaaagatttacctgatgtagaaggagatagaaaatttcaaatttctact ttagctactaaattaggagtaagaaatattgcttttttaggatctggattattatta gctaattatattggagctgtagtagctgctatttatatgcctcaagcttttagatct tctttaatgattcctgtacatgctattttagctttatgtttagtatttcaagcttgg ttattagaaaaagctaattatactaaagaagctatttctgcttattatcaatttatt tggaattttttttatgctgaatatttaatttttccttttatt
SEQ ID NO.17 AQUILEGIA HST DNA atgttatgtttttcttctccttctatttctattcctcctcattgttctactactact cattatagaaaaattcctattaattctacttttaaatctactaattttttatctaaa gcttctaataatttaactacttttggattttctagaaataaaaaatattctagatct attttatctagaaaatctagacatttttctatttgggcttcttctcaagtaggagct gctggatctgatgatcctttattaaaaaaaattcctgattttaaagatgctgtatgg agatttttaagacctcatactattagaggaactgctttaggatctattgctttagta tctagagctttaattgaaaatactcatttaattaaatggtctttattatttaaagct atttgtggagtatttgctttaatgtgtggaaatggatatattgtaggaattaatcaa atttatgatattggaattgataaagtaaataaaccttatttacctattgctgctgga gatttatctgtacaatctgcttggtctttagtaactttttttgctgtagctggagta tgtattgtagcttttaattttggaccttttattacttctttatattgtttaggatta tttttaggaactatttattctgtacctcctttaagaatgaaaagatatcctgtagct gcttttttaattattgctactgtaagaggatttttattaaattttggagtatatcat gctactagagctgctttaggattaacttttgaatggtcttatcctgtagcttttatt actacttttgtaactatgtttgctttagtaattgctattactaaagatttacctgat gtagaaggagatagaaaatttcaaatttctactttagctactaaattaggagtaaga aatattgctttattaggaactggattattattagctaattatattggagctattgta gctgctatttatttacctcaagcttttagaagaaatttaatgattcctgctcatact attttagctttaggattagtatttcaagcttgggctttagaacaagctaaatattct aaagaagctattttagatttttatagatttgtatggaatttattttattctgaatat tttttatttccttttatt
SEQ ID NO.18 BRASSICA NAPUS HST DNA ttagctgcttcttctcatcattatagaccttctgtacatttagctggaaaattatta tctagatctaaagatgctgatttaacttctttatcttcttcttgtatgagatctaaa tttgtatctactaattatagaaaaatttctattagagcttcttctcaagtaggagct gctggatctgatcctgtattagatagattagctagatttcaaaatgcttgttggaga tttttaagacctcatactattagaggaactgctttaggatctactgctttagtaact agagctttaattgaaaatactcatttaattaaatggtctttagtattaaaagcttta tctggattattagctttaatttgtggaaatggatatattgtaggaattaatcaaatt tatgatattggaattgataaagtaaataaaccttatttacctattgctgctggagat ttatctgtacaatctgcttggttattagtaattttttttgctattgctggattaact gtagtaggatttaattttggaccttttattacttgtttatattctttaggattattt ttaggaactatttattctgtacctccttttagaatgaaaagatttcctgtagctgct tttttaattattgctactgtaagaggatttttattaaattttggagtatatcatgct actagagctgctttaggattatcttttcaatggtctgctcctgtagcttttattact tcttttgtaactttatttgctttagtaattgctattactaaagatttacctgatgta gaaggagatagaaaatttcaaatttctactttagctactaaattaggagtaagaaat attgcttttttaggatctggattattattagtaaattatatttctgctatttcttta gctttttatatgcctcaagtatttagaggatctttaatgattcctgctcatatgatt ttagcttcttgtttagtatttcaaacttgggtattagaaaaagctaattatactaaa gaagctattgctggatattatagatttatttggaatttattttatgctgaatattta ttatttccttttttt
SEQ ID NO.19 VITIS VINIFERA HST DNA atgaaagtagatgctgtacaagcttctactcaagtaggagctgctggatctgatcct cctttaaataaattttctgtatttaaagatgcttgttggagatttttaagacctcat actattagaggaactgctttaggatctactgctttagtagctagagctttaattgaa aatcctaatttaattaaatggtctttattatttaaagctttttctggattattagct ttaatttgtggaaatggatatattgtaggaattaatcaaatttatgatatttctatt gataaagtaaataaaccttatttacctattgctgctggagatttatctgtacaatct gcttggtttttagtattattttttgctgtagctggagtattaattgtaggatctaat tttggatcttttattacttctttatattgtttaggattagtattaggaactatttat tctgtacctccttttagaatgaaaagatttcctgtagctgcttttttaattattgct actgtaagaggatttttattaaattttggagtatattatgctactagagctgcttta ggattaccttttatgtggtctgctcctgtagtatttattactacttttgtaacttta tttgctttagtaattgctattactaaagatttacctgatgtagaaggagatagaaaa tatcaaatttctactttagctactaaattaggagtaagaaatattgcttttttagga tctggattattattagtaaattatattggatctattttagctgctatttatatgcct caagcttttagattatctttaatgattcctgctcatgctattttagctgctggatta atttttcaagctagagtattagaacaagctaattatactaaagaagctatttctgat ttttatagatttatttggaatttattttatgtagaatatattatttttccttttatt
SEQ ID NO.20 PHYSCOMITRELLA PATENS HST DNA
atgggattaactgctattgtagtagatgtagctcaagcttcttcttcttctgtagct ttatctcaaggaagaggagctactagaagattacctggaggattagctttaggagat gcttttaaaggattaagaaaaagagaatatgctcaaggattacaatgtagagtaaga agagaaggaggatgtgcttctgaagctagagtatggaaagtaagatgttcttctgat tctgctggatctttaggaggagatttacctgcttctcaacctcaacaatctgaagta tctggaattagagatcctgctgctgcttctgctgcttcttttgctcctttacctcaa agaattgctttattttatgatgctttttggagatttttaagacctcatactattaga ggaacttttttaggaacttctgctttagtaactagagctttattagaaaatcctact ttaattaattgggctttattacctaaagctttaagaggattattagctttattatgt ggaaatggatttattgtaggaattaatcaaatttttgattctggaattgataaagta aataaaccttttttacctattgctgctggagatttatctgtacctgctgcttgggct ttagtaggaggattagctgctttaggagtaggattagtagctactaattttggacct ttaattactactttatatacttttggattatttttaggaactatttattctgtacct cctttaagattaaaacaatatcctgtacctgcttttatgattattgctactgtaaga ggatttttattaaattttggagtatattatgctactagagctgctttaggattatct tatgaatggtctccttctgtaatgtttattactatttttgtaactttatttgctact gtaattgctattactaaagatttacctgatattgaaggagataaaaaatttaatatt tctacttttgctactaatttaggagtaagaaaaatttcttttttaggagctggatta ttattagtaaattatattggagctattgtagctgctttttatttacctcaagctttt aaaactaaaattatggtaactggacatgctgtattaggattatctttaatttatcaa acttggttattagatactgctaaatattctaaagaagctatttctaatttttataga tttatttggaatttattttattctgaatatgctttatttccttttatt
SEQIDNO.21 DNA ENCODING MATURE ARABIDOPIS HST agaaaaatctcaatccgggcatgttctcaggttggtgctgctgagtctgatgatcca gtgctggatagaattgcccggttccaaaatgcttgctggagatttcttagaccccat acaatccgcggaacagctttaggatccactgccttggtgacaagagctttgatagag aacactcatttgatcaaatggagtcttgtactaaaggcactttcaggtcttcttgct cttatttgtgggaatggttatatagtcggcatcaatcagatctacgacattggaatc gacaaagtgaacaaaccatacttgccaatagcagcaggagatctatcagtgcagtct gcttggttgttagtgatattttttgcgatagcagggcttttagttgtcggatttaac tttggtccattcattacaagcctatactctcttggcctttttctgggaaccatctat tctgttccacccctcagaatgaaaagattcccagttgcagcatttcttattattgcc acggtacgaggtttccttcttaactttggtgtgtaccatgctacaagagctgctctt ggacttccatttcagtggagtgcacctgtggcgttcatcacatcttttgtgacactg tttgcactggtcattgctattacaaaggaccttcctgatgttgaaggagatcgaaag ttccaaatatcaaccctggcaacaaaacttggagtgagaaacattgcattcctcggt tctggacttctgctagtaaattatgtttcagccatatcactagctttctacatgcct caggtttttagaggtagcttgatgattcctgcacatgtgatcttggcttcaggctta attttccagacatgggtactagaaaaagcaaactacaccaaggaagctatctcagga tattatcggtttatatggaatctcttctacgcagagtatctgttattccccttcctc ta
SEQ ID NO.22 DNA ENCODING MATURE RICE HST cggcgcgacgccgtgcgggtttgctctcaagctggtgcagctggaccagccccatta tcgaagacattgtcagacctcaaggattcctgctggagatttttacggccacataca attcgaggaactgccttgggatccatgtcattagttgctagagctttgatagagaac ccccaactgataaattggtggttggtattcaaagcgttctatgggctcgtggcgtta atctgtggcaatggttacatcgttgggatcaatcagatctatgacattagaatcgat aaggtaaacaagccatatttaccaattgctgccggtgatctctcagttcagacagca tggttattggtggtattatttgcagctgcgggattttcaattgttgtgacaaacttt ggacctttcattacctctctatattgccttggtctatttcttggcaccatatactct gttcctccattcagacttaagagatatcctgttgctgcttttcttatcattgcaacg gtccgtggttttcttctcaactttggtgtgtactatgctactagagcagcactgggt cttacattccaatggagctcgcctgttgctttcattacatgcttcgtgactttattt gctttggtcattgctataaccaaagatctcccagatgttgaaggggatcggaagtat caaatatcaactttggcgacaaagctcggtgtcagaaacattgcatttcttggctct ggtttattgatagcaaattatgttgctgctattgctgtagcttttctcatgcctcag gctttcaggcgcactgtaatggtgcctgtgcatgctgcccttgccgttggtataatt ttccagacatgggttctggagcaagcaaaatatactaaggatgctatttcacagtac taccggttcatttggaatctcttctatgctgaatacatcttcttcccgttgatatag
SEQ ID NO.23 DNA ENCODING MATURE RICE VARIANT HST
cggcgcgacgccgtgcgggtttgctctcaagctggtgcagctggaccagccccatta tcgaagacattgtcagacctcaaggattcctgctggagatttttacggccacataca attcgaggaactgccttgggatccatagcattagttgctagagctttgatagagaac ccccaactgataaattggtggttggtattcaaagcgttctatgggctcgtggcgtta atctgtggcaatggttacatcgttgggatcaatcagatctatgacattagaatcgat aaggtaaacaagccatatttaccaattgctgccggtgatctctcagttcagacagca tggttattggtggtattatttgcagctgcgggattttcaattgttgtgacaaacttt ggacctttcattacctctctatattgccttggtctatttcttggcaccatatactct gttcctccattcagacttaagagatatcctgttgctgcttttcttatcattgcaacg gtccgtggttttcttctcaactttggtgtgtactatgctactagagcagcactgggt cttacattccaatggagctcgcctgttgctttcattacatgcttcgtgactttattt gctttggtcattgctataaccaaagatctcccagatgttgaaggggatcggaagtat caaatatcaactttggcgacaaagctcggtgtcagaaacattgcatttcttggctct ggtttattgatagcaaattatgttgctgctattgctgtagcttttctcatgcctcag gctttcaggcgcactgtaatggtgcctgtgcatgctgcccttgccgttggtataatt ttccagacatgggttctggagcaagcaaaatatactaaggatgctatttcacagtac taccggttcatttggaatctcttctatgctgaatacatcttcttcccgttgatatag
SEQ ID NO. 24 DNA Encoding Insect Cell Codon Optimized Mature Arabidopsis HST gggatccctcgtgcttgctcccaggtcggcgctgctgagtccgacgaccccgtgctg gaccgtatcgctcgtttccagaacgcttgctggcgtttcctgcgtccccacaccatc cgtggcaccgctctgggttcca'ccgccctggtgacccgtgctctgatcgagaacacc cacctgatcaagtggtccctggtgctgaaggctctgtccggtctgctggctctgatc tgcggtaacggttacatcgtgggtatcaaccagatctacgacatcggtatcgacaag gtgaacaagccctacctgcccatcgctgctggtgacctgtccgtgcagtccgcttgg ctgctggtcatcttcttcgctatcgctggtctgctggtcgtgggtttcaacttcggt cccttcatcacttccctgtactccctgggcctgttcctgggcaccatctactccgtg ccccccctgcgtatgaagcgtttccccgtggctgctttcctgatcatcgctaccgtg cgtggtttcctgctgaacttcggtgtctaccacgctacccgtgctgctctgggtctg cccttccagtggtccgctcccgtggctttcatcaccagcttcgtgaccctgttcgct ctggtgatcgctatcaccaaggacctgcccgacgtggagggtgaccgtaagttccag atctccaccctggctaccaagctgggtgtgcgtaacatcgctttcctcggttccggc ctgctgctcgtgaactacgtgtccgctatctccctggctttctacatgccccaggtg ttccgtggttccctgatgatccccgctcacgtgatcctggcttccggtctgatcttc cagacctgggtgctcgagaaggctaactacaccaaggaagctatctccggttactac cgcttcatctggaacctgttctacgctgagtacctgctgttccccttcctgtaa
SEQ ID No. 25 HPPD a/a sequence from Pseudomonas fluorescem strain 87-79 madqyenpmglmgfefiefasptpgtlepifeimgftkvathrsknvhlyrqgeinl ilnnqpdslasyfaaehgpsvcgmafrvkdsqqaynralelgaqpihietgpmel.nl paikgiggaplylidrfgegssiydidfvylegvdrnpvgaglkvidhlthnvyrgr maywanfyeklfnfrearyfdikgeytgltskamsapdgmiriplneesskgagqie eflmqfngegiqhvafltedlvktwdalkkigmrfrαtappdtyyemlegrlpnhgep vdqlqargilldgssiegdkrlllqifsetlmgpvffefiqrkgddgfgegnfkalf esierdqvrrgvlttd
SEQ ID NO.26 HPPD a/a sequence from Avena sativa mpptpatatgaaaaavtpehaarsfprvvrvnprsdrfpvlsfhhvelwcadaasaa grfsfalgaplaarsdlstgnsahaslllrsgalafIftapyapppqeaataaatas ipsfsadaartfaaahglavrsvgvrvadaaeafrvsvaggarpafapadlghgfgl aevelygdvvlrfvsypdetdlpflpgfervsspgavdygltrfdhvvgnvpemapv idymkgflgfhefaeftaedvgttesglnsvvlannseavllplnepvhgtkrrsqi qtyleyhggpgvqhialasndvlrtlremrartpmggfefmappqakyyegvrriag dvlseeqikecqelgvlvdrddqgvllqiftkpvgdrptfflemiqrigcmekdevg qeyqkggcggfgkgnfselfksiedyekslevkqsvvaqks
SEQ ID NO.27 HPPD a/a sequence from wheat mpptpttpaatgaaavtpeharprrmvrfnprsdrfhtlafhhvefwcadaasaagr fafalgaplaarsdlstgnsvhasqllrsgnlaflftapyangcdaataslpsfsad aarqfsadhglavrsialrvadaaeafrasvdggarpafspvdlgrgfgfaevelyg dvvlrfvshpdgrdvpflpgfegvsnpdavdygitrfdhvvgnvpelapaaayvagf tgfhefaefttedvgtaesglnsmvlannsegvllplnepvhgtkrrsqiqtflehh ggsgvqhiavassdvlrtlremrarsamggfdflppplpkyyegvrriagdvlseaq ikecqelgvlvdrddqgvllqiftkpvgdrptlflemiqrigcmekdergeeyqkgg cggfgkgnfselfksiedyeksleakqsaavqgs
SEQ ID NO. 28 HPPD a/a sequence from Shewanella collwelliana
Maseqnplgllgieftefatpdldfmhkvfidfgfsklkkhkqkdivyykqndinf1 lnnekqgfsaqfakthgpaissmgwrvedanfafegavargakpaadevkdlpypai ygigdsliyfidtfgddnniytsdfealdepiitqekgfievdhltnnvhkgtmeyw snfykdifgftevryfdikgsqtalisyalrspdgsfcipinegkgddrnqideylk eydgpgvqhlafrsrdivasldamegssiqtldiipeyydtifeklpqvtedrdrik hhqilvdgdedgyllqiftknlfgpifieiiqrknnlgfgegnfkalfesierdqvr rgvl
SEQ ID NO. 29 HPPD DNA sequence from P seudomonas fluorescein strain 87-79 atggccgaccaatacgaaaacccaatgggcctgatgggctttgaatttattgaattc gcatcgccgactccgggcaccctggagccgatcttcgagatcatgggcttcaccaaa gtcgcgacccaccgctccaagaatgtgcacctgtaccgccagggcgagatcaacctg atcctcaacaaccagcccgacagcctggcctcgtacttcgccgccgaacacggccct tcggtgtgcggcatggcgttccgggtcaaagactcgcagcaggcttacaaccgcgcg ttggaactgggcgcccagccgattcatatcgaaaccggcccgatggaactcaacctg ccggccatcaagggcatcggcggtgcgccgctgtacctgatcgaccgcttcggtgaa ggcagctcgatatatgacatcgacttcgtgtacctcgaaggtgtcgaccgcaacccg gtaggcgcgggcctcaaggtcatcgaccacctgacccacaacgtgtatcgcggccgc atggcctactgggccaacttctacgagaaactgttcaacttccgtgaagcacgctac ttcgatatcaagggcgaatacaccggccttacgtccaaggccatgagtgccccggac ggcatgatccgcatcccgctgaacgaggaatcgtccaagggcgccggccagatcgaa gagttcctgatgcagttcaacggcgagggcatccagcacgtggcgttcctcaccgaa gacctggtcaagacctgggatgcgttgaagaagatcggcatgcgcttcatgaccgcg ccgccggacacctactacgaaatgctcgaaggccgcctgccaaaccacggcgagccg gtggaccaactgcaggcgcgcggtattttgctggacggctcctcgatcgagggcgac aagcgcctgctgctgcagatcttctcggaaaccctgatgggcccggtgttcttcgaa ttcatccagcgcaaaggcgacgatgggtttggcgagggcaacttcaaggcgctgttc gagtcgatcgagcgcgaccaggtacgtcgcggtgtactgaccaccgac
SEQ ID NO.30 HPPD DNA sequence from Avena sativa atgccgcccacccccgccaccgccaccggcgccgccgcggccgccgtgactccagag cacgcggcccggagctttccccgagtggtccgcgtcaacccgcgcagcgaccgcttc cccgtgctctccttccaccacgtcgagctctggtgcgccgacgccgcctcagcggcc ggacgcttctccttcgcgctcggcgcgccgctcgccgcccggtccgacctctccacg gggaactccgcgcacgcctccctcctgctccgctcgggcgccctcgccttcctcttc acggcgccctacgcgccgccgccgcaggaggccgccacggccgcagccaccgcctcc atcccctccttctccgccgacgccgcgcggacgttcgccgccgcccacggcctcgcg gtgcgctccgtcggggtccgcgtcgctgacgccgccgaggccttccgcgtcagcgta gccggcggcgctcgcccggccttcgccccagccgacctcggccatggcttcggcctc gccgaggtcgagctctacggcgacgtcgtgctacgcttcgtcagctacccggacgag acagacctgccattcctgccagggttcgagcgcgtgagcagccccggcgccgtggac tacggcctcacgcggttcgaccacgtcgtgggcaacgtcccggagatggccccggtc atagactacatgaaaggcttcttggggttccacgagttcgccgagttcaccgccgag gacgtgggcacgaccgagagcgggctcaactcggtggtgctcgccaacaactccgag gccgtgctgctgccgctcaacgagcccgtgcacggcacaaagcgacggagccagata cagacgtacctggagtatcacggcgggcccggcgtgcagcacatcgcgctcgccagc aacgacgtgctcaggacgctcagggagatgcgggcgcgcacgcccatgggcggcttc gagtteatggcgccaccgcaggcgaaatactatgaaggcgtgcggcgcatcgcaggt gacgtgctctcggaagagcagatcaaggaatgccaggagctgggggtgctagtcgac agggatgatcaaggggtgttgctccaaatcttcaccaagccagtaggggacaggcca acgtttttcctggagatgatccaaagaatcgggtgcatggagaaggacgaggtcggg caagagtaccagaagggtggctgcggcgggtttggcaagggcaatttctccgagctg ttcaagtccattgaggactatgagaaatcccttgaggtcaagcaatctgttgtagct cagaaatcctag
SEQ ID No.31 HPPD cDNA sequence from Wheat atgccgcccacccccaccacccccgcagccaccggcgccgccgcggtgacgccggag cacgcgcggccgcgccgaatggtccgcttcaacccgcgcagcgaccgcttccacacg ctcgccttccaccacgtcgagttctggtgcgcggacgccgcctccgccgccggccgc ttcgccttcgcgctcggcgcgccgctcgccgccaggtccgacctctccacggggaac tccgtgcacgcctcccagctgctccgctcgggcaacctcgccttcctcttcacggcc ccctacgccaacggctgcgacgccgccaccgcctccctgccctccttctccgccgac gccgcgcgccagttctccgcggaccacggcctcgcggtgcgctccatagcgctgcgc gtcgcggacgctgccgaggccttccgcgccagcgtcgacgggggcgcgcgcccggcc ttcagccctgtggacctcggccgcggcttcggcttcgcggaggtcgagctctacggc gacgtcgtgctccgcttcgtcagccacccggacggcagggacgtgcccttcttgccg gggttcgagggcgtgagcaacccagacgccgtggactacggcctgacgcggttcgac cacgtcgtcggcaacgtcccggagcttgcccccgccgcggcctacgtcgccgggttc acggggttccacgagttcgccgagttcacgacggaggacgtgggcacggccgagagc gggctcaactcgatggtgctcgccaacaactcggagggcgtgctgctgccgctcaac gagccggtgcacggcaccaagcgccggagccagatacagacgttcctggaacaccac ggcggctcgggcgtgcagcacatcgcggtggccagcagcgacgtgctcaggacgctc agggagatgcgtgcgcgctccgccatgggcggcttcgacttcctgccacccccgctg ccgaagtactacgaaggcgtgcggcgcatcgccggggatgtgctctcggaggcgcag atcaaggaatgccaggagctgggggtgctcgtcgacagggacgaccaaggggtgttg ctacaaatcttcaccaagccagtaggggacaggccgacgttgttcctggagatgatc cagaggatcgggtgcatggagaaggacgagagaggggaagagtaccagaagggtggc tgcggcgggttcggcaaaggcaacttctccgagctgttcaagtccattgaagattac gagaagtcccttgaagccaagcaatctgctgcagttcagggatcatag
SEQ ID NO. 32 HPPD DNA sequence from Shewanella collwelliana gactttatgagtcgcacaggtatcgaagcgggctacatgaccttacatcaaaaaggc gtgccgcatggaccacaacctggtcgtactgaagcctcagtgggcaaaactgaaacc tatgagtatgcagtaatggtggacacctttgcaccactgcaactgacccagcatgtc aatgcgtgcatgagcaaagattacaaccgttcctggctagaagagtaaaagcgttca gccagtgctgaacatctaataaatataacaccagaggtgacaccgaagagtgccctt ggttgcaataagttgaaagaggataattacatggcaagcgaacaaaacccactgggt ctacttggtatcgaattcactgaatttgctacaccagatctagattttatgcataaa gtttttatcgactttggtttctcaaaacttaaaaaacacaagcagaaagatattgtt tactataaacaaaatgatattaactttttactcaacaatgaaaaacagggcttttca gcccagtttgccaaaacgcatggcccagccattagttctatgggctggcgtgtagaa gatgccaactttgcctttgaaggtgctgtagcccgtggggctaaacccgcagcagat gaggtgaaagatcttccctatcccgctatctatggcattggtgacagccttatctac tttatcgatacgtttggcgatgacaacaatatctacacttctgattttgaagcgtta gatgagcctatcatcacccaagagaaaggcttcattgaggtcgaccatctcaccaat aatgtccataagggcaccatggaatattggtcaaacttctacaaagacatttttggc tttacagaagtgcgttacttcgacattaagggctcacaaacagctcttatctcttac gccctgcgctcgccagatggtagtttctgcattccaattaacgaaggcaaaggcgat gatcgtaaccaaattgatgagtacttaaaagagtacgatggcccaggtgtccaacac ttagcgttccgtagccgcgacatagttgcctcactggatgccatggaaggaagctcc attcaaaccttggacataattccagagtattacgacactatctttgaaaagctgcct caagtcactgaagacagagatcgcatcaagcatcatcaaatcctggtagatggcgat gaagatggctacttactgcaaattttcaccaaaaatctatttggtccaatttttatc gaaatcatccagcgtaaaaacaatctcggttttggcgaaggtaattttaaagcccta tttgaatcgattgagcgtgatcaggtgcgtcgcggcgtactctaacaatcacccagt gatccaacctcaaaaaaccagcatcgcgctggtttttttattgcagcacaacaataa acctctacactagca
SEQ ID NO. 33 TMV translational enhancer nucleotide sequence tatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattactatt tacaattacac
SEQ ID NO. 34 Fusion of TMV and Arabidopsis HST coding sequence tatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattactatt tacaattacacatatggagctctcgatctcacaatcaccgcgtgttcggttctcgtc tctggcgcctcgtttcttagcagcttctcatcatcatcgtccttctgtgcatttagc tgggaagtttataagcctcccacgagatgttcgcttcacgagcttatcaacttcaag aatgcggtccaaatttgtttcaaccaattatagaaaaatctcaatccgggcatgttc tcaggttggtgctgctgagtctgatgatccagtgctggatagaattgcccggttcca aaatgcttgctggagatttcttagaccccatacaatccgcggaacagctttaggatc cactgccttggtgacaagagctttgatagagaacactcatttgatcaaatggagtct tgtactaaaggcactttcaggtcttcttgctcttatttgtgggaatggttatatagt cggcatcaatcagatctacgacattggaatcgacaaagtgaacaaaccatacttgcc aatagcagcaggagatctatcagtgcagtctgcttggttgttagtgatattttttgc gatagcagggcttttagttgtcggatttaactttggtccattcattacaagcctata ctctcttggcctttttctgggaaccatctattctgttccacccctcagaatgaaaag attcccagttgcagcatttcttattattgccacggtacgaggtttccttcttaactt tggtgtgtaccatgctacaagagctgctcttggacttccatttcagtggagtgcacc tgtggcgttcatcacatcttttgtgacactgtttgcactggtcattgctattacaaa ggaccttcctgatgttgaaggagatcgaaagttccaaatatcaaccctggcaacaaa acttggagtgagaaacattgcattcctcggttctggacttctgctagtaaattatgt ttcagccatatcactagctttctacatgcctcaggtttttagaggtagcttgatgat tcctgcacatgtgatcttggcttcaggcttaattttccagacatgggtactagaaaa agcaaactacaccaaggaagctatctcaggatattatcggtttatatggaatctctt ctacgcagagtatctgttattccccttcctctag SEQ ID NO. 35 HST Polypeptide Motif 1.
W(R/K)FLRPHTIRGT
SEQ ID NO. 36 HST Polypeptide Motif 2.
NG(Y/F)IVGINQI(Y/F)D
SEQ ID NO. 37 HST Polypeptide Motif 3.
IAITKDLP
SEQ ID NO. 38 HST Polypeptide Motif 4.
Y(R/Q)(F/W)(I/V)WNLFY
EXAMPLES
The average and distribution of herbicide tolerance or resistance levels of a range of primary plant transformation events are evaluated in the normal manner based upon plant damage, meristematic bleaching symptoms etc. at a range of different concentrations of herbicides. These data can be expressed in terms of, for example, GR50 values derived from dose/response curves having "dose" plotted on the x-axis and "percentage kill", "herbicidal effect", "numbers of emerging green plants" etc. plotted on the y-axis where increased GR50 values may, for example, correspond to increased levels of inherent inhibitor-tolerance (e.g increased Ki x kcat./ KniHPP value) and/or level of expression of the expressed HPPD and/or HST.
The following experiments are conducted using a variety of HST-inhibiting herbicides which are described in Tables A-F.
Figure imgf000040_0001
Figure imgf000040_0003
Table A. Compounds 1.1 - 1.4.
Figure imgf000040_0002
Figure imgf000041_0001
Table B. Compounds 2.1-2.34.
Figure imgf000042_0001
Figure imgf000042_0004
Table C. Compounds 3.1-3.6.
Figure imgf000042_0002
Compound R1 R2 R3 R4 R5 R6
4.1 F H H F Cl CH3
Table D. Compound 4.1
Figure imgf000042_0003
Compound Aα R1 R2 R4 R5 R6 ,
5.1 N Cl H H Cl -CH2CF2H Table E. Compound 5.1
Figure imgf000043_0001
Figure imgf000043_0002
Table F. Compounds 6.1
EXAMPLE 1. Cloning and expressing plant HST enzymes in insect cells and in E.coli
The full length HST coding sequences (minus the ATG start codon) are amplified, with flanking EcoRI sites, from Arabidopsis (SEQ ID 11) and Rice (SEQ ID 12 or SEQ ID 13) from cDNA libraries or made synthetically. Both these full length and also the truncated coding sequences (encoding the mature sequences starting from ARG 64) Arabidopsis SEQ ID 21, rice SEQ ID 22 and rice SEQ ID 23) were cloned into the EcoRI site of the pAcG3X vector (BD Biosciences Cat. No. 21415P) transformed into and then expressed in Sf9 (Spodopterafugiperda) insect cells (as described below) as a N-terminal GST fusion proteins having a factor Xa cleavage site. Similarly, SEQ ID 24, the insect cell codon optimized DNA sequence encoding the alternative truncated mature (ARG 69) Arabidopsis HST is cloned into the EcoRI site of pAcG3X and expressed in SfP cells as a N-terminal GST fusion protein. The Arabidopsis HST SWISSPROT accession number (protein) is Ql ACB3 and the Arabidopsis HST EMBL accession number (DNA): DQ231060.
Alternatively, Arabidopsis and Chlamydomonas mature HST coding sequences are cloned as GST N-terminal fusion enzymes and expressed in E.coli. EXAMPLE 2. Growth of Cells and Preparation of HST Enzyme Extracts.
E.coli. BL21A1 cells expressing mature Arabidopsis or Chlamydomonas HST as a GST N-terminal fusion proteins are grown, harvested, broken and membrane fractions expressing HST produced.
For example, Ing of recombinant DNA is used to transform BL21DE3 cells to obtain a plateful of individual colonies. One of these colonies is picked and used to inoculate an overnight culture of 100ml of Luria Broth (LB) supplemented with 50ug/ml of kanamycin at final concentration, grown at 37° C with shaking at 220rpm. Next morning, lOmls of the overnight culture is used to inoculate 11 of fresh sterile LB supplemented with 50ug/ml of kanamycin at final concentration, grown at 37° C with shaking at 220rpm until the OD reached 0.6 at 600nm, induced with the addition of 0. ImM IPTG and left to induce at 15° C overnight. The cells are harvested by centrifugation at 4600 rpm for 10 min at 4° C and the pellet stored at -80°C.
For example it is found that one litre of cells yields approximately 5g of wet cell pellet. E.coli cell pellet is then resuspended in 25 ml of 5OmM Tris, pH 7.5 supplemented with Roche EDTA- free protease inhibitor tablet (one tablet in 20OmIs of buffer). 10 ml of cells are lysed by sonication on ice. The resultant lysed cells are centrifuged at 300Og for lOmin to pellet the cell nuclei/debris etc. lOmls of supernatant is aspirated and centrifuged at 150,00Og for 60 min at 4° C. The pellet containing the membranes is resuspended in 2 ml of the above buffer. These samples are stored as lOOul aliquots at -80° C, after being diluted with addition of glycerol to 50% v/v.
The HST expression pAcG3X -derived transfer vectors (described above) are independently co-transfoπned into Sf9 suspension cells with FlashBac (Oxford Expression Technologies) parental baculovirus vector. Baculovirus amplification and HST protein expression is performed in accordance with the manufacturer's instructions Sf9 Suspension cell cultures are subcultured at a density of 1.0 EXP 6 cells / ml in 140 ml SfPOOII medium (Invitrogen Cat No.10902) in 500 ml Erlenmeyer flasks. After 24 hours culture at 270C shaking at 120rpm the cell density is measured and readjusted to 2.0 EXP 6 cells / ml in 140 ml. Volumes of amplified virus stock of known titre are added to prepared suspension flasks to give a multiplicity of infection of 10. Flasks are sealed and incubated at 270C shaking at 125rpm for 72 hours to allow adequate protein expression without cell lysis. Cells are harvested by dividing flask contents evenly between three 50 ml Falcon tubes and centrifuging at 900rpm for 4 minutes. Medium is discarded leaving a 3 ml cell pellet which is snap frozen in liquid nitrogen and maintained at -800C
The pellet from 25mls of Sf9 cells (after induction of expression for 4 days) is resuspended in lOmls of 5OmM Tris, pH 7.5 supplemented with Roche-EDTA free protease inhibitor tablet (1 tablet in 20OmIs of buffer) and homogenised using a hand held homogeniser. The resultant lysed cells are centrifuged at 3000g for lOmin to pellet the cell nuclei/debris etc. 1 OmIs of supernatant is aspirated and centrifuged at 15O5OOOg for 60 min at 4° C. The pellet containing the membranes is resuspended in ImI of above buffer and samples are stored as lOOul aliquots at -80° C, after first being diluted with addition of glycerol to 50% v/v.
Western blotting to monitor expression is with anti-GST HRP conjugated Ab (GE
Healthcare, 1 :5000 working dilution) incubation followed by ECL (GE Healthcare).
HST enzyme preparations for assay are also prepared directly from fresh plant material. For example HST enzyme preparations are from spinach. In the first step intact spinach chloroplasts are prepared from two lots of 500 g of fresh baby spinach leaves (e.g from the salad section of the local supermarket). Prepacked spinach is usually already washed, but if buying loose leaves these must be rinsed in water before proceeding. Stalks, large leaves and mid-ribs are removed. Each 500g lot of leaves is added to 1.5 1 of 'Grinding medium' in a 2L plastic beaker. Grinding medium is cold (4 0C) 50 mM Tricine/ NaOH buffer at pH 7.1 containing 330 mM glucose, 2 mM sodium isoascorbate, 5 mM MgC12 and 0.1% bovine serum albumen The beaker, kept at 40C, is placed under a Polytron 6000 blender, fitted with a 1.5"cutting probe and the mixture blended in short bursts of 5-8sec up to 8- 1OK rpm until all the leaves are macerated. The homogenate is filtered into a 5L beaker (embedded in an ice bucket) through four layers of muslin, and two layers of 50μ mesh nylon cloth. The filtrate is transferred to 250ml buckets of a Beckman GS-6 centrifuge and spun at 200 x g (3020rpm) for 2 min at 4° C. The supernatant is drained away and discarded to leave a sediment of chloroplasts. Chloroplasts are resuspended in a few ml of cold resuspension medium by gentle swirling and gentle use of a quill brush soaked in resuspension medium. Resuspension medium is 50 mM Hepes/ KOH pH 7.8 containing 330 mM sorbitol, 2 mM EDTA, 5mM KH2PO4, 2 mM MgC12 and 0.1% bovine serum albumen at 40C. The chloroplasts are resuspended in 5-10 ml of resuspension buffer, recentrifuged down and resuspended again in order to wash them. The chloroplasts are then once again centrifuged down and then broken by resuspension in about 5 ml of 50 mM Tricine-NaOH pH 7.8 to a protein concentration of about 40 mg/ ml. The solution is stored frozen at -8O0C in aliquots. This resuspension is defrosted and used directly in HST activity assays. Alternatively, chloroplasts are prepared resuspended in 50 mM Tris/ HCl buffer at pH 7.8 containing 330 mM sorbitol (alternative resuspension buffer) and layered on top of a percol gradient (comprising the same buffer containing 45% percol), spun down, the intact chloroplast fraction taken and washed 2 or 3 times in the alternative resuspension buffer and then spun down again, resuspended in breaking buffer (without sorbitol), flash frozen and stored in aliquots at - 80 0C.
EXAMPLE 3. Assay of HST enzymes.
Prenyltransferase (HST) activities are measured by determining the prenylation rates of [U-I4C]homogentisate using farnesyl diphosphate (FDP) as prenyl donor. 14C homogentisate is prepared from 14C tyrosine using L amino acid oxidase and HPPD. For inhibitor testing, compounds are dissolved in dimethylsulfoxide (DMSO). DMSO added at up to 2% v/v has no effect on assays. Control assays contain DMSO at the same concentration as in inhibitor containing assays.
Assays using spinach chloroplast extracts (100 μl final volume) contain up to 2 mg of chloroplast protein, 50 mM Tricine-NaOH pH 8.5, 50 mM MgCl2, 200 μM farnesyl diphosphate (FDP) and 26 μM 14C-homogentisate (167 dpm/pmol). Assays are run for about an hour at 28 0C. For inhibitor studies, haloxydine at a final concentration of 500 ppm is found to completely inhibit the reaction. Alternatively, stopping the reaction and carrying out solvent extraction at zero time also provides a 100% inhibition baseline reference. Lipophilic reactions products are extracted and analyzed essentially as described in the literature.
The recombinant Chlamydomonas HST expressed in E.coli membranes is assayed in standard reaction mixtures with 200 μM FDP and 100 μM 14C-homogentisate
(40 dpm/pmol) in 50 mM Tricine-NaOH pH 8.5, 20 mM MgCl2 . Assays are started with the addition of enzyme and run for ~ 20 min at 28 0C.
Recombinant Arabidopsis and Rice HSTs are expressed in insect cells. Assays are run as for Chlamydomonas HST except that assay temperature is 270C. Assays are stopped with 300ul of solvent mix (1:2, Chloroform: Methanol) and lOOul of 0.5% NaCl, agitated / mixed and spun at 13,000rpm in abenchtop eppendorf centrifuge for 5 minutes. 80ul of of the lower phase extract is loaded onto a TLC plate (silica Gel 60, 20cm x 20cm) FLA3000 system and run for 35 minutes in dichloromethane. The radioactivity is quantified using a Fuji Phosphoimager and band intensity integrated as quantitative measure of product amount. The bands corresponding to oxidised and reduced 2~methyl-6-farnesyl-l,4-benzoquinol (MFBQ) are identified and the total of the two (oxidised and reduced) band intensities is calculated in order to estimate the total amount of MFBQ product formation. Specific activities of 8 pmol MFBQ min"1 mg'1 protein (23 pmol) and 7 pmol MFBQ min"1 mg"1 protein (14 pmol) are , for example, estimated for the GST-fusion truncated Arabidopsis HST gene (SEQ ID # 3) expressed in membranes from insect cells 4 days and 5 days after transfection respectively. Similar results are noted from past literature on E. coli expressed Arabidopsis HST. Activity from the insect cell expressed GST-fusion truncated rice HST (SEQ ID# 4) is similar. Expression of the non-truncated HST coding sequences as GST fusions also gives HST activity. Expression of the insect cell optimised GST- fusion truncated Arabidopsis HST (SEQ ID#23) gives, for example, an approximately 3-10 fold increase in specific activity over the non-optimized genes.
Using the above assays percentage inhibition of the amount of MFBQ formed at a range of doses of inhibitors relative to the control amount obtained with no inhibitor present is reported (Table 1). The better inhibitors give greater percentage inhibition at lower doses.
It is found that for all of the HST enzyme preparations assayed under the prescribed reaction conditions formation of MFBQ is by no means the only reaction catalysed by HST from 14C homogentisate(HGA). In fact the major (-90%) radiolabeled products from the 14C HGA are not MFBQ. These unknown other products, are also extracted into chloroform/ methanol but, in dichloromethane TLC, are found to stay on or chromatagraph near the base line. Four apparent 14C -labelled bands (presumably corresponding to two quinone/ quinol pairs) are partly resolved in a second dimension of TLC using 12:3:5:0.5, dichloromethane:hexane:acetonitrile:foπnic acid. No such bands are seen in the absence of FPP (or indeed when FPP is replaced with pyrophosphate) and, presumptively, the bands correspond to products formed due to farnesylation and decarboxylation not being tightly coupled; .i,e. farnesylation in the absence of decarboxylation (giving rise to carboxylated MFBQ) and decarboxylation in the absence of farnesylation (giving rise to the methyl quinol/ quinone). Whatever their identity it is quite clear that these products more polar than MFBQ are all bone fide enzyme reaction products since, in the absence of FDP or using like non- transgenic (non-HST expressing) membranes, they are not formed. In addition it is found that inhibitors such as haloxydine inhibit the formation of these other products in a way that, as dose is varied, is apparently co-linear with inhibition of the formation of MFBQ. Thus 500 ppra haloxydine about completely inhibits the HST enzyme reaction and neither MFBQ nor any of the other products are formed.
Thus in an improved, more sensitive and convenient version of the above assay, the TLC step is dispensed with, treatment with 500 ppm haloxydine (or other inhibitor at a suitable concentration) is used as the 100% inhibition 'control' and a portion of the chloroform/ methanol extract is taken directly into a scintillation vial and counted.
These assays are routinely run at lOOμM 14C HGA, 250C for <20 min (i.e over a period for which the control assay rate remains linear) and at a range of test inhibitor concentrations used so that IC50s can be derived by curve fitting to the Hill equation (allowing the value of the slope, n, to vary).
Results.
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
TABLE 1. Observed percentage inhibition of the HST reaction (relative to controls) at various concentrations of various compounds using HST from various sources (as labelled). Assays based on TLC and estimated amount of MFBQ formed.
Figure imgf000051_0002
Figure imgf000052_0001
TABLE 2. Estimated IC50 values for inhibition of Arabidopsis HST based on total extraction (non TLC) assay. These estimates were from 3 point dose curves only conducted at 50 μM HPP.
EXAMPLE 4. Preparation of stable transgenic plants lines expressing a heterologous HST enzyme
For example the Arabidopsis HST SEQ ID # 11 is cloned behind a double 35s CMV promoter sequence and a TMV translational enhancer sequence and in front of the 3' terminator from the nos gene. This expression cassette is ligated into pMJBl (described in WO98/20144) and then into pBIN19 and then transformed into Agrobacterium titmefaciens strains LBA4404 prior to plant transformation.
For example, the full length Arabidopsis HST seq ID # 11 is fused to the TMV translational enhancer sequence (SEQ ID #33) by overlapping PCR and, at the same time, 5' Xhol site and a 3'KpnI site are added by PCR. Site-directed mutagenesis is performed to remove an internal Xhol site. The TMV/HPPD fusion is removed from the pBIN19 by digestion with Xhol/Kpnl and is replaced by the TMV/HST fusion (SEQ ID #34). The TMV/HST fusion is now cloned behind a double 35s promoter and in front of the 3' terminator from the nos gene. Again the modified pBIN19 vector (ςpBinAT HST') is then transformed into Agrobacterium tumefaciens strain LBA4404.
Likewise, vectors for plant transformation are constructed to comprise DNA sequences any HST and, for example, SEQ Ids nos. 12-20.
Alternatively vectors comprise DNA encoding HSTs from photosynthetic protozoans, higher and lower plants the sequences of which are derived from cDNA libraries using methods known to the skilled man. For example total RNA is prepared from 5- 20 -day-old plant seedlings using the method of Tri-Zol extraction (Life
Technologies). mRNA is obtained, for example, from Avena sativa using the Oligotex mRNA purification system (Qiagen). The 5' end of, for example, the A. sativa HST gene is identified using 5' RACE, performed using the Gene Racer kit (Invitrogen) with internal HST gene specific primers (based on HST consensus regions e.g SEQ No. 35, 36, 37 and 38). The 3' end of the gene is identified by 3' RACE, performed using Themoscript RT (Life Technologies) with appropriate oligo dT primer and an appropriate internal HST gene primer, followed by PCR All methodologies are performed according to protocols provided by the various stated manufacturers. Products obtained from the 5' and 3' RACE reactions are cloned into pCR 2.1 TOPO (invitrogen) and the cloned products sequenced using universal M 13 forward and reverse primers with an automated ABI377 DNA sequencer. Primers are then designed to the translation initiation and termination codons of the HST gene respectively. Both primers are used in conjunction with the One-step RTPCR kit (Qiagen or Invitrogen) to obtain full length coding sequences. Products obtained are cloned into pCR 2.1 TOPO, sequenced, and identified as HST by comparison with sequences known in the art (and for example the HST sequences herein). A master plate of Agrobacterium tumefaciens containing the binary vector pBinAT HST (described above) or analogous binary vector comprising a different HST is used to inoculate 10 ml LB containing 100 mg / 1 Rifampicin plus 50 mg / 1 Kanamycin using a single bacterial colony. This is incubated overnight at 280C shaking at 200 rpm. This entire overnight culture is used to inoculate a 50 ml volume of LA (plus antibiotics). Again this is cultured overnight at 280C shaking at 200 rpm. The Agrobacterium cells are pelleted by centrifuging at 3000 rpm for 15 minutes then resuspended in MS medium with 30 g / 1 sucrose, pH 5.9 to an OD (600 nM) = 0.6. This suspension is dispensed in 25 ml aliquots into petri dishes.
Clonal micro-propagated tobacco shoot cultures are used to excise young (not yet fully expanded) leaves. The mid rib and outer leaf margins are removed and discarded, the remaining lamina is cut into 1 cm squares. These are transferred to the Agrobacterium suspension for 20 minutes. Explants are then removed, dabbed on sterile filter paper to remove excess suspension then transferred to NBM medium (MS medium, 30 g / 1 sucrose, 1 mg / 1 BAP, 0.1 mg / 1 NAA, pH 5.9, solidified with 8 g / 1 Plantagar), with the abaxial surface of each explant in contact with the medium. Approximately 7 explants are transferred per plate, which are then sealed then maintained in a lit incubator at 250C, 16 hour photoperiod for 3 days.
Explants are then transferred to NBM medium containing 100 mg / 1 Kanamycin plus antibiotics to prevent further growth of Agrobacterium (200 mg / 1 timentin with 250 mg / 1 carbenicillin). Further subculture on to this same medium is then performed every 2 weeks.
As shoots start to regenerate from the callusing leaf explants these are removed to Shoot elongation medium (MS medium, 30 g / 1 sucrose, 8 g / 1 Plantagar, 100 mg / 1 Kanamycin, 200 mg / 1 timentin, 250 mg / 1 carbenicillin, pH 5.9). Stable transgenic plants readily root within 2 weeks. To provide multiple plants per event to ultimately allow more than one herbicide test per transgenic plant all rooting shoots are micropropagated to generate 3 or more rooted clones. Putative transgenic plants that are rooting and show vigorous shoot growth on the medium incorporating Kanamycin are analysed by PCR using primers that amplified a 500bp fragment within the Arabidopsis HST transgene. Evaluation of this same primer set on untransformed tobacco showed conclusively that these primers would not amplify sequences from the native tobacco HST gene.
To roughly evaluate comparative levels of ectopic HST expression independent PCR positive tobacco shoots have young leaves removed, cut into 1 cm squares and plated onto NBM medium incorporating 1 mg / 1 haloxydine. These leaf explants produce callus and ultimately regenerated shoots. Those explants over expressing HST regenerated green callus and shoots. Untransformed explants or those from transformants with limited HST expression produced bleached callus and stunted bleached shoots that ultimately died.
It is found that PCR positive events correspond with callus which yields green shoot proliferation (scores > = 3) in the presence of haloxydine and many more exhibit some level of tolerance that is greater than the untransformed control material.
Rooted transgenic TO plantlets are transferred from agar and potted into 50% peat, 50% John Innes soil no 3 or, for example, MetroMix® 380 soil (Sun Gro Horticulture, Bellevue, WA) with slow-release fertilizer in 3 inch round or 4 inch square pots and left regularly watered to establish for 8-12d in the glass house. Glass house conditions are about 24-270C day; 18-210C night and approximately a 14h (or longer in UK summer) photoperiod. Humidity is ~ 65% and light levels up to 2000 μmol/ m2 at bench level. Once new tissue emerges and plants they have reached the 2-4 leaf stage some of the clones from each event are sprayed with test chemicals dissolved in water with 0.2-0.25% X-77 surfactant and sprayed from a boom on a suitable track sprayer moving at 2 mph in a DeVries spray chamber with the nozzle about 2 inches from the plant tops. Spray volume is suitably 25 gallons per acre or, for example, 2001/ ha. Test chemicals are, for example, compound 2.3 at 500 g/ha. At the same time that transgenic plants are sprayed so too are w/t Samsun tobacco plants grown from seed as well as non-transgenic plants regenerated from tissue culture and non-transgenic tissue culture escapes. Damage is assessed versus unsprayed control plants of like size and development.
Compound Compound
3 3
EVENT at 50Og/ ha EVENT at 50Og/ ha
A1 0 E5 1
A12 1.5 E6 2.5
A4 0 E8 0
A6 0 F12 4
A9 0.5 F2 i 4
B1 2 F3 1
B3 2.5 F4 1
B8 5 F5 2
C11 2.5 F7 0
C12 0 G1 4
C4 2 G4 0
C8 5 G5 1
C9 0 G9 9
D2 10 H8 8
D4 2 W/T_SD 0
D5 0 W/T_SD 0
D6 2.5 W/T_SD 0
D8 3 W/T_TC 0
E1 2 W/T_TC 0
E10 0 W/T TC 0
Table 3. Arabidopsis HST transgenic tobacco plants assessed 11 DAT with compound 2.3 (designated compound 3 in the table). Compared to untreated controls all the plants are affected by treatment at 50Og/ ha and are smaller and growth is set back. However, unlike controls that show white meristems and that are essentially dead many of the HST transgenics show green meristems and are recovering and some show essentially no bleaching. Plants are scored on a scale from 0 to 10 with 0 meaning plant substantially bleached / burnt and meristem dead/ white and 10 meaning that the entire plant looks green and undamaged.
In addition to the above experiments two lines of Arabidopsis HST expressing transgenic plants, E9 and F6 were treated with haloxydine at 250 g/ha in a similar manner to the methods described above but at a slightly later growth stage (4-5 leaf). After assessment 8 DAT the w/t plants were 55 and 65% damaged, line F6 plants were 50 and 75% damaged whereas line E9 plants were only 20 and 40% damaged. At 25 DAT the w/t and F6 plants remained stunted, bleached and small (50-70% damage) whereas the E9 plants now appeared as healthy and similar sized to untreated control plants. Thus expression of the Arabidopsis HST confers resistance to haloxydine.
The plants are assessed at various times after treatment up to 28 DAT. Those events (e.g C8, G9, E9) showing the least damage from HST herbicides are grown on to flowering, bagged and allowed to self. The seed from selected events are collected sown on again in pots and tested again for herbicide resistance in a spray test for herbicide resistance. Single copy events amongst the Tl plant lines are identified by their 3 : 1 segregation ratio (for example, dependent on the construct, by both kanamycin selection and wrt herbicide resistance phenotype) and by quantitative RT- PCR.
EXAMPLE 5. Production and further testing of Tl and T2 transgenic plants transformed to express Arabidopsis HST , Avena HPPD or Pseudomonas HPPD TO transgenic tobacco plant lines B 8 and G9 described above in the foregoing examples are selfed. About 50 of the resultant seed from each selfing each line are planted out into a soil/ peat mixture in 3 inch pots, grown in the glass house for 7-10 d and sprayed with 500g/ ha of compound 2.3 (all as described in the foregoing examples). For each line about three quarters of the plants display visible resistance to the herbicide and of these a few plants (possible homozygotes at a single insertion event) appear the most resistant. A few of these more highly tolerant Tl plants are selfed again to produce batches of T2 seed.
6 Seed from w/t Samsun tobacco and 8 Tl seed from events B8 and G9 are also planted out in 3 inch pots , grown on for 7 to 12 d and then, as described in the foregoing examples, the plantlets spray tested for resistance to various chemicals and assessed at 14 DAT. Chemicals are formulated in 0.2% X77 and sprayed at a spray volume of 200 1/lia. Results are depicted in Table 4 below. The results clearly demonstrate the heritability of the herbicide resistance phenotype. The results also show that, aside from obvious non-transgenic segregants, the transgenic Arabidopsis HST Tobacco Tl plants display resistance to HST herbicides as exemplified using compounds 2.15 and 2.30 but that the phenotype is specific and the plants are not significantly tolerant to the other two herbicides tested, norflurazon and atrazine.
Figure imgf000058_0001
Table 4. Assessment of herbicide % damage to Tl progeny plants of Arabidopsis HST lines B8 and G9 at 14 DAT with various herbicides. Tobacco plants expressing the wild-type HPPD gene of Pseudomonas fluorescens strain 87-79 under operable control of the double enhanced 35S CMV promoter region , Nos3' terminator and TMV translational enhancer were provided as detailed in Example 4 of WO0246387. A TO event exhibiting tolerance to mesotrione was selfed to produce a single insertion Tl line (exhibiting 3:1 segregation of the herbicide tolerance and kanamycin selection phenotypes) which was again further selfed to provide the T2 line designated C2.
Seed of wild-type tobacco plants, C2 tobacco plants and of the Tl progeny of a further Arabidopsis-HST expressing tobacco line, D2 were planted out in 3 inch pots, grown on, sprayed with compounds 1.1, 2.30 and 3.6 at the rates shown in table 5. Percent damage scores were assessed at 7 DAT. Aside from the presumptive non- transgenic segregants the D2 line containing the Arabidopis HST expression construct offered the highest level of tolerance to the two HST herbicides with the Psuedomonas HPPD , C2 line, offering only marginal tolerance under the conditions of this test.
Figure imgf000059_0001
Table 5. Testing of wild-type (WT) Samsun, Cl and D2 tobacco lines versus various HST inhibitors. Results depict % damage at 7 DAT
Damage %
Line 5 DAT 10 DAT
367 25 10
332 30 10
404 45 20
426 45 20
351 50 25
Wt 55 25
Wt 55 60
Wt 55 60
Wt 50 35
Wt 60 60
Wt 60 30
Table 6 Testing of TO lines of Arabidopsis HST tobacco treated with mesotrione.
5 lines of tobacco transformed with Arabidopsis HST were less damaged 10 DAT with 10 g/ha mesotrione than like-treated wild-type lines. Expression of Arabidopsis HST confers a degree of tolerance to the HPPD herbicide, mesotrione.
EXAMPLE 6. Resistance of plants expressing Avena HPPD to HST herbicides.
Seed of segregating Tl lines of tobacco expressing the wild-type HPPD gene of Avena sativa under operable control of the double enhanced 35S CMV promoter region , Nos3' terminator and TMV translational enhancer were provided as described in WO0246387. About 30-40 Tl seed derived from selfing a mesotrione-tolerant TO event were grown up to 7-10 d old plantlets sprayed and assessed as described above and the results (at 6 DAT) are depicted in the Table 7 below. Under the conditions of the experiment the Avena HPPD appearsto offer a degree tolerance to both HST inhibitors 2.30 and 3.6.
Figure imgf000060_0001
Table 7. Testing of wild-type (WT) Samsun tobacco and an Avena HPPD expressing tobacco lines versus mesotrione, compound 2.30 and compound 3.6. Results depict percent damage at 6 DAT. EXAMPLE 7. Tobacco, transformation and selection of HST-expressing transformants on HST herbicides
A master plate of Agrobacterhim tiimefaciens containing the binary vector pBinAT HST (described in example 6) is used to inoculate 10 ml LB containing 100 mg / 1 Rifampicin plus 50 mg / 1 Kanamycin using a single bacterial colony. This is incubated overnight at 280C shaking at 200 rpm. This entire overnight culture is used to inoculate a 50 ml volume of LA (plus antibiotics). Again this is cultured overnight at 280C shaking at 200 rpm. The Agrobacierium cells are pelleted by centrifuging at 3000 rpm for 15 minutes then resuspended in MS medium with 30 g / 1 sucrose, pH 5.9 to an OD (600 nM) = 0.6. This suspension is dispensed in 25 ml aliquots into petri dishes.
Clonal micro-propagated tobacco shoot cultures are used to excise young (not yet fully expanded) leaves. The mid rib and outer leaf margins are removed and discarded, the remaining lamina is cut into 1 cm squares. These are transferred to the Agrobacterium suspension for 20 minutes. Explants are then removed, dabbed on sterile filter paper to remove excess suspension then transferred to NBM medium (MS medium, 30 g / 1 sucrose, 1 mg / 1 BAP, 0.1 mg / 1 NAA, pH 5.9, solidified with 8 g / 1 Plantagar), with the abaxial surface of each explant in contact with the medium. Approximately 7 explants are transferred per plate, which are then sealed then maintained in a lit incubator at 250C, 16 hour photoperiod for 3 days.
Explants are then transferred to NBM medium containing 0.5 mg / 1 Haloxydine plus antibiotics to prevent further growth of Agrobacterium (200 mg / 1 timentin with 250 mg / 1 carbenicillin). Further subculture on to this same medium is then performed every 2 weeks.
As shoots start to regenerate from the callusing leaf explants these are removed to Shoot elongation medium (MS medium, 30 g / 1 sucrose, 8 g / 1 Plantagar, lmg / 1 Haloxydine (or similar or concentration of any other HST herbicide at an appropriate discriminating concentration), 200 mg / 1 timentin, 250 mg / 1 carbenicillin, pH 5.9). Shoots that root and continue to proliferate are analysed for stable integration of the HST transgene by PCR. Ultimately these rooted shoots are transferred to soil and progressed under glasshouse conditions. Tl seed is produced from selected TO lines,
Thus it is found that the use of a HST gene in combination with a HST-inhibitor herbicide provides a means for the selection of transgenic plant tissue EXAMPLE 8. Preparation and testing of stable transgenic plants lines expressing a heterologous HPPD enzyme Transgenic lines of tobacco, soyabean and corn etc. can be engineered to express various heterologous HPPDs derived from, for example Avena (SEQ ID #26), Wheat (SEQ ID #27), Pseudomonas fluorescein (SEQ ID # 25) and Shewanella colwelliana (SEQ ID #28) as, for example, described in WO 02/46387.
The seed from selected events are collected sown on again in pots and tested again for herbicide resistance in a spray test for resistance to HPPD herbicide (for example mesotrione). Single copy events amongst the Tl plant lines are identified by their 3:1 segregation ratio (wrt kanamycin and or herbicide) and by quantitative RT-PCR. Seed from the thus selected Tl tobacco (var Samsun) lines are sown in 3 inch diameter pots containing 50% peat, 50% John Innes soil no 3. After growth to the 3 leaf stage, plants are sprayed, as described above, in order to test for herbicide tolerance relative to like- treated non-transgenic tobacco plants.
Control tobacco plants and transgenic Tl plants expressing either the Pseudomonas or the wheat HPPD gene are sprayed at 37, 111, 333 and 1000 g/lia rates of HST inhibitors and, for example, compound 2.3.
Plants are assessed and scored for % damage at 16 DAT.
RATE Pseudomonas Wheat w/t
Compound g/ha HPPD HPPD tobacco
Mesotrione
37 3 0 66
111 3 2 57
333 43 0 57
1000 67 13 54
Compound 37 0 0 17 2.3 111 3 0 40
333 6 3 47
1000 10 8 57 TABLE 8. Comparing % damage observed 16 DAT of w/t tobacco plants with transgenic plants expressing either Pseudomonas or Wheat HPPD
It is thus observed that expression of either HPPD gene provides tobacco with a high level of resistance to treatment with the HST inhibitor, 2.3 as well as to mesotrione. Treated w/t tobacco plants show white bleached meristems whereas transgenic HPPD expressing plants have green healthy meristems and new leaves and look almost undamaged. In this test plants were relatively large at the time of spraying and thus controls were not completely controlled.
EXAMPLE 9. Preparation of transgenic plants lines expressing different heterologous HST and HPPD enzymes and stacked combinations thereof. Glasshouse testing for herbicide tolerance
The full length Arabidopsis (plus start codon) HST seq ID 11 is cloned behind a double 35s CMV promoter sequence and a TMV translational enhancer sequence and in front of the 3' terminator from the nos gene as described previously. As described above this expression construct is cloned into a binary vector (pBIN 35S Arabidopsis HST) that is transformed into tobacco to produce populations of 30-50 transgenic events which are subdivided at the callus stage to produce 2-4 clonal plants from each transgenic 'event' which are then regenerated and transferred into soil before transfer to the glass hosue and testing. In just the same way the Chlamydomonas HST gene sequence (AM285678) is codon-optimised for tobacco and cloned behind the double Cauliflower mosaic virus 35S promoter and Tobacco mosaic virus enhancer sequences and in front of a nos gene terminator, cloned into a binary vector and transformed into tobacco to produce a population of TO plants.
Exactly as described in example 5 of WO 02/46387 the wheat HPPD gene sequence (Embl DD064495) is cloned behind an Arabidopsis Rubisco small subunit (SSU) promoter and in front of a nos gene terminator to produce an ' SSU Wheat HPPD nos expression cassette' which is cloned into a binary vector and transformed into tobacco to produce a population of 30-50 transgenic events.
A "pBin Arabidopsis HST/Wheat HPPD" vector is also built in order to provide a population of plants that co-express the HST and HPPD enzymes. The SSU Wheat HPPD nos cassette (described above) is cloned into the EcoRI site of the pBin 35S Arabidopsis HST vector (described above) to generate the HST/HPPD expression construct and binary vector. Again this is transformed into tobacco to produce a population of primary transformants.
Alternatively, transgenic plants expressing both HPPD and HST are produced by first transforming to express either a heterologous HST or HPPD and then the progeny tissue are subsequently transformed with a construct designed to express the other enzyme. For example, as described in WO 02/46387 tobacco plants are transformed to express wheat, Avena or Pseudomonas HPPD under expression control of the
Arabidopsis small subunit of rubisco promoter, TMV translational enhancer and nos gene 3 ' terminator. Examples of TO events highly tolerant to mesotrione are selfed on to make Tl seed. Approximately 100 of these Tl seeds from a single event are surface sterilised using 1% Virkon for 15 minutes then following washing in sterile water plated onto MS medium with 20 g / 1 sucrose, 100 mg / 1 Kanamycin, pH 5.8 solidified with 8 g / 1 plantagar. Individual plants are picked from the mixed population of hemizygous and homozygous plants that germinate, grown on in vitro and micropropagated to provide a clonal recombinant shoot culture. Leaves from these shoot cultures are subject to transformation using constructs and selection methods described previously. To initially evaluate whether co-expression of HPPD and HST results in elevated levels of plant resistance to mesotrione compared to expression of HPPD alone, shoot culture derived leaf explants from HPPD only and HPPD plus HST transformants are plated onto NBM medium containing a range of mesotrione concentrations between 0.1 to 5 mg / 1. Explants from transgenics combining HPPD and HST may exhibit green callus and more limited bleaching of regenerating shoots at higher mesotrione concentrations than the HPPD only 'background' explants from the clonal single plant, HPPD event derived material. 'ControPplantlets are regenerated from the untransformed (with HST) HPPD- expressing clonal background material derived from a single plant of a single event. TO HST transgenic plantlets that are additionally transformed with HST are selected against this background as described in the previous example on haloxydine, and are also regenerated. Plantlets are micropropagated into further clones, rooted and grown on in pots in the glass house as described in the previous examples.
cassettes described above 35 S Chlamydomonas HST gene. 2.30. The parameter "hi" "'blch" refers to the % bleaching all three constructs confers
Figure imgf000065_0001
(about 50% of the events being < 30% stunted) were observed in the populations transformed with either of the two HST genes. HST gene. Assessments parameter "ht" refers to the % in % bleaching observed at the 1.1. The highest number in plants transformed with
Figure imgf000066_0001
Figure imgf000067_0001
TABLE 11. TO populations of tobacco events containing , alternatively, the expression cassettes described above having 1 ) the Arabidopsis HST gene, 2) the wheat HPPD gene stacked with the Arabidopsis HST gene or 3) the Chlamydomonas HST gene. Assessments of herbicidal damage at various times after spray with 500 g of compound 2.30. The parameter "hi" refers to the % in height reduction relative to control plants, whilst the parameter "blch" refers to the % bleaching observed at the meristem relative to control plants. All three constructs provide tolerance to compound 2.30. The highest number of least damaged plants (more than 50% of the events < 30% stunted) were observed in plants transformed with the stacked combination of the HPPD and HST genes.
Figure imgf000068_0001
EXAMPLE 10: Construction of soybean transformation vectors.
A binary vector (17107) for dicot (soybean) transformation is, for example, constructed, with the Arabidopsis UBQ3 promoter driving expression of the
Chlamydomonas HST coding sequence (SEQ ID # 15), followed by Nos gene 3' terminator. The gene is codon optimized for soybean expression based upon the predicted amino acid sequence of the HST gene coding region. The amino acid sequence of the protein encoded by Chlamydomonas HST gene is provided in SEQ ID # 5. Optionally the transformation vector also contains two PAT gene cassettes (one with the 35S promoter and one with the CMP promoter, and both PAT genes are followed by the nos terminator) for glufosinate based selection during the transformation process. A similar binary vector (17108) is similarly constructed but also comprising an expression cassette expressing the soyabean codon-optimized Avena HPPD gene. In this case there is no PAT gene and selection is earned out using a HPPD herbicide or, as described herein, a HST herbicide.
Example 11: Soybean TO Plant establishment and selection.
Soybean transformation is achieved using methods well known in the art. TO plants were taken from tissue culture to the greenhouse where they are transplanted into saturated soil (Redi-Earth® Plug and Seedling Mix, Sun Gro Horticulture, Bellevue, WA) mixed with 1% granular Marathon® (Olympic Horticultural Products, Co., Mainland, PA) at 5-10 g/gal Redi-Earth® Mix in 2" square pots. The plants are covered with humidity domes and placed in a Conviron chamber (Pembina, ND) with the following environmental conditions: 240C day; 180C night; 23 hr photoperiod; 80% relative humidity. After plants became established in the soil and new growth appeared (-1-2 weeks), plants are sampled and tested for the presence of desired transgene by Taqman™ analysis using appropriate probes for the HST and/or HPPD genes, or promoters (for example prCMP and prUBq3). All positive plants and several negative plants are transplanted into 4" square pots containing MetroMix® 380 soil (Sun Gro Horticulture, Bellevue, WA). Sierra 17-6-12 slow release fertilizer is incorporated into the soil at the recommended rate. The negative plants serve as controls for the spray experiment. The plants are then relocated into a standard greenhouse to acclimatize (~1 week). The environmental conditions are: 270C day; 210C night; 12 hr photoperiod (with ambient light); ambient humidity. After acclimatizing (~1 week), the plants are ready to be sprayed with the desired herbicides.
Example 12 HPPD/ HST herbicide mixtures. Effect of adding small amounts of HPPD inhibitor on the herbicidal activity of HST herbicides
Tobacco (var Samsun) plantlets germinated aseptically in agar made up in 1/3 strength Murashige and Skoog salts medium along with various doses of herbicide. Bleaching damage to emerging plantlets is assessed 7 DAT. The plantlets are kept covered under clear perspex and grown at 18 0C (night) and 24° C (day) under a 16h day (~ 500-900 umol/ m2) , 8 h darkness regime. Herbicide affected plantlets are bleached white and grow less. Synergistic / antagonistic responses are calculated using the Colby formula (Colby, S. R. (Calculating synergistic and antagonistic responses of herbicide Combinations", Weeds, 15, p. 20-22, 1967).
% Injury
Plus Mesotrione plus Mesotrione
[Haloxydine] / Haloxydine (0.004ppm) (0.004ppm) ppm only (OBSERVED) (EXPECTED) (O-E)
37.5 100 100 100 0
18.8 100 100 100 0
9.4 90 100 94 6
4.7 70 100 80 20
2.4 50 90 67.5 22.5
1.2 35 70 58 12
0.6 20 50 48 2
0.3 10 50 41.5 8.5
0.75% v/v DMSO 0 35 35 0
TABLE 13. HST + HPPD herbicide effects on tobacco seedlings in agar. The % bleaching observed 7 DAT of germinating tobacco seeds in agar is assessed with various doses of haloxydine and 0.75% v/v DMSO in the presence or absence of 0.004 ppm mesotrione. At this dose the mesotrione by itself consistently gives 35% bleaching damage and the expected values for the damage in mixture with the various doses of haloxydine are therefore calculated accordingly as described by Colby (1967).
In an alternative herbicide test procedure tobacco (var Samsun) plantlets germinated aseptically in agar made up in 1/3 strength Murashige and Skoog salts medium are transferred after 4d to float on top of 2.9 ml of sterile liquid culture medium (half strength Murashige and Skoog medium containing 30 mM sucrose) in wells of 12 well plates. Test compounds are added at various doses and bleaching damage is assessed after 14-20 DAT. The plantlets are kept covered under clear perspex and grown at 18 0C (night) and 24° C (day) under a 16h day (~ 500-900 umol/ m2), 8 h darkness regime. Plantlets continue to grow and produce new tissue over the 14-20 DAT period but are bleached and grow less in the presence of controlling concentrations of herbicide. % Injury
Plus
Compound 2.13 Mesotrione plus Mesotrione
/ ppm compound 2.13 only (0.001 ppm) (0.0005ppm)
23 100 100 100
7.67 50 100 100
2.56 0 90 90
0.85 0 90 40
0.28 0 80 0
0.09 0 80 0
0.03 0 60 0
0.01 0 40 0
0.75% v/v DMSO 0 20 0
TABLE 14. HST + HPPD herbicide effects on tobacco seedlings growing on liquid. The % bleaching observed 20 DAT of tobacco seedlings on liquid culture medium is assessed versus the presence of various concentrations of the HST herbicide, compound 2.13 with 0.75% v/v DMSO in the presence or absence of 0.001 or 0.0005 ppm of mesotrione. At these doses the mesotrione by itself produced either minimal, 20% , or zero bleaching damage.
From the data provided it is apparent that addition of a low dose of mesotrione synergises the herbicidal effect of the HST inhibitor, haloxydine on agar grown tobacco plantlets.
Figure imgf000071_0001
Figure imgf000072_0001
Table 15b
TABLES 15a and 15b. HST + HPPD herbicide injury on tobacco seedlings growing on liquid culture medium. The % bleaching observed 14 DAT of tobacco seedlings on liquid culture medium is assessed versus the presence of various concentrations of the HST herbicides, haloxydine (Table 15a), and compound 2.15 (Table 15b), with 0.75% v/v DMSO in the presence or absence of 0.001 ppm mesotrione. At this dose mesotrione produced minimal (0-5%v) damage.
In liquid culture the synergising effect of mesotrione on the activity of HST inhibitor, 2.13 is even more apparent than that on haloxydine. Even addition of a dose of mesotrione that itself produces no visible damage at all results in levels of 40, 90 and 100% bleaching injury at doses of compound 2.13 where the expected level of control (according to Colby) is only 0, 0 and 50%. Similarly, at the higher dose of mesotrione, 80 or 90% bleaching is observed across a range of rates of compound 2.13 where only 20% is expected. Similarly, under similar conditions and in repeat experiments, there are clear synergistic effects of low amounts of mesotrione on the injury observed down haloxydine and compound 2.15 dose responses.
EXAMPLE 13. GLASS HOUSE WEED CONTROL BY MIXTURES OF HST AND HPPD HERBICIDES
Weed seeds are planted out in trays containing suitable soil (for example 50% peat, 50% John Innes soil no 3) and grown in the glass house conditions under 24-270C day; 18- 210C night and approximately a 14h (or longer in UK summer) photoperiod.
Humidity is ~ 65% and light levels up to 2000 μmol/ m2 at bench level. Trays are sprayed with test chemicals dissolved in water with 0.2-0.25% X-77 surfactant and sprayed from a boom on a suitable track sprayer moving at about 2 mph in a suitable track sprayer (for example a DeVries spray chamber with the nozzle about 2 inches from the plant tops). Spray volume is suitably 500 - 1000 1/ha. Sprays are carried out both pre-emergence and over small plants at about 7-12 d post-emergence
Plants are assessed 14 DAT and herbicidal damage is scored on a scale from 0 to 100%. The HST inhibitor compound 2.30 (designated compound A in table 16 and 17), haloxydine (compound 1.1) and compound 2.13 (designated compound AE in tables 16 and 17) are sprayed at rates between 0 and 50Og/ ha. Mesotrione is applied at a very low rate of lg/ha at which it causes essentially no (< 10% damage).
POST-EMERGENCE APPLICATION
Figure imgf000074_0001
TABLE 16. Posteinergence Control of Weeds by HST/ HPPD Herbicide Mixtures. Numbers reported correspond to the % damage observed.
Figure imgf000075_0001
TABLE 17. Pre-Emcrgence Weed Control by HPPD/ HST Herbicide Mixtures EXAMPLE 14. Further studies showing Synergy between HPPD and HST herbicides
In a further test, the results of which are depicted in tables 18 and 19, weed seeds are planted out in trays containing 50% peat/ 50% John Iniies no. 3 soil and grown in the glass house at 24- 27 C day; 18-21 C night and approximately a 15h photoperiod. Humidity is ~ 65% and light levels at bench level are up to 2mmol/ m2. Again all spray chemicals are dissolved in 0.2% X77 surfactant and sprayed from a boom on a track sprayer moving at 2 mph with the nozzle set about 2 inches above the plant tops. The spray volume is 5001/ ha. Sprays are carried out both pre-emergence and post- emergence over small plants at about 7-12d post-emergence. Plants are assessed at 14 DAT with herbicidal damage scored on a scale from 0 to 100%. The HPPD inhibiting herbicide is compound A22 (4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]-6- (trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1 ]oct-3-en-2-one) which is sprayed at 2g/ ha both alone and in mixture with various HST herbicides. Results and spray rates are depicted in Tables 18 and 19. Again the Colby formula has been used to calculate synergy scores observed following treatment with the mixture based on the results obtained with the single components alone. Positive synergy is observed between the HPPD herbicide and a wide variety of HST inhibitor herbicides applied both pre and postemergence across a variety of weeds.
5 2
5
5
5
75
3 8
84
27
75
45
so 141
TABLE 18. Postemergence weed control of a range of weeds. % control scores following sprays with a variety of HST inhibitors alone and in mixture with A22 (single replicate tests only) 7
75
15
75
55
5
TABLE 18 (continued). Postemergence weed control of a range of weeds. % control scores following sprays with a variety of HST inhibitors alone and in mixture with A22 (single replicate tests only) Compound 2 15 alone
625
15 625
Compound 2.15 + 2 g/ ha compound A22
-25 275
625 -25 175
15 625 -25 -25
|Compound3 5 alone
625
15 625
Compound 35 + 2 g/ ha compound A22 202 -25 -05
62 5 -25
15625 -25 -25
(compound 221 alone
625
15625
Compound 2 21 + 2 g/ ha compound A22
555 22 625
625 -25
15 625 -25 -25
Compound 2 14alone
625
15625
Compound 2 14 + 2 g/ ha compound A22
4405 2405 4575
625 15 55 52625
15625 -1 175
[Compound 2 19 alone
625
15625
Compound 2 19 + 2g/ ha compound A22
59 2 -10 25
625 6202 29 NC -2375 S 75
15625 -05
Compound 2 18 alone
625
15625
Compound218 + 2g/ha compound A22
293 33 125
625 -107
15625 -25
Compound 29 alone
625
15625
Compound 29 + 2 g/ ha compound A22 -06 -15 35 28 25
625 175 72 5
TABLE 19. Pre-emergence weed control of a range of weeds. % control scores at 14 DAT following sprays with a variety of HST inhibitors alone and in mixture with A22. |Compound 2.3 alone
625
15625
Compound 23 + 2g/ha compound A22 -15 S -306 825 28.75
625 22625 5775
15 625 NC 175 275 jcompound 28 alone
625
15625
Compound 2 8 + 2 g/ ha compound A22 -098 -287 1775 8.75
625 275 5055
15625 375
[Compound 217alone
625
15625
Compound 217 + 2g/ ha compound A22
2775 4775
62.5 -05 -05
15625 -1 jCompound 2 16 alone
625
15625
Compound 216 + 2g/ha compound A22
2 ■16 375
625 is -225 2875
15625 1 1 225
Compound 2 28 alone
625
15625
Compound 228 + 2 g/ ha compound A22
165
625 -11875 -31 25
15625 -1 -1 175
Compound 225 alone
625 1
15625
Compound 225 + 2 g/ ha compound A22
702 -595 -2 125
625 -199 11525 375
15 625 -1 -2 5 -2 5
Compound 220 alone
625
15625 1
Compound 2 20 + 2 g/ ha compound A22
-095 -108 -20875 5 25
62.5 2 67 S75
15625 16525
Compound 61 alone 2
625
15625
Compound 61 +2 g/ ha compound A22 so 7702
625 1055 26 525
TABLE 19. (continued) Prc-einergence weed control of a range of weeds . % control scores following sprays with a variety of HST inhibitors alone and in mixture with A22. The data provided in the above tables indicate that, in many cases, addition of even low (sub-lethal) levels of mesotrione improves weed control by HST inhibiting herbicides both pre and post emergence.

Claims

Claims
1. A method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide and/or hydroxyphenyl pyruvate dioxygenase (HPPD) inhibiting herbicide, wherein the crop plants comprise at least one recombinant polynucleotide which comprises a region which encodes an HST.
2. A method according to 1 , wherein the crop plants contain an additional recombinant polynucleotide which comprises a region which encodes a hydroxyphenyl pyruvate dioxygenase (HPPD).
3. A method of selectively controlling weeds at a locus comprising crop plants and weeds, wherein the method comprises application to the locus of a weed controlling amount of a pesticide composition comprising an homogentisate solanesyltransferase (HST) inhibiting herbicide, wherein the crop plants comprise at least one recombinant polynucleotide which comprises a region which encodes a HPPD enzyme.
4. A method according to any one of the previous claims, wherein the pesticide composition comprises an HST-inhibiting herbicide and a hydroxyphenyl pyruvate dioxygenase (HPPD) inhibiting herbicide
5. A method according to any one of the previous claims, wherein the HST inhibiting herbicide is selected from the group consisting of Formula (Ha), (lib), (lie), (lid), (He) and (Uf).
6. A method according to any one of claims 1, 2, 4 and 5, wherein the HPPD- inhibiting herbicide is selected from the group consisting of mesotrione, sulcotrione, isoxaflutole, tembotrione, topramezone, benzofenap, pyrazolate, pyrazoxyfen, pyrasulfotole, ketospiradox or the free acid thereof, 4-hydroxy-3- [[2-(2-methoxyethoxy)methyl] -6-(trifluoromethyl)-3 -pyridinyl] carbonyl] - bicyclo[3.2.1]oct-3-en-2-one, [2-chloro-3-(2-methoxyethoxy)-4- (methylsulfonyl)phenyl](l-ethyl-5-hydroxy-lH-pyrazol-4-yl)-methanone, α-
(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-(trifluoromethyl)- benzenepropanenitrile, and (2,3-dihydro-3,3,4-trimethyl-l,l- dioxidobenzo[b]thien-5-yl)(5-hydroxy-l-methyl-lH-pyrazol-4-yl)-methanone.
7. A method according to any one of the preceding claims, wherein the HST enzyme is derived from Arabidopsis thaliana, Glycine max, Oryza sativa or Chlamydomonas reinhardtii.
8. A method according to any one of the preceding claims, wherein the HST enzyme is selected from the group consisting of
(a) the HST enzymes depicted as SEQ ID NO. 1 to 10; and
(b) an HST which comprises one or more of the following polypeptide motifs : -
W-(R/K)-F-L-R-P-H-T-I-R-G-T; and/or N-G-(Y/F)-I-V-G-I-N-Q-I-(Y/F)-D; and/or
I-A-I-T-K-D-L-P; and/or Y-(R/Q)-(F/W)-(I/V)-W-N-L-F-Y.
9. A method according to any one of the previous claims wherein the crop plant comprises a further recombinant polynucleotide encoding a further herbicide tolerance enzyme.
10. A method according to claim 9, wherein the further herbicide tolerance enzyme is selected from the group consisting of, 5-enolpyravylshikimate-3- phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT),
Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPGO), Pliytoene desaturase (PD), dicamba degrading enzymes and aryloxy herbicide degrading enzymes.
11. A method according to any one of the previous claims, wherein the pesticide composition comprises one or more additional herbicides.
12. A method according to claim 11, wherein the one or more additional herbicides is selected from the group consisting of glyphosate (including agrochemically acceptable salts thereof); glufosinate (including agrochemically acceptable salts thereof); chloroacetanilides e.g alachlor, acetochlor, metolachlor, S-metholachlor; photo system II inhibitors e.g triazines such as ametryn, atrazine, cyanazine and terbuthylazine, triazinones such as hexazinone and metribuzin, and ureas such as chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; ALS -inhibitors e.g sulfonyl ureas such as amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, primisulfuron, prosulfuron, rimsulfuron, triasulfuron, trifloxysulfuron and tritosulfuron; diphenyl ethers e.g aciflurofen and fomesafen.
13. A method according to claim 12, wherein the one or more additional herbicides is glyphosate and/or a PS-II inhibiting herbicide.
14. A method according to any one of the preceding claims, further comprising application to the locus of an insecticide and/or a fungicide.
15. A recombinant polynucleotide which comprises a region which encodes an
HST-enzyme operably linked to a plant operable promoter, wherein the region which encodes an HST-enzyme does not include the polynucleotide sequence depicted in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 14 or SEQ ID NO. 15.
16. A recombinant polynucleotide according to claim 15, wherein the HST is selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO.10.
17. A recombinant polynucleotide comprising (i) a region which encodes a HST enzyme operably linked to a plant operable promoter and (ii) at least one additional region, which encodes a herbicide tolerance enzyme selected from the group consisting of hydroxyphenyl pyruvate dioxygenase (HPPD), 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase
(PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPGO), Phytoene desaturase (PD), dicamba degrading enzymes), and aryloxy herbicide degrading enzymes, operably linked to a plant operable promoter.
18. A recombinant polynucleotide according to claim 17, comprising (i) a region which encodes a HST enzyme and (ii) at least one additional region which encodes an HPPD.
19. A recombinant polynucleotide according to claim 17, comprising at least two additional regions encoding a herbicide tolerance enzyme.
20. A recombinant polynucleotide according to claim 19, wherein the polynucleotide comprises (i) a region which encodes a HST enzyme, (ii) a region which encodes a HPPD enzyme and (iii) a region which encodes a glyphosate tolerance enzyme.
21. A vector comprising a recombinant polynucleotide according to any one of claims 15 to 20.
22. A plant cell which is tolerant to an HST-inhibiting herbicide and/or an HPPD- inhibiting herbicide - said plant cell comprising a recombinant polynucleotide according to claims 15 to 20.
23. A plant cell according to claim 22, wherein the plant is selected from corn, soybean, wheat, barley, sugar beet, rice and sugarcane.
24. A HST-inhibitor and/or HPPD-inhibitor tolerant plant which comprises a plant cell according to claim 22 or claim 23.
25. Seed, or material derived therefrom, comprising a plant cell according to claim
22.
26. A method of providing a transgenic plant which is tolerant to HST-inhibiting and/or HPPD-inhibiting herbicides which comprises transformation of plant material with a recombinant polynucleotide which comprises a region which encodes an HST and, optionally, a region encoding a HPPD, selection of the transformed plant material using an HST-inhibitor and/or an HPPD inhibitor, and regeneration of that material into a morphological normal fertile plant.
27. A method according to 26, wherein the recombinant polynucleotide further comprises a region encoding the target for a non-HST inhibitor herbicide and/or a region encoding a protein capable of conferring on plant material transformed with the region resistance to insects, fungi and/or nematodes.
28. A morphologically normal fertile whole plant obtained by the method of any one of claims 26 or 27.
29. Use of a recombinant polynucleotide which comprises a region which encodes an HST enzyme as a selectable marker in plant transformation.
30. Use of a recombinant polynucleotide comprising a region which encodes an
HST enzyme in the production of plants which are tolerant to herbicides which act wholly or in part by inhibiting HST.
31. Use of HST inhibitors as selection agents in plant transformation
32. Use of a recombinant HST enzyme in in vitro screening of potential herbicides.
33. A herbicidal composition comprising a HPPD-inhibiting herbicide and a HST- inhibiting herbicide.
34. A herbicidal composition according to claim 33, wherein the HPPD-inhibiting herbicide is as defined in claim 6.
35. A herbicidal composition according to claim 33 or claim 34, wherein the HST- inhibiting herbicide is as defined in claim 5.
36. A herbicide composition according to any one of claim 33 to 35, wherein the molar ratio of the HPPD-inhibiting herbicide to the HST-inhibiting herbicide in the composition is from 100:1 to 1:100.
37. A herbicide composition according to any one of claim 36, wherein the molar ratio of the HPPD-inhibiting herbicide to the HST-inhibiting herbicide in the composition is from 1 :1 to 1 :20.
38. A herbicidal composition according to any one of claims 33 to claim 37, further comprising one or more additional pesticidal ingredient(s).
39. A herbicidal composition according to claim 38, wherein the one or more additional pesticidal ingredient(s) comprises a herbicide.
40. A herbicidal composition according to claim 39, wherein the additional herbicide is selected from the group consisting of glyphosate (including agrochemically acceptable salts thereof); glufosinate (including agrochemically acceptable salts thereof); chloroacetanilides e.g alachlor, acetochlor, metolachlor, S-metholachlor; photo system II inhibitors e.g triazines such as ametryn, atrazine, cyanazine and terbuthylazine, triazinones such as hexazinone and metribuzin, and ureas such as chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; ALS -inhibitors e.g sulfonyl ureas such as amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, primisulfuron, prosulfuron, rimsulfuron, triasulfuron, trifloxysulfuron and tritosulfuron; diphenyl ethers e.g aciflurofen and fomesafen.
41. A herbicidal composition according to claim 40, wherein the additional is herbicide selected from the group consisting of glyphosate, glufosinate, atrazine and S-metolachlor.
42. A method of selectively controlling weeds at a locus comprising crop plants and weeds comprising applying to the locus a weed controlling amount of a herbicidal composition according to any one of claims 33 to 41.
43. A method according to claim 42, wherein the HPPD-inhibiting herbicide present in the composition is applied to the locus at a rate which is normally sub-lethal to the weeds when the HPPD-inhibiting herbicide is applied alone.
44. A method according to claim 43, wherein the HST-inhibiting herbicide is applied from 10 to 2000 g/ha and the HPPD-inhibiting herbicide is applied from 5 to 1000 g ai/ha.
45. A method according to any one of claims 42 to 44, wherein the crop plants comprise at least one recombinant polynucleotide which comprises a region which encodes one or more herbicide tolerance enzymes which provide increased tolerance to the herbicides comprised within the herbicidal composition.
46. A method according to claim 45, wherein the herbicide tolerance enzyme is selected from the group consisting of HST, HPPD, EPSPS, GAT, Cytochrome
P450, PAT, ALS, PPGO, Phytoene desaturase (PD) and dicamba degrading enzymes.
47. A method according to any one of claims 42 to 46, wherein the crop plants are selected from the group consisting of corn, wheat, barley, rice, sorghum, soybean, sugar beet and sugar cane.
48. Use of a sub-lethal application of an HPPD-inhibiting herbicide to increase the weed controlling efficacy of an HST-inhibiting herbicide.
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