WO2004055191A1 - Expression of hydroxyphenylpyruvate dioxygenase in plastids of plants for herbicide tolerance - Google Patents

Abstract

The invention relates to transplastomic plants with enhanced tolerance for herbicides based on inhibitors of hydroxyphenylpyruvate dioxygenase (HPPD), a method for generating such plants, and the vectors used.

Description

EXPRESSION OF HYDROXYPHENYLPYRUVATE DIOXYGENASE IN PLASTIDS OF PLANTS FOR HERBICIDE TOLERANCE

This invention relates to transplastomic plants with enhanced tolerance for herbicides based on inhibitors of hydroxyphenylpyruvate dioxygenase (HPPD) , methods for generating such plants and the vectors used. The invention also covers a method of treating transplastomic plants in which a HPPD inhibitor is applied to clear weeds . Enzymes belonging to the HPPD family catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is converted into homogentisate, a reaction which requires the presence of iron (Fe²⁺) and oxygen (Crouch N. P. et al., Tetrahedron 1997 53, 20: 6993 - 7010). In this invention, the term HPPD is intended to include all wild-type, mutated and chimeric forms of the enzyme which are active. Many different forms of HPPD have been described in the literature, notably those in bacteria such as Pseudomonas (Rϋetschi et al., Eur. J. Biochem. 1992 205: 459 - 466, WO 96/38567), those of plants such as Arabidopsis (WO 96/38567, Genebank AF047834) and carrot (WO 96/38567, Genebank 87257), those of fungi such as Coccidioides (Genebank COITRP) , and those of mammals such as the human, the mouse and the pig- In this invention, the term "mutated HPPD" refers to any form of HPPD which is mutated in such a way that it is more tolerant of herbicides based on HPPD inhibitors than the corresponding parent wild-type HPPD. Advantageously, the mutated HPPD has been modified at the C-terminal as described in Patent Application WO 99/24585. Also advantageously, the mutated HPPD contains the W336 mutation as described in Patent Application WO 99/24585.

The term "chimeric HPPD" refers to any form of HPPD which is made up of parts of HPPDs of different origins, notably the chimeric HPPDs described in Patent Application WO 99/24586.

Advantageously, the HPPD is derived from Pseudomonas fluorescens (WO 96/38567).

A number of compounds are known to be able to inhibit this enzyme, all of which bind the enzyme to inhibit the conversion of HPP to homogentisate. Some of these compounds have been used as herbicides which cause first bleaching of the leaves of treated plants, and then their death (Pallet K. E. et al . , 1997 Pestic. Sci. 50: 83 - 84) . Among the herbicides which target HPPD described in the background art, there are notably: the isoxazoles (EP 418 185, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, US 5 424 276), in particular isoxaflutole (IFT) which is a corn-selective herbicide; the diketonitriles (DKT) (EP 496 630, EP 496 631), in particular 2-cyano-3-cyclopropyl-l- (2-S0₂ CH3-4-CF₃ Cl₂ phenyl) propane 1,3-dione and 2-cyano-3-cyclopropyl-l- (2- S0₂ CH3-4-2-3 Cl₂ phenyl) propane 1,3-dione; the triketones (EP 625 505, EP 625 508, US 5 506 195), in particular sulcotrione or mesotrione; and the pyrazolinates .

There are three main types of strategy for enhancing a plant's tolerance for herbicides (1): making it possible for the plant to detoxify the herbicide by providing it with an enzyme which can convert the herbicide (i.e. the parent compound and/or its active metabolites) into non-toxic breakdown products, e.g. the enzyes which confer tolerance for bromonoxynil or basta (EP 242 236, EP 337 899); by mutating the target enzyme so that it is more resistant to the herbicide (i.e. the parent compound and/or its active metabolites) but still functional, e.g. the enzymes which confer resistance to glyphosphate (EP 293 356, Padgette S. R. et al . , J. Biol. Chem. 1991 266:33, FR2 736 926); or by over-expressing the wild-type target enzyme so that it is produced in sufficient quantity to maintain a normal rate of catalytic activity despite the presence of its inhibitor (i.e. the herbicide).

It is this third strategy which has been described for the generation of plants which are resistant to HPPD inhibitors (WO 96/38567), constituting the first instance in which simple over-expression of a target enzyme was successfully used to render a plant resistant to the kind of concentration of herbicide encountered in an agricultural context. An agricultural level of tolerance for HPPD inhibitors was similarly obtained using chimeric forms of HPPD (WO 99/24586), and an even higher level of resistance was obtained with forms of HPPD carrying mutations in their terminal domains (WO 99/24586) .

Although HPPD is a cytoplasmic enzyme (Garcia I. et al., Biochem. J. 1997 325: 761 - 769), it has nevertheless been shown that the resistance conferred is greater if the exogenous HPPD is concentrated in plastids, specifically the chloroplasts . Thus, HPPD can be trafficked to chloroplasts while expressed from a construct in which an appropriate signal sequence (OTP, EP 508 909) is included before the coding region of the Pseudomonas fluorescens hppd gene which is in turn followed by an Agrobacterium tumefaciens terminator (WO 96/38567) .

Various expression systems have been described as suitable for enhancing the resistance of transfected plants to herbicides, and these include diverse regulatory elements, notably so-called "light-dependent" promoters (WO 99/25842) and, for monocotyledonous species, a regulatory sequence including the H3C4 histone promoter from corn and the first intron from rice actin (WO 99/34005) .

The results show on the one hand that homogentisate can be imported from the extracellular environment inside the cell where it is needed to compensate for the inhibition of endogenous HPPD and on the other that genetically modified plants with high levels of exogenous HPPD in plastids or in the cytoplasm are resistant to herbicides whose mode of action is based on the inhibition of HPPD.

Another way of generating plants which express HPPD in their plastids is direct transfection of the plastids themselves. In practice, direct plastid transfection is associated with a number of advantages, among which specific mention could be made of the following:

- The new sequence is incorporated by double homologous recombination into one or more of the copies of the circular plastid genome (or "plastome") present in every cell. The advantage is that the insertion point in the plastome can be precisely controlled by placing the transgene in the middle of the selected plastid sequence in the transfection vector. This fine targeting means that the "position effects" often seen in nuclear transgenesis are avoided. - Each cell ends up with many copies of the transgene. Depending on its maturity, a leaf cell can contain up to 10,000 copies of a small (120 to 160 kilobases) circular genome containing a significant proportion of highly repetitive DNA. In consequence, engineered plant cells may contain up to 20,000 copies of a gene of interest.

This makes marked over-expression possible: the products of transgenes can account for over 40% of all soluble protein inside a modified cell (De Cosa et al., 2001) .

- Plastid transfection has the other advantage that it reduces the environmental dissemination of transgenes since plastid-encoded traits are not transmitted via pollen thereby precluding the possibility of genetic contamination of wild-type plants.

Methods for transfecting plastids are clearly described in the following documents: US Patents 5 451 513, 5 545 817, 5 545 818 and 5 576 198; International Patent Applications WO 95/16783 and WO 97 / 32977; and the article of McBride et al. (1994). Plastid transfection using high velocity microprojectiles was first performed in the single-celled alga Chlamydomonas reinhardtii (Boynton et al., 1988) and the approach was later repeated in tobacco (Svab et al., 1990) .

The classic way of transfecting plastids involves bombarding leaves with microprojectiles carrying DNA molecules (Svab et al., 1993; US Patent Ser. ^■ N° 6 376 744) .

Today, stable plastid transfection is only routinely performed in the tobacco species N. tabacum (Svab and Maliga, 1990; Svab et al . , 1993) although there has been some progress recently in rice (Khan and Maliga, 1999) ,

Arabidopsis thaliana (Sikdar et al . , 1998), potato

(Sidorov et al., 1999), colza (Chaudhari et al., 1999) and tomato (Ruf et al . , 2001). Fertile transplastomic tobacco, tomato and potato plants have all been obtained.

Direct plastid transfection has been exploited for various ends, including tolerance for herbicide, resistance to insects, and for the production of large quantities of a protein. In tobacco, the overexpression of plastome-encoded genes conferring tolerance for herbicides such as glyphosphate (Daniell, 1998; WO 99/10513; Ye et al., 2000; WO 01/04331; WO 01/04327) and phosphinothricin (Basta) (Lutz et al., 2001) has been effectively used to enhance resistance to the relevant herbicides. Other applications have yielded transplastomic plants that are resistant to insects or which overproduce proteins of therapeutic value (McBride et al., 1995; US Patent 5 451 513; Staub et al., 2000; WO 99/10513) .

However, none of the work described in the background art has ever demonstrated that, for genes conferring herbicide tolerance, overexpression from the plastome gives a greater degree of resistance than nuclear overexpression. Document WO 01/04327 describes a method for transfecting plastids and also concerns transplastomic plants which express a gene conferring tolerance for glyphosphate. The results presented in this document indicate that herbicide resistance is comparable whether the transgene is nuclear or plastomic.

All the above-mentioned literature references are incorporated by reference in this Patent Application, in particular those references which stipulate the DNA sequences encoding wild-type, chimeric and mutated forms of HPPD, including any associated signal or transit peptides . DESCRIPTION OF DRAWINGS

Drawing 1: map of plasmid pCLTlll Drawing 2 : map of plasmid pCLT129 Drawing 3 : map of plasmid pCH43 DETAILED DESCRIPTION OF THE INVENTION The subject of this invention is a chimeric gene including at least one component chimeric gene which contains, linked to one another in a functional fashion in the direction of transcription, a promoter sequence which is active in plastids, a heterologous sequence encoding a hydroxyphenylpyruvate dioxygenase (HPPD) , and a terminator which is active in the plastids of plant cells .

Preferably, the HPPD sequence is selected from the group of wild-type, chimeric and mutated forms of HPPD. More preferably still, the sequence encoding the HPPD comes from Pseudomonas fluorescens .

The invention also covers a chimeric gene including at least one component chimeric gene including a HPPD- coding sequence, together with at least one other component chimeric gene which includes, linked to one another in an operational fashion in the direction . of transcription, a promoter sequence which is active in plastids, a sequence encoding a selective marker, and a terminator which is active in the plastids of plant cells.

The phrase "linked to . one another in an operational fashion" means that the specified elements of the component chimeric gene are linked to one another in such a way that they function as a unit to allow expression of the coding sequence. By way of example, a promoter is said to be linked to a coding sequence in an operational fashion if it is capable of promoting the expression of said coding sequence. A chimeric gene according to the invention can be assembled from the various components using techniques which are familiar to those skilled in the art, notably methods such as those described in Sambrook et al. (1989, Molecular Cloning, A Labora tory Manual, Nolan C, ed. , New York: Cold Spring Harbor Laboratory Press) . Exactly which regulatory elements are to be included in the chimeric gene will depend on the plant and the type of plastid in which they are to work: those skilled in the art are able to select which regulatory elements are going to work in a given plant.

Among the promoters that are active in plastids of plant cells, by way of example, special mention can be made of the promoter of the psbA gene which encodes the Dl polypeptide of PSII (Staub et al. 1993 EMBO Journal 12(2): 601 - 606), and the constitutive Prrn promoter which regulates the ribosomal RNA operon (Staub et al. 1992 Plant Cell 4: 39 - 45). As a general rule, any promoter resulting from a plastomic plant gene or a bacterial gene will work, and those skilled in the art will know which of the available promoters to select in order to obtain the desired mode of expression (i.e. either inducible or constitutive) . According to this invention, a preferred promoter is the Prrn promoter of tobacco which is associated with a 5' part of the 5' untranslated sequence of the rbcL gene (Svab et al. 1993 Proc. Natl. Acad. Sci. 90: 913 - 917). More preferably still, the promoter is the light- dependent promoter of the psbA gene which encodes the Dl polypeptide of PSII (Staub et al . 1993 EMBO Journal 12(2) : 601 - 606) . The selective marker is used to select for transfected plastids and cells, i.e. those that have incorporated the chimeric gene(s) into their plastome, and it also makes it possible to obtain fertile, homoplastic transplastomic plants. Among the genes that can be used as selective markers, by way of example, special mention can be made of two chimeric genes, namely the aadA gene which codes for an aminoglycoside 3"- adenyltransferase that confers resistance to spectinomycin and streptomycin (Svab et al . , 1993), and the neo gene which codes for a neomycin phosphotransferase (Carrer et al., 1993) that confers resistance to kanamycin. Other suitable candidate selective markers include genes that confer resistance to betain aldehyde such as the gene that codes for betain aldehyde dehydrogenase (Daniell et al., 2001), and also genes that confer herbicide tolerance such as the bar gene (White et al., 1990, Nucleic Acid Res. 18 [4]: 1062) which confers tolerance for bialaphos, and the EPSPS gene

(US 5 188 642) which confers resistance to glyphosate. Alternatively, reporter genes can be used, i.e. genes that code for readily identified enzymes such as GUS (β- glucuronidase) (Staub et al., 1993) or the Green Fluorescent Protein (GFP, Sidorov et al, 199) , genes coding for pigments, or for enzymes that regulate pigment production. Such genes are described in Patent Applications WO 91/02071, WO 95/06128, WO 96/38567, WO 97/04130 and WO 01/64023. Preferably, the gene coding for the selective marker is the aadA gene which codes for an aminoglycoside 3"- adenyltransferase that confers resistance to spectinomycin and streptomycin (Svab et al., 1993). Among the terminators which are active in plant cell plastids, by way of example, special mention could be made of the terminators of the psbA gene, the rbcL gene (which codes for the large sub-unit of rubisco) , and the rpsl β gene (which codes for a tobacco ribosomal protein) (Shinozaki et al . , 1986; Staub et al., 1993).

The invention also relates to a transformation vector adapted for the transformation of plant plastids, characterized in that it contains two sequences for homologous recombination (corresponding to sequences of the relevant plastome) flanking at least one chimeric gene according to the invention.

These sequences—one upstream (LHRS) and the other downstream (RHRS) of the component chimeric gene (s)—make double homologous recombination possible within an intergenic region of the plastome, comprising the contiguous regions LHRS and RHRS.

Preferably, the two homologous recombination sequences according to the invention are contiguous so that the chimeric gene is inserted at a non-coding (intergenic) sequence of the plastome. In a particular embodiment, this sequence is part of the operon of the plastid's ribosomal RNA. In another particular embodiment, the non-coding sequence includes the 3' end of the rbcL gene (which codes for the large sub-unit of rubisco) , with the other homologous sequence including the 5" end of the accD gene (which codes for one of the sub-units of acetyl-CoA carboxylase) . And more particularly still, the non-coding sequence including the 3' end of the rbcL gene (which codes for the large sub- unit of rubisco) corresponds to nucleotides 57755 through

59297 of the plastome of N. tabacum, var. Petit Havana, with the other homologous sequence including the 5 ' end of the accD gene (which codes for one of the sub-units of acetyl-CoA carboxylase) corresponding to nucleotides

59298 through 60526 of the same plastome.

This invention also relates to a method of generating transplastomic plants with enhanced tolerance for HPPD inhibitors, said method including the following steps:

- transformation of cells with a vector according to the invention, - selection of cells carrying transformed plastids,

- regeneration of plants from the transformed cells. According to the invention, what is meant by

"transplastomic" is plants that have assimilated a stable, functional chimeric gene in the plastome of their plastids, in particular their chloroplasts. The plastoma corresponds to the extranuclear genome, i.e. that found in intracellular organelles other than the nucleus, in particular, the chloroplasts.

The cells may be transformed using any method suitable for plant cells. Among the methods that could be used to generate transformed cells according to the invention, one possibility involves exposing the cells or tissues to be transformed to the transformation vector in the presence of polyethylene glycol (PEG) (Chang and Cohen 1979, Mol. Gen. Genet. 168 [1]: 111 - 115; Mercenier and Chassy, 1988, Biochimie 70 [4]: 503 - 517). Electroporation is another method which involves subjecting the cells or tissues to be transfected to an electric field in the presence of the vector (Andreason and- Evans, 1988, Biotechniques 6[7]: 650 - 666; Shigekawa and Dowe, 1989, Aust. J. Biotechnol. 3[1]: 56 - 62). Another possibility is based on micro-injecting the vectors directly into the cells or tissues (Gordon and Ruddle, 1985, Gene 33 [2]: 121 - 136). Plant cells and tissues can also be transformed using bacteria of the genus Agrobacterium, preferably by infection of said cells or tissues by either Agrobacterium tumefaciens (Knopf, 1979, Subcell Biochem. 6: 143 - 173; Shaw et al., 1983, Gene 23 [3]: 315 - 330) or Agrobacterium rhizogenes (Bevan and Chilton, 1982, Annu. Rev. Genet. 16: 357 - 384; Tepfer and Casse-Delbart, 1987, Microbiol. Sci. 4 [1] : 24 - 28) which have been genetically modified so that their T-DNA is specifically trafficked to the plastids. Preferably, the plant cells or tissues are transformed by means of Agrobacterium tumefaciens using the protocol described by Ishida et al . (1996, Mat. Biotechnol. 14 [6]: 745 - 750).

According to a preferred embodiment of the invention, the particle bombardment transformation method based on bombarding the target material with high- velocity particles will be used. In this method, the vector is adsorbed onto the surface of the particles with which the target tissues are bombarded (Bruce et al., 1989, Proc. Natl. Acad. Sci USA 86[24]: 9692 - 9696; Klein et al., 1992, Biotechnology 10 [3]: 296 - 289; US Patent N° 4 945 050) . Transformed cells can be selected using an HPPD inhibitor and/or another selection marker; the products used for the selection step can be applied either together or separately. Preferably, selection is carried out using a combination of an HPPD inhibitor together with streptomycin and/or spectinomycin.

The method of obtaining transplastomic plants which are tolerant to HPPD inhibitors according to the invention may include an extra selection step performed on the regenerated plants.

The invention also covers a transplastomic plant cell which is tolerant to herbicides whose mode of action is based on the inhibition of HPPD, characterized in that it comprises a chimeric gene that includes at least one sequence coding for HPPD and also, possibly, a heterologous sequence coding for a selective marker.

The invention also concerns a transplastomic plant which is tolerant to herbicides whose mode of action is based on the inhibition of HPPD, characterized in that it is regenerated from cells which have been transformed in the way described above.

The invention also covers a transplastomic plant which is tolerant to herbicides whose mode of action is based on the inhibition of HPPD, carrying a chimeric gene that includes at least one sequence coding for HPPD.

Another subject of the invention is a transplastomic plant which is tolerant to herbicides whose mode of action is based on the inhibition of HPPD according to the invention, carrying a chimeric gene that includes a sequence coding for HPPD as well as a sequence coding for a selective marker.

Heteroplastic transplastomic plants which are tolerant to herbicides whose mode of action is based on the inhibition of HPPD constitute one aspect of the invention. The word "heteroplastic" intends to describe a plant into which at least one cell contains a variety of different plastome profiles inside its plastids or a plant containing different populations of cells which contain the same plastome profile, wherein the populations are characterized by different types of plastomes .

Homplastic transplastomic plants which are tolerant to herbicides whose mode of action is based on the inhibition of HPPD constitute another aspect of the invention. What the word "homoplastic" means is that all the cells contain exactly the same kind of plastome and only that plastome. According to the invention, transplastomic plants are homoplastic when all their cells contain only copies of the transfected plastome without any copies of the non-transfected plastome. This status is generally achieved by selecting for copies of the plastome into which the chimeric gene is incorporated, notably by virtue of the association between said chimeric gene with a gene coding for a selective marker. Plastomes in which the marker is not incorporated are selected out when the transfected tissue is exposed to the relevant selective agent.

Transplastomic plants which are tolerant to herbicides targeting HPPD according to the invention may be monocotyledonous such as, for example, cereals such as wheat, sugar cane, rice and corn; alternatively, they may be dicotyledonous such as tobacco, soy, colza, cotton, beetroot, clover, etc.

The invention also covers parts of transplastomic plants and the progeny of these plants. What "part" means is any organ of the plant, be it above or below the ground. Organs that are above the ground are the stalk, the leaves and the flowers, the last including the male and female reproductive organs . Organs that are below the ground are mainly the roots but in some cases they may be tubers. By "progeny", what is mainly referred to is the seeds containing the embryos resulting from the reproductive process as it proceeds between these plants. By extension, the word "progeny" also applies to any seed formed in any of the successive generations following a cross in which at least one of the parents was a plant transfected according to the invention. Progeny may also be generated by the asexual reproduction of said transfected plants. The seeds according to the invention may be coated with an agrochemical formulation containing at least one active substance with one of the following activities: fungicide, herbicide, insecticide, nematocide, bactericide or virucide.

The invention also concerns a method for selective weed-killing based on the use of an HPPD inhibitor as above specified, on and around plants, notably cultivated plants, characterized in that the herbicide is applied to the area in which the transplastomic plants according to the invention are to be planted, are planted or were planted, prior to sowing, during growth and after harvest . This invention also concerns a method for controlling the growth of weeds in fields containing seeds or plants transfected with the chimeric gene acording to the invention, which method involves applying to the surface of said field a quantity of HPPD- inhibiting herbicide which is toxic for said weeds without significantly compromising the growth of the transplastomic seeds or plants according to the invention.

This invention also concerns a method for cultivating plants transfected according to the invention with a chimeric gene according to the invention, which method involves sowing seeds of said transfected plants over the surface of a field suitable for the cultivation of said plants, then applying to said surface of the field a quantity of HPPD-inhibiting herbicide which is toxic for said weeds without significantly compromising the growth of said transfected seeds or plants, and then harvesting the plants once they have reached the desired degree of maturity, after which the seeds may be recovered from the harvested plants. By "without significantly compromising the growth of the transplastomic seeds or plants", what is meant according to the invention is that the plants containing the expression system according to the invention, when exposed to a dose of herbicide which is toxic to weeds, experience mild or null phytotoxicity. By "a quantity of HPPD-inhibiting herbicide which is toxic for said weeds", what is meant according to the invention is that the dose of the herbicide applied is sufficient to kill the weeds. By "mild phytotoxicity", what is meant according to the invention is that the percentage of leaves bleached remains below 25%, preferably below 10% and more preferably still below 5%. It is also understood according to this invention that application of the same dose of herbicide to a plant which has not been transfected (i.e. not carrying the expression system according to the invention) but which is otherwise comparable would induce in said plant a greater degree of phytotoxicity than that observed in its transfected homolog (i.e. one which is carrying the expression system according to the invention) .

In both of the above-mentioned methods, application of the herbicide whose mode of action is based on the inhibition of HPPD can be carried out according to the invention before sowing, during growth and after harvest.

By "herbicide" in the sense of this invention, what is meant is a substance which is an active herbicide when applied either on its own or in association with an additive which modulates its activity; said additive could for example either enhance the herbicidal activity (synergistic action) or restrict it (a safener) . HPPD- inhibiting herbicides are specifically listed above. Of course, for practical usage, the above-mentioned herbicides are routinely combined with additives in formulations which are in widespread use in agriculture.

The HPPD-inhibiting herbicide can be selected from among the following: the isoxazoles, in particular isoxaflutole; the diketonitriles, in particular 2-cyano- 3-cyclopropyl-l- (2-S0₂ CH3-4-CF₃ phenyl) propane 1,3-dione and 2-cyano-3-cyclopropyl-l- (2-S0₂ CH3-4-2-3 Cl₂ phenyl) propane 1,3-dione; the triketones, in particular sulcotrione or mesotrione; and the pyrazolinates . The techniques of molecular biology are described in Ausubel (Ed.) Current Protocols in Molecular Biology, John Wiley and Sons Inc. (1994), and in Maniatis T, Fritsch E F, and Sambrook J, Molecular Cloning, A Labora tory Manual , Cold Spring Harbor Laboratory Press, New York (1989) . PCR amplification was carried out in a Perkin Elmer GeneAmp 9600 PCR machine. The amplification reactions for each sample involved 30 cycles with the following steps: denaturation at 94 °C for one minute; hybridization for 45 seconds at a temperature of 50 to 60°C (depending on the primers being used); and synthesis for one to two minutes (depending on the length of the sequence being synthesized) at 72°C. After 30 cycles, the temperature is lowered to 4°C and PCR products are purified by agarose gel electrophoresis .

The positions of the various DNA fragments derived from the Nicotiana tabacum plastome are indicated in accordance with the numbering system proposed by Shinozki et al (1986) and used at Genebank (Access N°: Z00044).

The invention is specifically illustrated by the following examples which are not in any way limiting. Example 1 : construction of vectors pCLTlll and pCLT!29 , designed to help genera te transplastomic plants which over-express HPPD

The two plasmids pCLTlll and pCLT129 both contain two contiguous, component genes, namely aadA and hppd, which are flanked by two DNA fragments, namely RHRS (Right Homologous Recombination Sequence) and LHRS (Left Homologous Recombination Sequence) , which mediate recombination of the chimeric gene into the plastid genome. In both of these plasmids, the LHRS (nucleotides 57755 through 59297 of the plastome of N. tabacum var. Petit Havana corresponds to the 3' end of the rbcL gene (which codes for the large sub-unit of rubisco) , and the RHRS (nucleotides 59298 through 60526 of the plastome of N. tabacum var. Petit Havana) corresponds to the 5' end of the accD gene (which codes for one of the sub-units of acetyl-CoA carboxylase) .

The aadA chimeric component gene ("AADA-111" SEQ ID Ν° 1) in the plasmid pCLTlll (derived from Stratagene plasmid pBSllSK) is a constructed chimeric gene made up of (going from the 5' end towards the 3' end): the promoter of the ribosomal RNA operon { "Prrn" : nucleotides 102,561 through 102,677 of the plastome of N. tabacum) ; a part of the 5 ' region of the rbcL gene which is transcribed but not translated ("5'ri->c-L": nucleotides 57,569 through 57,584 of the plastome of N. tabacum) ; the coding sequence of the aadA gene, the product of which confers resistance to streptomycin and spectinomycin (Svab and Maliga, 1993) ; and the terminator of the psbA gene ( " 3 'psbA" : nucleotides 146 through 533 of the plastome of N. tabacum var. Petit Havana) . The hppd chimeric component gene in the plasmid pCLTlll ("HPPD- 111, SEQ ID Ν° 5) is a constructed chimeric gene made up of (going from the 5' end towards the 3' end): the promoter of the psbA gene { " PpsbA" : nucleotides 1,596 through 1,819 of the plastome of N. tabacum var. PBD6) ; the coding sequence of the Pseudomonas fluorescens hppd gene (SEQ ID Ν° 1 in Patent Application WO 98/02562); and the terminator of the rbcL gene (" 3 ' rbcL" : nucleotides 59,036 through 59,246 of the plastome of N. tabacum var. PBD6) . Both chimeric component genes are inserted in the same direction with the hppd gene 5 ' to the aadA gene so that the hppd gene is flanked by two identical, directly repeating 3 ' rbcL sequences (from the LHRS and in the HPPD-111 sequence) (Drawing 1) .

The aadA chimeric component gene ("AADA-129" SEQ ID Ν° 3) in the plasmid pCLT129 (derived from Stratagene plasmid pBSllSK) is a constructed chimeric gene made up of (going from the 5' end towards the 3' end): the Prrn promoter; a part of the 5' region of the rbcL gene which is transcribed but not translated ( " 5 'rbcL" : nucleotides 57,569 through 57,584 of the plastome of N. tabacum) ; the coding sequence of the aadA gene; and the 3 'rbcL terminator.

The hppd chimeric component gene in the plasmid pCLT129 ("HPPD-129, SEQ ID Ν° 7) is a constructed chimeric gene made up of (going from the 5' end towards the 3' end) : the Prrn promoter; a part of the 5' region of the rbcL gene which is transcribed but not translated { " 5 'rbcL" : nucleotides 57,569 through 57,584 of the plastome of N. tajacum) ; the coding sequence of the Pseudomonas fluorescens hppd gene (the sequence of which is the same as that of the HPPD-111 chimeric component gene); and the 3 'psbA terminator. Both chimeric component genes are inserted in the same direction with the AADA- 129 sequence 5' to the HPPD-129 sequence so that the aadA gene is flanked by two nearly-identical 3 ' rbcL-Prrn sequences (the one differing from the other only at the junction between the junction between the 3 ' rbcL and Prrn sequences,) (Drawing 2). Example 2 : microprojectile-mediated transfection of the tobacco plastome

Νictoiana tabacum var. 'PBD6' plants were cultivated in sterile conditions on MS medium (Murashige T and Skoog F, 1962) (Sigma M-5519 at a concentration of 4.4 grams per liter) supplemented with sucrose (30 g/1) . The lower surface of leaves measuring 3 to 5 centimeters were bombarded using a gun built in the laboratory following the model described by Finer et al. (1992), the Particle Inflow Gun. DΝA (pCLTlll or pCLT129: 5 μg per shot) was adsorbed onto particles made of tungsten (M17 particles with a diameter of 1 μm) in the presence of CaCl₂ (0.8 to 1.0 M) and spermidine (14 to 16 πiM) . With their lower surfaces down, the thus treated leaves were then placed on MS medium supplemented with α-naphthalene acetic acid (ANA from Sigma: 0.05 mg/1) + 6-benzylaminopurine (BAP from Sigma: 2 mg/1) + sucrose (30 g/1) + phytagar (7 g/1) (MS [0.05-2] medium). Two to three days after bombardment, the treated leaves were cut up into squares with sides of 5 mm in length and placed—still lower surface down—on MS [0.05-2] medium containing spectinomycin hydrochloride (500 mg/1) . Some cultures also contained various concentrations of DKN. Calluses and shoots which were resistant to spectinomycin (and/or DKN) were regenerated on the same medium and then rooted in a medium containing MS at half-strength + sucrose (15 g/1) + spectinomycin (500 mg/1) (± DNK at a range of concentrations) to generate TO plants (corresponding to the first regeneration cycle referred to as Rl) . In order to promote homoplasty, a second cycle of regeneration was performed on MS [0.05-2] medium containing spectinomycin

(500 mg/1) and DKN (1 ppm) . The regenerated shoots (referred to as R2) were rooted and the resultant plants (TO) later transferred to a greenhouse. The first generation of seeds is referred to as the TI generation.

Two independent series of bombardment procedures were carried out on tobacco leaves with pCLTlll, respectively CLTlll-1 in which selection was performed using only spectinomycin (500 mg/1) , and CLT111-2 in which a mixture of both spectinomycin (300 mg/1) and DKN

(0.5 ppm) was used for selection.

Example 3 : identification of transplastomic lines CLT111 and CLT129

Screening the plants which were phenotypically resistant to spectinomycin or a combination of spectinomycin and DKN for transplastomic lines was achieved by means of the PCR based on a pair of specific primers capable of detecting expression cassettes which had been recombined into the plastome. This involved one of the primers recognizing a sequence of the wild-type plastid genome close to the recombination point, and the other recognizing a sequence unique to one of the transgenes (hppd or aadA) . The primers used were: ORBCL52 (5 ' atgtcaccacaaacagagactaaagc-3 ' ) (SEQ ID N° 9) and RHPP1 (5'-gagccgatcttcgagatca-3' ) (SEQ ID N° 10) which hybridize with the plastome respectively in the rbcL gene (nucleotides 57,595 to 57,620, outside of the LHRS) and in the hppd gene. The generation of a 2026 bp amplicon indicates a CLT111 transplastomic recombination, and one of 3106 bp is indicative of CLT129 recombination. A total of 15% of the calluses which regenerated on spectinomycin following the CLTlll-1 transfection cycle proved to be transplastomic; and after the second transfection cycle, CLT111-2, 28% of those which grew on a doubly selective medium containing spectinomycin + DKN were recombinant. We concluded that the double selection system combining both an antibiotic and a herbicide, compared with a system based on antibiotic alone, increased the frequency of plastome transfection and reduced the number of false positives.

The transplastomic nature of the various transfection events was confirmed by Southern Blot analysis of the DNA from the relevant plants and their progeny. DNA from the leaves of tobacco plants resulting from transfection with either pCLTlll or pCLT129 was extracted and digested with restriction enzymes {Nco I and Hind III), together with the DNA of wild-type tobacco for the purposes of comparison.

Apart from Line 4 of cycle CLTlll-1, all the lines analyzed presented the expected profile: an HPPD probe detected a band at 1298 bp in CLTIII lines, and at 6074 bp in the CLT129 lines, without any band detected in a non-transfected control. A LHRS probe detected a band at 6429 bp (corresponding to the wild-type plastome) in the non-transfected control; in the tranfected lines, the same probe detected a principal band at 2085 bp in CLTIII lines and one at 1863 bp in CTL129 lines, both of these being characteristic of insertion of the transgene between the RHRS and the LHRS. Nevertheless, the consistent presence of a secondary band at 6429 bp indicates that a fraction of the plastomes do not contain an integrated transgene and that none of the lines are perfectly homoplastic.

That the transgenes are transmitted down through the generations was checked by testing for the AADA marker by sowing on spectinomycin-containing media. Fully 100% of the seeds sown were found to be resistant to the antibiotic. Southern Blot analysis of the DNA of descendants revealed a profile that was in all respects identical to that of the originally regenerated plants. In particular, the progeny were similarly heteroplastic without any major change in the ratio of the numbers of copies of wild-type and transfected plastomes (as estimated by the relative intensity of the bands at 2085 and 6429 bp for CLTIII events, and of those at 1863 and 6429 bp for CLT129 events) . The phenotypic uniformity of these plants, all of which were highly resistant to spectinomycin, also confirms that there is little difference between the various lines in terms of the level of expression of the gene. This is in contrast to what is usually seen with nuclear transfection, in which case "position effects" are common. Example 4 : tolerance of transplastomic lines to a HPPD- inhibi ting herbicide

About five hundred TI seeds from CLTlll-1 or CLT111- 2 transfection events were sown on half-strength MS germination medium supplemented with a range of concentrations of DKN (1, 5, 10, 20 and 40 ppm) . Wild- type seeds and seeds from Line pCH43-4 were sown in parallel on the same medium in order to compare the transplastomic recombinants with nuclear recombinants and wild-type plants for resistance to DKN. The pCH43-4 line was generated by nuclear transfection with a chimeric gene made up of: a CsVMV promoter (WO 97/48819) defined in this Patent Application as SEQ ID N° 11; a signal peptide (OTP) (EP 508 909); the coding sequence of a hppd gene (SEQ ID N° 1 of Patent Application WO 96/38567); and the nos terminator of Agrobacterium tumefaciens. This construct was incorporated in the vector pCH43D (Drawing 3) . HPPD synthesized in the cytosol is eventually trafficked into the plastids by virtue of the signal peptide. pCH43-4 recombinants are resistant to the kind of levels of isoxaflutole (5-cycloprpylisoxazole-4-yl mesyl-4- trifluopromethylphenyl ketone or IFT) encountered in the agricultural context.

All plants from these seeds grew normally at doses of up to 10 ppm 2-cyclopropyl-3-2-mesyl-4- trifluoromethylphenyl-3oxopropanenitrile (DKN) . However, at this concentration, pCH43-4 nuclear recombinants showed significant signs of phytotoxicity; in contrast, some of the CLTIII lines grew perfectly normally in the presence of a DKN concentration of 40 ppm.

Experiments to test the tolerance of emerging plants to IFT were carried out in the greenhouse context. About 500 TI seeds from CLTIII recombinants were sown in vermiculite trays and then sprayed with an IFT solution (for a dose of 200 g/Ha) . In these conditions, the pCH43- 4 control showed significant signs of phytotoxicity whereas the transplastomic recombinants appeared perfectly normal.

Example 5 : comparison of the efficiency of the Prrn and PpsbA promoters

In order to investigate the efficiency of the promoter sequence upstream of the hppd gene, and its effect on the degree of tolerance for herbicide in the transplastomic plants, the following experiment was carried out.

Fragments of leaves from CLTIII and CLT129 recombinants were placed on a regeneration medium containing 1 ppm DKN. At this concentration, all the shoots growing out of the CLTIII recombinant were green, unlike those growing out of the CLT129 recombinant, not all of which are tolerant to DKN (some of the shoots were green while some from the same recombinant were white) .

The conclusion reached was that using a chloroplast- specific promoter, or a promoter that is _. both chloroplast-specific and light-inducible such as PpsbA, is particularly suitable for generating plants that are tolerant to HPPD-inhibiting herbicides. REFERENCES

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Claims

1. Chimeric gene including at least one chimeric component gene which contains, linked to one another in a functional fashion in the direction of transcription:

- a promoter sequence which is active in plastids,

- a heterologous sequence encoding a hydroxyphenylpyruvate dioxygenase (HPPD) , - a terminator which is active in the plastids of plant cells.

2. Chimeric gene according to Claim 1,

^• characterized in that the promoter sequence results in constitutive expression of the gene.

3. Chimeric gene according to Claim 1, characterized in that the promoter sequence results in expression of the gene in photosynthetic tissue.

4. Chimeric gene according to Claim 3, characterized in that the promoter sequence is the PpsbA promoter.

5. Chimeric gene according to Claims 1 to 4, characterized in that the sequence coding for HDDP is selected from among the group of wild-type, mutated or chimeric hppd genes .

6. Chimeric gene according to Claims 1 to 4, characterized in that the sequence coding for HDDP is derived from the genus Pseudomonas .

7. Chimeric gene according to Claims 1 to 4, characterized in that the sequence coding for HDDP is derived from Pseudomonas fluorescens .

8. Chimeric gene according to Claims 1 to 4, characterized in that the sequence coding for HDDP is derived from the genus Synechocystis .

9. Chimeric gene according to Claims 1 to 4, characterized in that the sequence coding for HDDP is taken from a plant.

10. Chimeric gene according to Claim 9, characterized in that the sequence coding for HDDP is derived from the genus Arabidopsis .

11. Chimeric gene according to Claim 9, characterized in that the sequence coding for HDDP is derived from an umbel.

12. Chimeric gene according to Claim 12, characterized in that the sequence coding for HDDP is derived from Daucus carotta .

13. Chimeric gene according to Claim 12, characterized in that the sequence coding for HDDP is derived from Zea mais .

14. Chimeric gene according to any of Claims 1 to 13 including in addition a second component chimeric gene which includes, linked to one another in an operational fashion in the direction of transcription:

- a promoter sequence which is active in plastids, - a sequence encoding a selective marker,

- a terminator which is active in the plastids of plant cells.

15. Chimeric gene according to Claim 14, characterized in that the promoter sequence of the second chimeric gene results in constitutive expression of the gene .

16. Chimeric gene according to Claim 15, characterized in that the promoter sequence is the Prrn promoter.

17. Chimeric gene according to Claim 14, characterized in that the coding sequences codes for aminoglycoside 3"-adenyltransferase.

18. Vector designed for the transformation of plant plastids, characterized in that it contains at least two sequences that are homologous to sequences in the plastome of the plant to be transformed, said homologous sequences flanking at least one chimeric gene according to any of Claims 1 to 13.

19. Vector designed for the transformation of plant plastids, characterized in that it contains at least two sequences that are homologous to sequences in the plastome of the plant to be transfected, said homologous sequences flanking at least one chimeric gene according to any of Claims 14 to 17.

20. Vector according to either of Claims 18 or 19, characterized in that the two sequences that are homologous to sequences in the plastome of the plant to be transfected correspond to contiguous sequences so that the chimeric gene is recombined into a non-coding region of the plastome.

21. Vector according to Claim 20, characterized in that said region corresponds to the plastomic ribosomal

RNA operon.

22. Vector according to Claim 20, characterized in that one of the two homologous sequences includes the 3' end of the rbcL gene which codes for the large sub-unit of rubisco, with the other homologous sequence including the 5' end of the accD gene which codes for one of the sub-units of acetyl-CoA carboxylase.

23. Vector according to Claim 22, characterized in that one of the two homologous sequences includes the 3 ' end of the rbcL gene (which codes for the large sub-unit of rubisco) corresponding to nucleotides 57755 through 59297 of the plastome of N. tabacum, cv. Petit Havana, with the other homologous sequence including the 5' end of the accD gene and corresponding to nucleotides 59298 through 60526 of the same plastome.

24. Method for generating transplastomic plants, including at least the following steps:

- transformation of plant cells with a vector designed for the transfection of plastids, - selection of cells carrying transformed plastids,

- regeneration of plants from the transformed cells .

25. Method for generating transplastomic plants according to Claim 24, characterized in that transformation of the cells is achieved by a method based on high-velocity microprojectiles bombardment (biolistics) .

26. Method for generating transplastomic plants according to Claim 24, characterized in that the vector designed for the transformation of plastids is a vector according to any of Claims 18, 20 or 23.

27. Method for generating transplastomic plants according to Claim 24, characterized in that the vector designed for the transformation of plastids is a vector according to any of Claims 19 to 23.

28. Method for generating transplastomic plants according to any of Claims 24 to 27, characterized in that the transformed plants are selected using a HPPD inhibitor.

29. Method for generating transplastomic plants according to Claim 27, characterized in that the transfected plants are selected using a HPPD inhibitor coupled with another selective agent.

30. Method for generating transplastomic plants according to Claim 29, in which a HPPD inhibitor and spectinomycin are applied at the same time.

31. Method for generating transplastomic plants according to Claim 29, in which a HPPD inhibitor and streptomycin are applied at the same time.

32. Method for generating transplastomic plants according to Claim 29, in which a HPPD inhibitor, streptomycin and spectinomycin are all applied at the same time.

33. Method for generating transplastomic plants according to Claim 27, including an extra selection step carried out on regenerated plants.

34. Method for generating transplastomic plants according to Claim 33, characterized in that the extra selection step involves exposure to a HPPD inhibitor.

35. Transplastomic plant cell tolerant to herbicides which target HPPD, characterized in that it contains at least one chimeric gene according to either of Claims 1 or 14.

36. Transplastomic plant tolerant to herbicides which target HPPD, characterized in that it is regenerated from cells transfected according to Claim 35.

37. Transplastomic plant tolerant to herbicides which target HPPD, carrying a chimeric gene according to Claim 1.

38. Transplastomic plant tolerant to herbicides which target HPPD, carrying a chimeric gene according to

Claim 14.

39. Transplastomic plant tolerant to herbicides which target HPPD, according to any of Claims 36 to 38, characterized in that the plant is heteroplastic.

40. Transplastomic plant tolerant to herbicides which target HPPD, according to any of Claims 36 to 38, characterized in that the plant is homoplastic.

41. Transplastomic plant tolerant to herbicides which target HPPD, according to any of Claims 36 to 40, characterized in that it is monocotyledonous such as a cereal, sugar cane, rice and corn, or dicotyledonous such as tobacco, soy, colza, cotton, beetroot and clover.

42. Seed of a transplastomic plant tolerant to herbicides which target HPPD, according to any of Claims 36 to 41.

43. Method for selective weeding using a HPPD- inhibiting herbicide, characterized in that the herbicide is applied to plants according to any of Claims 36 to 41.

44. Method for controlling the growth of weeds in a field in which seeds according to Claim 42 have been sown, or in which plants according to any of Claims 36 to 41 are growing, characterized in that it involves applying to the surface of said field a quantity of HPPD- inhibiting herbicide which is toxic for said weeds without significantly compromising the growth of the seeds or plants which have been transfected with the chimeric gene according to the invention.

45. Method for growing transplastomic plants plants according to any of Claims 36 to 41, characterized in that it involves sowing seeds of said transfected plants over the surface of a field suitable for the cultivation of said plants, then applying to said surface of said field a quantity of HPPD-inhibiting herbicide which is toxic for said weeds if said weeds are present, without significantly compromising the growth of said transfected seeds or plants, and then harvesting the plants once they have reached the desired degree of maturity, after which the seeds may be recovered from the harvested plants.

46. Method according to either of Claims 44 or 45, characterized in that the herbicide is applied prior to the sowing of the seeds, during growth and/or after harvest of the crop.

47. Method according to any of Claims 43 to 46, characterized in that the HPPD-inhibiting herbicide is selected from among the following: the isoxazoles, in particular isoxaflutole; the diketonitriles, in particular 2-cyano-3-cyclopropyl-l- (2-S0₂ CH3-4-CF₃ phenyl) propane 1,3-dione and 2-cyano-3-cyclopropyl-l- (2- S0₂ CH3-4-2-3 Cl₂ phenyl) propane 1,3-dione; the triketones, in particular sulcotrione or mesotrione; and the pyrazolinates .