BRPI0907359B1 - MUTATE HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD), NUCLEIC ACID SEQUENCE, CHIMERIC GENE, MUTTED TRANSIT PEPTIDE/HPPD FUSION PROTEIN, CLONING AND/OR EXPRESSION VECTOR, METHOD OF OBTAINING A PLANT RESISTANT TO AN HPDD INHIBITOR AND CONTROL METHOD SELECTION OF WEEDS IN AN AREA OR FIELD - Google Patents

Abstract

MUTATE HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD), NUCLEIC ACID SEQUENCE, CHIMERIC GENE, TRANSIT PEPTIDE FUSION PROTEIN,/MUTATED HPPD, CLONING AND/OR EXPRESSION VECTOR, METHOD OF OBTAINING A PLANT RESISTANT TO AN HPPD INHIBITOR, METHOD OF CONTROL OF WEEDS IN AN AREA OR FIELD, METHOD OF OBTAINING OIL OR BRAN AND USE OF A MUTATED HPPD. The present invention relates to a nucleic acid sequence encoding a mutated hydroxyphenylpyruvate dioxygenase (HPPD), a chimeric gene comprising this sequence as a coding sequence, and its use in obtaining plants that are resistant to HPPD-inhibiting herbicides.

Description

FIELD OF INVENTION

The present invention relates to a nucleic acid sequence encoding a mutated hydroxyphenylpyruvate dioxygenase (HPPD), a chimeric gene comprising this sequence as a coding sequence, and its use in obtaining plants that are resistant to HPPD-inhibiting herbicides.

BACKGROUND OF THE INVENTION

Hydroxyphenylpyruvate dioxygenases (HPPD; EC 1.13.11.27) are enzymes that catalyze the reaction in which parahydroxyphenylpyruvate (HPP), a degradation product of tyrosine, is transformed into homogentisate (HG), the plant precursor of tocopherol and plastoquinone ( Crouch, N. P. et al, 1997; Fritze et al, 2004). Tocopherol acts as a membrane-associated antioxidant. Plastoquinone acts, firstly, as an electron conductor between PSII and the cytochrome b6/f complex and, secondly, it is a redox cofactor of phytoene desaturase, which is involved in carotenoid biosynthesis.

Most plants synthesize tyrosine via arrogenate (Abou-Zeid et al, 1995; Bonner et al, 1995; Byng et al, 1981; Connely and Conn, 1986; Gaines et al, 1982). In these plants, HPP is derived solely from tyrosine degradation. On the other hand, in organisms such as the yeast Saccharomyces cerevisiae or the bacterium Escherichia coli, HPP is a tyrosine precursor and is synthesized through the action of an enzyme, prephenate dehydrogenase (hereafter PDH), which converts prephenate to HPP ( Lingens et al, 1967; Sampathkumar and Morrison, 1982). In these organisms, the production of HPP is therefore directly related to the biosynthetic process of aromatic amino acids (shikimate pathway) and not to the tyrosine degradation process.

Inhibition of HPPD generates uncoupling of photosynthesis, deficiency of light-absorbing accessory pigments and, most importantly, destruction of chlorophyll by UV radiation and reactive oxygen species, due to the lack of photoprotection normally provided by carotenoids (Norris et al, 1995). Photodegradation of photosynthetically active tissues leads to growth inhibition and plant death.

Some molecules that inhibit HPPD and that specifically bind to the enzyme in order to inhibit the transformation of HPP into homogentisate have proven to be very effective selective herbicides.

Most commercially available HPPD inhibitor herbicides belong to one of four chemical families: 1. Triketones, such as sulcotrione (i.e., 2-[2-chloro-4-(methylsulfonyl)benzoyl]-1,3-cyclo- hexanedione), mesotrione (i.e. 2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-cyclohexanedione), tembotrione (i.e. 2-[2-chloro-4-(methylsulfonyl)-3 -[(2,2,2-trifluoroethoxy)methyl]benzoyl]-1,3-cyclohexanedione); 2. Diketonitrils, such as 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)-propane-1,3-dione and 2-cyano-1-[4-(methylsulfonyl)-2-trifluoromethylphenyl ]-3-(1-methylcyclopropyl)propane-1,3-fione; 3. isoxazoles, such as isoxaflutole (i.e., (5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone); in plants, isoxaflutole is rapidly metabolized to DKN, a diketonitrile compound that exhibits HPPD-inhibiting property; and 4. pyrazolinates, such as topramezone (i.e., [3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1-methyl-1H -pyrazol-4-yl)methanone) and pyrasulfotol ((5-hydroxy-1,3-dimethylpyrazol-4-yl(2-mesyl-4-trifluoromethylphenyl)methanone).

These HPPD-inhibiting herbicides can be used on grasses and/or broadleaf weeds of crop plants that exhibit metabolic tolerance, such as corn (Zea mays), in which they are rapidly degraded (Schulz et al, 1993; Mitchell et al , 2001; Garcia et al, 2000; Pallett et al, 2001). In order to extend the scope of these HPPD-inhibiting herbicides, several efforts have been undertaken to confer agricultural-level tolerance on plants, particularly plants with or without subnormal metabolic tolerance.

In addition to the attempt to overcome the HPPD-mediated production of homogentisate (US patent 6,812,010), hyperexpression of the sensitive enzyme was carried out to produce amounts of the target enzyme in the plant that are sufficient with respect to the herbicide (publication WO 96/38567). Overexpression of HPPD resulted in improved pre-emergence tolerance to the diketonitrile (DKN) derivative of isoxaflutole (IFT), but tolerance was not sufficient for post-emergence treatment (Matringe et al, 2005).

A third strategy was to mutate HPPD in order to obtain a target enzyme that, while maintaining its catalyzing properties of transforming HPP into homogentisate, is less sensitive to HPPD inhibitors than native HPPD before mutation. This strategy has been successfully applied in the production of plants tolerant to 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)-propane-1,3-dione and 2-cyano-1- [4- (methylsulfonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-fione (patent EP496630), two HPPD inhibitor herbicides belonging to the diketonitrile family (publication WO 99/24585). The mutations Pro215Leu, Gly336Glu, Gly336Ile and, more specifically, Gly336Trp (mutated amino acid positions are indicated with reference to Pseudomonas HPPD of SEQ ID NO. 2) have been identified as mutations that are responsible for an increase in tolerance to pre-treatment. emergence with these diketonitrile herbicides without causing changes in enzyme activity. More recently, it was demonstrated that the introduction of a Pseudomonas HPPD gene into the plastid genome of tobacco and soybean is much more effective than nuclear transformation, also conferring tolerance to post-emergence application of isoxaflutole (Dufourmantel et al, 2007). In publication WO 04/024928, the inventors sought to increase the biosynthesis of prenylquinone (such as the synthesis of plastoquinones, tocopherols) in plant cells by increasing the flux of the HPP precursor into the cells of those plants. This was done by connecting the synthesis of the aforementioned precursor to the “shikimate” pathway through the overexpression of a PDH enzyme. They also observed that the transformation of plants with a gene that encodes a PDH enzyme makes it possible to increase the tolerance of the aforementioned plants to HPPD inhibitors.

Despite these successes achieved in the development of plants that exhibit tolerance to diketonitrile herbicides, it is still necessary to develop and/or improve the HPPD inhibitor tolerance system, particularly for HPPD inhibitors belonging to the triketone classes (such as sulcotrione, mesotrione and tembotrione) and pyrazolinates (such as topramezone and pyrasulfotol).

BRIEF DESCRIPTION OF THE INVENTION

The present invention therefore relates to new mutated HPPD enzymes that maintain their properties of catalyzing the conversion of parahydroxyphenylpyruvate (HPP) into homogentisate and that are less sensitive to HPPD inhibitors than the original unmutated HPPD, characterized by fact that such enzymes contain a mutation at position 336 (amino acid glycine in native HPPD), with reference to Pseudomonas HPPD of SEQ ID NO. 2, which is selected from the following mutations: Gly336Arg, Gly336His, Gly336Met, Gly336Phe, Gly336Asn, Gly336Cys, Gly336Val, Gly336Trp, Gly336Glu and Gly336Asp.

DETAILED DESCRIPTION OF THE INVENTION

In a specific embodiment, the mutation at position 336 with reference to the Pseudomonas HPPD of SEQ ID NO: 2 is selected from the following mutations: Gly336Arg, Gly336His, Gly336Met, Gly336Phe, Gly336Asn, Gly336Cys and Gly336Val, provided that the mutated HPPD other than the Gly334Ala-Gly336Arg double mutant (positions are given with reference to Pseudomonas HPPD of SEQ ID NO. 2).

In a more specific embodiment, the mutation at position 336 with reference to the Pseudomonas HPPD of SEQ ID NO. 2 is selected from the following mutations: Gly336His, Gly336Met, Gly336Cys and Gly336Phe.

In another specific embodiment, the HPPD enzyme is from a plant, particularly Arabidopsis thaliana, and contains a glycine mutation at position 422 with reference to the Arabidopsis HPPD amino acid sequence of SEQ ID NO: 4 (i.e., position 336 with reference to the amino acid sequence of Pseudomonas HPPD of SEQ ID NO: 2) which is selected from the following mutations: Gly336Arg, Gly336His, Gly336Met, Gly336Phe, Gly336Asn, Gly336Cys, Gly336Val, Gly336Trp, Gly336Glu and Gly336Asp.

In a more specific embodiment, the mutation at position 422 with reference to Arabidopsis HPPD of SEQ ID NO. 4 (i.e., at position 336 with reference to Pseudomonas HPPD of SEQ ID NO. 2) is selected from the mutations to follow: Gly336His, Gly336Asn, Gly3364Cys and Gly336Val and the mutated HPPD is of plant origin, particularly from Arabidopsis. It is observed that position 336 with reference to Pseudomonas HPPD of SEQ ID NO. 2 is position 422 with reference to Arabidopsis thaliana HPPD of SEQ ID NO. 4.

In a specific embodiment, the mutated HPPD according to the present invention is less sensitive than the original unmutated HPPD to an HPPD-inhibiting herbicide of the isoxazole, diketonitrile, triketone or pyrazolinate class.

In a specific embodiment, the mutated HPPD according to the present invention is less sensitive than the original unmutated HPPD to an HPPD-inhibiting herbicide selected from isoxaflutole, tembotrione, mesotrione, sulcotrione, pyrasulfotol, topramezone, 2-cyano-3 -cyclopropyl-1-(2-SO2CH3-4-CF3 phenyl)propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4- 2,3 Cl2 phenyl)propane-1, 3-dione.

In another specific embodiment, the mutated HPPD according to the present invention is less sensitive to an HPPD inhibitor of the triketone class (so-called triketone HPPD inhibitor), such as tembotrione, sulcotrione and mesotrione, particularly tembotrione, or of the class of pyrazolinates (called pyrazolinate HPPD inhibitor), such as pyrasulfotol and topramezone, than the original unmutated HPPD.

In a more specific embodiment, the mutated HPPD according to the present invention is less sensitive to a triketone HPPD inhibitor selected from tembotrione, sulcotrione and mesotrione, particularly tembotrione.

In another specific embodiment, the HPPD mutated according to the present invention contains a second mutation, in addition to the first mutation on the amino acid glycine at position 336 with reference to the Pseudomonas HPPD of SEQ ID NO. 2.

In a more specific embodiment, the second mutated amino acid is selected from the selected amino acids: Pro215, Gly298, Gly332, Phe333, Gly334 and Asn337, with reference to the Pseudomonas HPPD sequence of SEQ ID No. 2.

Furthermore, the present invention provides mutated HPPD enzymes that retain their properties of catalyzing the conversion of parahydroxyphenylpyruvate (HPP) to homogentisate and that are less sensitive to HPPD inhibitors of the triketone class such as tembotrione, sulcotrione and mesotrione. , or the class of pyrazolinates such as pyrasulfotol and topramezone, which are the original unmutated HPPD, characterized by the fact that they contain a mutation of the amino acid glycine at position 336 with reference to Pseudomonas HPPD of SEQ ID No. 2, as well as uses of these enzymes to make plants tolerant to these HPPD inhibitors, processes in which triketone or pyrazolinate herbicides are applied to plants expressing these mutant enzymes and plants tolerant to such HPPD inhibitors of the triketone or pyrazolinate class comprising in their genome a gene encoding certain HPPD enzymes that have been mutated at position 336 with reference to the Pseudomonas HPPD of SEQ ID NO. 2.

In a specific embodiment of the present invention, the mutated HPPD enzyme is less sensitive to an HPPD inhibitor of the triketone class such as tembotrione, sulcotrione and mesotrione than the original unmutated HPPD and is mutated at position 336 with reference to Pseudomonas HPPD of SEQ ID NO. 2 according to a mutation selected from the following mutations: Gly336Arg, Gly336Asp, Gly336Glu, Gly336His, Gly336Met, Gly336Phe, Gly336Trp, Gly336Asn, Gly336Cys and Gly336Val.

In a specific embodiment of the present invention, the mutated HPPD enzyme is less sensitive to an HPPD inhibitor of the triketone class such as tembotrione, sulcotrione and mesotrione than the original unmutated HPPD and is mutated at position 336 with reference to Pseudomonas HPPD of SEQ ID NO. 2 according to a mutation selected from the following mutations: Gly336His, Gly336Met, Gly336Phe and Gly336Cys.

Several HPPDs and their primary sequences have been described in the prior art, particularly HPPDs from bacteria such as Pseudomonas (Rüetschi et al, Eur. J. Biochem., 205, 459-466, 1992, publication WO 96/38567), from plants such as Arabidopsis (publication WO 96/38567, Genebank AF047834), carrot (publication WO 96/38567, Genebank 87257), Avena sativa (publication WO 02/046387), wheat (publication WO 02/046387), Brachiaria platyphylla (publication WO 02/046387), Cenchrus echinatus (WO publication 02/046387), Lolium rigidum (WO publication 02/046387), Festuca arundinacea (WO publication 02/046387), Setaria faberi (WO publication 02/046387), Eleusine indica (WO publication 02/046387) and sorghum (publication WO 02/046387), from Coccicoides (Genebank COITRP) or from mammals such as mice and pigs. The corresponding sequences described in the indicated references are incorporated herein by reference.

By aligning these known sequences, using the usual means of the art, such as the method described by Thompson, J. D. et al (CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment through Sequence Weighting, Positions-Specific Gap Penalties and Weight Matrix Choice, Nucleic Acids Research, 22; 4673-4680, 1994) and access to sequence alignment computer programs that are accessible via the Internet, for example, those skilled in the art are able to define sequence homologies with respect to a sequence reference and find key amino acids or define common regions.

In the case of the present invention, the reference sequence is the Pseudomonas sequence, with all definitions and indications of specific amino acid positions that are made with respect to the primary Pseudomonas HPPD sequence of SEQ ID NO. 2, except where specifically indicated . The attached Figure 1 illustrates an alignment of several HPPD sequences that are described in the prior art; These sequences are aligned with respect to the Pseudomonas HPPD sequence as the reference sequence and comprise the HPPD sequences of Streptomyces avermitilis (Genebank SAV11864), Daucus carota (Genebank DCU 87257), Arabidopsis thaliana (Genebank AF047834), Zea more, from Hordeum vulgare (Genebank HVAJ693), from Mycosphaerella graminicola (Genebank AF038152), from Coccicoides immitis (Genebank COITRP) and from Mus musculus (Genebank MU54HD). This figure provides the numbering of the amino acids in the Pseudomonas sequence and also the amino acids that are common to these sequences, where these amino acids are indicated by an asterisk. Based on this alignment, it is easy, by defining the Pseudomonas amino acid by its position and nature, to identify the position of the corresponding amino acid in another HPPD sequence. Figure 1 demonstrates that this can be done by aligning sequences originating from different plants, mammals and bacteria, which demonstrates that this alignment method, which is well known to those skilled in the art, can be generalized to any other sequence. An alignment of different HPPD sequences is also described in publication WO 97/49816.

In publication WO 99/24585, analysis of the tertiary structure of the Pseudomonas HPPD monomer shows the presence of a C-terminal part of the HPPDs, which is where the active site of the enzyme is located, linked to its N-terminal part by a peptide binding that guarantees the stability of the enzyme and its oligomerization (Pseudomonas HPPD is a tetramer while plant HPPDs are dimers). This structure was obtained using the usual methods of studying crystal X-ray diffraction. The binding peptide makes it possible to define the N-terminal end of the C-terminal part of the enzyme, where the aforementioned binding peptide is located between amino acids 145 and 157 in the case of Pseudomonas (cf. Figure 1). Two amino acids, which are found at positions 161 and 162 in the case of the Pseudomonas sequence (D = Asp161 and H = His162), will be observed in all sequences shown in the sequence alignment illustrated in the attached Figure 1. With reference to Pseudomonas HPPD, it is therefore possible to define the binding peptide as being located between about five and fifteen amino acids upstream from the amino acid Asp161. According to the present invention, “mutated HPPD” is understood as the replacement of at least one amino acid in the HPPD primary sequence by another amino acid. The expression “mutated amino acid” will be used below to designate the amino acid that is replaced by another amino acid, in order to designate the mutation site in the primary sequence of the protein. According to the present invention, the mutation is made on the amino acid glycine at position 336 with reference to the Pseudomonas sequence of SEQ ID No. 2, which is common to almost all identified HPPD sequences. Of the 240 HPPD sequences known so far, 238 contain a glycine at position 336 and only the HPPD sequences from Synechococcus sp JA-3-3Ab (Acc-No Q2JX04) and Synechococcus sp. JA-2-3B’a (2-13) (Acc-No Q2JPN8)) contain an alanine at this position. Gly336 is part of a consensus sequence “Gly-Phe-Gly-X-Gly-Asn-Phe” found in most HPPD sequences, where X can be any of the twenty amino acids, among HPPDs of different origins, which makes it possible to identify Gly336 without any difficulty in HPPDs from any source using the sequence alignment method. Gly336 with reference to the Pseudomonas sequence is, for example, Gly422 with reference to the Arabidopsis thaliana sequence of SEQ ID NO. 4 (see Figure 1), but herein reference will be made to Gly at reference position 336 by reference to the Pseudomonas sequence of SEQ ID No. 2 (except where specifically indicated), although the mutation may occur in any HPPD enzyme useful in accordance with the present invention, not necessarily in Pseudomonas HPPD.

The enzymatic activity of HPPDs can be measured using any method that makes it possible to measure the reduction in the amount of HPP or O2 substrates or to measure the accumulation of any of the products derived from the enzymatic reaction, that is, homogentisate or CO2. In particular, HPPD activity can be measured using the method described in Garcia et al (1997) or Garcia et al (1999), which are incorporated herein by reference.

According to the present invention, a triketone class HPPD inhibitor (or triketone HPPD inhibitor) indicates an HPPD inhibitor that has a triketone skeleton. As an example of this triketone HPPD inhibitor, one can mention the molecules sulcotrione (i.e. 2-[2-chloro-4-(methylsulfonyl)benzoyl]-1,3-cyclohexanedione), mesotrione (i.e. 2 -[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-cyclohexanedione) and tembotrione (i.e., 2-[2-chloro-4-(methylsulfonyl)-3-[(2,2,2- trifluoroethoxy)methyl]benzoyl]-1,3-cyclohexanedione).

According to the present invention, an HPPD of the pyrazolinate class (or pyrazolinate HPPD inhibitor) indicates an HPPD inhibitor that contains a pyrazole radical. As an example of such a pyrazolinate HPPD inhibitor, one can mention the molecules topramezone (i.e., [3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5 -hydroxy-1-methyl-1H-pyrazol-4-yl)methanone) and pyrasulfotol ((5-hydroxy-1,3-dimethylpyrazol-4-yl(2-mesyl-4-trifluoromethylphenyl)methanone).

In a further embodiment of the present invention, HPPD is mutated at a second amino acid position in addition to the Gly336 mutation. The presence of this second mutation may further increase tolerance to the same HPPD-inhibiting herbicide as that for which the first mutation confers tolerance, or may confer tolerance to a second HPPD-inhibiting herbicide. Examples of such mutations that confer tolerance to HPPD inhibitors, and particularly to diketonitrile and isoxaflutole, are described in publication WO 99/24585.

In a specific embodiment of the present invention, the second mutated amino acid is selected from the following reference amino acids, with reference to the Pseudomonas sequence of SEQ ID NO: 2: Pro215, Gly332, Phe333, Gly334 and Asn337, as well as Gly298 in Pseudomonas sequence (the latter has no counterpart in other HPPDs, see Fig. 1).

In one embodiment of the present invention, the second mutated amino acid is Pro215 with reference to the Pseudomonas sequence of SEQ ID NO: 2 and the mutation is particularly Pro215Leu.

The present invention also relates to a nucleic acid sequence, particularly an isolated DNA, encoding a mutated HPPD as described above.

The present invention also relates to a nucleic acid sequence which encodes a mutated HPPD enzyme and maintains its properties of catalyzing the conversion of parahydroxyphenylpyruvate (HPP) to homogentisate and which is less sensitive to HPPD inhibitors of the triketone class such such as tembotrione, sulcotrione and mesotrione, or from the class of pyrazolinates, such as pyrasulfotol and topramezone, which are HPPD without original mutations, characterized by the fact that it contains a mutation of the amino acid glycine at position 336 with reference to Pseudomonas HPPD of SEQ ID N ° 2.

In a more specific embodiment, the nucleic acid sequence according to the present invention encodes a mutated HPPD enzyme and is less sensitive to an HPPD inhibitor of the triketone class such as tembotrione, sulcotrione and mesotrione than the original unmutated HPPD and in that HPPD is mutated at position 336 with reference to Pseudomonas HPPD of SEQ ID NO. 2 according to a mutation selected from the following mutations: Gly336Arg, Gly336Asp, Gly336Glu, Gly336His, Gly336Met, Gly336Phe, Gly336Trp, Gly336Asn, Gly336Cys and Gly336Val.

In an even more specific embodiment, the nucleic acid sequence according to the present invention encodes a mutated HPPD enzyme and is less sensitive to an HPPD inhibitor of the triketone class, such as tembotrione, sulcotrione and mesotrione, than unmutated HPPD. original and in which the HPPD is mutated at position 336 with reference to the Pseudomonas HPPD of SEQ ID NO. 2 according to a mutation selected from the following mutations: Gly336His, Gly336Met, Gly336Phe and Gly336Cys.

According to the present invention, a “nucleic acid sequence” is understood to be a sequence of nucleotides that may be of the DNA or RNA type, preferably of the DNA type, particularly with two strands, whether of natural or synthetic origin, particularly one DNA sequence in which the codons encoding the mutated HPPD according to the present invention have been optimized according to the host organism in which it is to be expressed (such as by replacing codons with the most preferred or highest preferred codons in tables of codon usage of that host organism or the group to which that host organism belongs, compared to the original host), wherein these optimization methods are well known to those skilled in the art.

An “isolated DNA”, as used herein, means DNA that is not naturally occurring or no longer found in the natural environment in which it was originally present, such as a DNA coding sequence associated with other regulatory elements in a gene chimeric, a DNA transferred into another host cell, such as a plant cell, or an artificial synthetic DNA that contains a different nucleotide sequence compared to any known naturally occurring DNA.

The sequence encoding an original, unmutated HPPD that will be mutated according to the present invention can be of any origin. Particularly, it may be of bacterial origin. Advantageous examples that can be mentioned are bacteria of the Pseudomonas sp type, such as Pseudomonas fluorescens, or other cyanobacteria of the genus Synechocystis. The sequence may also be of plant origin, particularly derived from dicotyledonous plants, umbelliferous plants or other monocotyledonous plants. Advantageous examples that may be mentioned are plants such as tobacco, Arabidopsis, Daucus carotta, Zea mais (corn), wheat, barley, Avena sativa, Brachiaria platyphylla, Cenchrus echinatus, Lolium rigidum, Festuca arundinacea, Setaria faberi, Eleusine indica and sorghum. The coding sequences and the method of their isolation and cloning are described in the references mentioned above, the content of which is incorporated herein by reference. In a specific embodiment of the present invention, the HPPD has bacterial origin, particularly from Pseudomonas sp, more specifically from Pseudomonas fluorescens or from a plant origin, particularly from Arabidopsis thaliana.

The HPPD for making mutation(s) for the purpose of the present invention may be any naturally occurring HPPD or any of its active fragments or any of its variations in which some amino acids (one to ten amino acids) have been substituted, added or excluded for cloning purposes, to prepare a transit peptide fusion and the like, which maintains HPPD activity, that is, the property of catalyzing the conversion of parahydroxyphenylpyruvate into homogentisate.

According to the present invention, the HPPD may be a chimeric HPPD. The term “chimeric HPPD” is intended to indicate an HPPD that comprises elements originating from several HPPDs. These chimeric HPPDs are specifically described in WO 99/24586.

The mutation may be carried out in the nucleic acid sequence encoding the original unmutated HPPD by any means that is appropriate for replacing, in said sequence, the codon encoding the mutated amino acid with the codon corresponding to the amino acid it is to replace, wherein the aforementioned codons are widely described in the literature and well known to those skilled in the art.

Several molecular biological methods can be used to achieve this mutation.

A preferred method of preparing a mutated nucleic acid sequence in accordance with the present invention and the corresponding protein comprises conducting site-directed mutagenesis on codons encoding one or more amino acids that are selected in advance, including the reference position codon. Gly336 with reference to the Pseudomonas HPPD sequence of SEQ ID No. 2. Methods of obtaining such site-directed mutations are well known to those skilled in the art and widely described in the literature (particularly, Directed Mutagenesis: A Practical Approach, 1991, edited by M. J. McPherson, IRL Press) or are methods for which it is possible to employ commercial kits (such as the U. S. E. PHARMACIA mutagenesis kit). After site-directed mutagenesis, it is useful to select cells that contain a mutated HPPD that is less sensitive to an HPPD inhibitor using an appropriate selection aid. A selection method that is simple to implement is the determination of the dose of HPPD inhibitor that completely inhibits the original unmutated HPPD and that is lethal for cells that express this unmutated HPPD, subjecting the cells that have undergone mutation to that dose previously. determined, then isolate the cells that were mutated and withstood that lethal dose and then isolate and clone the gene that encodes the mutated HPPD. In view of a specific embodiment of the present invention and the solution sought, i.e., an HPPD that is less sensitive to a pyrazolinate or triketone HPPD inhibitor, the selection may be carried out as described above, using a triketone or an HPPD inhibitor. of pyrazolinate, particularly an HPPD inhibitor selected from tembotrione, mesotrione, pyrasulfotol, topramezone and sulcotrione.

In view of another embodiment of the present invention, that is, an HPPD that is additionally mutated about a second amino acid, in addition to the first mutation about the reference amino acid at position 336 with reference to the Pseudomonas HPPD sequence of SEQ ID NO. 2 , the second mutation can be obtained through site-directed mutagenesis, carried out simultaneously or successively to the first.

As an alternative to site-directed mutagenesis as described above, the second mutation can be obtained using random mutation methods (such as EMS or radiation treatment) associated with an appropriate selection aid. These mutation methods are well known to those skilled in the art and widely described in the literature (particularly, Sambrook et al, 1989). Selection methods can be carried out as described above.

The terminology DNA or protein “comprising” a certain sequence X, as used throughout the text, designates a DNA or protein that includes or contains at least the sequence be included at the 5' (or N-terminal) and/or 3' (or C-terminal) end, such as (the nucleotide sequence of) a selectable marker protein, (the nucleotide sequence of) a transit peptide, and /or a 5' leader sequence or a 3' trailing sequence. Similarly, the use of the term "comprises", "comprising" or "comprises" throughout the text and claims of the present application should be understood as indicating the inclusion of a indicated integer, step or group of numbers integers or steps, but not to the exclusion of any other integer, step or group of integers or steps.

The present invention therefore also relates to a method of preparing a nucleic acid sequence encoding a mutated HPPD according to the present invention, wherein said method is defined above.

The present invention also relates to the use, in a plant transformation method, of a nucleic acid encoding a mutated HPPD according to the present invention as a marker gene or as a coding sequence that makes it possible to confer tolerance to the plant to herbicides that are HPPD inhibitors and the use of HPPD inhibitors on plants comprising a nucleic acid sequence encoding a mutated HPPD in accordance with the present invention. In one embodiment of the present invention, in this use, the HPPD inhibitors are triketones or pyrazolinates, preferably tembotrione, mesotrione or sulcotrione. It is understood, of course, that such a sequence may also be used in combination with one or more other marker genes and/or sequence(s) encoding one or more proteins with useful agricultural properties.

Among the genes that encode proteins that confer useful agronomic properties to transformed plants, we can mention the DNA sequences that encode proteins that confer tolerance to certain herbicides, those that confer tolerance to certain insects, those that confer tolerance to certain diseases, etc. These genes are particularly described in publications WO 91/02071 and WO 95/06128. Among the DNA sequences that encode proteins that confer tolerance to certain herbicides on plant cells and transformed plants, mention may be made of the bar gene that confers tolerance to glufosinate herbicides, wherein the gene encodes an appropriate EPSPS that confers tolerance to herbicides that target EPSPS, such as glyphosate and its salts (in patents US 4,535,060, US 4,769,061, US 5,094,945, US 4,940,835, US 5,188,642, US 4,971,908, US 5,145,783 , US 5,310,667, US 5,312,910, US 5,627,061, US 5,633,435), the gene encoding glyphosate oxidoreductase (US patent 5,463,175).

Among the DNA sequences encoding an appropriate EPSPS that confers tolerance to herbicides that target EPSPS, more specific mention will be made of the gene encoding a plant EPSPS, particularly maize EPSPS, which contains two mutations, 102 and 106 , and which is described in Patent Application FR 2,736,926, hereinafter referred to as EPSPS double mutant, or the gene encoding an EPSPS isolated from Agrobacterium and which is described by SEQ ID NO. 2 and SEQ ID NO. 3 of the Patent North American No. 5,633,435, hereinafter referred to as CP4.

In the cases of DNA sequences encoding EPSPS and, more specifically, encoding the above genes, the sequence encoding these enzymes is conveniently preceded by a sequence encoding a transit peptide, particularly encoding the “optimized transit peptide” described. in US Patent 5,510,471 or US 5,633,448.

Among the DNA sequences that encode proteins of interest and confer innovative tolerance properties against insects, more specifically mention will be made of the Bt proteins widely described in the literature and well known to those skilled in the art. Mention will also be made of proteins extracted from bacteria such as Photorhabdus (publications WO 97/17432 and WO 98/08932).

The present invention also relates to a chimeric gene (or expression set) comprising a coding sequence, as well as heterologous regulatory elements, in the 5' and/or 3' position, at least in the 5' position, which are capable of functioning in a host organism, particularly plant cells or plants, with the coding sequence containing at least one nucleic acid sequence encoding a mutated HPPD as defined above.

The present invention therefore relates to a chimeric gene (or expression set) comprising a coding sequence as well as heterologous regulatory elements, in the 5' and/or 3' position, at least in the 5' position, which are capable of of functioning in a host organism, particularly plant cells or plants, with the coding sequence containing at least one nucleic acid sequence as defined above.

In a specific embodiment, the present invention relates to a chimeric gene as described above, wherein the host organism is selected from bacteria, yeast, Pichia, fungi, bacillovirus, plant cells and plants.

In another specific embodiment, the present invention relates to a chimeric gene as previously described, wherein the chimeric gene contains, at position 5' of the nucleic acid sequence encoding a mutated HPPD, a nucleic acid sequence encoding a plant transit peptide, wherein this sequence is arranged between the promoter region and the sequence encoding the mutated HPPD, so as to allow expression of a mutated transit peptide/HPPD fusion protein.

As a regulatory sequence that is a promoter in plant cells and plants, use can be made of any promoter sequence of a gene that is expressed naturally in plants, particularly a promoter that is expressed especially in plant leaves, such as "constitutive" promoters. of bacterial, viral or plant origin, or “light-dependent” promoters, such as those from a plant ribulose-bicarboxylase/oxygenase (RuBisCo) small subunit gene or any suitable known promoter that can be used. Among promoters of plant origin, mention will be made of histone promoters, as described in EP Application 0,507,698, or the rice actin promoter (US patent 5,641,876). Among the promoters of a plant virus gene, mention will be made of the cauliflower mosaic virus (CAMV 19S or 35S) or the circovirus promoter (patent AU 689,311).

One can also make use of a regulatory promoter sequence specific to specific regions or tissues of plants, such as seed-specific promoters (Datla, R. et al, 1997), especially the napin promoter (EP 255,378), the phaseolin promoter, the glutenin promoter, the heliantinin promoter (publication WO 92/17580), the albumin promoter (publication WO 98/45460), the oleosin promoter (publication WO 98/45461), the SAT1 promoter or the SAT3 promoter (PCT/US98/06978 ).

One can also make use of an inducible promoter conveniently selected from phenylalanine ammonium lipase (PAL), HMG-CoA reductase (HMG), chitinase, glucanase, proteinase inhibitor (PI), PR1 family gene, nopaline synthase promoters (us) and vspB (US patent 5,670,349, Table 3), HMG2 promoter (US patent 5,670,349), apple betagalactosidase (ABG1) promoter and apple aminocyclopropane carboxylate synthase (ACC synthase) promoter (publication WO 98/45445 ).

According to the present invention, use can 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”), such as translation activator from tobacco mosaic virus (TMV) described in publication WO 87/07644, from tobacco rust virus (TEV) described by Carrington & Freed, 1990, for example, or introns such as the maize adh1 intron or actin intron 1 of rice.

As a regulatory terminus or polyadenylation sequence, use may be made of any corresponding sequence of bacterial origin, such as the nos terminus of Agrobacterium tumefaciens, of viral origin, such as the CaMV 35S terminus, or of plant origin, such as the of histone described in EP Application 0,633,317.

“Host organism” is understood to mean any unicellular or multicellular organism into which the chimeric gene according to the present invention can be introduced for the purpose of producing mutated HPPD. These organisms are, particularly, bacteria, such as E. coli, yeasts, particularly the genus Saccharomyces or Kluyveromyces, Pychia, fungi, particularly Aspergillus, bacillovirus or, preferably, plant cells and plants.

“Plant cell” is understood, according to the present invention, to be any cell that is derived from or found in a plant and that is capable of forming undifferentiated tissues such as callus, differentiated tissues such as embryos, parts of plants, plants or seeds, or be part of them.

“Plant”, according to the present invention, is understood to be any differentiated multicellular organism that is capable of photosynthesis, particularly a monocotyledonous or dicotyledonous organism, more especially cultivated plants whether or not intended for animal or human nutrition, such as corn or white corn, wheat, Brassica spp plants such as Brassica napus or Brassica juncea, soybeans, rice, sugar cane, beetroot, tobacco, cotton, vegetables such as cucumber, leek, carrot, tomato, lettuce, pepper, melon, watermelon etc.

In one embodiment, the present invention relates to the transformation of plants. Any promoter sequence of a gene that is naturally expressed in plants or any hybrid or combination of promoter elements of genes naturally expressed in plants, including Agrobacterium or plant virus promoters, or any promoter that is suitable for controlling transcription of a gene of herbicide tolerance, can be used as the promoter regulatory sequence in plants according to the present invention. Examples of such suitable promoters are described above.

According to the present invention, it is also possible to use, in combination with the promoter regulatory sequence, other regulatory sequences that are located between the promoter and the coding sequence, such as intron sequences or transcription activators (amplifiers). Examples of such appropriate regulatory sequences are described above.

Any corresponding sequence of bacterial origin, such as the nos-terminal of Agrobacterium tumefaciens, or of plant origin, such as a histone terminus as described in EP application 0,633,317, can be used as a terminal transcription (and polyadenylation) regulatory sequence. .

In a specific embodiment of the present invention, a nucleic acid sequence encoding a transit peptide is employed 5' to the nucleic acid sequence encoding a mutated HPPD, wherein this transit peptide is disposed between the promoter region and the sequence which encodes the mutated HPPD, so as to allow expression of a mutated HPPD/transit peptide fusion protein, wherein the mutated HPPD is defined above. The transit peptide makes it possible to direct the mutated HPPD to plastids, more especially chloroplasts, where the fusion protein is split between the transit peptide and the mutated HPPD when the latter enters the plastid. The transit plastid may be a single peptide, such as an EPSPS transit peptide (described in U.S. Patent No. 5,188,642) or a ribulose biscarboxylase/plant oxygenase small subunit transit peptide (RuBisCO ssu), wherein appropriate it includes some amino acids from the N-terminal part of the mature RuBisCO ssu (EP 189,707) or may be a fusion of several transit peptides such as a transit peptide comprising a first plant transit peptide that is fused to a part of the N-terminal sequence of a mature protein having a plastid site, which part, in turn, is fused to a second plant transit peptide as described in Patent EP 508,909 and, more especially, the optimized transit peptide comprising a sunflower RuBisCO ssu transit peptide fused 22 amino acids from the N-terminal end of corn RuBisCO ssu, fused, in turn, to the corn RuBisCO ssu transit peptide, as described, with its coding sequence, in Patent EP 508,909.

The present invention also relates to a mutated HPPD/transit peptide fusion protein and a nucleic acid or chimeric gene that can be expressed in plants that encodes such a fusion protein, wherein the two elements of such a fusion protein are as defined above. .

The present invention also relates to a cloning and/or expression vector for transforming a host organism, wherein said vector contains at least one chimeric gene as defined above. In addition to the above chimeric gene, this vector contains at least one origin of reproduction. This vector may be a plasmid, cosmid, bacteriophage or a virus that has been transformed through the introduction of the chimeric gene in accordance with the present invention. These transformation vectors, which depend on the host organism to be transformed, are well known to those skilled in the art and widely described in the literature. The transformation vector that is used, particularly, for the transformation of plant cells or plants can be a virus, which can be used for the transformation of developed plants and which additionally contains its own reproduction and expression elements. According to the present invention, the vector for transforming plant cells or plants is preferably a plasmid, such as an armless Agrobacterium Ti plasmid.

The present invention also relates to host organisms, particularly plant cells or plants, which are transformed and which contain a chimeric gene comprising a sequence encoding a mutated HPPD as defined above, and to the use of the plants according to the present invention in a field growing a crop and harvesting a plant product, such as soybeans or corn, wherein, in one embodiment, said use involves applying HPPD-inhibiting herbicides to said plants to control weeds. In one embodiment of the present invention, in this use, the HPPD inhibitors are triketones or pyrazolinates, preferably tembotrione, mesotrione or sulcotrione, particularly tembotrione.

The present invention therefore relates to a host organism, particularly a plant cell or plant, characterized in that it contains at least one chimeric gene as described above, or at least one nucleic acid sequence as described above.

In a specific embodiment, the present invention relates to a plant cell or plant characterized by the fact that it contains at least one nucleic acid sequence that encodes a mutated HPPD enzyme and maintains its properties of catalyzing the conversion of para-hydroxyphenylpyruvate ( HPP) in homogentisate and which is less sensitive to an HPPD inhibitor than the original unmutated HPPD, characterized by the fact that it contains a mutation at position 336 (glycine amino acid in native HPPD) with reference to Pseudomonas HPPD of SEQ ID NO. 2 which is selected from the following mutations: Gly336Arg, Gly336His, Gly336Met, Gly336Phe, Gly336Asn, Gly336Cys and Gly336Val, provided that the mutated HPPD is not the Gly334Ala-Gly336Arg double mutant (positions are given with reference to Pseudomonas HPPD from SEQ ID NO. 2).

In another more specific embodiment, the present invention relates to a plant cell or plant characterized by the fact that it contains at least one nucleic acid sequence encoding a mutated HPPD as described above, wherein the mutation at position 336 with reference the Pseudomonas HPPD of SEQ ID NO: 2 is selected from the following mutations: Gly336His, Gly336Met, Gly336Cys and Gly336Phe, particularly Gly336His.

In another embodiment, the present invention relates to a plant cell or plant characterized by the fact that it contains at least one nucleic acid sequence encoding a mutated HPPD that maintains its properties of catalyzing the conversion of parahydroxyphenylpyruvate (HPP ) in homogentisate and which is less sensitive to an HPPD inhibitor than the original unmutated HPPD, wherein the HPPD enzyme is from a plant, particularly Arabidopsis thaliana, and contains a mutation on glycine at position 422 with reference to the amino acid sequence of Arabidopsis HPPD of SEQ ID NO. 4 (i.e., position 336 with reference to the amino acid sequence of Pseudomonas HPPD of SEQ ID NO. 2) selected from the following mutations: Gly336Arg, Gly336His, Gly336Met, Gly336Phe, Gly336Asn , Gly336Cys, Gly336Val, Gly336Trp, Gly336Glu and Gly336Asp.

In an even more specific embodiment, the present invention relates to a plant cell or plant characterized in that it contains at least one nucleic acid sequence encoding a mutated HPPD as described above, wherein the mutation at position 336 with reference the Pseudomonas HPPD of SEQ ID No. 2 is selected from the following mutations: Gly336His, Gly336Asn, Gly336Cys and Gly336Val and the mutated HPPD is of plant origin, particularly from Arabidopsis. It is observed that position 336 with reference to Pseudomonas HPPD of SEQ ID NO. 2 is position 422 with reference to Arabidopsis thaliana HPPD of SEQ ID NO. 4.

In a specific embodiment, the present invention relates to a plant cell or plant, characterized in that it contains at least one nucleic acid sequence encoding a mutated HPPD as described above, wherein the mutated HPPD in accordance with the present invention is less sensitive than the original unmutated HPPD to an HPPD-inhibiting herbicide from the class of isoxazoles, diketonitriles, triketones or pyrazolinates.

In a more specific embodiment, the present invention relates to a plant cell or plant, characterized in that it contains at least one nucleic acid sequence encoding a mutated HPPD as described above, wherein the mutated HPPD is less sensitive than the original unmutated HPPD is an HPPD inhibitor herbicide selected from isoxaflutole, tembotrione, mesotrione, sulcotrione, pyrasulfotol, topramezone, 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-CF3 phenyl)propane- 1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4,2,3 Cl2 phenyl)propane-1,3-dione.

In another specific embodiment, the present invention relates to a plant cell or plant, characterized in that it contains at least one nucleic acid sequence encoding a mutated HPPD as described above, wherein the mutated HPPD is less sensitive to an HPPD inhibitor from the triketone class such as tembotrione, sulcotrione and mesotrione, particularly tembotrione, or from the pyrazolinate class such as pyrasulfotol and topramezone, which is the original unmutated HPPD.

In a more specific embodiment, the present invention relates to a plant cell or plant characterized in that it contains at least one nucleic acid sequence encoding a mutated HPPD as described above, wherein the mutated HPPD is less sensitive to a triketone HPPD inhibitor selected from tembotrione, sulcotrione and mesotrione, particularly tembotrione.

In another specific embodiment, the present invention relates to a plant cell or plant, characterized in that it contains at least one nucleic acid sequence encoding a mutated HPPD as described above, wherein the mutated HPPD in accordance with The present invention contains a second mutation, in addition to the first mutation on the amino acid glycine at position 336 with reference to Pseudomonas HPPD of SEQ ID NO. 2.

In a specific embodiment, the present invention relates to a plant cell or plant characterized in that it contains at least one nucleic acid sequence encoding a mutated HPPD as described above, wherein the second mutated amino acid is selected from the selected amino acids: Pro215, Gly298, Gly332, Phe333, Gly334 and Asn337, with reference to the Pseudomonas HPPD sequence of SEQ ID No. 2.

The present invention further relates to a plant cell or plant characterized by the fact that it contains at least one nucleic acid sequence that encodes a mutated HPPD enzyme and maintains its catalyzing properties for the conversion of parahydroxyphenylpyruvate (HPP) into homogentisate. and which is less sensitive to HPPD inhibitors of the triketone class such as tembotrione, sulcotrione and mesotrione, or of the pyrazolinate class such as pyrasulfotol and topramezone, than the original unmutated HPPD, characterized by the fact that it contains a mutation of the amino acid glycine at position 336 with reference to Pseudomonas HPPD of SEQ ID NO. 2.

In a more specific embodiment, the present invention relates to a plant cell or plant characterized by the fact that it contains at least one nucleic acid sequence encoding a mutated HPPD enzyme and is less sensitive to an HPPD inhibitor of the triketone class. or pyrazolinates in which the original unmutated HPPD is mutated at position 336 with reference to the Pseudomonas HPPD of SEQ ID NO: 2 according to a mutation selected from the following mutations: Gly336Arg, Gly336Asp, Gly336Glu, Gly336His, Gly336Met, Gly336Phe, Gly336Trp, Gly336Asn, Gly336Cys and Gly336Val.

In another specific embodiment, the present invention relates to a plant cell or plant characterized in that it contains at least one nucleic acid sequence as described above and, in addition, a gene that is functional in plants, which allows the overexpression of an enzyme PDH (prephenate dehydrogenase).

The present invention also relates to plants that contain transformed cells, particularly plants that are regenerated from the transformed cells. Regeneration may be achieved by any appropriate method, where the method depends on the nature of the species, as described, for example, in the references above. The following patents and patent applications may be particularly mentioned with respect to methods of transforming plant cells and regenerating plants: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187. 073, EP 267,159, EP 604,662, EP 672,752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5 ,484,956 , US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442,174, EP 486,233, EP 486,234, EP 539. 563, EP 674,725 , WO 91/02071 and WO 95/06128.

The present invention also relates to transformed plants or parts thereof, which are derived through cultivation and/or crossing of the above regenerated plants and to the seeds of the transformed plants.

The present invention also relates to end products such as bran or oil which are obtained from the plants, their part or seeds according to the present invention.

The transformed plants that can be obtained according to the present invention can be of the monocotyledonous type, such as cereals, sugar cane, rice and corn or white corn, or of the dicotyledonous type, such as tobacco, soybeans, Brassica spp, plants such as such as oilseed rape, cotton, sugar beet, clover etc.

The present invention relates to a method of transforming host organisms, particularly plant cells or plants, by integrating into these organisms at least one nucleic acid sequence or a chimeric gene as defined above, in which it is possible to obtain- the transformation is carried out by any known appropriate means, which means are widely described in the specialized literature and, particularly, the references mentioned in the present application, more especially using the vector according to the present invention.

A series of methods involves bombarding cells, protoplasts or tissues with particles to which DNA sequences are attached. Another series of methods comprises the use, as a means of transferring to the plant, a chimeric gene that is inserted into a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium tumefaciens.

Agrobacterium rhizogenes. Other methods can be used, such as microinjection, electroporation or another type of direct precipitation using PEG. Those skilled in the art can select any appropriate method of transforming the selected host organism, particularly the plant cell or plant. As examples, soybean processing technology has been extensively described in Examples 1 to 3 of EP 1186666, incorporated herein by reference. For rice, transformation mediated by agrobacteria (Hiei et al, 1994, and Hiei et al, 1997, incorporated herein by reference), electroporation (US Patent 5,641,664 and US Patent 5,679,558, incorporated herein by reference) may be carried out. present by reference) or bombing (Christou et al, 1991, incorporated herein by reference). An appropriate technology for transforming monocotyledonous plants, particularly rice, is described in publication WO 92/09696, incorporated herein by reference. For cotton, agrobacteria-mediated transformation (Gould, J. H. and Magallanes-Cedeno, M., 1998 and Zapata, C., 1999, incorporated herein by reference), polybrene and/or treatment-mediated transformation (Sawahel, W. A., 2001, incorporated herein by reference).

In a specific embodiment of the present invention, the mutated HPPD is targeted to the chloroplast. This can be done by integrating a nucleic acid sequence encoding a mutated HPPD fusion protein and transit peptide as described above.

Alternatively, mutated HPPD can be expressed directly in chloroplasts using chloroplast genome transformation. A suitable method comprises bombarding leaf sections by particles coated with DNA and integrating the introduced gene encoding the protein according to the present invention by means of homologous recombination. Appropriate selection vectors and systems are known to those skilled in the art. An example of means and methods that can be used for such integration into the chloroplast genome of tobacco strains is provided in publication WO 06/108830, the content of which is incorporated herein by reference. When polypeptides are targeted directly to the chloroplast using chloroplast genome transformation, a transit peptide sequence is generally not required. The present invention also relates to a method of obtaining a plant resistant to an HPPD inhibitor, characterized by the fact that the plant is transformed with a chimeric gene as previously described.

The present invention also relates to a method of obtaining a plant resistant to an HPPD inhibitor, characterized by the fact that the plant is transformed with a chimeric gene comprising a coding sequence as well as a heterologous regulatory element in the 5' position. and, optionally, in the 3' position, which are capable of functioning in a host organism, wherein the coding sequence contains at least one nucleic acid sequence as described above.

In a specific embodiment of the present invention, in this method, the HPPD inhibitor is a triketone or pyrazolinate herbicide, preferably tembotrione, mesotrione or sulcotrione, particularly tembotrione.

In another specific embodiment, the present invention relates to a method of obtaining a plant resistant to an HPPD inhibitor as described above, characterized by the fact that the plant is additionally transformed, simultaneously or successively, with a functional gene in that plant that allows the hyperexpression of a PDH enzyme (prephenate dehydrogenase).

The present invention also relates to a method of selectively removing weeds from plants, particularly plant crops, with the aid of an HPPD inhibitor, particularly a herbicide as defined above, wherein this method is characterized by the fact that this herbicide is applied to plants that have been transformed in accordance with the present invention, either before planting the crop, before crop emergence or after crop emergence.

In a specific embodiment of the present invention, in this method, the HPPD inhibitor is a triketone or pyrazolinate herbicide, preferably tembotrione, mesotrione or sulcotrione, particularly tembotrione.

The present invention also relates to a method of controlling weeds in an area or field containing transformed seeds as previously described in the present patent application, wherein said method comprises applying to said area of the field a dose of a herbicide. HPPD inhibitor that is toxic to said weeds, without significantly affecting seeds or plants that contain a nucleic acid sequence or a chimeric gene as previously described in the present patent application.

In a specific embodiment of the present invention, in this method, the HPPD inhibitor is a triketone or pyrazolinate herbicide, preferably tembotrione, mesotrione or sulcotrione, particularly tembotrione.

The present invention also relates to a method of cultivating plants that have been transformed with a chimeric gene in accordance with the present invention, wherein said method comprises planting seeds comprising a chimeric gene in accordance with the present invention, in a area of a field which is suitable for cultivation of said plants and the application, if weeds are present, of a dose, which is toxic to weeds, of a herbicide which targets the HPPD defined above to the said area of said field, without significantly affecting said transformed seeds or said transformed plants, and then harvesting the cultivated plants or parts of plants when they reach the desired stage of maturity and, where appropriate, separating the seeds from the harvested plants.

In a specific embodiment of the present invention, in this method, the HPPD inhibitor is a triketone or pyrazolinate herbicide, preferably tembotrione, mesotrione or sulcotrione, particularly tembotrione.

In the above methods, the herbicide targeting HPPD can be applied in accordance with the present invention, either before planting the crop, before crop emergence or after crop emergence.

The present invention also relates to a process of obtaining oil, particularly soybean oil or meal, which comprises cultivating a crop, particularly a soybean crop, that expresses a mutated HPPD according to the present invention in a field, optional treatment of this crop with an HPPD-inhibiting herbicide, harvesting of the grains, grinding of the grains to produce bran and extraction of the oil. Furthermore, seeds or grains of plants, whether whole, broken or chopped, which contain the chimeric gene according to the present invention are part of the present invention.

The present invention therefore relates to a method of obtaining oil or meal comprising cultivating a transformed plant as described above, optionally treating that plant with an HPPD-inhibiting herbicide, harvesting the grains and grinding the grains to manufacture bran and extract the oil.

In specific embodiments, the above methods in accordance with the present invention involve an HPPD inhibitor herbicide selected from isoxaflutole, tembotrione, mesotrione, pyrasulfotol, sulcotrione, topramezone, 2-cyano-3-cyclopropyl-1-(2-SO2CH3- 4-CF3phenyl)propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-2,3 Cl2 phenyl)propane-1,3-dione.

In other specific embodiments, the above methods according to the present invention involve an HPPD-inhibiting herbicide of the triketone class, such as tembotrione, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone, particularly selected from tembotrione, sulcotrione and mesotrione, more specifically tembotrione.

Within the meaning of the present invention, it is understood that “herbicide” is a substance active as a herbicide on its own or a substance that is combined with an additive that alters its effectiveness, such as an agent that increases its activity (an agent synergistic) or that limits its activity (a security agent). Naturally, it must be understood that, for their application in practice, the above herbicides are combined, in a way that is intrinsically known, with the formulation adjuvants that are customarily used in agricultural chemistry.

When the plant that has been transformed in accordance with the present invention contains one or more different genes for tolerance against other herbicides (such as a gene encoding an EPSPS that has or has not been mutated and confers on the plant tolerance against glyphosate herbicides or a pathogen gene or bar that confers tolerance against glufosinate herbicides) or when the transformed plant is naturally sensitive to another herbicide (such as tolerance against sulfonylurea), the method according to the present invention may comprise the simultaneous or chronologically coordinated application of a glufosinate inhibitor. HPPD in combination with said herbicide or combination of herbicides, such as glyphosate and/or glufosinate and/or sulfonylurea herbicides.

The present invention also relates to the use of the chimeric gene encoding a mutated HPPD according to the present invention as a marker gene during the transformation of a plant species, based on the selection of the HPPD-inhibiting herbicides mentioned above.

The present invention also relates to a method of obtaining a plant resistant to a triketone or pyrazolinate HPPD inhibitor, characterized by the fact that the plant is transformed with a chimeric gene that expresses in the plant an HPPD mutated in the amino acid glycine at position 336. with reference to the amino acid sequence of Pseudomonas HPPD of SEQ ID NO. 2.

In a specific embodiment, the present invention relates to the aforementioned method of obtaining a plant resistant to a triketone or pyrazolinate HPPD inhibitor, characterized by the fact that the HPPD mutation is selected from Gly336Arg, Gly336Asp, Gly336Glu, Gly336His , Gly336Met, Gly336Phe, Gly336trp, Gly336Asn, Gly336Cys and Gly336Val.

In another specific embodiment, the present invention relates to the aforementioned method of obtaining a plant resistant to a triketone HPPD inhibitor selected from tembotrione, mesotrione and sulcotrione.

In another specific embodiment, the present invention relates to the aforementioned method of obtaining a plant resistant to a triketone or pyrazolinate HPPD inhibitor, characterized by the fact that the plant is additionally transformed, simultaneously or successively, with a functional gene in this plant that allows the hyperexpression of an enzyme PDH (prephenate dehydrogenase).

The present invention also relates to a method of controlling weeds in an area or field, wherein the method comprises planting in that area or field transformed plants resistant to a triketone or pyrazolinate HPPD inhibitor that has been obtained in accordance with the method described above or transformed seeds originating therefrom and in applying a dose that is toxic to weeds of said triketone or pyrazolinate HPPD inhibitor without significantly affecting said transformed seeds or said transformed plants.

The present invention also relates to a method of obtaining oil or bran comprising cultivating either a pyrazolinate or triketone HPPD inhibitor that has been obtained in accordance with the method described above or a transformed seed that originates from that plant, optional treatment of this plant or seed with an HPPD triketone or pyrazolinate inhibitor, harvesting the grains and grinding the grains to make bran and extract the oil.

The present invention also relates to the use of an HPPD mutated in the amino acid glycine at position 336 with reference to the amino acid sequence of the Pseudomonas HPPD of SEQ ID No. 2 to make plants tolerant to a triketone or pyrazolinate HPPD inhibitor.

The present invention also relates to the use of a mutated HPPD as described above, characterized by the fact that the HPPD mutation is selected from Gly336Arg, Gly336Asp, Gly336Glu, Gly336His, Gly336Met, Gly336Phe, Gly336trp, Gly336Asn, Gly336Cys and Gly336Val.

The present invention also relates to the use of a mutated HPPD as described above, characterized in that the HPPD inhibitor is a triketone HPPD inhibitor selected from tembotrione, mesotrione and sulcotrione.

The present invention also relates to a host organism, particularly plant cells or plants, which contain a chimeric gene comprising a sequence encoding a mutated HPPD in accordance with the present invention and which also contains a functional gene in that host organism which allows for overexpression of an enzyme prephenate dehydrogenase (abbreviated in this PDH).

In the expression “gene that is functional in plants, allowing the overexpression of a PDH enzyme”, the term “PDH” should be interpreted as indicating any natural or mutated PDH enzyme and exhibits the PDH activity of converting prephenate to HPP. Particularly, the aforementioned PDH enzyme can originate from any type of organism. An enzyme with PDH activity can be identified using any method that makes it possible to measure the drop in the amount of prephenate substrate or to measure the accumulation of a product derived from the enzymatic reaction, that is, HPP or one of the cofactors NADH or NADPH. Particularly, PDH activity can be measured using the method described in Example 4.

Many genes that encode PDH enzymes are described in the literature and their sequences can be identified on the website http://www.ncbi.nlm.nih.gov/entrez/. Particularly known is the gene that encodes the PDH enzyme from the yeast Saccharomyces cerevisiae (Accession no. S46037) as described in Mannhaupt et al (1989), from a bacterium of the genus Bacillus, particularly the species B. subtilis (Accession no. P20692) as described in Henner et al (1986), from a bacterium of the genus Escherichia, particularly the species E. coli (Accession no. KMECTD) as described in Hudson et al (1984), or from a bacterium of the genus Erwinia, particularly the species E herbicola (Accession no. S29934), as described in Xia et al (1992).

The present invention further relates to a method of obtaining a host organism, particularly a plant cell or a plant, resistant to an HPDD inhibitor by integrating into that organism at least one nucleic acid sequence or a chimeric gene. as defined above and through its further transformation, simultaneously or successively, with a functional gene in that host organism that allows the hyperexpression of a PDH (prephenate dehydrogenase) enzyme.

In a specific embodiment, the present invention relates to a method of obtaining a host organism, particularly a plant cell or plant, resistant to a triketone or pyrazolinate HPDD inhibitor, particularly tembotrione, mesotrione or sulcotrione.

Means and methods that may be used to obtain host organisms, particularly a plant cell or a plant, transformed with a gene that allows the hyperexpression of an HPPD enzyme, and with a gene that allows the hyperexpression of a PDH enzyme are extensively described. in publication WO 04/024928, the content of which is incorporated herein by reference.

Reference in this specification to any prior publication (or information derived therefrom) or to any subject matter which is known is not and shall not be construed as an acknowledgment, admission or any form of suggestion that the prior publication (or information) or matter known is part of the common general knowledge in the field of the present invention.

BRIEF DESCRIPTION OF FIGURES

Fig. 1: Alignment of HPPD sequences from Streptomyces avermilitis, Daucus carota, Arabidopsis thaliana, Zea mais, Hordeum vulgare, Mycosphaerella graminicola, Coccicoides immitis, Mus musculus and Pseudomonas fluorescens. The numbering of amino acids is carried out according to the Pseudomonas sequence and an asterisk designates the amino acids that are common to these sequences.

SEQUENCE LISTING

SEQ ID NO. 1: nucleic acid sequence encoding HPPD from Pseudomonas fluorescens.

SEQ ID NO. 2: amino acid sequence of HPPD from Pseudomonas fluorescens.

SEQ ID NO. 3: nucleic acid sequence encoding HPPD from Arabidopsis thaliana.

SEQ ID NO. 4: amino acid sequence of HPPD from Arabidopsis thaliana.

SEQ ID NO. 5: nucleic acid sequence encoding HPPD from Mus musculus.

SEQ ID NO. 6: Amino acid sequence of HPPD from Mus musculus.

SEQ ID NO. 7: nucleic acid sequence encoding HPPD from Coccidioides immitis.

SEQ ID NO. 8: amino acid sequence of HPPD from Coccidiodides immitis.

SEQ ID NO. 9: nucleic acid sequence encoding HPPD from Mycosphaerella graminicola.

SEQ ID NO. 10: amino acid sequence of HPPD from Mycosphaerella graminicola.

SEQ ID NO. 11: nucleic acid sequence encoding HPPD from Hordeum vulgare.

SEQ ID NO. 12: amino acid sequence of HPPD from Hordeum vulgare.

SEQ ID NO. 13: nucleic acid sequence encoding Zea plus HPPD.

SEQ ID NO. 14: Zea plus HPPD amino acid sequence.

SEQ ID NO. 15: nucleic acid sequence encoding HPPD from Daucus carota.

SEQ ID NO. 16: amino acid sequence of HPPD from Daucus carota.

SEQ ID NO. 17: nucleic acid sequence encoding HPPD from Streptomyces avermilitis.

SEQ ID NO. 18: amino acid sequence of HPPD from Streptomyces avermilitis.

SEQ ID NO. 19: kerfiOOI primer sequence.

SEQ ID NO. 20: kerfi002 primer sequence.

SEQ ID NO. 21: kerfi003 primer sequence.

SEQ ID NO. 22: kerfi004 primer sequence.

SEQ ID NO. 23: kerfi007 primer sequence.

SEQ ID NO. 24: kerfi008 primer sequence.

SEQ ID NO. 25: kerfiO11 primer sequence.

SEQ ID NO. 26: kerfi012 primer sequence.

SEQ ID NO. 27: kerfi014 primer sequence.

SEQ ID NO. 28: kerfi016 primer sequence.

SEQ ID NO. 29: kerfi019 primer sequence.

SEQ ID NO. 30: kerfi020 primer sequence.

SEQ ID NO. 31: kerfi015 primer sequence.

SEQ ID NO. 32: kerfi018 primer sequence.

EXAMPLES

The various aspects of the present invention will be better understood with the aid of the experimental examples that follow. All methods or operations that are described below in these examples are provided by way of example and correspond to a selection that is drawn up from the different methods that are available to achieve the same or similar result. This choice has no effect on the quality of the result and, consequently, those skilled in the art can use any appropriate method to achieve the same or similar result. Most methods for manipulating DNA fragments are described in

Current Protocols in Molecular Biology, Volumes 1 and 2, Ausubel, F. M. et al, published by Greene Publishing Associates & Wiley Interscience (1989) or in Molecular Cloning, T. Maniatis, E. F. Fritsch, J. Sambrook, 1982, or in Sambrook, J. and Russell, D., 2001, Molecular Cloning: A Laboratory Manual (third edition).

EXAMPLE 1 PREPARATION OF MUTATED HPPD GENERAL DESCRIPTION

The Arabidopsis thaliana AtHPPD coding sequence (1335 bp) (Genebank AF047834; WO 96/38567) was initially cloned into the expression vector pQE-30 (QIAGEN) between the BamHI and HindIII restriction sites.

The coding sequence for PfHPPD from Pseudomonas fluorescens (1174 bp) (Rüetschi et al, Eur. J. Biochem., 205, 459-466, 1992, WO 96/38567) was initially cloned into the exclusive NcoI site of the expression vector pKK233- 2 (Pharmacia) which provides a start codon.

The vectors pQE-30-AtHPPD and pKK233-2-PfHPPD were used for PCR-mediated connection of an NcoI restriction site and a sequence encoding an N-terminal His6 tag to the 5' ends and an XbaI restriction site. to the 3' ends of AtHPPD and PfHPPD.

The AtHPPD gene PCR product was isolated from an agarose gel, cut with NcoI and XbaI restriction enzymes, purified with the MinElute® PCR Purification Kit (Qiagen) and cloned into the cut pSE420(RI)NX vector with the same restriction enzymes.

With reference to the PfHPPD gene, the PCR product was isolated from an agarose gel and cloned into the PCR®2.1-TOPO® vector. It was excised from this vector with the restriction enzymes NcoI and XbaI, isolated from an agarose gel and cloned into the pSE420(RI)NX vector cut with the same restriction enzymes. Both pSE420(RI)NX-AtHPPD and -PfHPPD were subjected to PCR-mediated site-directed mutagenesis to change a defined codon in corresponding sites of the two genes. The corresponding codon encodes Gly336 in WT PfHPPD and Gly422 in WT AtHPPD.

The codons that have undergone mutation in the coding sequences are analyzed using the Pyrosequencing® method.

PCR-MEDIATED BINDING OF A SEQUENCE ENCODING AN N-TERMINAL HIS6 TAG AND NCO I AND XBA I RESTRICTION SITES

The PCR reaction for each gene (AtHPPD and PfHPPD) was conducted in 24 wells of a 96-well PCR plate, respectively. As the forward and reverse primers for this reaction differ in size by 18 (AtHPPD) and 22 bp (PfHPPD), an annealing temperature gradient from 40.9°C to 64.5°C was performed, where each well is subjected to another annealing temperature within this range. When the primers anneal on the single-stranded template for the first time, a 5' overlap is produced on the new strand, until its complementary strand is synthesized and this overlap formed by the 5' region of the first primer is part of the template. The coding sequences were extended in this way at both ends, introducing a sequence encoding an N-terminal His6 tag and a restriction site at both ends. Reaction mixtures contain 500 ng of pQE-30-At HPPD pQE-30 DNA (1 μl of plasmid maxiprep) or 1 μg of pKK233-2-PfHPPD DNA (0.75 μl of plasmid maxiprep), 1 μl of kerfi001 and kerfi002, respectively, for AtHPPD or kerfi003 and kerfi004, respectively, for AtHPPD or kerfi003 and kerfi004, respectively, for PfHPPD (all primer solutions have a concentration of 10 pmol*μl-1), 25 μl of mixture Master HotStarTaq (Qiagen) and HyPure® molecular biology grade water to a final volume of 50 μl. The PCR program is defined as follows:

Step 2 is repeated twenty times.

The PCR reactions were subjected to agarose gel electrophoresis which produced clear bands corresponding to fragments of around 1500 bp (AtHPPD) or 1100 bp (PfhPPD). Lanes were cut from the gel and DNA was purified using the QIAquick® Gel Extraction Kit (Qiagen).

CLONING IN VECTOR PCR®2.1-TOPO® (INVITROGEN)

The pCR®2.1-TOPO® vector (3931 bp) was used for one-step cloning of Taq polymerase-amplified PCR products exhibiting 3'-adenosine (A) overlaps. The vector, in turn, was linearized and exhibited isolated 3'-thymidine (T) overlaps at its ends. Topoisomerase I was covalently linked to these 3'-thymidines, which served as a covalent link between the vector and the PCR product. To select bacterial cells that carry the vector, ampicillin or kanamycin can be used. The vector had an XbaI restriction site in its various cloning sites and an NcoI restriction site in the KanR gene.

The DNA solutions obtained from each gel extraction were used for TOPO TA cloning, respectively. After transformation of E. coli TOP10 cells, each reaction generated three white colonies (A1-A3, P1-P3) that were used to inoculate 5 ml of LB medium/amp.

To determine whether the vectors from these colonies carried the correct inserted fragment, plasmid DNA was prepared from 4 ml of pCR®2.1-TOPO®-AtHPPD cultures A1-A3 and PfHPPD cultures P1-P3 using the QIAprep® Centrifugation (Qiagen). DNA solutions obtained from these plasmid preparations were subjected to restriction digestion with HindIII and XhoI which was then analyzed on a 1% agarose gel. Both HindIII and XhoI have a single restriction site in the pCR®2.1-TOPO®-AtHPPD/PfHPPD vector, respectively. Restriction digestion of clone A1 DNA produced the expected bands representing a 1461-bp fragment (AtHPPD coding sequence) and the 3831-bp vector fragment; restriction digestion of P3 produced the expected bands representing a 1206 bp fragment (PfHPPD coding sequence) and the 3831 bp vector fragment on the agarose gel.

DNA obtained through plasmid maxipreparation using the QIAfilter® Maxi Kit (Qiagen) and a subsequent NaAc and EtOH precipitation from 100 ml of liquid LB/amp culture A1 (AtHPPD) or P3 (PfHPPD) was used to determine the DNA sequence of the corresponding HPPD gene inserted into the pCR®2.1-TOPO® vector. DNA sequencing was conducted with primers M13 uni (-21) and M13 rev (-29) by Eurofins MWG GmbH.

Sequencing confirmed the correct DNA sequence of AtHPPD and PfHPPD in the pCR®2.1-TOPO® vector, including the restriction sites at both ends of the coding sequences.

CLONING IN PSE420(RI)NX

The pSE420(RI)NX cloning and expression vector (5261 bp) is based on the pSE420 plasmid from Invitrogen. Modifications of this vector include the addition of a kanamycin tolerance gene and the removal of most of the superlinker region (multiple cloning sites).

The plasmid has the trp-lac (trc) promoter and the lacIq gene that provides the lac repressor in each 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 through induction with isopropyl β-D-1-thiogalactopyranoside (IPTG).

The AtHPPD and PfHPPD genes were cloned into the pSE420(RI)NX vector between the NcoI and XbaI restriction sites.

PCR-BASED SITE-DIRECTED MUTAGENESIS

Template DNA (pSE420(RI)NX-AtHPPD and pSE420(RI)NX- PfHPPD) was isolated from liquid culture of E. coli TOP10 by performing a plasmid miniprep. The DNA solutions obtained through these minipreparations were diluted to a concentration of 0.05 μg*μl-1. PCR-based site-directed mutagenesis requires two chemically synthesized DNA primers (forward and reverse primer) that are complementary to the same region of DNA, each of which to a template strand of double-stranded DNA. These primers contain the desired mutation in their center and cover a region of about twenty to thirty nucleotides of the template, including the mutation site and ten to fifteen bases on each of its sides. The mutation site covers three nucleotides that vary 46/60 independently in the primers in order to obtain every possible codon at the selected site.

In circular PCR mutagenesis, a plasmid template is completely copied by reproducing rolling circles from the 3' OH end of a primer that is incorporated into the growing strand. Each new DNA molecule then carries one or more altered nucleotides that were contained in the primer. High-fidelity DNA polymerase is used to reduce the possibility of additional unwanted mutations.

Oligonucleotide primer pairs kerfi007/kerfi008 (AtHPPD) and kerfi011/kerfi012 (PfHPPD) were dissolved in water to a concentration of 10 pmol*μl-1. For the PCR reaction by mutagenesis, 50 ng of template plasmid from minipreparations of pSE420(RI)NX- AtHPPD or pSE420(RI)NX-PfHPPD were used, diluted to a concentration of 0.05 μg*μl-1. The reaction mixture was composed as follows:

The PCR program was the same for AtHPPD and PfHPPD mutagenesis and the elongation time was set at seven minutes, considering that it takes one minute to reproduce 1 kb of plasmid DNA.

Step 2 is repeated eighteen times.

After the PCR reaction, the reactions were kept on ice to cool to room temperature.

Then, PCR reaction mutant plasmids were selected using the Dpn I restriction endonuclease. Only dam-methylated DNA is degraded by the Dpn I restriction enzyme whose GMe6ATC restriction site is relatively abundant. The plasmid templates that were produced by bacteria have been methylated and are therefore degraded. DNA amplified by PCR, however, remains intact.

1 μl of Dpn I restriction enzyme (10 U*μl-1) was added to the PCR reactions and the solutions were mixed by pipetting up and down. After one minute of centrifugation (13,200 rpm), the reactions were incubated at 37 °C for one hour.

The mutant plasmids contained coordinated nicks at the 5' end of each primer and could be transformed directly into competent cells. To concentrate mutant plasmids, a NaAC and EtOH precipitation was conducted and the DNA was resuspended in 10 μl of HyPure® Molecular Biology Grade Water. 3 μl of these plasmid solutions were subsequently used to transform electrocompetent E. coli K-12 MG1655 cells and, in the case of AtHPPD, 1 μl was used to transform electrocompetent E. coli TOP10 cells.

For AtHPPD, a total of 62 E. coli K-12 MG1655 clones were obtained, which were grown for subsequent analysis of the mutated codon in Costar® 2 ml deep 96-well plates. To obtain a higher number of clones, E. coli TOP10 was used as an alternative host for cloning plasmids in mutagenesis. Transformation of TOP10 E. coli cells with plasmids that underwent mutagenesis generated several hundred clones.

With reference to PfHPPD, a total of 252 E. coli K-12 MG1655 clones were obtained and were grown for analysis as described for clones transformed with AtHPPD plasmids.

EXAMPLE 2 PYROSEQUENCING® REACTIONS FOR VERIFICATION OF POINT MUTATIONS

Pyrosequencing® technology was used to verify point mutations by determining the nucleotide sequence of a defined short section of DNA. A PCR reaction was performed before amplification of a short DNA fragment containing the section to be sequenced. The template amplified by PCR needs to be single-stranded and covalently linked to a biotin molecule at its 5' end. Biotin served to non-covalently bind the template to streptavidin which was bound to a cross-linked agarose stationary phase (sepharose).

AMPLIFICATION OF BIOTINYLATED DNA FRAGMENTS

The PCR reaction was conducted in 96-well PCR plates. The reaction mixture contains 1 μl of forward primer solution (kerfi016 for AtHPPD, kerfi020 for PfHPPD; 10 pmol*μl-1), 1 μl of reverse primer solution (contains a biotin modification at its 5' ends; kerfi019 for AtHPPD, kerfi014 for PfHPPD; 10 pmol*μl-1), 2 μl of liquid bacterial culture from a clone grown in a deep well plate, 25 μl of HotStarTaq® Master Mix, and 21 μl of HyPure® Molecular Biology Grade Water .

The PCR programs for AtHPPD and PfHPPD showed differences in annealing temperatures, which were set at 55 °C and 60 °C, respectively.

PYROSSEQUENCING® REACTION

The Pyrosequencing® reaction (Biotage) was conducted in 96-well plates. To each 45 μl of PCR reaction, 40 μl of binding buffer (10 mM Tris-HCl; 2 M NaCl; 1 mM EDTA; 0.1% Tween 20), 3 μl of streptavidin sepharose beads were added. (patented composition - GE Healthcare BioScience AB) and 12 μl of ddH2O. These mixtures were shaken for ten minutes in the 96-well PCR plate.

Using a “vacuum preparation tool”, each solution was then deposited through a small filter attached to a small metal tube, while the streptavidin beads, now bound to the biotinylated PCR product, were retained on the filters through of suction. According to this principle, the filters were then immersed in 70% ethanol for five seconds to wash the DNA and remove the primers, dNTPs and other components of the PCR reaction. The procedure was repeated with 0.2 M NaOH to denature dsDNA and leave only the biotinylated DNA strand attached to the streptavidin beads. After a final wash of the DNA in wash buffer, the “vacuum priming tool” was held over a PSQ® 96 plate that contained 40 μl of annealing buffer and 0.1 μl of Pyrosequencing® primer solution (100 pmol *μl*; kerfi018 for At HPPD/kerfi015 for PfHPPD) per well. The vacuum was then turned off and each filter was immersed in its corresponding well to dissolve the DNA that was retained by the filter. The plate was then incubated at 80 °C for two minutes to resolve secondary structures eventually formed on the DNA templates. While the solutions were cooled to room temperature, the Pyrosequencing® primers hybridized to their binding sites on the template.

The remaining components of the Pyrosequencing® reactions (620 μl of enzyme mixture, 620 μl of substrate mixture, and 130 μl of each dNTP solution) were placed in separate wells of a cartridge. The cartridge and PSQ® plate were then placed inside the PyroMark® ID.

The Pyrosequencing® instrument automatically added enzyme and substrate to the reaction mixture before initiating the sequencing reaction through the addition of the first dNTP. To determine the DNA sequence downstream in the primer flow, a round of SQA is conducted. The order of nucleotides added to the reaction mixture is defined in advance. You can use ID PyroMark® software to translate Pyrogram® traces into the DNA sequence.

RESULTS

The PCR-amplified fragment of AtHPPD is 239 bp in size and biotin is linked to the non-coding strand; the PfHPPD fragment comprises 142 bp and biotin is linked to the coding strand.

The mutated codon in AtHPPD is located three bases downstream from the primer sequence of kerfi018. The first three bases sequenced are adenines, followed by the mutated codon. The coding strand of the AtHPPD fragment is synthesized by DNA polymerase, in such a way that the sequence can be translated directly into the amino acid sequence.

Screening of 438 AtHPPD colonies emitted 146 mutant genes, 181 wild-type genes (GGC codon at position 422), and 111 no longer had sequencing reactions or ambiguous results.

The production of mutant clones through transformation of mutant plasmids into E. coli K-12 MG1655 or E. coli TOP10 was therefore successful in 33% of cases. Codons encoding all amino acids except lysine could be obtained. Genes containing codons for glutamic acid, histidine, isoleucine, threonine, tryptophan, and tyrosine were present in TOP10 E. coli clones from which DNA was prepared and transformed into MG1655 E. coli K-12 cells. If possible, synonymous codons were selected considering codon usage in E. coli K-12. No codon used at a frequency below 10% was selected and most of the selected codons are used at a frequency greater than 35% (codon usage database; E. coli K-12: http://www .kazusa.or.jp/codon/cgi- bin/showcodon.cgi?species=83333).

From primer kerfi015, the non-coding strand of the PfHPPD fragment is synthesized by DNA polymerase, in such a way that the nucleotide sequence needs to be translated into the reverse complement before it can be translated into the amino acid sequence. The mutated codon immediately follows the primer and is therefore represented by the first three bases sequenced in the reaction.

Selection of 252 PfHPPD colonies yielded 119 mutant genes, 73 unchanged genes, TGG codon at position 336) and sixty unsuccessful sequencing reactions or ambiguous results.

The production of mutant clones through the transformation of mutant plasmids into E. coli K-12 MG1655 cells was therefore successful in 47% of cases. The codons that encode all amino acids, except alanine, could be used. If possible, codon synonyms were selected considering codon usage in E. coli K-12 as described above for AtHPPD codons.

EXAMPLE 3 HPPD ACTIVITY TEST

HPPD produces homogentisate and CO2 from 4-HPP and O2. The enzyme is incubated with its substrate 4-HPP in the presence or absence of an inhibitor. L-ascorbic acid is present in the reductant form to retain iron in the active site in the ferrous form and catalase is present to degrade toxic H2O2. After an incubation time of one hour, the reaction is stopped by adding 2,4-dinitrophenylhydrazine (DNP). DNP forms a hydrazone derivative with the remaining 4-HPP molecules in the test mixture that appears amber-brown in color under alkaline pH. The amount of unconsumed 4-HPP is measured photometrically at 405 nm.

For the preparation of inhibitor standard solutions, Tembotrione (Mw = 440.82) and DKN (Mw = 359.3) are dissolved in DMSO to a concentration of 10 mM. This standard solution is first diluted twenty times in 25% DMSO to a concentration of 0.5 mM. Additional dilutions are made with ddH2O to obtain the inhibitory solutions used in the test (5 μM, 10 μM and 20 μM). The corresponding inhibitor solution represents half the volume of the test mixture, which means that its active concentration is reduced twice again. This results in inhibitor concentrations of 2.5 μM, 5 μM, and 10 μM. A 2% DMSO solution provides half of the test mixture in uninhibited reactions to normalize a possible inhibitory effect of DMSO.

The test is designed for an HPPD concentration of 444 nM on a monomeric basis and a 4-HPP concentration of 500 μM. This corresponds to 44.4 pmol of HPPD and 50 nmol of 4-HPP in a 100 μl test mixture, which results in about a thousand-fold excess of substrate with respect to the enzyme. The calculated theoretical molecular weight of an AtHPPD subunit is 49.515 kD, which results in 2.2 μg of HPPD per test mixture. The calculated theoretical molecular weight of a PfHPPD subunit is 41.205 kD, which results in 1.8 μg of HPPD per test mixture. The enzyme solution provides a quarter of the volume of the test mixture, such that standard enzyme solutions are produced by diluting AtHPPD solutions to 88 μg*ml-1 with 50 mM TRIS buffer; PfHPPD solutions are diluted to 72 μg*ml-1.

Inhibitor concentrations (2.5 μM, 5 μM, and 10 μM) provide five-fold, ten-fold, and twenty-fold excess of inhibitor compared to the amount of enzyme. A buffer and substrate solution is prepared that provides one-fourth of the test mixture. 2.5 ml of buffer and substrate solution contain 1 ml of 1 M TRIS buffer, 500 μl of 10 mM 4-HPP solution, 500 μl of 200 mM L-ascorbic acid solution, 13 μl of Catalase solution and 487 μl of ddH2O. The test is conducted in 96-well microplates with a Greiner F bottom and all reactions are carried out in three copies. Controls are run six times per plate and contain 25 μl of 50 mM TRIS instead of HPPD solution (corresponding to consumption of 0% 4-HPP) or a buffer and substrate solution that contains 500 μl of 1 M TRIS instead of 500 μl of 10 mM 4-HPP (corresponding to 100% HPP consumption). The reaction is initiated by adding 25 μl of HPPD solution to a mixture of 50 μl of the corresponding inhibitor solution or 50 μl of 2% DMSO and 25 μl of buffer and substrate solution. The reaction is kept running for one hour at room temperature. The reaction is stopped and 4-HPP staining is induced by adding 50 μl of 0.04% DNP and 3.8 N HCl solution. After fifteen minutes, the addition of 100 μl of 5 N KOH generates a change in color of the hydrazone derivative. Photometric measurement with a BMG FLUOstar Galaxy microplate reader is conducted immediately at 405 nm and the data obtained is used for analysis of HPPD activities in the presence and absence of an inhibitor.

RESULTS

AtHPPD mutants at position 422 with reference to the Arabidopsis HPPD amino acid sequence of SEQ ID NO: 4 (i.e., Gly422Ala, Arg, Asn, Asp, Cys, Glu, His, Leu, Met, Phe, Pro, Ser , Tyr and Val) were tested together with the WT enzyme in the HPPD activity test (it is observed that Gly422 with reference to the Arabidopsis HPPD amino acid sequence of SEQ ID NO. 4 corresponds to Gly336 with respect to the reference sequence of Pseudomonas of SEQ ID NO. 2). All enzymes were active, but only the activities of the Gly422Ala, Asn, Asp, Cys, His, Met, Phe, Tyr, and Val mutants were within or above the range (>70%) of the WT enzyme. The WT enzyme retained 35% of its activity in the presence of 2.5 μM tembotrione; only the Gly422Asn, Cys, His and Val mutants retained higher activities than aria mem 39, 44, 51 and 43%, respectively. Activities were further reduced under higher concentrations of Tembotrione. Only the mutant Gly422His exhibited a residual activity of about 40% in the presence of 5 and 10 μM Tembotrione while all other enzymes exhibited activities comparable to the WT enzyme at these inhibitor concentrations, which range from about 20 to 10%, respectively ( Table 1).

The PfHPPD mutants Gly336Arg, Asp, Gln, Glu, His, Leu, Lys, Met, Phe, Thr, Trp and Pro were tested together with the WT enzyme. With the exception of the Gly336Pro mutant, whose unexhibited activity ranged below 70% of the WT activity, the activities of the Gly336 mutants were within or above the range of the WT enzyme (>75%). The WT enzyme retained only 5% of its activity in the presence of 2.5 μM Tembotrione, while the Gly336Asp, Arg, Gln, Glu, His, Met, Phe and Trp mutants retained activities above 14%. The highest residual activities were those of Gly336His (26%) and Gly336Phe (33%). Interestingly, the Gly336His mutant exhibited residual activities of 13 and 11.2% in the presence of 5 and 10 μM Tembotrione, respectively, while the activities of Gly336Phe were reduced to 12.4 and 2.5%, respectively. The Gly336Met mutant exhibited residual activities of 7 and 10%, respectively, at these inhibitor concentrations, whereas the activity of the WT enzyme was reduced to zero (Table 1).

TABLE 1 RELATIVE ACTIVITY (IN PERCENTAGE) OF PF HPPD AND THPPD MUTANTS IN THE PRESENCE AND ABSENCE OF TEMBOTRIONA

Activities are normalized by setting uninhibited enzyme activity to 100%. Pseudomonas fluorescens HPPD: * Mutation in gly at position 422 with reference to the amino acid sequence of Arabidopsis HPPD of SEQ ID NO. 4 (corresponds to Gly336 with reference to the amino acid sequence of Pseudomonas HPPD of SEQ ID NO. 2).

EXAMPLE 4 PDH ACTIVITY TESTING

Prephenate dehydrogenase activity was measured at 25 °C by spectrophotometric monitoring at 340 nm of the formation of NADH or NADPH in a solution containing 50 mM tris-HCl, pH 8.6, 300 μM prephenate and 1 mM NAD or NADP in a total volume of 200 μl.

EXAMPLE 3 CONSTRUCTION OF CHIMERIC GENES FOR EVALUATION OF HDDP PF WITH AND WITHOUT MUTATION IN SMOKE A. Construction of chimeric genes:

The vector that is used to elaborate the constructs whose HPPD (wild type or mutants) to be expressed in PBD6-type tobacco plants is called pRP-RD224. This vector was initially designed for the cloning of all Pseudomonas HPPD mutants by simply replacing the truncated HPPD gene of this vector between the KpnI and BstEII sites. Its construction from the binary vector pBI121 (Clontech) is extensively described in WO 99/24585.

The pRP-RD224 clone therefore has the following structure: RB/Nos promoter/NPTII/Nos terminal/dual histone promoter/tev/otp/HPPD truncated/Nos terminal/LB where “truncated HPPD” designates the sequence that encodes the truncated PfHPPD of about five hundred base pairs in subsequent order to facilitate the selection of transformed colonies that have integrated the mutant HPPDs (WO 99/24585).

PRP-RD224 MUTANTS

The DNAs of the vectors that carry the HPPDs with and without mutation were digested with KpnI and BstEII, purified and then ligated into the vector pRP-RD224, which had been digested with KpnI and BstEII and purified. Transformants that had integrated the mutated HPPD gene were selected by insert size through digestion with KpnI and BstEII. The resulting clones are called pRP-RD224, to which the type of mutation that was driven in HPPD is added; In this way, the following clones were created: pRP RD224 Pf (for the unmutated enzyme), pRP RD224 PfH336 (for the enzyme that contains a histidine at position 336), pRP RD224 PfM336 (for the enzyme that contains a methionine at position 336) and pRP RD224 PfF336 (for the enzyme containing a phenylalanine at position 336).

EXAMPLE 4 CONSTRUCTION OF A CHIMERIC GENE THAT HYPEREXRESSES PDH

The construction of a chimeric gene that overexpresses PDH comprises the assembly, in the direction of transcription, of a “double histone” promoter (PdH4) as described in Patent Application No. EP 0,507,698, the translation amplifying sequence of the virus of smoke corrosion (TEV) described in Carrington and Freed (1990), a sequence coding for an optimized transit peptide (OTP) as described in Patent Application No. EP 0,508,909, the coding part of the yeast PDH gene described in Manhaupt et al (1989) and the nos terminal of the nopaline synthase gene described in Bevan et al (1983). The set was then cloned into the pRD 224 binary vector, which contains a kanamycin tolerance gene (NPTII), to generate the pRD 224-PDH vector.

This binary vector was then used to transform the Agrobacterium strain EHA 105 and to generate the Agrobacterium strain EHA 105-pRD 224-PDH. This Agrobacterium strain was used to transform tobacco plants transformed with the chimeric genes as described in Example 3.

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Claims (11)

1. Mutated hydroxyphenylpyruvate dioxygenase (HPPD) which maintains its properties of catalyzing the conversion of para-hydroxyphenylpyruvate (HPP) into homogentisate, and which is less sensitive to an HPPD inhibitor than the original unmutated HPPD, characterized by containing a mutation in the amino acid glycine at position 336, with reference to the amino acid sequence of Pseudomonas HPPD of SEQ ID NO: 2, which is Gly336His. 2. Mutated hydroxyphenylpyruvate dioxygenase (HPPD), according to claim 1, characterized by the fact that the mutated HPPD contains a second mutation. 3. Mutated hydroxyphenylpyruvate dioxygenase (HPPD), according to claim 2, characterized by the fact that the second mutated amino acid is selected from the selected amino acids: Pro215, Gly298, Gly332, Phe333, Gly334 and Asn337, with reference to the sequence of Pseudomonas HPPD of SEQ ID NO:2. 4. Nucleic acid sequence characterized by the fact that said sequence is identified by SEQ ID NO: 1, wherein nucleotides 1006-1008 correspond to the His codon in the encoded mutated HPPD, as defined in any one of claims 1 to 3. 5. Chimeric gene comprising a coding sequence as well as a heterologous regulatory element in the 5' position and, optionally, in the 3' position, which are capable of functioning in a host organism, characterized by the fact that the coding sequence contains a sequence of nucleic acid as defined in claim 4. 6. Chimeric gene according to claim 5, characterized in that it contains, in position 5' of the nucleic acid sequence encoding a mutated HPPD, a nucleic acid sequence encoding a plant transit peptide, wherein this sequence is disposed between the promoter region and the sequence encoding the mutated HPPD, so as to allow expression of a mutated transit peptide/HPPD fusion protein. 7. Mutated transit peptide/HPPD fusion protein wherein the mutated HPPD is as defined in any one of claims 1 to 3. 8. Cloning and/or expression vector for transforming a host organism characterized by containing a chimeric gene, as defined in claim 5 or 6. 9. Method of obtaining a plant resistant to an HPDD inhibitor characterized by the fact that the plant is transformed with a chimeric gene, as defined in claim 5 or 6. 10. Method for selecting transformed seeds or transformed plants, characterized in that it comprises applying to said transformed seeds or transformed plants containing a nucleic acid sequence, as defined in claim 4, or a chimeric gene, as defined in claim 5 or 6, of a dose of an HPPD inhibitor herbicide that is toxic to a seed or plant that contains a wild-type HPPD nucleic acid or gene, without significantly affecting seeds or plants that contain a nucleic acid sequence as defined in claim 4, or a chimeric gene as defined in claim 5 or 6. 11. Method according to claim 9 or 10, characterized in that the HPPD inhibitor is tembotrione.