WO2013092555A1 - Plantes tolérantes à des herbicides inhibiteurs de hppd - Google Patents

Plantes tolérantes à des herbicides inhibiteurs de hppd Download PDF

Info

Publication number
WO2013092555A1
WO2013092555A1 PCT/EP2012/075906 EP2012075906W WO2013092555A1 WO 2013092555 A1 WO2013092555 A1 WO 2013092555A1 EP 2012075906 W EP2012075906 W EP 2012075906W WO 2013092555 A1 WO2013092555 A1 WO 2013092555A1
Authority
WO
WIPO (PCT)
Prior art keywords
hppd
plants
seq
protein
plant
Prior art date
Application number
PCT/EP2012/075906
Other languages
English (en)
Inventor
Fabien Poree
Bernd Laber
Nathalie Knittel-Ottleben
Gudrun Lange
Arno Schulz
Rüdiger Hain
Original Assignee
Bayer Intellectual Property Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Intellectual Property Gmbh filed Critical Bayer Intellectual Property Gmbh
Publication of WO2013092555A1 publication Critical patent/WO2013092555A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Definitions

  • the present invention relates to nucleic acid sequences encoding a
  • HPPD hydroxyphenylpyruvate dioxygenase
  • HPPDs are enzymes which catalyse the reaction in which para- hydroxyphenylpyruvate (abbreviated herein as HPP), a tyrosine degradation product, is transformed into homogentisate (abbreviated herein as HG), the precursor in plants of tocopherol and plastoquinone (Crouch N.P. et al. (1997), Tetrahedron, 53, 20,
  • Tocopherol acts as a membrane-associated antioxidant. Plastoquinone, firstly acts as an electron carrier between PSII and the cytochrome b6/f comple and secondly, is a redox cofactor for phytoene desaturase, which is involved in the biosynthesis of carotenoids.
  • nucleic acid sequences from various organisms present in the NCBI database were annotated as coding for a putative protein having an HPPD domain including the sequence disclosed under the C1 B587 accession number given in the UniProtKB/TrEMBL database as well as under the YP_002779958 accession number given in the NCBI protein database, as well as under BAH51013 accession number given in the EMBL protein database.
  • HPPD proteins and their primary sequences have been described in the state of the art, in particular the HPPD proteins of bacteria such as Pseudomonas (Ruetschi et al., Eur. J. Biochem., 205, 459-466, 1992, WO 96/38567), Kordia
  • WO201 1076892 of protists such as Blepharisma (WO201 1076882), of euryarchaeota such as Picrophilus (WO201 1076885) of plants such as Arabidopsis (WO 96/38567, Genebank AF047834), carrot (WO 96/38567, Genebank 87257), Avena sativa (WO 02/046387), wheat (WO 02/046387), Brachiaria platyphylla (WO 02/046387),
  • Cenchrus echinatus (WO 02/046387), Lolium rigidum (WO 02/046387), Festuca arundinacea (WO 02/046387), Setaria faberi (WO 02/046387), Eleusine indica (WO 02/046387), Sorghum (WO 02/046387), Coccicoides (Genebank COITRP),of Coptis japonica (WO 06/132270), Chlamydomonas reinhardtii (ES 2275365), or of mammals such as mouse or pig.
  • the corresponding sequences disclosed in the indicated references are hereby incorporated by reference.
  • HPP is a tyrosine precursor, and it is synthesized by the action of an enzyme, prephenate dehydrogenase (hereinafter referred to as PDH), which converts prephenate to HPP (Lingens et al. (1967) European J. Biochem 1 : 363-374; Sampathkumar and Morrisson (1982), Bioch Biophys Acta 701 : 204-21 1 ).
  • PDH prephenate dehydrogenase
  • HPPD inhibitor herbicides belong to one of these six chemical families:
  • the triketones e.g. sulcotrione [i.e. 2-[2-chloro-4-(methylsulfonyl)benzoyl]-1 ,3- cyclohexanedione], mesotrione [i.e. 2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1 ,3- cyclohexanedione]; tembotrione [i.e. 2-[2-chloro-4-(methylsulfonyl)-3-[(2,2,2,-tri- fluoroethoxy)methyl] benzoyl]-1 ,3-cyclo-hexanedione]; tefuryltrione [i.e.
  • isoxazoles e.g. isoxaflutole [i.e.(5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)- 4-(trifluoromethyl)phenyl]methanone].
  • isoxaflutole is rapidly metabolized in plants.
  • pyrazolinates e.g. topramezone [i.e. [3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4- (methylsulfonyl) phenyl](5-hydroxy-1 -methyl-1 H-pyrazol-4-yl)methanone], and pyrasulfotole [i.e. (5-hydroxy-1 ,3-dimethylpyrazol-4-yl(2-mesyl-4- trifluaromethylphenyl)methanone]; pyrazofen [i.e. 2-[4-(2,4-dichlorobenzoyl)-1 ,3- dimethylpyrazol-5-yloxy]acetophenone]. 5) N-(1 ,2,5-oxadiazol-3-yl)benzamides; and
  • HPPD-inhibiting herbicides can be used against grass and/or broad leaf weeds in crop plants that display metabolic tolerance, such as maize (Zea mays) in which they are rapidly degraded (Schulz et al. (1993). FEBS letters, 318, 162-166; Mitchell et al. (2001 ) Pest Management Science, Vol 57, 120-128; Garcia et al. (2000)
  • a third strategy was to mutate the HPPD in order to obtain a target enzyme which, while retaining its properties of catalysing the transformation of HPP into
  • homogentisate is less sensitive to HPPD inhibitors than is the native HPPD before mutation.
  • Gly336lle, and more particularly Gly336Trp positions of the mutated amino acid are indicated with reference to the Pseudomonas HPPD
  • Gly336Trp positions of the mutated amino acid are indicated with reference to the Pseudomonas HPPD
  • the inventors have sought to increase the prenylquinone biosynthesis (e.g., synthesis of plastoquinones, tocopherols) in the cells of plants by increasing the flux of the HPP precursor into the cells of these plants. This has been done by connecting the synthesis of said precursor to the "shikimate" pathway by overexpression of a PDH enzyme. They have also noted that the transformation of plants with a gene encoding a PDH enzyme makes it possible to increase the tolerance of said plants to HPPD inhibitors.
  • prenylquinone biosynthesis e.g., synthesis of plastoquinones, tocopherols
  • HPPD protein Pseudomonas fluorescens HPPD protein and its use for obtaining plants which are tolerant to HPPD inhibitor herbicides is disclosed.
  • a method to generate plants tolerant to HPPD inhibitors by overexpressing not only a gene coding for a tolerant HPPD, as for example from Avena sativa, but also in combination with several plant genes coding for an HST (homogentisate solanesyltransferase) protein.
  • HST homogentisate solanesyltransferase
  • HPPD inhibitors belonging to the classes of the triketones e.g.sulcotrione, mesotrione, tembotrione, benzobicyclon and bicyclopyrone
  • the pyrazolinates e.g., topramezone and pyrasulfotole
  • N-(1 ,2,5-Oxadiazol-3-yl)benzamides WO
  • the present invention therefore relates to the generation of transgenic plants containing a gene encoding an HPPD protein obtainable or obtained from an organism belonging to Rhodococcus opacus, and variants or mutants thereof, more especially to a gene from an organism belonging to the strain Rhodococcus opacus B4, variants or mutants thereof coding for an HPPD enzyme showing the properties of catalysing the conversion of para-hydroxyphenylpyruvate to homogentisate and which plants are less sensitive to one or more HPPD inhibitors compared to plants not containing any such HPPD encoding transgene.
  • genes from rhodococcus opacuscoding for HPPD proteins were selected as excellent HPPD-inhibitor tolerant candidates due to their high divergences in the amino acids composition at positions relevant for HPPD inhibitor tolerance as determined experimentally and structurally in the HPPD protein compared to the sensitive Arabidopsis thaliana HPPD protein which was taken as the HPPD-inhibitor herbicide sensitive reference molecule.
  • the present invention therefore relates to the generation of transgenic plants containing a gene obtainable or obtained from an organism belonging to Rhodococcus opacus, especially from the strain Rhodococcus opacus B4, variants or mutants thereof, coding for an HPPD enzyme showing the properties of catalysing the conversion of para-hydroxyphenylpyruvate to homogentisate and which are less sensitive to one or more HPPD inhibitors compared to plants not containing any such HPPD transgene.
  • this invention relates to an HPPD protein named herein "the HPPD protein of this invention” or "the rhodococcus opacus HPPD protein", which is an HPPD protein with at least 96 %; at least 97 %; at least 98 %, or at least 99 % amino acid sequence identity to the amino acid sequence of SEQ ID No. 17 from amino acid position 2 to 401 , particularly to the amino acid sequence of any one of SEQ ID Nos. 17, 18, 19, 20, 21 , and 22 preferably SEQ ID No. 22.
  • the invention relates to an HPPD protein named herein "the HPPD protein of this invention” or "the rhodococcus opacus HPPD protein", which is an HPPD protein with at least 96 %; at least 97 %; at least 98 %, or at least 99 % amino acid sequence identity to the amino acid sequence of SEQ ID No. 17 from amino acid position 2 to 401 , particularly to the amino acid sequence of any one of SEQ ID Nos. 17, 18, 19, 20, 21 , and 22 preferably SEQ ID No. 22, and in which any amino acids from position 207 to position 401 of SEQ ID No. 17 can be amended by any naturally occurring amino acid, preferably it can be any conservative substitution.
  • the invention relates to an HPPD protein named herein "the HPPD protein of this invention” or "the rhodococcus opacus HPPD protein", which is an HPPD protein with at least 96 %; at least 97 %; at least 98 %, or at least 99 % amino acid sequence identity to the amino acid sequence of SEQ ID No. 17 from amino acid position 2 to 401 , particularly to the amino acid sequence of any one of SEQ ID Nos. 17, 18, 19, 20, 21 , and 22 preferably SEQ ID No. 22, and having one or more of the following amino acids at the position defined by its number (relating to the number of SEQ ID No. 17) given in brackets, i.e. His(205), Ser(248), Asn(263), Gln(287), His(288), Tyr(317), Gln(354), Phe(367), Glu(369), Gly(380), and Asn(383).
  • the HPPD protein of this invention or "the rhodococcus
  • the invention relates to an HPPD protein named herein "the HPPD protein of this invention” or "the rhodococcus opacus HPPD protein", which is an HPPD protein with at least 96 %; at least 97 %; at least 98 %, or at least 99 % amino acid sequence identity to the amino acid sequence of SEQ ID No. 17 from amino acid position 2 to 401 , particurlarly to the amino acid sequence of any one of SEQ ID Nos. 17, 18, 19, 20, 21 , and 22 preferably SEQ ID No. 22, and at the respective positions given in the second column of Table (i) the originally occuring amino acids can substituted by any of the amino acids listed in column 3 of Table (i).
  • Ala 232 Trp lie, Leu, Ser, Arg, Lys, His, Asp, Glu, Pro, Gly, Asn
  • the invention relates to an HPPD protein named herein "the HPPD protein of this invention” or "the rhodococcus opacus HPPD protein", which is an HPPD protein with at least 96 %; at least 97 %; at least 98 %, or at least 99 % amino acid sequence identity to the amino acid sequence of SEQ ID No. 17 from amino acid position 2 to 401 , particurlarly to the amino acid sequence of any one of SEQ ID Nos. 17, 18, 19, 20, 21 , and 22 preferably SEQ ID No. 22 and at the respective positions given in the second column of Table (ii) the originally occuring amino acids can substituted by any of the amino acids listed in column 3 of Table (ii).
  • the invention relates to an HPPD protein named herein "the HPPD protein of this invention” or "the rhodococcus opacus HPPD protein", which is an HPPD protein with at least 96 %; at least 97 %; at least 98 %, or at least 99 % amino acid sequence identity to the amino acid sequence of SEQ ID No. 17 from amino acid position 2 to 401 , particurlarly to the amino acid sequence of any one of SEQ ID Nos. 17, 18, 19, 20, 21 , and 22 preferably SEQ ID No. 22, and at the respective positions given in the second column of Table (iii) the originally occuring amino acids can substituted by any of the amino acids listed in column 3 of Table (iii).
  • This invention includes a protein with amino acids substituted, deleted or added compared to the sequence of SEQ ID No. 17 from amino acid position 2 to amino acid position 401 , such as a transit peptide fusion protein, or a protein with amino acid changes in the sequence of SEQ ID No. 17 that retains the enzymatic function of an HPPD protein, and that still confers HPPD tolerance when expressed in plants, preferably HPPD tolerance of comparable range to that conferred by the protein of SEQ ID No. 17.
  • pyrazolinates particularly any one of mesotrione, tembotrione, isoxaflutole or bicyclopyrone is applied on such plants, more particularly when applied post- emergence.
  • This also includes a protein comprising an active portion of the sequence of SEQ ID No.17, which portion confers HPPD inhibitor tolerance when expressed in plants.
  • This includes a protein with substantially the same amino acid sequence as the sequence of SEQ ID No.17, such as a protein with the amino acid sequence of any one of SEQ ID Nos. 17 to 22.
  • HPPD proteins of this invention are HPPD proteins comprising the amino acid sequence of SEQ ID No. 17 from amino acid position 2 to 401 , but wherein 1 -20, 1 -15, 1 -10, or 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acids have been deleted or have been substituted by other amino acids, particularly such protein which retains HPPD enzymatic activity and which confers tolerance to HPPD inhibitor herbicides when expressed in a host plant.
  • HPPD proteins with at least 88 % sequence identity to SEQ ID No. 17 which are encoded by a DNA sequence found in the genome sequence of a microorganism, particularly a microorganism of rhodococcus opacus, especially from the strain rhodococcus opacus B4 .
  • an HPPD protein of this invention is a HPPD protein which confers herbicide tolerance to plants when expressed in such plants, wherein such tolerance is observed to one or more HPPD inhibitor selected from the group consisting ofmesotrione, tembotrione, topramezone, pyrasulfotole,
  • HPPD protein is a HPPD rhodococcus opacus protein, such as a protein comprising the sequence of SEQ ID No. 17 from amino acid position 2 to 401 . This includes the mutant or variant HPPD proteins as described further below.
  • the present invention includes and provides an antibody capable of specifically binding a substantially purified protein comprising an amino acid sequence selected from the group consisting of SEQ ID Nos. 17, 18, 19, 20, 21 , and 22 preferably SEQ ID No. 22, or derived sequences thereof according to amino acid replacement as disclosed in one or more of tables (i), (ii), or (iii), above.
  • a further aspect of the invention concerns antibodies, single-chain antigen binding molecules, or other proteins that specifically bind to one or more of the protein or peptide molecules of the invention and their homologs, fusions or fragments.
  • the antibody specifically binds to a protein having the amino acid sequence set forth in SEQ ID Nos. 17-22 or a fragment thereof, or derived sequences thereof according to amino acid replacement as disclosed in one or more of tables (i), (ii), or (iii), above.
  • the antibody specifically binds to a fusion protein comprising an amino acid sequence selected from the amino acid sequence set forth in SEQ ID Nos. 17-22 or a fragment thereof. In another embodiment the antibody specifically binds to a fusion protein comprising an amino acid sequence selected from the amino acid sequence set forth in SEQ ID Nos. 17-22 or a fragment thereof, or derived sequences thereof according to amino acid replacement as disclosed in one or more of tables (i), (ii), or (iii), above.
  • Antibodies of the invention may be used to quantitatively or qualitatively detect the protein or peptide molecules of the invention, or to detect post translational modifications of the proteins. As used herein, an antibody or peptide is said to
  • this invention relates to an HPPD nucleic acid or DNA, named herein "the HPPD nucleic acid/DNA of this invention", which is a nucleic acid or DNA encoding an HPPD of this invention as defined above.
  • This includes a DNA which comprises a nucleotide sequence selected from the group consisting of the sequence of SEQ ID No. 1 from nucleotide position 4 to nucleotide position 1206, the sequence of SEQ ID No. 2 from nucleotide position 7 to nucleotide position 1209, or the sequence of SEQ ID No. 3 from nucleotide position 139 to nucleotide position 1341 , or the sequence of SEQ ID No.
  • nucleotide position 382 to nucleotide position 1584 or the sequence of SEQ ID No. 15 from nucleotide position 379 to nucleotide position 1581 , or the sequence of SEQ ID No. 16 from nucleotide position 376 to nucleotide position 1578 , or the sequence of SEQ ID No. 29 from nucleotide position 4 to 1206, or the sequence of SEQ ID No. 30 from nucleotide position 4 to 1206, or the sequence of SEQ ID No. 31 from nucleotide position 4 to 1206, or the sequence of SEQ ID No. 32 from nucleotide position 4 to 1206, or the sequence of SEQ ID No.
  • the nucleic acid constituting the test sequence preferably has a TM
  • the TM values of the sequences are preferably within 5° C of each other. More preferably the hybridization is performed under relatively stringent hybridization conditions as defined below.
  • a denatured test or inventive sequence is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of between 60 and 65° C in 5xSSC containing 0.1 % SDS followed by rinsing of the support at the same temperature but with O. l xSSC.
  • the hybridization involves a fragment of the sequence selected from the group consisting of of SEQ ID Nos. 1 , 2, 3, 12, 15, 16, 29, 30, 31 , 32, 33, 34, 35, 36, and 37the hybridization conditions may be less stringent, as will be obvious to the skilled person.
  • HPPD DNA of this invention are DNA sequences encoding an HPPD protein of the invention which DNA sequences have been adapted for expression in microorganisms or plants, such as by replacing native codons by codons more preferred in a host cell, or wherein certain restriction sites have been added or removed for ease of cloning, or DNA sequence with a certain number of added, replaced or deleted nucleotides.
  • This also includes isolated DNA sequences and variant, mutant or synthetic DNAs or nucleic acids as described further below.
  • the Rhodococcus opacus HPPD DNA of this invention is expressed in plants under the control of a promoter that allows expression of exogenous genes in plants.
  • a signal transit peptide such as a transit peptide is located, preferably a plastid transit peptide, such as a chloroplast transit peptide of about 30 to 125 amino acids, preferably 125 amino acids, most preferably present as most preferably a double transit peptide, such as an optimized transit peptide of which the first part is originated from sunflower (Helianthus annuus) and the second part from Zea mays (described in US patent 5,188,642) or a plastid transit peptide of that of the plant ribulose biscarboxylase/oxygenase small subunit (RuBisCO ssu), where appropriate including a few amino acids of the N-terminal part of the mature RuBisCO sss
  • this invention includes a DNA encoding an HPPD protein of this invention which is derived or is obtainable from SEQ ID No. 1 and is optimized for the expression in E. coli, such as a codon-optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 2 from nucleotide position 7 to nucleotide position 1209 (including the positions defined).
  • this invention includes a DNA encoding an HPPD protein of this invention which is derived from SEQ ID No. 1 and is optimized for the expression in plants, such as a codon-optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 15 from nucleotide position 379 to nucleotide position 1581 (including the positions defined).
  • this invention includes a DNA encoding an HPPD protein of this invention which is derived from SEQ ID No. 1 and is optimized for the expression in plants, such as a codon-optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 37 from nucleotide position 376 to nucleotide position 1578 (including the positions defined).
  • this invention includes a DNA encoding an HPPD protein of this invention which is derived from SEQ ID No. 1 and is optimized for the expression in plants, such as a codon-optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 29, 30, 31 , 32, 33, 34, 35, or 36 from nucleotide position 4 to nucleotide position 1206 (including the positions defined).
  • the HPPD of the invention such as the HPPD comprising the amino acid sequence of SEQ ID No. 17 from amino acid position 2 to amino acid position 401 , or the HPPD comprising the amino acid sequence of any one of SEQ ID Nos.
  • HPPD inhibitor herbicides selected from the group consisting of isoxazoles, diketonitriles, triketones N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol- 4-yl)- or N-(triazol-3-yl)arylcarboxamides, and pyrazolinates, or or preferably less sensitive to one or more HPPD inhibitor herbicide(s) selected from the group consisting of isoxaflutole, tembotrione, mesotrione, sulcotrione, pyrasulfotole, topramezone, 2-cyano-3-cyclopropyl-1 -(2-S02CH3-4-CF3phenyl)propane-1 ,3-dione and 2-cyano-3-cyclopropyl-1 -(2-S02CH 3 -4-2,3 C phenyl)propane-1 ,3-dione
  • this invention includes a DNA encoding an HPPD protein of this invention which is optimized for the expression in E. coli, such as a codon-optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 2 from nucleotide position 7 to nucleotide position 1209 (including the positions defined) which encodes an HPPD less sensitive than the host plant endogenous HPPD to at least one HPPD inhibitor herbicide selected from the group consisting of isoxazoles, diketonitriles, triketones, N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N- (triazol-3-yl)arylcarboxamides, and pyrazolinates, preferably selected from the group consisting of tembotrione, mesotrione, bicyclopyrone, tefuryltrione, isoxaflutole, diketonitrile, pyrasulfoto
  • this invention includes a DNA encoding an HPPD protein of this invention which is optimized for the expression in plants, such as a codon-optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 15 from nucleotide position 379 to nucleotide position 1581 (including the positions defined) which encodes an HPPD less sensitive than the host plant endogenous HPPD to at least one HPPD inhibitor herbicide selected from the group consisting of isoxazoles, diketonitriles, triketones, N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol- 4-yl)- or N-(triazol-3-yl)arylcarboxamides or pyrazolinates, preferably selected from the group consisting of tembotrione, mesotrione, bicyclopyrone, tefuryltrione, isoxafiutole, diketonitrile, pyrasulfotole
  • this invention includes a DNA encoding an HPPD protein of this invention which is optimized for the expression in plants, such as a codon-optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 29, 30, 31 , 32, 33, 35 or 36 from nucleotide position 4 to nucleotide position 1206
  • HPPD inhibitor herbicide selected from the group consisting of isoxazoles, diketonitriles, triketones, N-(1 ,2,5-oxadiazol-3- yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamides or pyrazolinates, preferably selected from the group consisting of tembotrione, mesotrione,
  • bicyclopyrone bicyclopyrone, tefuryltrione, isoxafiutole, diketonitrile, pyrasulfotole, topramezone, sulcotrione, pyrazolate and benzofenap.
  • this invention includes a DNA encoding an HPPD protein of this invention which is optimized for the expression in plants, such as a codon-optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 37 from nucleotide position 376 to nucleotide position 1578 (including the positions defined) which encodes an HPPD less sensitive than the host plant endogenous HPPD to at least one HPPD inhibitor herbicide selected from the group consisting of isoxazoles, diketonitriles, triketones, N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol- 4-yl)- or N-(triazol-3-yl)arylcarboxamides or pyrazolinates, preferably selected from the group consisting of tembotrione, mesotrione, bicyclopyrone, tefuryltrione, isoxafiutole, diketonitrile, pyrasulfotole
  • this invention relates to plants, plant parts, plant cells, and progenies of these plants comprising a DNA encoding an HPPD protein of the invention which is optimized for the expression in E. coli, or is optimized for the expression in plants such as a codon-optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 2 from nucleotide position 7 to nucleotide position 1209 (including the positions defined) or of SEQ ID No. 15 from nucleotide position 379 to nucleotide position 1581 1581 or of SEQ ID Nos. 29, 30, 31 , 32, 33, 34, 35, or 36 from nucleotide position 4 to 1206 (including the positions defined) or of SEQ ID No.
  • nucleotide position 376 to 1578 which encodes an HPPD less sensitive than the host plant endogenous HPPD to at least one HPPD inhibitor herbicide selected from the group consisting of isoxazoles, diketonitriles, triketones, N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, and pyrazolinates, preferably selected from the group consisting of tembotrione, mesotrione, bicyclopyrone, tefuryltrione, isoxaflutole, diketonitrile, pyrasulfotole, topramezone, sulcotrione, pyrazolate and benzofenap.
  • HPPD inhibitor herbicide selected from the group consisting of isoxazoles, diketonitriles, triketones, N-(1 ,2,5-oxadiazol-3-
  • Such plants include but are not limited to field crops, fruits and vegetables such as canola, sunflower, tobacco, sugarbeet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, 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.
  • fruits and vegetables such as canola, sunflower, tobacco, sugarbeet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, soya spp, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses,
  • this invention relates to plants, plant parts, plant cells, and progenies of these plants comprising a DNA encoding an HPPD protein of the invention which is optimized for the expression in E. coli, or optimized for the expression in plants such as a codon-optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 2 from nucleotide position 7 to nucleotide position 1209 (including the positions defined) or of SEQ ID No. 15 from nucleotide position 379 to nucleotide position 1589 (including the positions defined) of SEQ ID No.
  • nucleotide position 4 to nucleotide position 1206 (including the positions defined), or of SEQ ID No. 37 from nucleotide position 376 to nucleotide position 1578 (including the positions defined) which encodes an HPPD less sensitive than the host plant endogenous HPPD to at least one HPPD inhibitor herbicide selected from the group consisting of isoxazoles, diketonitriles, triketones, N-(1 ,2,5- oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamides, and pyrazolinates, preferably selected from the group consisting of tembotrione, mesotrione, bicyclopyrone, tefuryltrione, isoxaflutole, diketonitrile, pyrasulfotole, topramezone, sulcotrione, pyr
  • HPPD inhibitor herbicide selected from the group consisting of
  • the HPPD protein of the invention comprises the sequence of SEQ ID No. 20, SEQ ID No. 21 or SEQ ID No. 22 all of which are less sensitive to one or more HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, triketones (named triketone HPPD inhibitor), such as
  • tembotrione sulcotrione mesotrione, bicyclopyrone, tefuryltrione, particularly tembotrione, or from the group consising of diketonitriles (like isoxafiutoie) or from the group consisting of pyrazolinates (named pyrazolinate HPPD inhibitor), such as pyrasulfotole, pyrazolate, topramezone, benzofenap compared to the endogenous unmutated HPPD of a plant, particularly the host plant wherein such HPPD of the invention is expressed or is to be expressed.
  • pyrazolinate HPPD inhibitor such as pyrasulfotole, pyrazolate, topramezone, benzofenap compared to the endogenous unmutated HPPD of a plant, particularly the host plant wherein such HPPD of the invention is expressed or is to be expressed.
  • the enzymatic activity of HPPD proteins can be measured by any method that makes it possible either to measure the decrease in the amount of the HPP or O2 substrates, or to measure the accumulation of any of the products derived from the enzymatic reaction, i.e. homogentisate or CO2.
  • the HPPD activity can be measured by means of the method described in Garcia et al. (1997), Biochem. J. 325, 761 -769 or Garcia et al. (1999), Plant Physiol. 1 19, 1507-1516, which are incorporated herein by reference.
  • an HPPD inhibitor of the class/group of triketones means an HPPD inhibitor having a triketone skeleton.
  • triketone HPPD inhibitor one can cite the molecules sulcotrione [i.e.
  • an HPPD of the class of pyrazolinates means an HPPD inhibitor having a pyrazole radical.
  • pyrazolinates HPPD inhibitor one can cite the molecules topramezone [i.e. [3- (4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1 -methyl-1 H- pyrazol-4-yl)methanone] and pyrasulfotole [(5-hydroxy-1 ,3-dimethylpyrazol-4-yl(2- mesyl-4-trifluaromethylphenyl)methanone].
  • the present invention also relates to a nucleic acid sequence, particularly an isolated DNA, preferably a plant-expressible chimeric gene, which encodes the Rhodococcus opacus HPPD of the invention and adapted sequences thereof.
  • the present invention also relates to a nucleic acid sequence encoding an HPPD enzyme of this invention which retains its properties of catalysing the conversion of para-hydroxyphenylpyruvate to homogentisate and which is less sensitive to one or more HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5- oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamides, triketones, such as tembotrione, sulcotrione and mesotrione, or from the group consisting of pyrazolinates such as pyrasulfotole and topramezone, tefuryltrione, bicyclopyrone, benzobicyclon than the endogenous unmutated plant HPPD, and of which the encoded amino acid sequence shows a sequence identity to SEQ ID No. 17 of at least 96 %, particularly at least 97%, more
  • the nucleic acid sequence of the invention encodes an HPPD enzyme which is less sensitive to one or more HPPD inhibitor herbicides belonging to the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol- 4-yl)- or N-(triazol-3-yl)arylcarboxamides, triketones, such as tembotrione, sulcotrione, mesotrione, bicyclopyrone, and tefuryltrione, belonging to the group consisting of isoxazoles, such as isoxaflutole, belonging to the group consisting of pyrazolinates (named pyrazolinate HPPD inhibitor), such as pyrasulfotole, pyrazolate, topramezone, benzofenap, and/or belonging to the group consisting of diketones, such as diketonitrile than the host plant endogenous HPPD.
  • HPPD inhibitor herbicides belonging to the group consisting
  • nucleic acid sequence is understood as being a nucleotide sequence which can be of the DNA or RNA type, preferably of the DNA type, and in particular double-stranded, whether it be of natural or synthetic origin, in particular a DNA sequence in which the codons which encode the HPPD according to the invention have been optimized in accordance with the host organism in which it is to be expressed (e.g., by replacing codons with those codons more preferred or most preferred in codon usage tables of such host organism or the group to which such host organism belongs, compared to the original or source organism).
  • isolated nucleic acid/DNA/protein refers to a nucleic acid
  • acid/DNA/protein which is not naturally occurring (such as an artificial or synthetic DNA with a different nucleotide sequence than the naturally occurring DNA, or a modified protein) or which is no longer in the natural environment wherein it was originally present, e.g., a DNA coding sequence associated with a heterologous regulatory element (such as a bacterial coding sequence operably linked to a plant- expressible promoter) in a chimeric gene, a DNA transferred into another host cell, such as a transgenic plant cell.
  • a heterologous regulatory element such as a bacterial coding sequence operably linked to a plant- expressible promoter
  • the tolerance level measurement is analyzed using the method extensively described in WO 2009/14407 as described below using a N-(1 ,2,5-oxadiazol-3-yl)benzamides, N- (tetrazol-4-yl)- or N-(triazo!-3-yl)arylcarboxamides, triketone, an isoxazole, or a pyrazolinate HPPD inhibitor, particularly less sensitive to one or more HPPD inhibitor herbicide(s) selected from the group consisting of tembotrione, meso
  • the coding regions encoding HPPD comprise a nucleotide sequence encoding proteins with the amino acid sequences as set forth in SEQ ID Nos 17, 18, 19, 20, 21 , and 22 such as the nucleotide sequences of SEQ ID Nos 1 , 2, 3, 12, 15, 16, 29, 30, 31 , 32, 33, 34, 35, 36, and 37.
  • variants of these nucleotide sequences including insertions, deletions and substitutions thereof may be also be used to the same effect.
  • homologues to the mentioned nucleotide sequences from species different from Blepharisma can be used.
  • a protein with "substantially the same amino acid sequence" to a protein as described in present invention refers to a protein with at least 96 %, particularly at least 97 %, preferably at least 98 % sequence identity with a protein according to the invention, wherein the percentage sequence identity is determined by using the "blosum62 scoring matrix" in the GAP program of the Wisconsin package of GCG (Madison, Wisconsin, USA), version 10.0 (GCG defaults used).
  • GCG Garnier scoring matrix
  • sequence identity when related to DNA sequences, is determined by using the "nwsgapdna scoring matrix" in the GAP program of the Wisconsin package of GCG (Madison, Wisconsin, USA), version 10.0 (GCG defaults used).
  • sequence identity of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (x100) divided by the number of positions compared.
  • a gap i.e. a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues.
  • the alignment of the two sequences is performed by the
  • Nucleotide sequences homologous to the nucleotide sequences encoding an HPPD enzyme according to the invention may be identified by in silico analysis of genomic sequence data.
  • Homologous nucleotide sequence may also be identified and isolated by hybridization under stringent conditions using as probes identified nucleotide sequences encoding HPPD enzymes according to the invention or parts thereof. Such parts should preferably have a nucleotide sequence comprising at least 40 consecutive nucleotides from the coding region of HPPD encoding genes sequences according to the invention, preferably from the coding region of SEQ ID No. 1 , SEQ ID No. 2, SEQ ID No. 3. SEQ ID No. 12, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31 , SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34,
  • the probes may however comprise longer regions of nucleotide sequences derived from the HPPD encoding nucleic acids, such as about 50, 60, 75, 100, 200 or 500 consecutive nucleotides from any of the mentioned HPPD genes.
  • the probe should comprise a nucleotide sequence coding for a highly conserved region which may be identified by aligning the different HPPD proteins.
  • Stringent hybridization conditions as used herein means that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence.
  • Examples of stringent hybridization conditions are overnight incubation in a solution comprising 5xSSC (150 mM NaCI, 15 mM tri sodium-citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1 x SSC at approximately 65 °C, preferably twice for about 10 minutes.
  • Other hybridization and wash conditions are well known and are exemplified in Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989), particularly chapter 1 1 .
  • Such variant sequences may also be obtained by DNA amplification using
  • oligonucleotides specific for HPPD genes encoding enzymes as primers such as but not limited to oligonucleotides comprising about 20 to about 50 consecutive nucleotides selected from the nucleotide sequences of SEQ ID Nos. 1 , 2, 3, 12, 15, 16, 29, 30, 31 , 33, 34, 35, 36, and 37 or their complement.
  • variant HPPD enzymes which are amino acid sequences similar to the HPPD amino acid sequence of SEQ ID No. 17 wherein one or more amino acids have been inserted, deleted or substituted.
  • variants of an amino acid sequence refer to those polypeptides, enzymes or proteins which have a similar catalytic activity as the amino acid sequences described herein, notwithstanding any amino acid substitutions, additions or deletions thereto.
  • the variant amino acid sequence has a sequence identity of at least about 96%, or 97 or 98% or 99% with the amino acid sequence of SEQ ID No. 17.
  • a polypeptide comprising the variant amino acid sequence has HPPD enzymatic activity. Methods to determine HPPD enzymatic activity are well known in the art and include assays as extensively described in WO 2009/144079 or in
  • substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which an amino acid residue contained in an HPPD protein of this invention is replaced with another naturally- occurring amino acid of similar character, for example Gly « >Ala, Val ⁇ »lle ⁇ >Leu, Asp* >Glu, Lys ⁇ Arg, Asn ⁇ »Gln or Phe ⁇ >Trp ⁇ >Tyr.
  • Substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue which is present in an HPPD protein of the invention is substituted with an amino acid with different properties, such as a naturally-occurring amino acid from a different group (eg. substituting a charged or hydrophobic amino acid with alanine.
  • Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed. Amino acid deletions will usually be of the order of about 1 -10 amino acid residues, while insertions may be of any length. Deletions and insertions may be made to the N-terminus, the C-terminus or be internal deletions or insertions.
  • Similar amino acids refers to amino acids that have similar amino acid side chains, i.e. amino acids that have polar, non-polar or practically neutral side chains.
  • Non- similar amino acids refers to amino acids that have different amino acid side chains, for example an amino acid with a polar side chain is non-similar to an amino acid with a non-polar side chain.
  • Polar side chains usually tend to be present on the surface of a protein where they can interact with the aqueous environment found in cells (“hydrophilic" amino acids).
  • amino acids that have polar side chains are arginine, asparagine, aspartate, cysteine, glutamine, glutamate, histidine, lysine, serine, and threonine (all hydrophilic, except for cysteine which is hydrophobic).
  • amino acids that have non-polar side chains are alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan (all hydrophobic, except for glycine which is neutral).
  • the invention also relates to the use, in a method for transforming plants, of a nucleic acid which encodes an HPPD according to the invention as a marker gene or as a coding sequence which makes it possible to confer to the plant tolerance to herbicides which are HPPD inhibitors, and the use of one or moreHPPD inhibitor(s) on plants comprising a nucleic acid sequence encoding a HPPD according to the invention.
  • HPPD inhibitors to be used are selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N- (triazol-3-yl)arylcarboxamides, triketones, pyrazolinates, preferably selected from the group consisting of tembotrione, mesotrione, sulcotrione, bicyclopyrone, and tefuryltrione. It is, of course, understood that this sequence can also be used in combination with (an) other gene marker(s) and/or sequence(s) which encode(s) one or more protein with useful agricultural properties.
  • herbicides have different longevities in the field, and some herbicides persist and are effective for a relatively long time after they are applied to a field while other herbicides are quickly broken down into other and/or non-active compounds.
  • An ideal treatment system would allow the use of different herbicides so that growers could tailor the choice of herbicides for a particular situation.
  • HPPD protein or gene of the invention is advantageously combined in plants with other genes which encode proteins or RNAs that confer useful agronomic properties to such plants.
  • genes which encode proteins or RNAs that confer useful agronomic properties on the transformed plants mention can be made of the DNA sequences encoding proteins which confer tolerance to one or more herbicides that, according to their chemical structure, differ from HPPD inhibitor herbicides, and others which confer tolerance to certain insects, those which confer tolerance to certain diseases, DNAs that encodes RNAs that provide nematode or insect control, etc.
  • Such genes are in particular described in published PCT Patent Applications WO 91/02071 and WO95/06128.
  • EPSPS EPSPS which confer tolerance to the herbicides which have EPSPS as a target
  • sequence encoding these enzymes is advantageously preceded by a sequence encoding a transit peptide, in particular the "optimized transit peptide" described in US Patent 5,510,471 or 5,633,448.
  • plants being tolerant to glyphosate and at least one ALS
  • acetolactate synthase inhibitor More specifically plants containing genes encoding a GAT (Glyphosate-N-Acetyltransferase) polypeptide and a polypeptide conferring resistance to ALS inhibitors are disclosed.
  • GAT Glyphosate-N-Acetyltransferase
  • transgenic tobacco plants containing mutated Arabidopsis ALS/AHAS genes were disclosed.
  • plants containing genes encoding 2,4-D-monooxygenases conferring tolerance to 2,4-D (2,4-dichlorophenoxyacetic acid) by metabolisation are disclosed.
  • plants containing genes encoding Dicamba monooxygenases conferring tolerance to dicamba (3,6-dichloro-2- methoxybenzoic acid) by metabolisation are disclosed.
  • the Cry1 A-type proteins or toxic fragments thereof preferably the CrylAc protein or hybrids derived from the CrylAc protein (e.g., the hybrid Cry1 Ab-Cry1 Ac protein described in US 5,880,275) or the Cryl Ab or Bt2 protein or insecticidal fragments thereof as described in EP451878, the Cry2Ae, Cry2Af or Cry2Ag proteins as described in WO02/057664 or toxic fragments thereof, the Cry1 A.105 protein described in WO 2007/140256 (SEQ ID No.
  • the present invention also relates to a chimeric gene (or expression cassette) which comprises a coding sequence as well as heterologous regulatory elements, at the 5' and/or 3' position, at least at the 5' position, which are able to function in a host organism, in particular plant cells or plants, with the coding sequence containing at least one nucleic acid sequence which encodes an HPPD as previously defined.
  • the present invention relates to a chimeric gene as previously described, wherein the host organism is selected from bacteria, yeast, Pichia, fungi, baculovirus, in vitro cells, protoplasts, plant cells, plants, plant parts, and plant seeds thereof.
  • the present invention relates to a chimeric gene as previously described, wherein the chimeric gene contains in the 5' position of the nucleic acid sequence which encodes a HPPD according to the invention, a nucleic acid sequence which encodes a plant transit peptide, with this sequence being arranged between the promoter region and the sequence encoding the HPPD according to the invention so as to permit expression of a transit peptide/HPPD fusion protein.
  • the present invention relates to the use of HPPD inhibitor herbicides on plants, plant parts, or plant seeds comprising HPPD tolerant gene according to the invention, or to the use of HPPD inhibitor herbicides on soil where such plants, plant parts or seeds are to be grown or sown, either alone or in combination with one or more other known herbicides acting in a different matter to HPPD inhibitors.
  • the employed HPPD inhibitor herbicide is selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamides,
  • triketones such as tembotrione, sulcotrione mesotrione, bicyclopyrone, tefuryltrione, particularly tembotrione, of the class diketone such as diketonitrile of the class of isoxazoles such as isoxaflutole or of the class of pyrazolinates (named pyrazolinate HPPD inhibitor), such as pyrasulfotole, pyrazolate, topramezone, benzofenap, even more specifically present invention relates to the application of tembotrione, mesotrione, diketonitrile, bicyclopyrone, tefuryltrione, benzofenap, pyrasulfotole, pyrazolate and sulcotrione to such HPPD inhibitor tolerant plants, plant parts or plant seeds.
  • triketone HPPD inhibitor such as tembotrione, sulcotrione mesotrione, bicyclopyrone, tefuryltri
  • promoter sequence As a regulatory sequence which functions as a promoter in plant cells and plants, use may be made of any promoter sequence of a gene which is naturally expressed in plants, in particular a promoter which is expressed especially in the leaves of plants, such as for example "constitutive" promoters of bacterial, viral or plant origin, or "light- dependent" promoters, such as that of a plant ribulose-biscarboxylase/oxygenase (RuBisCO) small subunit gene, or any suitable known promoter-expressible which may be used.
  • a promoter sequence of a gene which is naturally expressed in plants in particular a promoter which is expressed especially in the leaves of plants, such as for example "constitutive" promoters of bacterial, viral or plant origin, or "light- dependent" promoters, such as that of a plant ribulose-biscarboxylase/oxygenase (RuBisCO) small subunit gene, or any suitable known promoter-expressible which may be used.
  • promoters of plant origin mention will be made of the histone promoters as described in EP 0 507 698 A1 , the rice actin promoter (US 5,641 ,876), or a plant ubiquitin promoter (US 5,510,474).
  • promoters of a plant virus gene mention will be made of that of the cauliflower mosaic virus (CaMV 19S or 35S, Sanders et al. (1987), Nucleic Acids Res. 15(4):1543-58.), the circovirus (AU 689 31 1 ) or the Cassava vein mosaic virus (CsVMV, US 7,053,205).
  • a promoter sequence specific for particular regions or tissues of plants can be used to express the HPPD proteins of the invention, such as promoters specific for seeds (Datla, R. et al. (1997), Biotechnology Ann. Rev. 3, 269-296), especially the napin promoter (EP 255 378 A1 ), the phaseolin promoter, the glutenin promoter, the helianthinin promoter (WO 92/17580), the albumin promoter (WO 98/45460), the oleosin promoter (WO 98/45461 ), the SAT1 promoter or the SAT3 promoter (PCT/US98/06978).
  • promoters specific for seeds such as promoters specific for seeds (Datla, R. et al. (1997), Biotechnology Ann. Rev. 3, 269-296), especially the napin promoter (EP 255 378 A1 ), the phaseolin promoter, the glutenin promoter, the helianthinin promoter (WO 92/17580), the albumin promoter (WO
  • an inducible promoter advantageously chosen from the phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG), chitinase, glucanase, proteinase inhibitor (PI), PR1 family gene, nopaline synthase (nos) and vspB promoters (US 5 670 349, Table 3), the HMG2 promoter (US 5 670 349), the apple beta-galactosidase (ABG1 ) promoter and the apple aminocyclopropane carboxylate synthase (ACC synthase) promoter (WO 98/45445).
  • promoter use may also be made, in combination with the promoter, of other regulatory sequences, which are located between the promoter and the coding sequence, such as transcription activators ("enhancers"), for instance the translation activator of the tobacco mosaic virus (TMV) described in Application WO 87/07644, or of the tobacco etch virus (TEV) described by Carrington and Freed (1990), J. Virol. 64: 1 590-1597, for example, or introns such as the adhl intron of maize or intron 1 of rice actin.
  • transcription activators for instance the translation activator of the tobacco mosaic virus (TMV) described in Application WO 87/07644, or of the tobacco etch virus (TEV) described by Carrington and Freed (1990), J. Virol. 64: 1 590-1597
  • introns such as the adhl intron of maize or intron 1 of rice actin.
  • the gene of the invention is present in plants in multiple, preferably two copies, each of these controlled by a different plant expressible promoter.
  • the chimeric gene of the invention can be combined with any further chimeric gene coding for an HPPD protein, preferably these different genes are controlled by different regulatory elements being active in plants.
  • the chimeric gene of the invention can be combined with a CYP450 Maize monooxygenase (nsfl gene) gene being under the control of an identical or different plant expressible promoter.
  • a regulatory terminator or polyadenylation sequence use may be made of any corresponding sequence of bacterial origin, such as for example the nos terminator of Agrobacterium tumefaciens, of viral origin, such as for example the CaMV 35S terminator, or of plant origin, such as for example a histone terminator as described in published Patent Application EP 0 633 317 A1 .
  • the term "gene”, as used herein refers to a DNA coding region flanked by 5' and/or 3' regulatory sequences allowing a RNA to be transcribed which can be translated to a protein, typically comprising at least a promoter region.
  • DNA/protein comprising the sequence X and “DNA/protein with the sequence comprising sequence X”, as used herein, refer to a DNA or protein including or containing at least the sequence X in their nucleotide or amino acid sequence, so that other nucleotide or amino acid sequences can be included at the 5' (or N- terminal) and/or 3' (or C-terminal) end, e.g., a N-terminal transit or signal peptide.
  • the term “comprising”, as used herein, is open-ended language in the meaning of "including”, meaning that other elements then those specifically recited can also be present.
  • DNA encoding a protein comprising sequence X refers to a DNA comprising a coding sequence which after transcription and translation results in a protein containing at least amino acid sequence X.
  • a DNA encoding a protein need not be a naturally occurring DNA, and can be a semi-synthetic, fully synthetic or artificial DNA and can include introns and 5' and/or 3' flanking regions.
  • nucleotide sequence refers to the sequence of a DNA or RNA molecule, which can be in single- or double-stranded form.
  • HPPD proteins according to the invention may be equipped with a signal peptide according to procedures known in the art, see, e.g., published PCT patent application WO 96/10083, or they can be replaced by another peptide such as a chloroplast transit peptide (e.g., Van Den Broeck et al.
  • tolerance means the relative levels of inherent tolerance of the HPPD screened according to a visible indicator phenotype of the strain or plant transformed with a nucleic acid comprising the gene coding for the respective HPPD protein in the presence of different concentrations of the various HPPD inhibitors.
  • Dose responses and relative shifts in dose responses associated with these indicator phenotypes are conveniently expressed in terms, for example, of GR50 (concentration for 50% reduction of growth) or MIC (minimum inhibitory concentration) values where increases in values correspond to increases in inherent tolerance of the expressed HPPD, in the normal manner based upon plant damage, meristematic bleaching symptoms etc.
  • GR50 values derived from dose/response curves having "dose” plotted on the x-axis and “percentage kill", “herbicidal effect”, “numbers of emerging green plants” etc. plotted on the y-axis where increased GR50 values correspond to increased levels of inherent tolerance of the expressed HPPD.
  • Herbicides can suitably be applied pre-emergence or post emergence.
  • tolerance level of the nucleic acid or gene encoding an HPPD protein according to the invention, or the HPPD protein of the invention is screened via transgenesis, regeneration, breeding and spray testing of a test plant such as tobacco, or a crop plant such as soybean or cotton and according to these results, such plants are at least 2-4 times more tolerant to HPPD inhibitors like tembotrione, mesotrione, diketonitrile and/or bicyclopyrone, than plants that do not contain any exogenous gene encoding an HPPD protein, or than plants that contain a gene comprising an Arabidopsis thaliana HPPD-encoding DNA, under control of the same promoter as the HPPD DNA of the invention.
  • HPPD inhibitors like tembotrione, mesotrione, diketonitrile and/or bicyclopyrone
  • “Host organism” or “host” is understood as being any unicellular or multicellular heterologous organism into which the nucleic acid or chimeric gene according to the invention can be introduced for the purpose of producing HPPD according to the invention.
  • These organisms are, in particular, bacteria, for example E. coli, yeasts, in particular of the genera Saccharomyces or Kluyveromyces, Pichia, fungi, in particular Aspergillus, a baculovirus or, preferably, plant cells and plants.
  • Plant cell is understood, according to the invention, as being any cell which is derived from or found in a plant and which is able to form or is part of undifferentiated tissues, such as calli, differentiated tissues such as embryos, parts of plants, plants or seeds. This includes protoplasts and pollen, cultivated plants cells or protoplasts grown in vitro, and plant cells that can regenerate into a complete plant.
  • Plant is understood, according to the invention, as being any differentiated multicellular organism which is capable of photosynthesis, in particular a
  • Transgenic plants refer to plants comprising a foreign or heterologous gene stably inserted in their genome.
  • the invention relates to the transformation of plants.
  • Any promoter sequence of a gene which is expressed naturally in plants, or any hybrid or combination of promoter elements of genes expressed naturally in plants, including Agrobacterium or plant virus promoters, or any promoter which is suitable for controlling the transcription of a herbicide tolerance gene in plants can be used as the promoter sequence in the plants of the invention (named "plant-expressible promoter" herein). Examples of such suitable plant-expressible promoters are described above.
  • plant-expressible promoters are operably-linked to a coding sequence encoding an HPPD protein of the invention to form a chimeric HPPD gene of this invention.
  • any corresponding sequence of bacterial or viral origin such as the nos terminator from Agrobacterium tumefaciens, or of plant origin, such as a histone terminator as described in application EP 0 633 317 A1 , may be used as transcription termination (and polyadenylation) regulatory sequence.
  • a nucleic acid sequence which encodes a transit peptide is employed 5' (upstream) of the nucleic acid sequence encoding the exogenous HPPD according to the invention, with this transit peptide sequence being arranged between the promoter region and the sequence encoding the exogenous HPPD so as to permit expression of a transit peptide-HPPD fusion protein, such as the protein of SEQ ID No. 20, SEQ ID No. 21 , or SEQ ID No. 22.
  • the transit peptide makes it possible to direct the HPPD into the plastids, more especially the
  • the transit peptide may be a single peptide, such as an EPSPS transit peptide (described in
  • the present invention also relates to the transit peptide-HPPD fusion protein and a nucleic acid or plant-expressible chimeric gene encoding such fusion protein, wherein the two elements of this fusion protein are as defined above.
  • the present invention also relates to a cloning, transformation and/or expression vector, which vector contains at least one chimeric gene as defined above. In addition to the above chimeric gene, this vector can contain an origin of replication.
  • This vector can be a plasmid or plasmid portion, a cosmid, or a bacteriophage or a virus which has been transformed by introducing the chimeric gene according to the invention. Transformation vectors are well known to the skilled person and widely described in the literature.
  • the transformation vector which can be used, in particular, for transforming plant cells or plants may be a virus, which can be employed for transforming plant cells or plants and which additionally contains its own replication and expression elements.
  • the vector for transforming plant cells or plants is preferably a plasmid, such as a disarmed Agrobacterium Ti plasmid.
  • the present invention also relates to the host organisms, in particular plant cells, seeds or plants, which comprise a chimeric gene which comprises a sequence encoding an HPPD protein of the invention, such as a protein comprising the amino acid sequence of SEQ ID Nos 17, 18, 19, 20, 21 , or 22 as defined above, and the use of the plants or seeds of the invention in a field to grow a crop and harvest a plant product, e.g., soya spp, rice, wheat, barley or corn grains or cotton bolls, where in one embodiment said use involves the application of an HPPD inhibitor herbicide to such plants to control weeds.
  • a chimeric gene which comprises a sequence encoding an HPPD protein of the invention, such as a protein comprising the amino acid sequence of SEQ ID Nos 17, 18, 19, 20, 21 , or 22 as defined above
  • the HPPD inhibitors are N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, triketones or pyrazolinates, preferably tembotrione, mesotrione, topramezone or sulcotrione, bicyclopyrone, pyrasulfotole, pyrazolate, benzofenap and tefuryltrione, particularly tembotrione.
  • the present invention relates to a host organism, in particular a plant cell, seed, or plant, characterized in that it contains at least one HPPD chimeric gene as described above, or at least an HPPD nucleic acid sequence as previously described.
  • the present invention relates to a plant cell, seed, or plant characterized in that it contains at least a nucleic acid sequence which encodes an HPPD protein of this invention which retain its properties of catalysing the conversion of para-hydroxyphenylpyruvate to homogentisate and which makes this plant more tolerant to one or more HPPD inhibitor herbicide(s) selected from the group consisting of particularly to N-(1 ,2,5-oxadiazol-3-yl)benzamides,
  • triketones or pyrazolinates, preferably to one or more HPPD inhibitor heribcide(s) selected from the group consisting of tembotrione, mesotrione, topramezone or sulcotrione, bicyclopyrone, pyrasulfotole, pyrazolate, benzofenap and tefuryltrione, particularly to tembotrione.
  • HPPD inhibitor heribcide(s) selected from the group consisting of tembotrione, mesotrione, topramezone or sulcotrione, bicyclopyrone, pyrasulfotole, pyrazolate, benzofenap and tefuryltrione, particularly to tembotrione.
  • the present invention relates to a plant cell, seed, or plant characterized in that it contains at least a nucleic acid sequence which encodes an HPPD of this invention which retain its properties of catalysing the conversion of para-hydroxyphenylpyruvate to homogentisate and which is less sensitive to one or more HPPD inhibitor herbicide(s) than the host plant endogenous HPPD, such as the HPPD from Arabidopsis thaliana, particularly the HPPD comprising the amino acid sequence of SEQ ID No. 28 (from the amino acid position 126 to the amino acid position 568).
  • the present invention relates to a host plant cell, seed or host plant characterized in that it contains at least a nucleic acid sequence which encodes an HPPD of the invention, compared to the host plant endogenous HPPD, is less sensitive to one or more HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, isoxazoles, diketonitriles, triketones or pyrazolinates, more especially to one or more HPPD inhibitor herbicide(s) selected from the group consisting of isoxafiutole, tembotrione, mesotrione, sulcotrione, pyrasulfotole, bicyclopyrone, tefuryltrione, topramezone, 2-cyano-3-cyclopropyl-1 -(2-S)
  • the present invention relates to a plant cell, seed, or plant characterized in that it contains at least a nucleic acid sequence encoding an HPPD of the invention as previously described, and in addition a chimeric gene comprising a plant-expressible promoter as described above, operably-linked to a nucleic acid sequence encoding a PDH (prephenate dehydrogenase) enzyme (US 2005/0257283).
  • PDH prephenate dehydrogenase
  • the present invention also relates to the plants which contain transformed cells, in particular the plants which are regenerated from the transformed cells, and progeny plants or seeds thereof, comprising the chimeric HPPD gene of the invention.
  • the regeneration can be obtained by any appropriate method, with the method depending on the nature of the species, as described, for example, in the above references.
  • the following patents and patent applications may be cited, in particular, with regard to the methods for transforming plant cells and regenerating plants: US 4,459,355,
  • the present invention also relates to the transgenic plants or part thereof, which are derived by cultivating and/or crossing the above transgenic plants, and to the seeds of the transgenic plants, comprising the HPPD chimeric gene of the invention.
  • the present invention also relates to the end products such as the meal or oil which are obtained from the plants, part thereof, or seeds of the invention.
  • the transformed plants which can be obtained in accordance with the invention can be of the monocotyledonous type, such as wheat, barley, sugarcane, rice, onion, and corn or maize, or of the dicotyledonous type, such as tobacco, soya spp, alfalfa
  • Brassica spp. plants such as oilseed rape, cotton, sugarbeet clover, vegetables, etc.
  • the invention relates to a method for transforming host organisms, in particular plant cells or plants, by integrating in such organisms at least one nucleic acid sequence or one chimeric gene as previously defined, wherein it is possible to obtain the transformation by any appropriate known means, which means are amply described in the specialist literature and, in particular, the references cited in the present application, e.g., by using the vector according to the invention.
  • One transformation method in accordance with this invention comprises bombarding cells, protoplasts or tissues with solid or liquid particles to which DNA is attached, or containing DNA.
  • Another transformation method comprises using, as mean for transfer into the plant, a chimeric gene which is inserted into an Agrobacterium tumefaciens Ti plasmid or an Agrobacterium rhizogenes Ri plasmid.
  • Other methods may be used, such as microinjection or electroporation or otherwise direct gene transfer using PEG.
  • the skilled person can select any appropriate method for transforming the host organism of choice, in particular the plant cell or the plant.
  • the technology for soybean transformation has been extensively described in the examples 1 to 3 disclosed in EP 1 186666 A1 , incorporated herein by reference.
  • Agrobacterium-mediated transformation Hiei et al. (1994) Plant J 6:271 -282, and Hiei et al.
  • the HPPD of the invention is targeted into the chloroplast. This may be done by fusing a nucleic acid sequence which encodes a transit peptide to the nucleic acid sequence encoding the HPPD protein of the invention to obtain a nucleic acid encoding a fusion protein as described above.
  • the HPPD of the invention may be expressed directly in the plastids, such as the chloroplasts, using transformation of the plastid, such as the chloroplast genome.
  • a suitable method comprises the bombardment of plant cells or tissue by solid particles coated with the DNA or liquid particles comprising the DNA, and integration of the introduced gene encoding the protein of the invention by
  • the present invention also relates to a method for obtaining a plant to an HPPD inhibitor, characterized in that the plant is transformed with a chimeric HPPD gene of the invention as previously described.
  • the present invention also relates to a method for obtaining a plant tolerant to an HPPD inhibitor, characterized in that the plant contains a chimeric HPPD gene of the invention which comprises a coding sequence as well as a heterologous regulatory element in the 5' and optionally in the 3' positions, which are able to function in a host organism, characterized in that the coding sequence comprises at least a nucleic acid sequence defining a gene encoding an HPPD of the invention as previously described.
  • the HPPD inhibitor in the above method is selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol- 4-yl)- or N-(triazol-3-yl)arylcarboxamides, triketone or pyrazolinate herbicide, preferably selected from the group consisting of tembotrione, mesotrione,
  • bicyclopyrone tefuryltrione pyrasulfotole, pyrazolate, diketonitrile, benzofenap, or sulcotrione, particularly it is tembotrione.
  • a method for obtaining a plant tolerant to an HPPD inhibitor as described above is also provided, characterized in that a plant is obtained comprising a first transgene which is a chimeric HPPD gene of the invention, and a second transgene, which is a chimeric gene comprising a plant-expressible promoter operably-linked to a nucleic acid encoding a PDH (prephenate dehydrogenase) enzyme.
  • a first transgene which is a chimeric HPPD gene of the invention
  • a second transgene which is a chimeric gene comprising a plant-expressible promoter operably-linked to a nucleic acid encoding a PDH (prephenate dehydrogenase) enzyme.
  • a plant comprising such two transgenes can be obtained by transforming a plant with one transgene, and then re-transforming this transgenic plant with the second transgene, or by transforming a plant with the two transgenes simultaneously (in the same or in 2 different transforming DNAs or vectors), or by crossing a plant comprising the first transgene with a plant comprising the second transgene, as is well known in the art.
  • the invention also relates to a method for selectively removing weeds or preventing the germination of weeds in a field to be planted with plants or to be sown with seeds, or in a plant crop, by application of an HPPD inhibitor to such field or plant crop, in particular an HPPD inhibitor heribicide as previously defined, which method is characterized in that this HPPD inhibitor herbicide is applied to plants which have been transformed in accordance with the invention, either before sowing the crop (hereinafter named p re-planting application), before emergence of the crop
  • the invention also relates to a method for controlling in an area or a field which contains transformed seeds as previously described in the present invention, which method comprises applying, to the said area of the field, a dose of an HPPD inhibitor herbicide which is toxic for the said weeds, without significantly affecting the seeds or plants which contain the HPPD nucleic acid or the chimeric HPPD gene of the invention as previously described in the present invention.
  • the present invention also relates to a method for cultivating the plants which have been transformed with a chimeric gene according to the invention, which method comprises planting seeds comprising a chimeric gene of the invention, in an area of a field which is appropriate for cultivating the said plants, and in applying, if weeds are present, a dose, which is toxic for the weeds, of a herbicide whose target is the above- defined HPPD to the said area of the said field, without significantly affecting the said transformed seeds or the said transformed plants, and in then harvesting the cultivated plants or plant parts when they reach the desired stage of maturity and, where appropriate, in separating the seeds from the harvested plants.
  • the herbicide whose target is the HPPD enzyme can be applied in accordance with the invention, either before sowing the crop, before the crop emerges or after the crop emerges.
  • the present invention also relates to a process for obtaining oil, particularly soya spp, corn or cotton oil, or meal, comprising growing a crop, particularly a soya spp crop, expressing an HPPD protein of the invention optionally treating such crop with an HPPD inhibitor herbicide, harvesting the grains and milling the grains to make meal and extract the oil. Also the seeds or grains, either whole, broken or crushed, comprising the chimeric gene of the invention are part of this invention. Therefore, the present invention relates to a method for obtaining oil or meal comprising growing a transformed plant as described above, optionally treating such plant with an HPPD inhibitor herbicide, harvesting the grains and milling the grains to make meal and extract the oil.
  • HPPD inhibitor herbicide(s) selected from the group consisting of isoxaflutole, tembotrione, mesotrione, pyrasulfotole, sulcotrione, bicyclopyrone, tefuryltrione, topramezone, 2-cyano-3-cyclopropyl-1 -(2-methylsulphonyl-4-trifluoromethylphenyl)- propane-1 ,3-dione and to 2-cyano-1 -[4-(methylsulphonyl)-2-trifluoromethylphenyl]-3- (1 -methylcyclopropyl)propane-1 ,3-dione.
  • HPPD inhibitor herbicide(s) selected from the group consisting of isoxaflutole, tembotrione, mesotrione, pyrasulfotole, sulcotrione, bicyclopyrone, tefuryltrione, topramezone, 2-cyano-3-cyclopropyl-1 -(2-methyl
  • HPPD inhibitor herbicide(s) selected from the group consisting of triketones, such as tembotrione, sulcotrione and mesotrione, or of the class of pyrazolinates, such as pyrasulfotole and topramezone, particularly selected from the group consisting of tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione and mesotrione, more particularly it is tembotrione.
  • triketones such as tembotrione, sulcotrione and mesotrione
  • pyrazolinates such as pyrasulfotole and topramezone
  • pyrasulfotole and topramezone particularly selected from the group consisting of tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione and mesotrione, more particularly it is tembotrione.
  • herbicide is understood as being a herbicidally active substance on its own or such a substance which is combined with an additive which alters its efficacy, such as, for example, an agent which increases its activity (a synergistic agent) or which limits its activity (a safener). It is of course to be understood that, for their application in practice, the above herbicides are combined, in a manner which is known per se, with the formulation adjuvants which are customarily employed in agricultural chemistry.
  • HPPD inhibitor herbicides like those of the class of N-(1 ,2,5-oxadiazol-3- yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamides,
  • triketones such as tembotrione, sulcotrione and mesotrione, or of the class of pyrazolinates, such as pyrasulfotole and topramezone, particularly selected from tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione and mesotrione, more particularly tembotrione, have an outstanding herbicidal activity against a broad spectrum of economically important monocotyledonous and dicotyledonous annual harmful plants.
  • the active substances also act efficiently on perennial harmful plants which produce shoots from rhizomes, wood stocks or other perennial organs and which are difficult to control.
  • the present invention therefore also relates to a method of controlling undesired plants or for regulating the growth of plants in crops of plants comprising an HPPD according to the invention, where one or more HPPD inhibitor herbicides of the class of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, triketones, such as tembotrione, sulcotrione and mesotrione, or of the class of pyrazolinates, such as pyrasulfotole and topramezone, particularly selected from tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione and mesotrione, more particularly tembotrione are applied to the plants (for example harmful plants such as monocotyledonous or dicotyledonous weeds or undesired crop plants), to the seeds (for example grains
  • one or more HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol- 4-yl)- or N-(triazol-3-yl)arylcarboxamides, triketones, such as tembotrione, sulcotrione and mesotrione, pyrazolinates, such as pyrasulfotole and topramezone, particularly selected from the group consisting of tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione and mesotrione, more particularly it is tembotrione can be applied for example pre-planting (if appropriate also by incorporation into the soil), pre-emergence or post-emergence.
  • topramezone particularly selected from tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione and mesotrione, more particularly tembotrione are hereby mentioned, without this mentioning being intended as a limitation to certain species only: Monocotyledonous harmful plants of the genera: Aegilops, Agropyron, Agrostis, Alopecurus, Apera, Avena, Brachiaria, Bromus, Cenchrus, Commelina, Cynodon, Cyperus, Dactyloctenium, Digitaria, Echinochloa, Eleocharis, Eleusine,
  • Emex Erysimum, Euphorbia, Galeopsis, Galinsoga, Galium, Hibiscus, Ipomoea, Kochia, Lamium, Lepidium, Lindernia, Matricaria, Mentha, Mercurialis, Mullugo, Myosotis, Papaver, Pharbitis, Plantago, Polygonum, Portulaca, Ranunculus, Raphanus, Rorippa, Rotala, Rumex, Salsola, Senecio, Sesbania, Sida, Sinapis, Solanum, Sonchus, Sphenoclea, Stellaria, Taraxacum, Thlaspi, Trifolium, Urtica,
  • transgenic crops according to the invention comprising an HPPD protein, DNA or chimeric gene according invention and which may also show one more further herbicide resistances against herbicides that differ from HPPD inhibitor herbicides
  • novel plants with modified properties can be generated with the aid of recombinant methods (see, for example, EP-A-0221044 A1 , EP-A-0131624 A1 ). For example, the following have been described in several cases:
  • glufosinate type cf., for example, EP-A-0242236, EP-A-242246) or of the glyphosate type (WO 92/00377) or of the sulfonylurea type (EP-A-0257993, US-A-5013659),
  • transgenic crop plants for example corn, cotton or soya spp, which are capable of producing Bacillus thuringiensis toxins (Bt toxins), or hybrids or mutants thereof, which make the plants resistant to certain pests (EP-A-0193259), transgenic crop plants with a modified fatty acid composition (WO 91/13972), genetically modified crop plants with novel constituents or secondary
  • transgenic crop plants which are distinguished by higher yields or better quality
  • transgenic crop plants which are distinguished by a combination of novel properties such as a combination of the abovementioned novel properties ("gene stacking").
  • the generation of plant cells with a reduced activity for a gene product can be achieved for example by the expression of at least one corresponding antisense RNA, a sense RNA for achieving a cosuppression effect, or a combination of both an antisense and sense RNA forming a double-stranded silencing RNA molecule (RNAi), or by the expression of at least one correspondingly constructed ribozyme, which specifically cleaves transcripts of the abovementioned gene product.
  • RNAi double-stranded silencing RNA molecule
  • DNA molecules which comprise all of the coding sequence of a gene product including any flanking sequences which may be present, or else DNA molecules which only comprise parts of the coding sequence, it being necessary for these parts to be long enough to bring about an antisense effect in the cells. It is also possible to use DNA sequences which have a high degree of homology with the coding sequences of a gene product, but which are not entirely identical.
  • the obtained protein may be localized in any compartment of the plant cell.
  • sequences are known to the skilled person (see, for example, Braun et al.,(1992), EMBO J. ,1 1 , 3219-3227; Wolter et al.(1988), Proc. Natl. Acad. Sci. USA 85, 846-850; Sonnewald et al. (1991 ), Plant J. ,1 ,95-106).
  • the nucleic acid molecules can also be expressed in the organelles of the plant cells.
  • the transgenic plant cells can be regenerated by known techniques to give intact plants.
  • the transgenic plants may be plants of any plant species, including monocotyledonous or dicotyledonous plants.
  • HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5- oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamides, triketones, such as tembotrione, sulcotrione and mesotrione, or of the class of pyrazolinates, such as pyrasulfotole and topramezone, particularly selected from the group consisting of tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione and mesotrione, more particularly it is tembotrione in transgenic crops which are also resistant to growth regulators such as, for example, 2,4-D or dicamba, or against herbicides which inhibit essential plant enzymes, for example acetolactate synthases (ALS), EPSP syntha
  • ALS acetolactate synthases
  • the invention therefore also relates to the use of herbicides applied to this HPPD tolerant plants according to the invention for controlling harmful plants (i.e. weeds) which also extends to transgenic crop plants comprising a second or more herbicide resistance(s) beside the resistance against one or more HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol- 4-yl)- or N-(triazol-3-yl)arylcarboxamides, triketones, such as tembotrione, sulcotrione and mesotrione, of the class of isoxazoles such as isoxaflutole or of the class of pyrazolinates, such as pyrasulfotole and topramezone, particularly selected from the group consisting of tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione and mesotrione,
  • WP wettable powders
  • SP water-soluble powders
  • EC emulsifiable concentrates
  • EW emulsions
  • SC suspension concentrates
  • SC oil- or water-based dispersions
  • CS capsule suspensions
  • DP dusts
  • seed-dressing products granules for application by broadcasting and on the soil, granules (GR) in the form of
  • microgranules spray granules, coated granules and adsorption granules, water- dispersible granules (WG), water-soluble granules (SG), ULV formulations, microcapsules and waxes.
  • WG water- dispersible granules
  • SG water-soluble granules
  • ULV formulations microcapsules and waxes.
  • formulation auxiliaries required such as inert materials, surfactants, solvents and further additives, are also known and are described, for example, in: Watkins,
  • Wettable powders are preparations which are uniformly dispersible in water and which, besides the active substance, also comprise ionic and/or nonionic surfactants (wetters, dispersers), for example polyoxyethylated alkylphenols, polyoxyethylated fatty alcohols, polyoxyethylated fatty amines, fatty alcohol polyglycol ether sulfates, alkanesulfonates, alkylbenzenesulfonates, sodium lignosulfonate, sodium
  • ionic and/or nonionic surfactants for example polyoxyethylated alkylphenols, polyoxyethylated fatty alcohols, polyoxyethylated fatty amines, fatty alcohol polyglycol ether sulfates, alkanesulfonates, alkylbenzenesulfonates, sodium lignosulfonate, sodium
  • the herbicidally active substances are ground finely, for example in customary apparatuses such as hammer mills, blower mills and air-jet mills, and mixed with the formulation auxiliaries, either simultaneously or subsequently.
  • Emulsifiable concentrates are prepared by dissolving the active substance in an organic solvent, for example butanol, cyclohexanone, dimethylformamide, xylene or else higher-boiling aromatics or hydrocarbons or mixtures of the organic solvents with addition of one or more ionic and/or nonionic surfactants (emulsifiers).
  • organic solvent for example butanol, cyclohexanone, dimethylformamide, xylene or else higher-boiling aromatics or hydrocarbons or mixtures of the organic solvents with addition of one or more ionic and/or nonionic surfactants (emulsifiers).
  • emulsifiers which may be used are: calcium alkylarylsulfonates such as calcium dodecylbenzenesulfonate, or nonionic emulsifiers such as fatty acid polyglycol esters, alkylarylpolyglycol ethers, fatty alcohol polyglycol ethers, propylene oxide/ethylene oxide condensates, alkyl polyethers, sorbitan esters such as, for example, sorbitan fatty acid esters or polyoxyethylene sorbitan esters such as, for example,
  • Dusts are obtained by grinding the active substance with finely divided solid materials such as, for example, talcum, natural clays such as kaolin, bentonite and pyrophyllite, or diatomaceous earth.
  • finely divided solid materials such as, for example, talcum, natural clays such as kaolin, bentonite and pyrophyllite, or diatomaceous earth.
  • Suspension concentrates can be water- or oil-based. They can be prepared for example by wet-grinding by means of commercially available bead mills, if appropriate with addition of surfactants as already listed above for example in the case of the other formulation types.
  • Emulsions for example oil-in-water emulsions (EW)
  • EW oil-in-water emulsions
  • Granules can be prepared either by spraying the active substance onto adsorptive, granulated inert material, or by applying active substance concentrates to the surface of carriers such as sand, kaolinites or granulated inert material with the aid of stickers, for example polyvinyl alcohol, sodium polyacrylate or else mineral oils. Suitable active substances can also be granulated in the manner which is customary for the production of fertilizer granules, if desired as a mixture with fertilizers. Water-dispersible granules are generally prepared by customary methods such as spray drying, fluidized-bed granulation, disk granulation, mixing with high-speed stirrers, and extrusion without solid inert material.
  • the agrochemical preparations comprise from 0.1 to 99% by weight, in particular from 0.1 to 95% by weight, of compounds according to the invention.
  • the active substance concentration is, for example,
  • the active substance concentration can amount to approximately 1 to 90, preferably 5 to 80% by weight.
  • Formulations in the form of dusts comprise from 1 to 30% by weight of active substance, preferably in most cases from 5 to 20% by weight of active substance, and sprayable solutions comprise approximately from 0.05 to 80, preferably from 2 to 50% by weight of active substance.
  • the active substance content depends partly on whether the active compound is in liquid or solid form, and on the granulation auxiliaries, fillers and the like which are being used.
  • the active substance content is between 1 and 95% by weight, preferably between 10 and 80% by weight.
  • the active substance formulations mentioned comprise, if appropriate, the auxiliaries which are conventional in each case, such as stickers, wetters, dispersants, emulsifiers, penetrations, preservatives, antifreeze agents, solvents, fillers, carriers, colorants, antifoams, evaporation inhibitors, and pH and viscosity regulators.
  • Active substances which can be applied to HPPD tolerant plants according to the present invention in combination with one or more HPPD inhibitor herbicide(s) of the class of ) N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, triketones, such as tembotrione, sulcotrione and mesotrione, or of the class of pyrazoiinates, such as pyrasuifotole and topramezone, particularly selected from tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione and mesotrione, more particularly tembotrione in mixed formulations or in the tank mix are, for example, known active substances which are based on the inhibition of, for example, acetolactate synthase, acetyl-CoA carboxylase, cellulose synthase,
  • Known herbicides or plant growth regulators which can be combined with the compounds according to the invention are, for example, the following active substances (the compounds are either designated by the common name according to the International Organization for Standardization (ISO) or by a chemical name, if appropriate together with the code number) and always comprise all use forms such as acids, salts, esters and isomers such as stereoisomers and optical isomers.
  • ISO International Organization for Standardization
  • chemical name if appropriate together with the code number
  • acetochlor acibenzolar, acibenzolar-S-methyl, acifluorfen, acifluorfen-sodium, aclonifen, alachior, aiiidochlor, alloxydim, alloxydim-sodium, ametryne, amicarbazone, amidochlor, amidosulfuron, aminocyclopyrachlor, aminopyralid, amitrole, ammonium sulfamate, ancymidol, anilofos, asulam, atrazine, azafenidin, azimsulfuron,
  • aziprotryne BAH-043, BAS-140H, BAS-693H, BAS-714H, BAS-762H, BAS-776H, BAS-800H, beflubutamid, benazolin, benazolin-ethyl, bencarbazone, benfluralin, benfuresate, bensulide, bensulfuron-methyl, bentazone, benzfendizone,
  • chlorflurenol chlorflurenol-methyl, chloridazon, chlorimuron, chlorimuron-ethyl, chlormequat-chloride, chlornitrofen, chlorophthalim, chlorthal-dimethyl, chlorotoluron, chlorsulfuron, cinidon, cinidon-ethyl, cinmethylin, cinosulfuron, clethodim, clodinafop clodinafop-propargyl, clofencet, clomazone, clomeprop, cloprop, clopyralid, cloransulam, cloransulam-methyl, cumyluron, cyanamide, cyanazine, cyclanilide, cycloate, cyclosulfamuron, cycloxydim, cycluron, cyhalofop, cyhalofop-butyl, cyperquat, cyprazine, cypra
  • oxaziclomefone oxyfluorfen, paclobutrazole, paraquat, paraquat dichloride, pelargonic acid (nonanoic acid), pendimethalin, pendralin, penoxsulam, pentanochlor, pentoxazone, perfluidone, pethoxamid, phenisopham, phenmedipham,
  • phenmedipham-ethyl picloram, picolinafen, pinoxaden, piperophos, pirifenop, pirifenop-butyl, pretilachlor, primisulfuron, primisulfuron-methyl, probenazole, profluazol, procyazine, prodiamine, prifluraline, profoxydim, prohexadione,
  • prohexadione-caicium prohydrojasmone, prometon, prometryn, propachlor, propanil, propaquizafop, propazine, propham, propisochlor, propoxycarbazone,
  • pyribenzoxim pyributicarb, pyridafol, pyridate, pyriftalid, pyriminobac, pyriminobac- methyl, pyrimisulfan, pyrithiobac, pyrithiobac-sodium, pyroxasulfone, pyroxsulam, quinclorac, quinmerac, quinoclamine, quizalofop, quizalofop-ethyl, quizalofop-P, quizalofop-P-ethyl, quizalofop-P-tefuryl, rimsulfuron, saflufenacil, secbumeton, sethoxydim, siduron, simazine, simetryn, SN-106279, sulf-allate (CDEC),
  • sulfentrazone sulfometuron, sulfometuron-methyl, sulfosate (glyphosate-trimesium), sulfosulfuron, SYN-523, SYP-249, SYP-298, SYP-300, tebutam, tebuthiuron, tecnazene, tepraloxydim, terbacil, terbucarb, terbuchlor, terbumeton, terbuthylazine, terbutryne, TH-547, thenylchlor, thiafluamide, thiazafluron, thiazopyr, thidiazimin, thidiazuron, thiencarbazone, thiencarbazone-methyl, thifensulfuron, thifensulfuron- methyl, thiobencarb, tiocarbazil, tralkoxydim, tri-allate, tria
  • the application rate required of the HPPD inhibitor herbicide of the class of ) N-(1 ,2,5- oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamides, triketones, such as tembotrione, sulcotrione and mesotrione, or of the class of pyrazolinates, such as pyrasulfotole and topramezone, particularly selected from tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione and mesotrione, more particularly tembotrione to be applied to areas where HPPD tolerant plants according to the present invention are growing varies as a function of the external conditions such as temperature, humidity, the nature of the herbicide used and the like. It can vary within wide limits, for example between 0.001 and 1 .0 kg/ha and more of active substance, but it is preferably between 0.00
  • these mixtures may cause crop injury, based on the presence of the non HPPD inhibitor herbicide(s).
  • appropriate safeners may be added. These safeners, which are employed in antidotically active amounts, reduce the phytotoxic side effects of herbicides/pesticides used, for example in economically important crops, such as cereals (wheat, barley, rye, corn, rice, millet), alfalfa, sugar beet, sugarcane, oilseed rape, cotton and soya spp. , preferably corn, cotton, sugarbeet, or soya spp.
  • the safeners are preferably selected from the group consisting of: compounds of the formula S-l)
  • ⁇ is a natural number from 0 to 5, preferably from 0 to 3;
  • RA 1 is halogen, (CrC 4 )-alkyl, (CrC 4 )-alkoxy, nitro or (Ci-C4)-haloalkyl;
  • WA is an unsubstituted or substituted divalent heterocyclic radical from the group consisting of partially unsaturated or aromatic five-membered heterocycles having 1 to 3 hetero ring atoms of the type N or O, where at least one nitrogen atom and at most one oxygen atom is present in the ring, preferably a radical from the group consisting of (WA 1 ) to (WA 4 ),
  • VV (VV) ( A 2 ) (WA 3 ) (W A «)
  • m A is 0 or 1 ;
  • RA 2 is ORA 3 , SRA 3 or NR A 3 RA 4 or a saturated
  • RA 3 is hydrogen or an unsubstituted or substituted aliphatic hydrocarbon radical having preferably a total of 1 to 18 carbon atoms;
  • RA 4 is hydrogen, (Ci-Cr,)-alkyl, (Ci-Ce)-alkoxy or substituted or unsubstituted phenyl;
  • RA 5 is H, (Ci-C 8 )-alkyl, (Ci-C8)-haloalkyl), (Ci-C 4 )-alkoxy-(Ci-C 8 )-alkyl, cyano or
  • R A 9 is hydrogen, (CrC 8 )-alkyl, (CrC 8 )-haloalkyl, (Ci-C 4 )-alkoxy- (Ci-C 4 )-alkyl, (Ci-C 6 )-hydroxyalkyl, (C 3 -Ci 2 )-cycloalkyl or tri-(Ci-C 4 )-alkylsilyl;
  • RA 6 , RA 7 , RA 8 are identical or different and are hydrogen, (Ci-C 8 )-alkyl,
  • dichlorophenylpyrazolecarboxylic acid preferably compounds such as ethyl 1 -(2,4-dichlorophenyl)-5-methylpyrazole-3-carboxylate (S1 -2), ethyl
  • compounds of the type of the triazolecarboxylic acids preferably compounds such as fenchlorazole(-ethyl ester), i.e. ethyl 1 -(2,4-dichlorophenyl)-5-trichloro- methyl-(1 H)-1 ,2,4-triazole-3-carboxylate (S1 -6), and related compounds, as described in EP-A-174 562 and EP-A-346 620;
  • 5-phenyl-2-isoxazoline-3-carboxylate (S1 -8) and related compounds, as described in WO 91/08202, or ethyl 5,5-diphenyl-2-isoxazolinecarboxylate (S1 -9) ("isoxadifen- ethyl") or n-propyl 5,5-diphenyl-2-isoxazolinecarboxylate (S1 -10) or ethyl 5-(4-fluorophenyl)-5-phenyl-2-isoxazoline-3-carboxylate (S1 -1 1 ), as described in the patent application WO-A-95/07897.
  • RB 1 is halogen, (Ci-C-4)-alkyl, (Ci-Ci)-alkoxy, nitro or (Ci-C4)-haloalkyl;
  • ⁇ is a natural number from 0 to 5, preferably from 0 to 3;
  • RB 3 is hydrogen or an unsubstituted or substituted aliphatic hydrocarbon radical having preferably a total of 1 to 18 carbon atoms;
  • RB 4 is hydrogen, (d-CeJ-alkyl, (Ci-Ce)-alkoxy or substituted or unsubstituted phenyl;
  • TB is a (Ci- or C2)-alkanediyl chain which is unsubstituted or substituted by one or two (Ci-Ci)-alkyl radicals or by [(Ci-C3)-alkoxy]carbonyl; preferably:
  • Rc 1 is (Ci-Ci)-alkyl, (Ci-C 4 )-haloalkyl, (C 2 -C 4 )-alkenyl, (C 2 -C 4 )-haloalkenyl, (C3-C7)- cycloalkyl, preferably dichloromethyl;
  • Rc 2 , Rc 3 are identical or different and are hydrogen, (Ci-C 4 )-alkyl, (C2-C 4 )-alkenyl, (C 2 -C 4 )-alkynyl, (Ci-C 4 )-haloalkyl, (C 2 -C 4 )-haloalkenyl, (Ci-C 4 )-alkylcarbamoyl-(Ci-C )- alkyl, (C2-C 4 )-alkenylcarbamoyl-(Ci-C 4 )-alkyl, (CrC 4 )-alkoxy-(Ci-C 4 )-alkyl !
  • PPG-1292 N-allyl-N-[(1 ,3-dioxolan-2-yl)methyl]dichloroacetamide from PPG Industries
  • DKA-24 N-allyl-N-[(allylaminocarbonyl)methyl]dichloroacetamide from Sagro- Chem
  • TI-35 1 -dichloroacetylazepane from TRI-Chemical RT
  • RD 1 is CO-N R D 5 RD 6 or N HCO-RD 7 ;
  • RD 2 is halogen, (Ci-C4)-haloalkyl, (Ci-C-4)-haloalkoxy, nitro, (Ci-C-4)-alkyl, (C1-C4)- alkoxy, (Ci-C4)-alkylsulfonyl, (Ci-C4)-alkoxycarbonyl or (Ci-C-4)-alkylcarbonyl;
  • RD 3 is hydrogen, (Ci-C 4 )-alkyl, (C 2 -C 4 )-alkenyl or (C 2 -C 4 )-alkynyl;
  • RD 4 is halogen, nitro, (Ci-C4)-alkyl, (Ci-C4)-haloalkyl, (Ci-C4)-haloalkoxy, (C3-C.3)- cycloalkyl, phenyl, (Ci-C4)-alkoxy, cyano, (Ci-C4)-alkylthio, (Ci-C4)-alkylsulfinyl, (Ci- C4)-alkylsulfonyl, (Ci-C4)-alkoxycarbonyl or (Ci-C4)-alkylcarbonyl; RD 5 is hydrogen, (Ci-C 6 )-alkyl, (C 3 -C 6 )-cycloalkyl, (C 2 -C 6 )-alkenyl, (C 2 -C 6 )-alkynyl, (C5-C6)-cycloalkenyl, phenyl or 3- to 6-membered heterocyclyl
  • heteroatoms from the group consisting of nitrogen, oxygen and sulfur where the seven last-mentioned radicals are substituted by VD substituents from the group consisting of halogen, (Ci-C6)-alkoxy, (d-Cf -haloalkoxy, (Ci-C-2)-alkylsulfinyl, (C1-C2)- alkylsulfonyl, (C3-C6)-cycloalkyl, (Ci-Ci)-alkoxycarbonyl, (Ci-C ⁇ )-alkylcarbonyl and phenyl and, in the case of cyclic radicals, also (Ci-Ci)-alkyl and (Ci-C4)-haloalkyl;
  • RD 6 is hydrogen, (Ci-Ce)-alkyl, (C2-Ce)-alkenyl or (C2-Ce)-alkynyl, where the three last-mentioned radicals are substituted by VD radicals from the group consisting of halogen, hydroxy, (CrCi)-alkyl, (d-C ⁇ -alkoxy and (Ci-C4)-alkylthio, or
  • RD 7 is hydrogen, (Ci-C4)-alkylamino, di-(Ci-C4)-alkylamino, (Ci-Ce)-alkyl, (C3-C6)- cycloalkyl, where the 2 last-mentioned radicals are substituted by VD substituents from the group consisting of halogen, (Ci-C-4)-alkoxy, halogen-(Ci-C6)-alkoxy and (C1-C4)- alkylthio and, in the case of cyclic radicals, also (Ci-C 4 )-alkyl and (Ci-C 4 )-haloalkyl; no is 0, 1 or 2;
  • v D is 0, 1 , 2 or 3; from among these, preference is given to compounds of the type of the
  • N-acylsulfonamides for example of the formula (S-V) below, which are known, for example, fro
  • RD 7 is (Ci-Ce)-alkyl, (C3-C6)-cycloa!kyl, where the 2 last-mentioned radicals are substituted by VD substituents from the group consisting of halogen, (CrCi)-alkoxy, halogen-(Ci-Ce)-alkoxy and (Ci-C4)-alkylthio and, in the case of cyclic radicals, also (Ci-C 4 )-alkyl and (Ci-C 4 )-haloalkyl;
  • RD 4 is halogen, (Ci-C 4 )-alkyl, (C,-C 4 )-alkoxy, CF 3;
  • v D is 0, 1 , 2 or 3;
  • acylsulfamoylbenzamides for example of the formula (S-VI) below, which are known, for example, from WO 99/16744,
  • RD 8 and R D 9 independently of one another are hydrogen, (d-CeJ-alkyl, (Cr-Ce)- cycloalkyl, (C3-Ce)-alkenyl, (C3-C6)-alkynyl,
  • RD 4 is halogen, (CrC 4 )-alkyl, (CrC 4 )-alkoxy, CF 3 from among these in particular
  • RK 2 independently of one another are halogen, (Ci-C4)-alkyl, (Ci-C-4)-alkoxy, (Ci-C 4 )-haloalkyl, (Ci-C 4 )-alkylamino, di-(Ci-C 4 )-alkylamino, nitro;
  • a K is COORK 3 or COORK 4
  • RK 3 , RK 4 independently of one another are hydrogen, (Ci-C 4 )-alkyl, (Ci-Ce)- alkenyl, (C2-C 4 )-alkynyl, cyanoaikyi, (Ci-C 4 )-haloalkyl, phenyl, nitrophenyl, benzyl, halobenzyl, pyridinylalkyl or alkylammonium,
  • n K 1 is 0 or 1 ,
  • ⁇ 2 , n 3 independently of one another are 0, 1 or 2 preferably: methyl (diphenylmethoxy)acetate (CAS Reg. No. : 41 858-1 9-9),
  • RL 1 is halogen, (Ci-C 4 )-alkyl, (Ci-C 4 )-haloalkyl, (Ci-C 4 )-alkoxy, (Ci-C 4 )-haloalkoxy, nitro, (CrC4)-alkylthio, (CrC4)-alkylsulfonyl, (Ci-C 4 )-alkoxycarbonyl, optionally substituted phenyl, optionally substituted phenoxy,
  • RL 2 is hydrogen or (Ci-C 4 )-alkyl
  • Ri 3 is hydrogen, (Ci-C8)-alkyl, (C2-C 4 )-alkenyl, (C2-C 4 )-alkynyl or aryl, where each of the carbon-containing radicals mentioned above is unsubstituted or substituted by one or more, preferably by up to three, identical or different radicals from the group consisting of halogen and alkoxy; or salts thereof, M) active compounds from the class of the 3-(5-tetrazolylcarbonyl)-2-quinolones, for example
  • RN 1 is halogen, (Ci-C 4 )-alkyl, methoxy, nitro, cyano, CF3, OCF3 Y, Z independently of one another are O or S,
  • ⁇ is an integer from 0 to 4,
  • RN 2 is (Ci-Ci6)-alkyl, (C2-C6)-alkenyl, (C3-C6)-cycloalkyl, aryl, benzyl, halobenzyl, RN 3 is hydrogen, (Ci-Ce)alkyl,
  • a mixture with other known active compounds, such as fungicides, insecticides, acaricides, nematicides, bird repellents, plant nutrients and soil structure improvers is likewise possible.
  • Some of the safeners are already known as herbicides and accordingly, in addition to the herbicidal action against harmful plants, also act by protecting the crop plants.
  • the weight ratios of herbicide (mixture) to safener generally depend on the herbicide application rate and the effectiveness of the safener in question and may vary within wide limits, for example in the range from 200:1 to 1 :200, preferably from 100:1 to 1 :100, in particular from 20:1 to 1 :20.
  • the safeners may be formulated analogously to the compounds to be safened or their mixtures with other herbicides/pesticides and be provided and used as a finished formulation or as a tank mix with the herbicides.
  • the required application rate of the compound to be safened varies depending, inter alia, on external conditions such as temperature, humidity and the type of herbicide used. It can vary within wide limits, for example between 0.001 and 10 000 g/ha or more of active substance; however, it is preferably between 0.5 and 5000 g/ha, particularly preferably between 0.5 and 1000 g/ha and very particularly preferably between 0.5 and 500 g/ha.
  • the transgenic plant of the invention contains one or more other genes for tolerance towards other herbicides (as, for example, a gene which encodes a mutated or unmutated EPSPS which confers on the plant tolerance to glyphosate herbicides or a pat or bar gene conferring tolerance to glufosinate herbicides), or when the transgenic plant is naturally resistant to another herbicide (such as sulfonylurea tolerance), the method according to the invention can comprise the simultaneous or chronologically staggered application of one or more HPPD inhibitor herbicide(s) in combination with the said herbicide or herbicide combination, for example glyphosate and/or glufosinate and/or sulfonylurea herbicides, 2,4-D and/or dicamba.
  • the invention also relates to the use of the chimeric gene encoding the HPPD of the invention as a marker gene during the transformation of a plant species, based on the selection on the abovementioned HPPD inhibitor herbicides.
  • the present invention also relates to a method for obtaining a plant resistant to one or more HPPD inhibitor herbicide(s) selected from the group consisting of a N-(1 ,2,5- oxadiazol-3-yl)benzamide(s), N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamide(s), a triketone(s), and pyrazolinate(s), characterized in that the plant is transformed with a chimeric gene expressing in the plant an HPPD of the invention as defined herein.
  • HPPD inhibitor herbicide(s) selected from the group consisting of a N-(1 ,2,5- oxadiazol-3-yl)benzamide(s), N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamide(s), a triketone(s), and pyrazolinate(s), characterized in that the plant is transformed with a chimeric gene expressing in
  • the invention relates to said method for obtaining a plant resistant to one or more HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamide(s), N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamide(s), triketone(s), and pyrazolinate(s), characterized in that the HPPD protein of the invention comprises SEQ ID No.
  • the invention relates to said method for obtaining a plant resistant to one or more HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, and triketone(s), such as tembotrione, mesotrione, diketonitrile, isoxaflutole, sulcotrione, tefuryltrione, and bicyclopyrone.
  • HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, and triketone(s), such as tembotrione, mesotrione, diketonitrile, isoxaflutole,
  • the invention relates to said method for obtaining a plant resistant to one or more HPPD inhbitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, triketones and pyrazolinates, characterized in that the plant also comprises a plant-expressible chimeric gene encoding a PDH (prephenate
  • dehydrogenase enzyme, or an enzyme with at least PDH.
  • the invention also relates to a method for controlling weeds in an area or a field, which method comprises planting in this area or field transformed plants resistant to one or more HPPD inhibitor herbicide(s) selected from the group consisting of
  • the invention also relates to a method for obtaining oil or meal comprising growing a transformed plant resistant to one or more HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N- (triazol-3-yl)arylcarboxamides, triketones and pyrazolinates which has been obtained according to the method described above, or a transformed seed which originates from such plant, optionally treating such plant or seed with a triketone or a
  • HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N- (triazol-3-yl)arylcarboxamides, triketones and pyrazolinates which has been obtained according to the method described above, or a transformed seed which originates from such plant, optionally treating
  • pyrazolinate HPPD inhibitor harvesting the grains and milling the grains to make meal and extract the oil.
  • the invention also relates to the use of an HPPD of the invention as described above, characterized in that the HPPD inhibitor herbicide(s) is/are selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, triketones, preferably selected from the group consisting of tembotrione, mesotrione, topramezone, bicyclopyrone, tefuryltrione and sulcotrione.
  • the HPPD inhibitor herbicide(s) is/are selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamides, N-(tetrazol-4-yl)- or N-(triazol-3- yl)arylcarboxamides, triketones, preferably selected from the group consisting of tembotrione, mesotrione, topram
  • the present invention also relates to a host organism, in particular plant cells or plants, which contain a chimeric gene comprising a sequence encoding an HPPD according to the invention, and which also contain a gene functional in this host organism allowing overexpression of a prephenate dehydrogenase (abbreviated herein as PDH) enzyme.
  • PDH enzyme refers to any natural or mutated PDH enzyme exhibiting the PDH activity of conversion of prephenate to HPP.
  • said PDH enzyme can originate from any type of organism.
  • An enzyme with PDH activity can be identified by any method that makes it possible either to measure the decrease in the amount of prephenate substrate, or to measure the accumulation of a product derived from the enzymatic reaction, i.e. HPP or one of the cof actors NADH or NADPH.
  • HPP a product derived from the enzymatic reaction
  • NADH NADH
  • NADPH one of the cof actors NADH or NADPH.
  • Many genes encoding PDH enzymes are described in the literature, and their sequences can be identified on the website http://www.ncbi.nlm.nih.gov/entrez/.
  • the invention further relates to a method for obtaining a host organism, particularly a plant cell or a plant, resistant to one or more HPPD inhibitor herbicide(s) by
  • the invention relates to a method for obtaining a host organism, particularly a plant cell or a plant, resistant to one or more HPPD inhibitor herbicide(s) selected from the group consisting of N-(1 ,2,5-oxadiazol-3-yl)benzamide, N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamide, triketones and pyrazolinates, particularly resistant to one or more HPPD inhibitor herbicide(s) selected from the group consisting of tembotrione, mesotrione, topramezone, bicyclopyrone,
  • A, B, and C tobacco plants
  • D and E tobacco and soybean plants
  • F, G, H, and I soybean plants
  • J cotton plants.
  • H6 sequence coding for an His TAG
  • OTP optimized transit peptide
  • BAR Bialaphos resistant, WO 87/05629
  • PAT phosphinothricin N-Acetyltransferase, EP 257542
  • 2mEPSPS gene coding for the double mutant (Thr102lle and Pro106Ser) EPSPS (5-enolpyruvylshikimate synthase) from Zea mays (US 20030027312)
  • 2mAHAS gene coding for the double mutant ALS (acetolactate synthase) from Arabidopsis (Pro197Ala and Trp574Leu; US 5378824
  • HA histone promoter from
  • FMP45e gene coding for FMP45 optimized for the expression in E coli with an sequence coding for an His TAG at its 5 ' extremity
  • FMP45d gene coding for FMP45 optimized for the expression in dicotyledoneous plants
  • FMP45n is the native gene sequence coding for FMP45, LB, left border, RB, right border.
  • SEQ ID No. 2 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for E. coli , containing at the 5' end a nucleic acid sequence encoding an aspartate.
  • SEQ ID No. 3 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for E. coli, containing at the 5' end a nucleic acid sequence encoding a HIS tag (according to SEQ ID No. 4), followed by a nucleic acid stretch encoding 2 serines and 1 glycine, followed by a nucleic acid stretch encoding a protein binding site thrombin (according to SEQ ID No. 6) followed by a nucleic acid stretch endoding a glycine and methionine, followed by a nucleic acid stretch encoding a S-tag (according to SEQ ID No.
  • nucleic acid stretch encoding proline, aspartate, leucine, glycine, and threonine followed by a nucleic acid stretch encoding the recognition site of an enterokinase (according to SEQ ID No. 10), followed by a nucleic acid encoding aspartate in front of the AUG start codon subsequently followed by a nucleic acid sequence encoding for an aspartate.
  • SEQ ID No. 4 Nucleic acid sequence encoding 6 consecutive histidines.
  • SEQ ID No. 5 Amino acid sequence derived from SEQ ID No. 4.
  • SEQ ID No. 6 Nucleic acid stretch encoding a protein binding site thrombin.
  • SEQ ID No. 7 Amino acid sequence derived from SEQ ID No. 6.
  • SEQ ID No. 8 Nucleic acid stretch encoding a S-tag.
  • SEQ ID No. 9 Amino acid sequence derived from SEQ ID No. 8.
  • SEQ ID No. 1 Amino acid sequence derived from SEQ ID No. 10.
  • SEQ ID No.12 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for E. coli containing at the 5' end a nucleic acid sequence encoding an optimized transit peptide (according to SEQ ID No. 13) and an aspartate.
  • SEQ ID No. 13 Nucleic acid sequence encoding an optimized transit peptide.
  • SEQ ID No. 14 Amino acid sequence derived from SEQ ID No. 13.
  • SEQ ID No. 15 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for dicotyledonous plants containing at the 5' end a nucleic acid sequence encoding an optimized transit peptide
  • SEQ ID No. 16 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD containing at the 5' end a nucleic acid sequence encoding an optimized transit peptide (according to SEQ ID No. 13).
  • SEQ ID No. 17 Rhodococcus opacus B4 HPPD amino acid sequence derived from
  • SEQ ID No. 18 Protein encoded by SEQ ID No. 2.
  • SEQ ID No. 21 Protein encoded by SEQ ID No. 15.
  • SEQ ID No. 22 Protein encoded by SEQ ID No. 16.
  • SEQ ID No. 25 Nucleic acid sequence encoding Arabidopsis thaliana HPPD
  • nucleic acid encoding an alanine and 6 histidine amino acids
  • SEQ ID No. 26 Protein encoded by SEQ ID No. 25.
  • SEQ ID No. 27 Nucleid acid sequence encoding Arabidopsis thaliana HPPD
  • SEQ ID No. 28 Protein of SEQ ID No. 24 plus the OTP sequence (SEQ ID No. 14) located at the N-terminal extremity of the protein.
  • SEQ ID No. 29 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for Zea mays plants
  • SEQ ID No. 30 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for Brassica napus plants
  • SEQ ID No. 31 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for Beta vulgaris plants
  • SEQ ID No. 32 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for Gossypium hirsutum plants
  • SEQ ID No. 33 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for Glycine max plants
  • SEQ ID No. 34 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for Hordeum vulgare plants
  • SEQ ID No. 35 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for Oryza sativa plants
  • SEQ ID No. 37 Nucleic acid sequence encoding rhodococcus opacus B4 HPPD optimized for dicotyledonous plants containing at the 5' end a nucleic acid sequence encoding an optimized transit peptide
  • FMP45n represents the native gene sequence coding for the HPPD protein FMP45.
  • FMP45e represents the gene sequence optimized for the expression in E. coli coding for the HPPD protein FMP45.
  • FMP45d represents the gene sequence otpimized for the expression in
  • dicotyledeneous plants such as for example in soybean, cotton, oil seed rape and sugarbeet coding for the HPPD protein FMP45.
  • FMP45da represents the gene sequence optimized for the expression in
  • FMP45m represents the gene sequence otpimized for the expression in Zea mays plants coding for the HPPD protein FMP45.
  • Rhodococcus opacus B4 HPPD (named FMP45) of SEQ ID No. 19 and of the Arabidopsis thaliana HPPD identified by SEQ ID No. 26.
  • the Arabidopsis thaliana AtHPPD coding sequence (1335 bp; Genebank AF047834; WO 96/38567) was initially cloned into the expression vector pQE-30 (QIAGEN, Hilden, Germany) in between the restriction sites of BamHI and Hindlll. The obtained vector was called "pQE30-AtHPPD".
  • Rhodococcus opacus B4 HPPD sequence (1206 bp) coding for the protein listed under the accession number C1 B587 at UniProtKB/TrEMBL was modified and synthesized using an Escherichia coli K12 optimized codon usage (GeneArt, Regensburg, Germany, proprietary software) and cloned in a modified pBluescript vector (GeneArt, Regensburg, Germany).
  • nucleic acid sequence coding for an aspartate was inserted.
  • two additional cytosine base pairs were added in order to obtain a sequence corresponding to the recognition site of the restriction enzyme Ncol and downstream to the stop codon the sequences corresponding to the recognition site of the restriction enzyme BamHI were added.
  • the resulting vector "pBluescript-FMP45e" was digested with the restriction enzymes Ncol and BamHI, the band migrating not to the length of the size of the vector approximately 3000 bp corresponding to the DNA was separated on an agarose gel per electrophoresis.
  • DNA coding for the HPPD was purified using the MinEluteTM Gel Extraction Kit (Qiagen, Hilden, Germany) and cloned into the pET32a (Novagen, Darmstadt, Germany) vector (see below) previously cut with the same restriction enzymes.
  • the cloning of the gene coding for FMP45 into pET32a was made in frame in order to obtain a fusion between a upstream N-terminal His-tag (composed of six histidine amino acids (also called "Hise”)) and the protein FMP45 in E. coli cells.
  • the plasmid possesses the trp-lac (trc) promoter and the lacl q gene that provides the lac repressor in every E. coli host strain.
  • the lac repressor binds to the lac operator (lacO) and restricts expression of the target gene; this inhibition can be alleviated by induction with Isopropyl ⁇ -D-l -thiogalactopyranoside (IPTG).
  • the resulting vector was called "pET32a-FMP45e” (see Figurel ) and it was used to transform Escherichia coli BL21 cells (Merck, Darmstadt, Germany ).
  • AtHPPD (Arabidopsis thaliana HPPD) that was used as reference see
  • HPPD HPPD-induced HPPD
  • E. coli K-12 BL21 containing pQE30-AtHPPD or pET32a-FMP45e Cells were allowed to grow until OD reached 0.5, then
  • pre-starter culture 2 mL of TB medium (100 g*mL "1 carbenicillin) were inoculated with 50 ⁇ _ of an E. coli K-12 BL21 glycerol stock. The pre-starter culture was incubated at 37 °C with shaking at 140 rpm for 15 h. 200 ⁇ of the pre-starter culture was used to initiate the starter culture (5ml_ TB supplement with 100 Mg*L "1 ), which was incubated 3 h at 37°C.
  • Lysis of cells were lysed using Lysozyme, an enzyme that cleaves the 1 ,4-
  • the lysis buffer contained Benzonase ® Nuclease, an endonuclease that hydrolyzes all forms of DNA and RNA without damaging proteins and thereby largely reduces viscosity of the cell lysate. Lysis under native conditions was carried out on ice.
  • the cleared cell lysate (10 mL) obtained after centrifugation of the lysis reaction was loaded onto a Ni-NTA Fast Start Column from the QIAexpress ® Ni-NTA Fast Start Kit (Qiagen, Hilden, Germany) and purification was carried out according to the instruction manual.
  • the Hise-tagged protein was eluted with 2.5 mL of elution buffer. Desalting of HPPD solutions by gel filtration
  • HPPD solutions eluted from the desalting column were frozen at -80 °C in 1 mL aliquots.
  • the gel was immersed in Coomassie Brilliant Blue R-250 Staining Solution.
  • Coomassie Brilliant Blue R-250 Destaining Solution For staining of protein bands, the gel was immersed in Coomassie Brilliant Blue R-250 Destaining Solution until protein bands appear blue on a white gel.
  • HPPD activity was checked by the standard spectrophotmetric assay (method extensively described in WO 2009/144079) Determination of HPPD in vitro kinetic properties
  • the assay mixtures contained in a volume of 1 ml 150 mM Tris-HCI buffer at pH 7.8, 10 mM sodium ascorbate, 650 units of bovine catalase (Sigma C30 (Sigma-Aldrich, Kunststoff, Germany), 34 mg protein/ml, 23,000 units/mg), and appropriate amounts of HPP, purified HPPD enzyme and HPPD inhibitors.
  • K m , Vmax, and k ca t value determination HPP concentrations in the assay mixture were varied between 10 and 400 ⁇ .
  • Ki K on
  • K.i value determination 2 mM HPP 2 mM HPP was used. All assays were started by the addition of HPPD enzyme to the assay mixture and stopped at a series of times between 0 and 240 s by addition of 200 ⁇ of the reaction mixture to reaction assay tubes containing 20 ⁇ 10% perchloric acid. Precipitated protein was pelleted by a 5 minute centrifugation at 10,000 g. 100 ⁇ of the supernatant were loaded onto a 250 x 4mm Knauer (Berlin, Germany) Eurospher 100-5 C18-column equilibrated with 10% methanol, 0.1 % trifluoroacetic acid (buffer A).
  • the column was eluted, also at 1 .5 ml/min, using a 4 minute wash with buffer A, followed by a 3 min wash with 95% methanol and by a further 2 minute wash with buffer A.
  • the elution of HGA (homogentisic acid) and HPP (hydroxyphenylpyruvate) was monitored at 292 nm. HGA elutes at around 5 minutes and HPP elutes later.
  • a standard set of concentrations of HGA were used to provide a standard curve in order to calibrate the 292 nm absorbance of the HGA peak versus HGA concentration.
  • Mechanism A competitive inhibition, for tight-binding inhibitors (Cha, S. (1975) Tight- binding inhibitors - I. Kinetic behaviour. Biochemical Pharmacology 24, 2177-2185) using the ID Business Solutions Ltd. XLfit software suite
  • Table 2 in vitro tolerance measurement of both HPPD enzymes from Arabidopsis (SEQ ID No. 26) and Rhodococcus opacus B4 (SEQ ID No. 19) to the HPPD inhibitor herbicide tembotrione.
  • the given numbers represent the percentage of inhibition of the enzyme activity at different concentrations of the HPPD inhibitor herbicide tembotrione compared to the activity in absence of the HPPD inhibitor herbicide.
  • plso-value means the log value of the concentration of inhibitor necessary to inhibit 50% of the enzyme activity in molar concentration.
  • plso-values for HPPD inhibitors were determined from dose-response plots of HPPD activity versus inhibitor concentration using the assay extensively described in
  • Table 4 Determination of percentage of inhibition in presence of 5.0x10 "6 M inhibitors compared to the activity measured in absence of the inhibitor for the HPPD originated from Arabidopsis thaliana (SEQ ID No. 26) and from Rhodococcus opacus B4
  • SEQ ID No. 19 showed superior level of tolerance to all tested HPPD inhibitors than the plant at all tested HPPD inhibitor concentrations than observed by employing the HPPD “SEQ ID No. 26” under identical experimental conditions.
  • Table 5 Determination of pl50 of HPPD from Rhodococcus opacus B4 (SEQ ID No. 19) and HPPD from Arabidopsis thaliana (SEQ ID No. 26) for tembotrione and diketonitrile using the HPLC method. The amount of homogentisate form in the reaction vial is evaluation after the reaction being stop after 3 minutes.
  • Table 6 Determination of specific activity of of HPPD from Rhodococcus opacus B4 (SEQ ID No. 19) and HPPD from Arabidopsis thaliana (SEQ ID No. 26) using the spectophometric method. Samples were incubated, and the reaction was stopped after 24 min. The specific activity wa estimated by g of protein.
  • the vector pSE420(RI)NX was restricted with the same enzymes. The insert and the vector were ligated to generate the vector pSE420(RI)NX-FMP45e.
  • the DNA corresponding to vector pET32a-FMP45e generated above was restricted with the enzymes Mscl and BamHI in order to separate the DNA fragment corresponding to the gene FMP45e from the vector.
  • the vector pSE420(RI)NX was restricted with the enzymes Eco53KI and BamHI. The isolated insert and vector were ligated and the resulting vector was called pSE420(RI)NX-His-Tag-FMP45e.
  • Vectors will be used to transform E coli BL21 . Following the standard protocols described above, proteins will be produced. However, the protein will not be purified using His-Tag affinity column, but the HPPD activity will be directly estimated in the protein raw extract obtained from induced bacteria.
  • Example 4 Construction of chimeric genes for the evaluation HPPD inhibitor herbicide tolerance in tobacco plants. A) Construction of the chimeric genes
  • the vector pRP-RD224 (extensively described in WO 2009/144079) containing the sequence coding for the OTP was used for PCR-mediated attachment upstream of the nucleic acid sequence corresponding to the recognition site of the restriction enzyme Xhol and downstream of the nucleic acid sequence corresponding to the recognition site of the restriction enzyme Ncol.
  • the obtained PGR product was cloned in the vector pCR®-Blunt ll-TOPO® (Invitrogen, Düsseldorf, Germany) following the user manual instruction.
  • the resulting vector was called "pCR-TOPO-OTP". The insertion of the correct sequence was confirmed per standard DNA sequencing.
  • the DNA corresponding to the OTP was digested with the restriction enzymes Ncol and Xhol, separated per appropriate gel electrophoresis and cloned into the plasmid pRT100 (Toepfer (1987), Nucleic Acids Res 15:5890) previously and correspondingly digested with Nco I and Xhol restriction enzymes.
  • the plasmid pRT100 is containing the CaMV35S promoter and CaMV35S terminator.
  • the resulting vector was subsequently digested with the restriction enzymes Ncol and Xbal.
  • the vector pET32a-FMP45e (see Figurel ) was subjected to the restriction enzymes Ncol and BamHI in order to obtain the DNA fragment corresponding to the SEQ ID No. 2.
  • the resulting vector was digested by employing the restriction enzyme Sbfl to subclone the
  • CaMV35S::OTP::FMP45e::CaMV35-term cassette (see Figure2) into the binary vector pBin19 (Bevan (1984), Nucleic Acids Res. 12:871 1 -8721 .) previously digested with the same enzyme and dephosphorylated. The resulting vector was called "pBSN19- 35S-OTP-FMP45e".
  • the vectors pQE-30-AtHPPD was used for PCR-mediated attachment of an Ncol restriction site and of a sequence encoding an N-terminal Hise-Tag to the 5' ends and a Xbal restriction site to the 3' ends of AtHPPD.
  • the PGR product of the AtHPPD (Arabidopsis thaliana HPPD) gene was isolated from an agarose gel, cut with the restriction enzymes Ncol and Xbal, purified with the MinEluteTM PGR Purification Kit (Qiagen, Hilden, Germany) and cloned into the pSE420(RI)NX vector cut with the same restriction enzymes.
  • the generated vector was called "pSE420(RI)NX-AtHPPD” and was digested with the restriction enzymes Ncol and Xbal and cloned into the previously opened vector pRT100 (Toepfer et al. (1987), Nucleic Acids Res 15:5890) containing the CaMV35S promoter and CaMV35S terminator.
  • the generated vector was called "pRT100- AtHPPD”.
  • the vector pCR-TOPO-OTP was digested with the restriction enzymes Ncol and Xhol, and the DNA band corresponding to the OTP was cloned in the previously opened vector pRT100-AtHPPD with the above mentioned restriction enzymes.
  • the resulting vector was subsequently digested with restriction enzyme Hindll! and the expression cassette of interest was cloned into the previously opened and dephosphorylated binary vector pBin19.
  • the resulting vector was called "AtHPPDbv".
  • binary vectors for dicotyledoneous plants were constructed with the 2xCaMV35S promoter or 2xCsVMV
  • the transformation vector also contains a PAT gene cassette in which the gene is driven by a CaVM35S promoter and followed by a CaMV35S terminator for glufosinate based selection during the transformation process and a 2mEPSPS gene cassette in which the gene is driven by an histone promoter from Arabidopsis thaliana to confer tolerance to the herbicide glyphosate to the transformed plants (see Fig.3 F, Fig. 3 G, Fig.3 H, and Fig.3 I).
  • the binary vectors "pB!N19-35S-OTP-FMP45e and AtHPPDbv were used to transform Agrobacterium tumefaciens (ATHV derived from EHA101 ) competent cells selected on YEB media supplemented with the antibiotics kanamycin and rifampicin (extensively described in the patent application US2005925808A).
  • the binary vectors comprising 2x35S-TEV-OTP-FMP45d and 2xCsVMV-OTP-FMP45d (as described above) were employed to transform Agrobacterium tumefaciens (ATHV derived from EHA101 ) competent cells selected on YEB media supplemented with the antibiotics kanamycin and rifampicin (extensively described in the patent application US005925808A).
  • Agrobacterium strains containing the binary vectors of interest (“pBIN19-35S- OTP-FMP45e or AtHPPDbv) were used to transform leaf discs from tobacco Nicotiana tabacum L. cv SR1 NN plants, having approximately a size of 5x5mm 2 as extensively described in Horsch et al. (1985), Science 227 ; 1229-1231 .
  • the leaf disks were co-cultivated for 2 days with Agrobacterium tumefaciens cells containing any of either the binary vectors as described above "pBIN19-35S-OTP- FMP44e or AtHPPDbv. Then the leaf disks were transferred to a media allowing the regeneration of shoots for 6 weeks on MS (Musharige and Skoog, (1962), Physiol Plant 15(3): 473-497) media supplemented with BAP (1 mg/mL; Benzylaminopurine), carbenicillin (250 mg/mL), cefotaxine (250 mg/mL), kanamycin (75 mg/mL) and tembotrione (10-6 M) or with BAP (1 mg/mL; Benzylaminopurine), carbenicillin (250 mg/mL), cefotaxine (250 mg/mL), glyphosate (1 mM) and tembotrione (10-6 M), depending on the resistance provided by the specific vector. Regenerated calli were transferred on media
  • Rhodococcus opacus B4 or the protein of SEQ ID No. 37 comprising the HPPD sequence of Rhodococcus opacus B4 were transferred to a media inducing root growth for 6 to 12 weeks.
  • Example 5 Glasshouse trials to evaluate tolerance to HPPD inhibitor herbicides of transgenic tobacco plants expressing a gene coding for tolerant HPPD protein
  • 3 wild type (wt) tobacco plants will also be treated with an equivalent amount of tembotrione under identical conditions.
  • the symptoms in tranformed plants will be evaluated in comparison to the response observed on the wild type tobacco plants sprayed at the same time and under the same conditions as the tobacco plants containing the transgenes.
  • the treated plants evaluated as "0" are looking like the untreated tobacco plants.
  • the plants evaluated as “1” display temporarly light bleaching phenotype due to the application of the herbicides.
  • the plants evaluated as “2” display permanent light to strong bleaching symptoms.
  • Finally the plants evalutated as "3" are looking like wild type tobacco plants submitted to the same treatment
  • Seeds obtained from 6 primary tolerant transformants were collected and sawn on soil (ED73 mixed with sand and osmocote Pro) in the glasshouse (28/20°C).
  • - tembotrione at 100 gAI/ha prepared from a WP20 (wettable powder 20%) formulation supplemented with ammonium sulfate and methyl ester raps oil, or
  • blind formulation made from a WP20 formulation without active ingredient (Al) supplemented with ammonium sulfate and methyl ester raps oil,
  • the treated plants evaluated as "0" are looking like the untreated tobacco plants.
  • the plants evaluated as “1” display temporarly light bleaching phenotype due to the application of the herbicides.
  • the plants evaluated as “2” display permanent light to strong bleaching symptoms.
  • Finally the plants evalutated as "3" are looking like wild type tobacco plants submitted to the same treatment
  • Seeds obtained from 2 primary tolerant transformants (TO plants of ) CaMV35S- OTP- FMP45e were collected and sawn on soil (ED73 mixed with sand and osmocote Pro) in the glasshouse (28/20°C).
  • the treated plants evaluated as "0" are looking like the untreated tobacco plants.
  • the plants evaluated as “1” display temporarly light bleaching phenotype due to the application of the herbicides.
  • the plants evaluated as “2” display permanent light to strong bleaching symptoms.
  • Finally the plants evalutated as "3" are looking like wild type tobacco plants submitted to the same treatment
  • the treated plants evaluated as "0" are looking like the untreated tobacco plants.
  • the plants evaluated as “1” display temporarly light bleaching phenotype due to the application of the herbicides.
  • the plants evaluated as “2” display permanent light to strong bleaching symptoms.
  • Finally the plants evalutated as "3" are looking like wild type tobacco plants submitted to the same treatment
  • plants expressing FMP45 are tolerant to HPPD inhibitors.
  • Example 6 Construction of binary vectors to express dicotyledoneous optimized variant in plants and glasshouse trial to evaluate tolerance of tobacco plants containing such variant
  • the vector pFCO1 17 (WO201 1/09460) was derived from pSF49, a descendant of pBL150u2 (EP508909).
  • the bar cassette has first been cloned into pSF49 (Not!/ Avrll), to obtain pFC020.
  • the cassette contains lox sites for bar removal (c re/I ox system) in the event and some meganucleases sites (l-Scel, l-Crel, l-Ceul, l-Seel) for further gene insertion at the same locus by homologous recombination.
  • pFC020 contains convenient restriction sites for epsps cloning (Sbfl/ Swal) and HPPD cloning (Mscl/ Xhol).
  • EPSPS is under the control of Ph4A7, promoter of Arabidopsis thaliana histone H4 gene (Chaboute M, et al., (1987), Plant Mol Biol, 8:179-191 ).
  • the expression of the w336 mutated HPPD from Pseudomonas fluorescens (Boudec P.
  • TPotpc a fragment of the promoter region from the Cauliflower Mosaic Virus 35S (CaMV 35S) transcript, followed by ENtev, an enhancer sequence of tobacco etch virus (Carrington J.c. and Freed D.D. (1990), J. Virol., 64, 1590-1597).
  • HPPD proteins are targeted into the chloroplast via the optimized transit peptide TPotpc (Lebrun et al (1996); US5510471 ).
  • the TPotpc- HPPDPfw336 sequence is codon optimized in order to fit dicotyledeneous plants such as for example soybean, cotton, sugarbeets, and oil seeds raps usage codon.
  • a gene with codon usage optimized for the expression in dicotyledoneous plants such as for example soybean, cotton, sugarbeets, and oil seeds raps coding for the HPPD protein FMP45 was designed, and named FMP45d (SEQ ID No. 37) and a gene with codon usage optimized for the expression in dicotyledoneous plants such as for example soybean, cotton, sugarbeets, and oil seeds raps coding for FMP45 including an additional aspartate in position 2 (SEQ ID No 18, compared to orginal SEQ ID No. 17) was designed and named FMP45da (SEQ ID No. 15).
  • FMP45d SEQ ID No. 37
  • FMP45da SEQ ID No. 15
  • Cauliflower Mosaic Virus 35S transcript followed by ENtev, an enhancer sequence of tobacco etch virus (Carrington J.c. and Freed D.D. (1990), J. Virol., 64, 1590-1597), followed by the gene coding for mutant Pseudomonas fluorescens HPPD G336W.
  • the final vector containing a gene coding for double mutant EPSPS, a gene coding for the PAT/BAR and a gene encoding the HPPD FMP45 was called pFCO-FMP45 (see Fig.3 F, Fig. 3 G, Fig.3 H, and Fig.3 I).
  • the binary vectors were respectively called and can be used for example to transform dicotyledenous plants, such as tobacco plants as described above. Sufficiently grown transformant plants are then tested for their tolerance to HPPD inhibitor herbicides, such as tembotrione. The development of the observed symptoms in response to the herbicidal treatment is evaluated and compared to the response of wild type plants under the same conditions.
  • rooted plants containing the T-DNA Prom2xCaMV35S-TEV-OTP- FMP45d-TerCaMV35S will be transferred to the greenhouse under standard growth conditions.
  • the TO plants will be treated with a mixture containing an equivalent to 100 g tembotrione /ha prepared from a WP20 (wettable powder 20%) formulation supplemented with ammonium sulfate and methyl ester raps oil.
  • WP20 wettable powder 20%
  • the symptoms due to the application of the herbicides will be evaluated.
  • the plants will be classified in four categories.
  • the treated plants evaluated as "0" are looking like the untreated tobacco plants.
  • the plants evaluated as "1 " display temporarely a light bleaching phenotype.
  • the plants evaluated as "2” display permanent light to strong bleaching symptoms.
  • Finally the plants evalutated as "3" are looking like wild type tobacco plants submitted to the same treatment.
  • Example 7 Cloning of gene FMP44n and FMP44m coding for FMP45 HPPD in a vector to transform Zea mays plants
  • FMP45n SEQ ID No. 1
  • FMP45m SEQ ID No. 29 genes will be cloned under the control of a rice ubiquitin promoter and transferred into an approriate maize transformation vector. This vector will be used to transform Agrobacterium
  • Immature embryos of maize of the Hi Type II hybrid line (Armstrong et al. (1991 ), Maize Genet. Coop. News, 65:92-93) are aseptically isolated from greenhouse-grown ears, 10-16 days after pollination.
  • the embryos are infected and co-cultivated for 3 days with Agrobacterium tumefaciens cultures carrying an HPPD gene on the binary vector.
  • Agrobacterium tumefaciens cultures carrying an HPPD gene on the binary vector After co-cultivation, the embryos are grown on selective callus inducing medium containing phosphinoticin (5 mg/l) and sub-cultured every 2 weeks till production of type II embryogenic callus. The callus is then grown on selective regeneration medium for the development of transgenic maize plants (Frame et al.
  • Example 8 Soybean TO plant establishment and selection.
  • the vectors according to Example 6 will be employed for soybean transformation as described in WO201 1/09460.
  • Binary vectors for soybean transformation is, for example, constructed with the CaMV35 promoter driving the expression of the gene FMP45d (SEQ ID No. 37), FMP45da (SEQ ID No. 15) with a codon usage optimized for the expression in dicotyledoneous plants or FMP45n (SEQ ID No. 16) with a native codon usage and at the 5'extremity was added a sequence coding for an OTP, and further upstream a sequence TEV (Tobacco etch virus) to improve the stability of the mRNA in plants followed by the CaMV35S terminator.
  • FMP45d SEQ ID No. 37
  • FMP45da SEQ ID No. 15
  • FMP45n SEQ ID No. 16
  • TEV tobacco etch virus
  • the transformation vector also contains a PAT gene cassette in which the gene is driven by a CaVM35S promoter and followed by a CaMV35S terminator for glufosinate based selection during the transformation process and a 2mEPSPS gene cassette in which the gene is driven by an histone promoter from Arabidopsis thaliana to confer tolerance to the herbicide glyphosate to the transformed plants (see Fig.3 F, Fig. 3 G, Fig.3 H, and Fig.3 I). .
  • Soybean transformation is achieved using methods well known in the art, such as the one described using the Agrobacterium tumefaciens mediated transformation soybean half-seed explants described by Paz et al. (2006), Plant cell Rep. 25:206.
  • Transformants were identified using Isoxaflutole or tembotrione as selection marker. The appearance of green shoots was observed, and documented as an indicator of tolerance to the herbicide isoxaflutole or tembotrione. The tolerant transgenic shoots will show normal greening comparable to wild-type soybean shoots not treated with isoxaflutole or tembotrione, whereas wild-type soybean shoots treated with the same amount of isoxaflutole or tembotrione will be entirely bleached. This indicates that the presence of FMP45 protein enables the tolerance to HPPD inhibitor herbicides, like isoxaflutole or tembotrione.
  • Tolerant green shoots will be transferred to rooting media or grafted. Rooted plantlets will be transferred to the glasshouse after an acclimation period.
  • Plants containing the transgene will be then sprayed with HPPD inhibitor herbicides, as for example with tembotrione at a rate of 100g Al/ha. Ten days after the application the symptoms due to the application of the herbicide will be evaluated and compared to the symptoms observed on a wild type plants under the same conditions.
  • a binary vector for cotton transformation is, for example, constructed with the
  • CaMV35 promoter driving the expression of the gene FMP45d (SEQ ID No. 37), with a codon usage optimized for the expression in dicotyledoneous plants or the gene FMP45da (SEQ ID No. 15) with a codon usage optimized for the expression in dicotyledoneous plants or the gene FMP45n (SEQ ID No. 16) with a native codon usage and at the 5 ' extremity was added a sequence coding for an OTP, and further upstream a sequence TEV (Tobacco Etch Virus) to improve the stability of the mRNA in plants followed by the CaMV35S terminator.
  • TEV tobacco Etch Virus
  • the transformation vector also contains a PAT gene cassette in which the gene is driven by a CaVM35S promoter and followed by a CaMV35S terminator for glufosinate based selection during the transformation process and a 2mEPSPS gene cassette in which the gene is driven by an histone promoter from Arabidopsis thaliana to confer tolerance to the herbicide glyphosate to the transformed plants (see Fig.3 J).
  • Example 10 Cotton TO plant establishment and selection. Cotton transformation is achieved using methods well known in the art, especially preferred method in the one described in the PCT patent publication
  • Regenerated plants are transferred to the glasshouse. Following an acclimation period, sufficiently grown plants are sprayed with HPPD inhibitor herbicides as for example tembotrione equivalent to 100 gAI/ha supplemented with ammonium sulfate and methyl ester raps oil. Seven days after the spray application, the symptoms due to the treatment with the herbicide are evaluated and compared to the symptoms observed on wild type cotton plants subjected to the same treatment under the same conditions.
  • Example 1 1 Construction of binary transformation vectors to generate plants tolerant to four herbicides with distinct modes of action.
  • a binary vector for dicotyledoneous plant transformation is, for example, constructed with the CaMV35 promoter driving the expression of the gene FMP45d (SEQ ID No. 37), with a codon usage optimized for the expression in dicotyledoneous plants or of the gene FMP45n (SEQ ID No. 16) with a native codon usage and at the 5'-extremity is added a sequence coding for an OTP followed by the CaMV35S terminator.
  • the transformation vector also contains a PAT gene cassette in which the gene is driven by a CaVM35S promoter and followed by a CaMV35S terminator to confer tolerance to glufosinate to the plant expressing the gene, a 2mEPSPS gene cassette coding for the double mutant (Thr102lle and Pro106Ser) EPSPS in which the gene is driven by an histone promoter from Arabidopsis to confer tolerance to the herbicide glyphosate to the transformed plants, and an Arabidopsis thaliana 2mAHAS gene cassette encoding a tolerant ALS enzyme (Acetolactate synthase, Pro197Ala, Trp574Leu) driven by a CaMV35S promoter to confer tolerance to herbicides from the sulfonylurea or imidazolinone classes to the plant expressing this gene (see Fig.3 D and Fig.3 E).
  • a PAT gene cassette in which the gene is driven by a CaVM35S promoter and followed by a CaMV
  • the gene cassettes will be finally cloned into the vector pHoe6/Ac (US 6,316,694), and the final vectors will be called pHoe6/FMP45d/PAT/EPSPS/AHAS and
  • pHoe6/FMP45n/PAT/EPSPS/AHAS is used to transform dicotyledoneous plants via Agrobacterium tumefaciens mediated state of the art methods.
  • TO plants are transferred to soil, and after an acclimation period, sufficiently grown plants are sprayed successively with an herbicide from the HPPD inhibitor class, then with glyphosate, then with glufosinate and finally with an an ALS inhibitor herbicide, preferably with a compound selected from the group of sulfonylureas or

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Physiology (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention concerne des séquences d'acides nucléiques codant une hydroxyphénylpyruvate dioxygénase (EC 1.13.11.27, ci-après dénommée "HPPD"), obtenues de Rhodococcus opacus, ainsi que les protéines ainsi codées. Elle concerne un gène chimère comprenant une telle séquence d'acides nucléiques; et l'utilisation des ces séquences d'acides nucléiques, protéines ou gènes chimères pour obtenir des plantes tolérantes à un ou plusieurs herbicides inhibiteurs de la HPPD.
PCT/EP2012/075906 2011-12-22 2012-12-18 Plantes tolérantes à des herbicides inhibiteurs de hppd WO2013092555A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP11195295 2011-12-22
EP11195295.8 2011-12-22
US201161581127P 2011-12-29 2011-12-29
US61/581,127 2011-12-29

Publications (1)

Publication Number Publication Date
WO2013092555A1 true WO2013092555A1 (fr) 2013-06-27

Family

ID=48667746

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2012/075906 WO2013092555A1 (fr) 2011-12-22 2012-12-18 Plantes tolérantes à des herbicides inhibiteurs de hppd

Country Status (2)

Country Link
AR (1) AR089250A1 (fr)
WO (1) WO2013092555A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105409250A (zh) * 2013-07-15 2016-03-16 微软技术许可有限责任公司 用于多个sim卡的智能用户界面

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010079117A2 (fr) * 2009-01-07 2010-07-15 Bayer Cropscience Ag Plantes transplastomiques dépourvues du marqueur de sélection
WO2011076892A1 (fr) * 2009-12-23 2011-06-30 Bayer Cropscience Ag Plantes tolérantes aux herbicides inhibiteurs de hppd

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010079117A2 (fr) * 2009-01-07 2010-07-15 Bayer Cropscience Ag Plantes transplastomiques dépourvues du marqueur de sélection
WO2011076892A1 (fr) * 2009-12-23 2011-06-30 Bayer Cropscience Ag Plantes tolérantes aux herbicides inhibiteurs de hppd

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE UniProt [Online] 26 May 2009 (2009-05-26), "SubName: Full=4-hydroxyphenylpyruvate dioxygenase; EC=1.13.11.27;", XP002670446, retrieved from EBI accession no. UNIPROT:C1B587 Database accession no. C1B587 *
DUFOURMANTEL NATHALIE ET AL: "Generation and characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance", PLANT BIOTECHNOLOGY JOURNAL, BLACKWELL, OXFORD, GB, vol. 5, no. 1, 1 January 2007 (2007-01-01), pages 118 - 133, XP002531775, ISSN: 1467-7644, DOI: 10.1111/J.1467-7652.2006.00226.X *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105409250A (zh) * 2013-07-15 2016-03-16 微软技术许可有限责任公司 用于多个sim卡的智能用户界面
CN105409250B (zh) * 2013-07-15 2019-11-26 微软技术许可有限责任公司 用于多个sim卡的智能用户界面

Also Published As

Publication number Publication date
AR089250A1 (es) 2014-08-06

Similar Documents

Publication Publication Date Title
US8853496B2 (en) Plants tolerant to HPPD inhibitor herbicides
EP2516633B1 (fr) Plantes tolérantes à des herbicides inhibant l'HPPD
EP2516630B1 (fr) Plantes tolérantes à des herbicides inhibant l'HPPD
US8859856B2 (en) Plants tolerant to HPPD inhibitor herbicides
EP2516635B1 (fr) Plantes tolérantes à des herbicides inhibant l'hppd
WO2013092552A1 (fr) Plantes tolérantes à des herbicides inhibiteurs de hppd
WO2013092551A1 (fr) Plantes tolérantes à des herbicides inhibiteurs de hppd
WO2013092555A1 (fr) Plantes tolérantes à des herbicides inhibiteurs de hppd

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12809761

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12809761

Country of ref document: EP

Kind code of ref document: A1