WO2002020741A1 - Hydroxy-phenyl pyruvate dioxygenase fused with a signal peptide, dna sequence and use for obtaining plants containing herbicide-tolerant plants - Google Patents

Abstract

The invention concerns a nucleic acid sequence coding for a hydroxy-phenyl pyruvate-dioxygenase (HPPD) fused with a signal peptide, a chimeric gene containing said sequence as encoding sequence and its use for obtaining plants resistant to certain herbicides. The inventive fusion protein consists of a HPPD fused at its C-terminal or N-terminal end with a signal protein sequence enabling the HPPD to be directed into the cell compartments other than the cytoplasm or the plastids.

Description

Hydroxy-phenyl pyruvate dioxygenase fused to a signal peptide DNA sequence and production of plants containing such a gene, herbicide tolerant.

The present invention relates to a nucleic acid sequence encoding a hydroxy-phenyl pyruvate dioxygenase (HPPD) fused to a signal peptide, a chimeric gene containing this sequence as coding sequence and its use for obtaining plants resistant to certain herbicides.

Hydroxy-phenyl pyruvate dioxygenase are enzymes that catalyze the reaction of conversion of para-hydroxy-phenyl pyruvate (HPP) to homogentisate. This reaction occurs in the presence of iron (Fe ²⁺⁾ in the presence of oxygen (Crouch NP et al., Tetrahedron, 53, 20, 6993-7010, 1997).

It also has some inhibitory molecules of this enzyme, which attach themselves to the enzyme to inhibit transformation of the HPP into homogentisate. Some of these molecules have found employment as herbicides since inhibition reaction in plants leads to whitening of the leaves of treated plants, and the death of said plants (Pallett KE et al., 1997 Pestic. Sci. 50 83-84). Such herbicides having HPPD as target described in prior art are especially isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, US 5,424,276 ), in particular isoxaflutole, a selective maize herbicide, diketonitriles (EP 496 630, EP 496 631), in particular 2-cyano-3-cyclopropyl-l- (2-Sθ2 CH3-4-CF3 phenyl) propane l, 3-dione and 2-cyano-3-cyclopropyl-l- (2-Sθ2 CH3-4-2,3 Cl2 phenyl) propane-1, 3-dione, triketones (EP 625 505, EP 625 508, US 5,506,195), in particular sulcotrione or mesotrione, or the pyrazolinates. Tests to confirm that HPPD is the target of diketonitriles (DKN) and to demonstrate that HPPD is, at least at certain doses, the single target of diketonitriles were performed in the laboratory by germinating seeds Arabidopsis three types of environments in sterile-vitro:

1 Murashig and Skoog medium (Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and has bioassays with tobacco tissue culture. Physiol. Plant. 15, 473-479), germination control experience

2 MS medium plus DKN at a dose of 3 DPA MS medium plus DKN at the same dose + homogentisate at a concentration of 5 mM.

It is very clear that the medium 1 is normally germination, each seedling developing two well green cotyledons. The development is then normally. On medium 2, germination takes place but the seedling emerges is white, the two cotyledons having no pigmentation. Seedlings then die within a few days. On the middle 3, germination is normally the cotyledons are green. Seedlings grow quickly but the amount of Homogentisate in reducing environment, the first symptoms appear and bleaching plant growth stop, they eventually die as in the test conducted on the middle 2.

This confirms that the HPPD is the target of DKN in planta and appears to be the only target. This also shows that Homogentisate is transported from the culture medium to the cell site where it is necessary for the proper functioning of the cell and the survival of the plant. For making plants tolerant to herbicides, there are three main strategies: (1) detoxifying the herbicide with an enzyme from converting the herbicide or its active metabolite, non-toxic degradation products, such as enzymes tolerance to bromoxynil or to basta (EP 242 236, EP 337 899); (2) mutation of the target enzyme into a functional enzyme less sensitive to the herbicide, or its active metabolite, such as tolerance to glyphosate enzymes (EP 293 356, Padgette SR et al., J. Biol. Chem, 266, 33, 1991). or (3) overexpression of the sensitive enzyme, so as to produce in the plant sufficient amounts of target enzyme in terms of kinetic constants of this enzyme with respect to the herbicide so as to have sufficient functional enzyme despite the presence of the inhibitor.

This third strategy which has been described for successfully obtaining plants tolerant to HPPD inhibitors (WO 96/38567), it being understood that for the first time a simple overexpression of the sensitive enzyme target strategy (non-mutated ) was used successfully for conferring on plants tolerance to an agronomic level to a herbicide.

While the plants are HPPD enzymes cytoplasmic localization (Garcia, I., et al 1997. J. Biochem 325:.. .. 761-769), it was also shown that the best tolerance was achieved when the HPPD exogenous had accumulated in the plastids, particularly chloroplasts.

These results show firstly that Homogentisate can be transported from the external environment to the site intracellular where it is necessary to _complement. inhibition of endogenous HPPD, and secondly that the accumulation of an exogenous HPPD in the cell at the level of plastids or cytoplasm allows the same type of complementation possible to obtain genetically modified plants tolerant to herbicides that inhibit HPPD.

It has now been found that the addressing of an exogenous HPPD in other cellular compartments as the cytoplasm or plastids, possible to improve the tolerance to HPPD inhibitor herbicides with respect to a single cytoplasmic targeting.

The present invention therefore relates to a fusion protein consisting of an HPPD fused at its C-terminus or N-terminus to a protein signal sequence enabling the addressing of the HPPD in cellular compartments other than the cytoplasm or plastids (in particular chloroplasts).

By HPPD is meant according to the invention any native HPPD enzyme, mutated or chimeric, having HPPD activity. Many HPPD are described in the literature, including bacteria such as Pseudomonas HPPD (Rϋetschi et al., Eur. J. Biochem., 205, 459-466, 1992, WO 96/38567), of plants like Arabidopsis ( WO 96/38567, Genebank AF047834) or of carrot (WO 96/38567, Genebank 87257), of Coccicoides (Genebank COITRP) or of mammals such as the mouse or pig.

By mutated HPPD is meant according to the invention the HPPD mutated to obtain tolerance properties HPPD inhibitor herbicides improved compared with the corresponding native HPPD. Advantageously, the mutated HPPD is an HPPD mutated in its C-terminal portion, as described in patent application WO 99/24585.

By chimeric HPPD is meant an HPPD comprising elements coming from different HPPD, including chimeric HPPDs described in patent application WO 99/24586. Advantageously, the HPPD is an HPPD of Pseudomonas fluorescens (WO

96/38567). Advantageously, the mutated HPPD comprises the mutation W336 as described in patent application WO 99/24585.

It is known in the literature that the addition of a coding sequence corresponding to an additional peptide at the N-or C-terminus of the protein will enable the transport of the latter to another cellular compartment as the cytoplasm in which the mRNA is translated. This peptide said signal peptide or transit peptide as appropriate, according to its sequence will allow routing to the other compartment can be for example the plastid (membrane or stroma), the thylakoid lumen or thylakoid membrane, to the reticulum reticulum, into the vacuole, the cell wall, mitochondria, the golgi apparatus, the nucleus, the nuclear membrane, without being exhaustive

By protein sequence signal means according to the invention any signal or transit peptide allowing the addressing of HPPD in cellular compartments other than the cytoplasm or plastids (chloroplasts in particular).

The role of such protein sequences are in particular described in issue 38 of the journal Plant Molecular Biology (1998) devoted largely to transport proteins into the various compartments of the plant cell (Sorting of proteins to vacuoles in plant cells pp 127- 144; the nuclear pore complex ppl45-162; protein translocation into and across the chloroplastic envelope membranes pp 91-207; multiple pathways for the targeting of thylakoid proteins in chloroplasts pp 209-221; mitochondrial protein import in plants pp 311-338).

Advantageously, the protein sequence signal allows addressing of HPPD to the vacuolar compartment (vacuole). Such peptides are largements described in the literature (Neuhaus JM and JC Rogers Sorting of proteins to vacuoles in plant cellsPlant Molecular Biology 38: 127-144, 1998). Preferably, the peptide of the vacuolar protein described in JM MsaCiB

Ferullo et al (Plant Molecular Biology 33: 625-633, 1997) (SEQ ID NO 1 or 2) which is now known to be localized in the vacuole, fused to the C-terminal part of the HPPD.

Preferably, the fusion protein is described by the sequence identifier No. 3 or 4 (SEQ ID NO 3 or 4) with its coding sequence. The present invention also relates to a nucleic acid sequence encoding the fusion protein according to the invention. The present invention also relates to the sequences capable of hybridizing selectively with the nucleic acid sequence above, the sequences homologous to the above sequence, the fragments of said sequences, and the sequences which differ from the sequences above -Dessus but that due to the degeneracy of the genetic code, encode the same fusion protein.

According to the present invention, the term "nucleic acid sequence" means a nucleotide or polynucleotide sequence can be DNA or RNA type, preferably of the DNA type, in particular double-stranded.

By "sequence capable of selectively hybridizing to" refers according to the invention those sequences which hybridize with the sequences above at a level supperior background noise significantly. The background noise may be related to the hybridiqtion other DNA sequences present, in particular other cDNAs present in a cDNA library. The signal level generated by the interaction between the sequence capable of hybridizing selectively and the sequences defined by the above sequence ID according to the invention is generally 10 times, preferably 100 times more intense than that of the interaction of the other DNA sequences generating the background noise. The level of interaction may be measured, for example, by labeling the probe with radioactive elements, such as ³² P. Selective hybridization is generally obtained using very stringent medium conditions (for example 0.03 M NaCl and sodium citrate 0.03 M to about 50 ° C-60 ° C). Hybridization can of course be made by usual methods of the art (eg Sambrook et al, 1989, Molecular Cloning: A Labratory Manual).

By "homologous" is meant according to the invention a nucleic acid fragment exhibiting one or more sequence modifications relative to the nucleotide sequence encoding the fusion protein according to the invention. These modifications may be obtained according to the usual mutation techniques, or alternatively by choosing the synthetic oligonucleotides employed in the preparation of said sequence by hybridization. In view of the multiple combinations of nucleic acids which may lead to the expression of a same amino acid, the differences between the reference sequence according to the invention and the corresponding homologue may be substantial. Advantageously, the degree of homology will be at least 70% compared to the reference sequence, preferably at least 80%, more preferably at least 90%. These modifications are generally and preferably neutral, i.e. they do not affect the primary sequence of the fusion protein. This definition also applies to the protein sequence of the fusion protein, the mutations being in this case at the amino acid level and degree of homology being defined relative to the primary sequence of the fusion protein.

The methods for measuring and identifying homologies between nucleic acid sequences are well known to those skilled in the art. May be employed for example the PILEUP or BLAST programs (in particular Altschul & al, 1993, J. Mol Evol 36 .290-300;... Altschul & al, 1990, J. Mol Biol 215 .403-10..). The methods for measuring and identifying homologies between polypeptides or proteins are also known to the skilled person. For example, we can use the "package" UWGCG and BESTFITT program to calculate homology (Deverexu et al., 1984, Nucleic Acid Res. 12, 387-395).

By "fragment" is meant according to the invention fragments of the DNA sequences according to the invention, i.e. the sequences above for which parts have been deleted but which conserve the function of said sequences, c that is to say the addressing fonbction for petide signal and HPPD activity for the HPPD protein.

The invention also relates to the use of a nucleic acid sequence encoding the fusion protein according to the invention in a method for the transformation of plants, as a marker gene or as a coding sequence for imparting to the plant tolerance to HPPD inhibitor herbicides. This sequence may of course also be used in combination with other (s) gene (s) marker (s) and / or sequence (s) coding region (s) for one or more agronomic properties.

The present invention also relates to a chimeric gene (or expression cassette) comprising a coding sequence as well as regulatory elements in the 5 'and 3' heterologous operable in a host organism, in particular plant cells or plants, coding sequence comprising at least one nucleic acid sequence encoding a fusion protein as defined above.

Host organism is understood to mean any mono- or multicellular organism, lower or higher, wherein the chimeric gene of the invention can be introduced, for the production of mutated HPPD. This is in particular bacteria, e.g., E. coli, yeast, particularly Saccharomyces or Kluyveromyces, Pichia, fungi, in particular Aspergillus, a baculovirus, or preferably plant cells and plants.

"Plant cell" is understood according to the invention any cell derived from a plant and capable of constituting undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of plants, plants or seeds.

The term "plant" of the invention any differentiated multicellular organism capable of photosynthesis, in particular monocotyledons or dicotyledons, more particularly crop plants intended or not for animal or human food, such as corn, wheat, rapeseed, soybean, rice, sugarcane, beet, tobacco, cotton, etc.

The regulatory elements necessary for expression of the nucleic acid sequence encoding a fusion protein according to the invention are well known to those skilled in the art depending on the host organism. They include promoter sequences, transcription activators, terminator sequences, including start and stop codons. The means and methods for identifying and selecting the regulatory elements are well known in the art and widely described in the literature.

The invention more particularly the transformation of plants. Promoters As promoter regulatory sequence in plants, there may be used any promoter sequence of a gene which is naturally expressed in plants, in particular a promoter which is expressed in particular in the leaves of plants, such as so-called constitutive promoters of bacterial, viral or plant origin or of said light-dependent promoters such as that of a gene of the small subunit of ribulose biscarboxylase / oxygenase (RuBisCO) plant or any known suitable promoter can be used. Among the promoters of plant origin histone promoters may be mentioned as described in EP 0507698, or the rice actin promoter (US 5,641,876). Among the promoters of a plant virus gene, it may be mentioned of the cauliflower mosaic (19S or 35S CaMn) or the promoter of circovirus (AU 689 311).

Preferably, the promoter is selected from light-dependent promoters (WO 99/25842), the rice actin promoter, histone promoters and promoters consitués by melting a histone promoter and the first rice actin intron (WO 99/34005). transcription elements

According to the invention, can also be used in combination with the COMTII promoter according to the invention, other regulation sequences which are situated between the promoter and the coding sequence, such as transcription activators ( "enhancer") , such as the translation activator of the tobacco mosaic virus (NMT) described in WO 87/07644, or of the tobacco etch virus (TEN) described by Carrington & Freed. terminator

As regulatory terminator or polyadenylation sequence, there may be used any corresponding sequence of bacterial origin, such as for example the nos terminator of Agrobacterium tumefaciens, of viral origin, such as CAMn 35S terminator, or of plant origin, for example a histone terminator as described in application EP 0,633,317.

The present invention also relates to a cloning vector and / or expression vector for transforming a host organism containing at least one chimeric gene as defined above. This vector further comprises the chimeric gene above, at least one origin of replication. This vector may comprise a plasmid, a cosmid, a bacteriophage or a virus, transformed by introduction of the chimeric gene according to the invention. Such transformation vectors according to the host organism to be transformed are well known in the art and widely described in the literature. For transformation of plant cells or plants, it will include a virus that can be used for the transformation of developed plants and further containing its own elements for replication and expression. Preferably, the vector for transforming plant cells or plants according to the invention is a plasmid.

According to another embodiment of the invention, the vector further comprises the chimeric gene according to the invention at least a second chimeric gene functional in the same host organism, comprising a nucleic acid sequence encoding an HPPD for addressing in a cellular compartment other than the cellular compartment in which the first chimeric HPPD gene is addressed. For example, if the first chimeric gene according to the invention allows addressing of the HPPD in the vacuole, the second chimeric gene may be another chimeric gene according to the invention enabling the addressing of the HPPD in the lumen of the thylakoid or thylakoid membrane, in the endoplasmic reticulum ,, cell wall or mitochondria, or a chimeric gene as described in the prior art permetant addressing HPPD either in the cytoplasm or chloroplast .

According to a preferred embodiment of the invention, the second chimeric gene allows addressing of the HPPD in chloroplast. It further comprises the regulatory elements defined above, allowing the expression of the coding sequence in a host organism, a nucleic acid sequence encoding a peptide fusion protein transit / HPPD.

The transit peptide makes it possible to address the chimeric HPPD in plastids, particularly chloroplasts, the fusion protein being cleaved between the transit peptide and the chimeric HPPD the passage of the membrane plastids. The transit peptide may be simple, such as an EPSPS transit peptide (described in US Patent 5,188,642) or a transit peptide of the small subunit of ribulose biscarboxylase / oxygenase (RuBisCO ssu) of a plant, optionally comprising a few amino acids of the N-terminal part of the ssu mature RuBisCO (EP 189 707) or a multiple transit peptide comprising a first plant transit peptide fused to a portion of the N-terminal sequence a mature protein is located in plastids, fused to a second plant transit peptide as described in EP 508 909, more particularly the optimized transit peptide comprising a transit peptide of the sunflower RuBisCO ssu fused to 22 amino acids the N-terminus of the RuBisCO ssu fused to the maize RuBisCO ssu transit peptide of maize as described with its coding sequence in EP 508 909.

Such chimeric genes are in particular described in patent applications WO 96/38567, WO 99/25842, WO 99/24585, WO 99/24586, WO 99/34005.

According to another embodiment of the invention, the vector further comprises the chimeric gene according to the invention and optionally a second chimeric gene encoding an HPPD, another chimeric gene encoding another gene of interest. It may be a gene encoding a selection marker such as a gene conferring on the transformed plant new agronomic properties, or a gene for improving the agronomic quality of the transformed plant. Selection markers include genes encoding selectable markers include antibiotic resistance genes, the genes for tolerance to herbicides (bialaphos, glyphosate or isoxazoles), genes encoding readily identifiable reporter enzymes as the enzyme GUS, genes encoding pigments or enzymes regulating the production of pigments in the transformed cells. Such selectable marker genes are described in patent applications EP 242 236, EP 242 246, GB 2197653, WO 91/02071, WO 95/06128, WO 96/38567 and WO 97/04103. Genes of interest

Among the genes conferring new agronomic properties to the transformed plants include genes conferring tolerance to certain herbicides, those conferring resistance to certain insects, those conferring tolerance to certain diseases, etc. Such genes are in particular described in patent applications WO 91/02071 and WO 95/06128.

herbicide tolerance Among the genes which impart tolerance to certain herbicides, mention may be made the Bar gene conferring tolerance to bialaphos, the gene encoding a suitable EPSPS imparting resistance to herbicides having EPSPS as target, such as glyphosate and its salts (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, FR 2736926), the gene encoding glyphosate oxidoreductase (US 5,463,175).

Among the genes encoding a suitable EPSPS imparting resistance to herbicides having EPSPS as target, are more particularly the gene encoding a plant EPSPS, in particular corn, having two mutations 102 and 106, described in patent application GB 2736926, referred to below EPSPS double mutant, or the gene encoding an EPSPS isolated from Agrobacterium described by sequence ID 2 and ID 3 of US patent 5,633,435, referred to below CP4.

In the case of genes encoding EPSPS, and more particularly for the above genes, the sequence encoding these enzymes is advantageously preceded by a sequence encoding a transit peptide, in particular for the transit peptide as defined above above. Resistance to Insects Among the proteins of interest imparting novel insect resistance properties are more particularly Bt proteins widely described in literature and well known to those skilled in the art. Others include proteins extracted from bacteria such Photorabdus (WO 97/17432 & WO 98/08932). Resistance to diseases Among the proteins or peptides of interest which confer novel properties of resistance to diseases may be mentioned include chitinases, glucanases and oxalate oxidase, all these proteins and their coding sequences being widely described in the literature, or the antibacterial peptides and / or antifungal agents, in particular peptides of less than 100 amino acids rich in cysteine ​​as thionines or of plant defensins, particularly the lyric peptides of all origins comprising one or more disulphide bridges between the cysteines and regions comprising basic amino acids, in particular the following lytic peptides: androctonin (WO 97/30082 and WO 99/09189), drosomycin (WO 99/02717) or thanatin (WO 99/24594).

According to a particular embodiment of the invention, the protein or peptide of interest is chosen from fungal elicitor peptides, in particular elicitins (Kamoun et al, 1993; Panabières & al, 1995). Changing the quality

One can also cite the genes modify the constitution of modified plants, especially the content and quality of certain essential fatty acids (EP 666 918) or the content and quality of proteins, in particular in the leaves and / or seeds said plants. Particular examples are the genes encoding proteins enriched in sulfur-containing amino acids (Korit, AA et al, Eur J. Biochem (1991) 195, 329-334;.. WO 98/20133; WO 97/41239; WO 95 / 31554; WO 94/20828; WO 92/14822). These proteins enriched in sulfur amino acids also have the function of trapping and storing cysteine ​​and / or excess methionine, avoiding potential toxicity problems associated with overproduction of these sulfur amino acids by trapping. Can also be made of genes encoding peptides rich in sulfur-containing amino acids and more particularly cysteine, said peptides also having antibacterial and / or antifungal activity. Are more particularly of plant defensins, as lytic peptides of any origin, in particular the following lytic peptides: androctonin the drosomicin or thanatin.

The invention also relates to a transforming host organisms method, in particular plant cells by integration of at least one nucleic acid sequence or a chimeric gene as defined above, which transformation may be obtained by any appropriate known means widely described in the literature and in particular the references cited in this application, especially by the vector of the invention.

A series of methods consists in bombarding cells, protoplasts or tissues with particles to which are attached the DNA sequences. Another series of methods consists in using as means of transfer into the plant a chimeric gene inserted into an Agrobacterium tumefaciens Ti plasmid or an Agrobacterium rhizogenes Ri. Other methods may be used such as microinjection or electroporation, or alternatively direct precipitation using PEG. The skilled person will choose the appropriate method depending on the nature of the host organism, in particular of the plant cell or plant.

The present invention also relates to host organisms, in particular plant cells or plants, transformed and containing a chimeric gene comprising a coding sequence for a fusion protein defined above. Preferably, the chimeric gene is stably integrated in the host organism genome.

According to a particular embodiment of the invention, the host organism, in particular the plant cell, further comprises the chimeric gene according to the invention at least a second chimeric gene functional in the same host organism, comprising a sequence of nucleic acid encoding an HPPD for addressing in a cellular compartment other than the cellular compartment in which the first chimeric HPPD gene is addressed as defined above.

According to another particular embodiment of the invention, the host organism, in particular the plant cell, further comprises the chimeric gene according to the invention and optionally a second chimeric gene encoding an HPPD, another gene chimera encoding another gene of interest such that edéfini above.

Different chimeric genes can be integrated into the genome of the host organism by transformation using a vector of the invention comprising different chimeric genes as defined above.

Different chimeric genes may also be stably integrated into the genome of the host organism by cotransformation using several vectors each comprising at least one chimeric gene to be integrated into the host organism genome.

The present invention also relates to the transformed plants into the genome of which at least one chimeric gene according to the invention is stably integrated. Plants according to the invention contain transformed cells as defined above, in particular the plants regenerated from the transformed cells above. The regeneration is obtained by any appropriate method which depends on the nature of the species, as for example described in the above references. For processes for transforming plant cells and of regenerating plants, we might mention the following patents and patent applications: 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 the transformed plants derived from the cultivation and / or crossing the above regenerated plants, as well as the seeds of transformed plants.

According to a particular embodiment of the invention, transformed plants further comprise the chimeric gene of the invention at least a second chimeric gene functional in the same host organism, comprising a nucleic acid sequence encoding an HPPD for addressing in a cellular compartment other than the cellular compartment in which the first chimeric HPPD gene is addressed as defined above.

According to another embodiment of the invention, transformed plants further comprise the chimeric gene of the invention, and optionally a second chimeric gene encoding an HPPD, another chimeric gene encoding another gene of interest such defined above.

The different chimeric genes in the transformed plants according to the invention can originate from either the same transformed parent plant, and in this case the plant is derived from a single transformation method / regeneration with the different chimeric genes in the same vector or by cotransformation using several vectors. It can also be obtained by crossing parent plants each containing at least one chimeric gene, one of the parent plants comprising at least one chimeric gene according to the invention.

Transformed plants obtainable according to the invention may be of the monocotyledonous type, such as for example cereals, sugar cane, rice and maize or dicotyledons such as tobacco, soybean, rapeseed, cotton, beets, clover, etc.

The invention also relates to a selective weeding of plants method, in particular of crops, using an HPPD inhibitor, in particular a herbicide defined previously, characterized in that this herbicide is applied to transformed plants according to the invention, both pre-plant, preemergence in post-emergence of the crop. The present invention also relates to a weed control method in an area of ​​a field comprising seeds or plants transformed with the chimeric gene of the invention, which method comprises applying to the said area of ​​the field a toxic dose the said weeds of an HPPD-inhibiting herbicide, without however substantially affecting the seeds or plants transformed with the said chimeric gene according to the invention.

The present invention also relates to a method for culturing transformed plants according to the invention with a chimeric gene according to the invention which method comprises sowing the seeds of the said transformed plants in an area of ​​a field suitable for growing said plants, applying to said area of ​​said field a dose toxic to weeds of a herbicide whose target is defined above in case of presence of weeds HPPD, without substantially affecting the said seeds or the said plants processed, then in harvesting the cultivated plants when they reach the desired maturity and optionally in separating the seeds from the harvested plants. In both the above methods, the application of the herbicide having HPPD as the target can be made according to the invention, both pre-plant, preemergence in post-emergence of the crop. By herbicide as defined in the present invention means a single herbicide active ingredient or in combination with an additive which modifies its effectiveness such as an agent increasing the activity (synergist) or limiting the activity (safener). The HPPD inhibitors herbicides are particularly defined before. Of course, for practical application, the above herbicides are combined so in known formulations of adjuvants commonly used in agrochemicals

When the transformed plant according to the invention comprises a further tolerance gene to another herbicide (such as a gene encoding a mutated EPSPS or not giving the plant tolerance to glyphosate), or when the transformed plant is naturally insensitive to another herbicide, the method according to the invention may comprise simultaneous or staggered application in time of a HPPD inhibitor in combination with said herbicide, for example glyphosate.

The invention also relates to the use of the chimeric gene encoding a fusion protein according to the invention as a marker gene in the cycle "transformation-regeneration" of a plant species and selection on the above herbicide.

When the chimeric gene according to the invention is combined in one vector to another chimeric gene encoding a protein of interest which confer novel agronomic properties other than herbicide tolerance gene (insect resistance, disease resistance, modification of quality), the application of the herbicidal HPPD inhibitor on the transformed plants and their progeny, selects in the field, plants derived from a cross between a parent plant comprising the two chimeric genes and a non-transformed plant who have kept the chimeric genes.

The various aspects of the invention will be better understood using the experimental examples below. All the methods or operations described below in these examples are given as examples and correspond to a choice made among the various methods available to achieve the same result. This choice does not affect the quality of the result and consequently any suitable method may be used by the skilled person to achieve the same result. Most engineering methods of DNA fragments are described in "Current Protocols in Molecular Biology" Volumes 1 and 2, Ausubel FM et al, published by Greene Publishing Associates and Wiley Interscience (1989) Molecular cloning or T .Maniatis, EFFritsch, J.Sambrook 1982. Example 1: Construction of different genes for expression in tobacco

A) Clone O: a derivative of pRP O described in WO 96/38567 containing an expression cassette of HPPD, double histone promoter - TEV - HPPD gene - nos terminator. To obtain the clone O, pRP O was digested with Sac I and Pvu II, the chimeric gene was purified and then ligated into pPZP 212 previously digested with Sac I and Sma I described in P. Hajdukiewicz et al Plant Molecular Biology 25: 989 -994 1994.

B) Clone Q: a derivative of pRP Q described in WO 96/38567 containing an expression cassette of HPPD, double histone promoter - TEV OTP - - HPPD gene - nos terminator. For the Q clone, pRP Q was digested with Sac I and Pvu II, the chimeric gene was purified and then ligated into pPZP 212 previously digested with Sac I and Sma I, pPZP 212.

C) Clone vacuole: the vacuole peptide used in the context of this work is described in the following article: FeruUo JM et al Plant Molecular Biology 33: 625-633, 1997

Vacuole transit peptide: This peptide was performed using two successive PCR.

The first is carried out in order to synthesize the vacuole transit peptide. The oligonucleotides chosen have the following sequence:

VAC 1: 5 'AAGATCAAGG ACAAAATTCA TGGTGAAGGT GCAGATGGTG AAAAGAAAAA

GAAGAAGGAG AAGAAGAAAC ATG 3 'VAC 2: 5' ATCACTGTCA CTGCTGCTGC TATCATGGCC ATGTTCATGA CCCTCTCCAT

GTTTCTTCTT CTCCTTCTTC 3 'These oligonucleotides hybridize to one another in the region represented in bold and thus make it possible to reconstitute the vacuole transit peptide having 123 base pairs. The reaction was performed in a PCR apparatus with the HYBAID PERKIN ELMER Taq polymerase in its buffer under standard conditions, that is to say for 50 .mu.l of reaction mixture are dNTPs to 200 .mu.m, Taq polymerase 2.5 units and 0 , 5 .mu.l of each oligonucleotide lOμM. One PCR cycle was performed, it consists of 5 min at 95 ° C and 1 min at 64 ° C then 10 min at 72 ° C.

The second PCR was used to add the restriction sites Xcm I and Sal I needed to cloning in pRP O. The oligonucleotides used have the sequences: MAV 3: 5 'ACGTCCAGGT GCGTCGTGGT GTATTGACCG CCGATAAGAT CAAGGACAAA ATTC 3' VAC 4: 5 'ACGTGTCGAC TTAATCACTG TCACTGCTGC TG 3 '

These oligonucleotides make it possible to obtain an amplified fragment of 169 base pairs. The reaction was performed in a PCR apparatus with the HYBAID PERKIN ELMER Taq polymerase in its buffer under standard conditions, that is to say for 50 .mu.l of reaction mixture are dNTPs to 200 .mu.m, Taq polymerase 2.5 units, 0 , 5 .mu.l of each oligonucleotide at lOμM and lμl of the first PCR. The amplification program used is 4 min at 95 ° C then 35 cycles <30 sec 95 ° C, 30 sec 66 ° C, 1 min 72 ° C> followed by 10 min at 72 ° C. n pRP O - vacuole: pRP derivative O containing an expression cassette of HPPD, double histone promoter - TEV - HPPD gene - vacuole transit peptide - nos terminator. To construct the plasmid pRP O was digested with Xcm I and Sal I and then ligated to the vacuole transit peptide previously purified and digested with Xcm I and Sal I. n - clone vacuole: a drift pRP O - vacuole containing a cassette expression of HPPD, double histone promoter - TEV - HPPD gene - vacuole transit peptide - nos terminator. To obtain the cloned vacuole, pRP O - vacuole was digested with Sac I and Pvu II, the chimeric gene was purified and then ligated into pPZP 212 previously digested with Sac I and Sma I.

Example 2: Expression of Pseudomonas fluorescens HPPD in tobacco

A) Tobacco Transformation "Petit havana" for obtaining plants: kanamycin selection.

The chimeric genes described above were transferred into tobacco "Petit havana" according to the transformation and regeneration procedures already described in European Application EP No. 0,508,909.

1) Transformation:

The vector is introduced into the non-oncogenic Agrobacterium tumefaciens EHA 105 strain.

2) Regeneration: The regeneration of the tobacco "Petit Havana" from foliar explants is carried out on a Murashig and Skoog medium (MS) comprising 30 g / l of sucrose and 350 mg / l cefotaxime and 200 mg / ml kanamycin. The leaf explants are removed from greenhouse plants and processed according to the foliar disc technique (Science 1985, Vol 227, pl229-1231) trais in successive stages:

- the first comprises the induction of shoots on an MS medium supplemented with 30g / l of sucrose containing 0.05 mg / l of naphthylacetic acid (ANA) and 2 mg / l of benzylaminopurine (BAP) for 15 days and 200 mg / ml kanamycin.

- The green shoots formed during this stage are then developed by culture on an MS medium supplemented with 30 g / l of sucrose and 200 mg / ml kanamycin, but containing no hormone, for 10 days.

- then removed the developed shoots and cultured on an MS rooting medium with half the content of salts, vitamins and sugars 200 mg / ml kanamycin and containing no hormone. After about 15 days, the rooted shoots are planted.

The tolerance of the transformed plants is studied by sowing on soil treated with isoxaflutole.

B) Tobacco Processing "Petit havana" for evaluation built in terms of tolerance during the transformation / regeneration phase: kanamycin selection and diketonitrile.

1) Processing is carried out as above. 2) Regeneration of tobacco "Petit Havana" from foliar explants is carried out on a Murashige and Skoog medium (MS) comprising 30 g / l of sucrose as well as

350mg / l cefotaxime and 200 mg / 1 kanamycin and varying doses of the 2-cyano-3- cyclopropyl-l- (2-methylsulfonyl-4-trifluoromethylphenyl) propan-l, 3-dione, 2.5 ppm,

3.125 ppm, 3.75 ppm. The leaf explants are removed from greenhouse plants and processed according to the foliar disc technique (Science 1985, Vol 227, pl229-1231) in two successive steps:

- the first comprises the induction of shoots on an MS medium supplemented with 30g / l of sucrose containing 0.05 mg / l of naphthylacetic acid (ANA) and 2 mg / l of benzylaminopurine (BAP) for 19 days and the varying doses kanamycin + herbicide.

- The second is a subculture of leaf discs (on which calli and shoots were formed) on an MS medium supplemented with 30 g / l of sucrose containing 0.01 mg / l of naphthylacetic acid (ANA) and 2 mg / l benzylaminopurine (BAP) for 3 days and kanamycin + the varying doses of herbicide; measuring tolerance is performed at that time.

3) Measurement of the tolerance-vitro seedlings herbicide. The experiments were performed per share of isoxaflutole (or more precisely matching the diketonitrile isoxafiutole) on shoots.

If during processing, the shoots appear this means that they have incorporated the gene for tolerance to kanamycin; if they have incorporated the gene for tolerance to kanamycin it also means that they have incorporated the gene for expression of HPPD dde Pseudomonas fluorescens and its final location in the cytoplasm, or the chloroplast or the vacuole. Indeed these two genes are linked or built on the T-DNA. If the shoots are white this means that they are from a cell that has incorporated the gene for tolerance to kanamycin (otherwise the growth does not appear) and HPPD tolerance gene but that is not enough to induce tolerance to the herbicide. The percentage of green plantlets with respect to the number of seedlings (white and green) is therefore a measure of the tolerance induced by the gene.

This experiment was performed using different concentrations of HPPD inhibitor, the RPA 202248 (which is the diketonitrile of isoxafiutole), 2.5 ppm, 3.125 ppm and 3.75 ppm. The counting was done by counting all white or green shoots and all the green shoots, results in the table below.

Tabulated summary of the tolerance induced by the different location HPPD

Location of HPPD% green shoots 2.5ppm 3,125ppm cytoplasm plastids 3.1 3.7 17 2 8.6 10.1 9 vacuole

These results confirm that the addressing of the HPPD in the vacuole allows to obtain a percentage of neighboring green shoots that observed for addressing in chloroplasts (especially 3.125 ppm), and in any case higher than the percentage obtained with a cytoplasmic targeting, although native HPPD is located in the cytoplsame.

Claims

1. A fusion protein consisting of a HPPD fused at its C- terminus or N-terminus to a protein signal sequence enabling the addressing of the HPPD in cellular compartments other than the cytoplasm or plastids.

2. A fusion protein according to claim 1, characterized in that the HPPD is an HPPD enzyme native, mutated or chimeric, having HPPD activity.

3. A fusion protein according to one of claims 1 or 2, characterized in that the HPPD is selected from bacteria such as Pseudomonas HPPD, plants like Arabidopsis or carrot, Coccicoides, or mammalian cells such as mouse or pig.

4. A fusion protein according to one of claims 1 to 3, characterized in that the HPPD is a Pseudomonas fluorescens HPPD.

5. A fusion protein according to one of claims 1 to 3, characterized in that the HPPD is an HPPD mutated in its C-terminal part.

6. A fusion protein according to claim 5, characterized in that the mutated HPPD comprises the mutation W336.

7. A fusion protein according to one of claims 1 to 4. characterized in that the HPPD is a chimeric HPPD comprising elements coming from different HPPD.

8. A fusion protein according to one of claims 1 to 7, characterized in that the protein sequence signal allows addressing of HPPD to the thylakoid lumen or thylakoid membrane, to the endoplasmic reticulum, to the vacuole the cell wall, mitochondria, the golgi apparatus, the nucleus, the nuclear membrane.

9. A fusion protein according to claim 8, characterized in that the protein signal sequence allows the addressing of the HPPD to the vacuolar compartment

(Vacuole).

10. A fusion protein according to claim 9, characterized in that the protein signal sequence is described by SEQ ID NO 1.

11. A fusion protein according to claim 10, characterized in that the protein signal sequence is fused to the C-terminal part of the HPPD.

12. A fusion protein according to one of claims 1 to 11, characterized in that it is described by the sequence identifier No. 4 (SEQ ID NO 4).

13. A nucleic acid sequence encoding the fusion protein according to one of claims 1 to 12.

14. A chimeric gene (or expression cassette) comprising a coding sequence as well as regulatory elements in the 5 'and 3' heterologous operable in a host organism, in particular plant cells or plants, the coding sequence comprising at least one nucleic acid sequence encoding a fusion protein according to claims 1 to 12.

15. Chimeric gene according to Claim 14, characterized in that the host organism is chosen from plant cells or plants.

16. A chimeric gene according to one of claims 14 or 15, characterized in that the nucleic acid sequence represented by SEQ ID NO 3.

17. A cloning vector and / or expression vector for transforming a host organism containing at least one chimeric gene according to claims 14 to 16.

18. Vector according to claim 17, characterized in that it further comprises the chimeric gene according to one of claims 14 to 16 at least a second chimeric gene functional in the same host organism, comprising a nucleic acid sequence encoding an HPPD for addressing in a cellular compartment other than the cellular compartment in which the first chimeric HPPD gene is addressed.

19. Vector according to one of claims 17 or 18, characterized in that the second chimeric gene allows addressing of the HPPD in chloroplast comprising a nucleic acid sequence encoding a fusion protein transit peptide / HPPD.

20. Vector according to one of claims 17 to 19, characterized in that it further comprises the chimeric gene according to one of claims 14 to 16, and optionally a second chimeric gene encoding an HPPD, another gene chimera encoding another gene of interest.

21. A host organism transformed characterized in that they contain a chimeric gene according to one of claims 14 to 16.

22. Host organism according to Claim 21, characterized in that it further comprises the chimeric gene according to one of claims 14 to 16 at least a second chimeric gene functional in the same host organism, comprising a nucleic acid sequence encoding an HPPD for addressing in a cellular compartment other than the cellular compartment in which the first chimeric HPPD gene is addressed.

23. Host organism according to Claim 22, characterized in that the second chimeric gene allows addressing of the HPPD in chloroplast comprising a nucleic acid sequence encoding a peptide fusion protein transit / HPPD.

24. A host organism according to one of claims 21 to 23, characterized in that it further comprises the chimeric gene according to one of claims 14 to 16, and optionally a second chimeric gene encoding an HPPD, another chimeric gene encoding another gene of interest.

25. A host organism according to one of claims 21 to 24, characterized in that it is a plant cell.

26. Host organism according to Claim 25, characterized in that the plant cells are chosen from plant cells of monocots such as the cereals, sugar cane, rice and maize or dicotyledons such as tobacco, soybean, rapeseed, cotton, beets, clover.

27. Plants transformed, * into the genome of which at least one chimeric gene according to one of Claims 14 to 16 is stably integrated.

28. Plant according to claim 27, characterized in that it contains transformed cells according to one of Claims 25 or 26.

29. Plant according to one of claims 27 or 28, characterized in that it is regenerated from the transformed cells according to one of Claims 25 or 26.

30. A transformed plant according to claim 28, characterized in that it is derived from the cultivation and / or crossing the regenerated plants according to claim 29.

31. A transformed plant according to one of claims 27 to 30, characterized in that it further comprises the chimeric gene according to one of claims 14 to 16, at least one second functional chimeric gene comprising a nucleic acid sequence encoding an HPPD for addressing in a cellular compartment other than the cellular compartment in which the first chimeric HPPD gene is addressed.

32. A transformed plant according to claim 31, characterized in that the second chimeric gene allows addressing of the HPPD in chloroplast comprising a sequence of nucleic acid encoding a peptide fusion protein transit / HPPD.

33. A transformed plant according to one of claims 27 to 32, characterized in that it further comprises the chimeric gene according to one of claims 14 to 16, and optionally a second chimeric gene encoding an HPPD, another chimeric gene encoding another gene of interest.

34. A transformed plant according to one of claims 27 to 33, characterized in that it is of monocots such as the cereals, sugar cane, rice and maize or dicotyledons such as tobacco, soybean, rapeseed , cotton, beets, clover.

35. Seeds of transformed plants according to one of claims 27 to 34.

36. A method for selective weeding of plants, in particular of crops, using an HPPD inhibitor, characterized in that this herbicide is applied to transformed plants according to one of claims 27 to 34, as preplant, preemergence in emergent culture.

37. weed control method in an area of ​​a field comprising seeds or plants transformed with the chimeric gene according to one of claims 27 to 35, which process comprises applying to the said area of ​​the field a toxic dose for said weeds an HPPD-inhibiting herbicide, without substantially affecting the seeds or plants transformed.

38. A method of cultivating the transformed plants according to one of claims 27 to 34, which method comprises sowing the seeds of the said transformed plants according to claim 35 in a surface of a field suitable for growing said plants, applying on said area of ​​said field a dose toxic to weeds of a herbicide having HPPD as the target in case of presence of weeds without substantially affecting the said seeds or the said transformed plants, then in harvesting the cultivated plants when they reach the desired maturity and optionally in separating the seeds from the harvested plants.