AU760662B2 - DNA sequence of a gene of hydroxy-phenyl pyruvate dioxygenase and production of plants containing a gene of hydroxy-phenyl pyruvate dioxygenase and which are tolerant to certain herbicides - Google Patents

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AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANT: Rhone-Poulenc Agro ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys Level 3 303 Coronation Drive Milton, QLD, 4064 INVENTION TITLE: DNA sequence of a gene of hydroxy-phenyl pyruvate dioxygenase and production of plants containing a gene of hydroxy-phenyl pyruvate dioxygenase and which are tolerant to certain herbicides The following statement is a full description of this invention, including the best method of performing it known to me/us: Q:\OPER\VPA\2302734D.215 2/8/2000 1A DNA sequence of a hydroxyphenylpyruvate dioxygenase gene and obtainment of plants comprising a hydroxyphenylpyruvate dioxygenase gene, which are tolerant to certain herbicides The present invention relates to a hydroxyphenylpyruvate dioxygenase (HPPD) gene, a chimeric gene comprising this gene as coding sequence and its use to obtain plants resistant to certain herbicides.

Certain herbicides have been disclosed, such as the isoxazoles described especially in the French Patent Applications 95 06800 and 95 13570 and especially isoxaflutole, a selective maize herbicide, diketonitriles such as those described in European 15 Applications 0 496 630, 0 496 631, in particular 2-cyano-3-cyclopropyl-l- 2

-SO

2

CH

3 -4-CF 3 -phenyl)propane- S1,3-dione and 2-cyano-3-cyclopropyl-l-(2-SO 2

CH

3 -4- 2,3Cl 2 phenyl)propane-1,3-dione, triketones described in European Applications 0 625 505 and 0 625 508, in particular sulcotrione. However, a tolerance gene to such herbicides has not been described.

Hydroxyphenylpyruvate dioxygenase is an enzyme which catalyses the conversion reaction of parahydroxyphenylpyruvate into homogentisate.

In addition, the amino-acid sequence of hydroxyphenylpyruvate dioxygenase from Pseudomonas sp.

P.J. 874 has been described, without there being a 2 description of its role in the tolerance of the plants to herbicides (Rietschi et al.: Eur. J. Biochem. 205, 459-466, 1992). This document does not give a description of the gene coding for this protein.

There have now been discovered the sequence of a gene of this type and that such a sequence could, once incorporated into plant cells, produce an overexpression or an activation of HPPD in the plants giving to the latter a worthwhile tolerance to certain novel herbicides, such as those of the isoxazoles family or that of the triketones.

An object of the present invention is an isolated DNA sequence of a gene of plant origin which encodes a hydroxyphenylpyruvate dioxygenase (HPPD) and which hybridizes with the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

More particularly, this sequence can originate from monocotyledonous or dicotyledonous plants, especially of Arabidopsis or of Umbelliferae, such as, for example, the carrot (Daucus carotta). It can be native or wild or possibly mutated while at the same time fundamentally retaining a property of herbicidal tolerance against HPPD inhibitors, such as herbicides of the isoxazoles family or that of the triketones.

S.

S

3 The invention likewise relates to a process of isolating the above gene, characterized in that: as primers, some oligonucleotides from the amino-acid sequence of an HPPD are chosen, starting from these primers, amplification fragments are synthesized by PCR the gene is isolated by creation and screening of a genomic bank and the gene is cloned.

Preferably, primers from the HPPD sequence of a bacterium of the genus Pseudomonas are used.

Particularly preferably, they are from Pseudomonas fluorescens.

The invention also relates to the use of a 15 gene coding for HPPD in a process for the transformation of plants, as a marker gene or as a coding sequence allowing tolerance to certain herbicides to be conferred on the plant. It can likewise be used, in association with other marker 20 genes and/or coding sequence, for an agronomic property.

The coding gene can be of any origin, native or wild or possibly mutated, while at the same time fundamentally retaining a property of herbicidal tolerance against inhibitors of HPPD, such as herbicides of the isoxazoles family or that of the triketones. As coding sequence, especially that according to the invention such as described above, can 4 be used.

The transformation of plant cells can be achieved by any appropriate known means. A series of methods consists in bombarding cells or protoplasts with particles to which are coupled the DNA sequences.

Another series of methods consists in using, as means of transfer into the plant, a chimeric gene inserted into a Ti plasmid of Agrobacterium tumefaciens or Ri plasmid of Agrobacterium rhizogenes.

An object of the present invention is also a chimeric gene for the genetic transformation of plants comprising, in the transcription direction, at least one promoter regulation sequence that is operable in plants, a heterologous coding sequence which encodes hydroxyphenylpyruvate dioxygenase (HPPD) and which hybridizes with the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3 and at least one terminator or polyadenylation regulation sequence.

The promoter regulation sequence used can be 20 any promoter sequence of a gene which is naturally expressed in plants, in particular a promoter of bacterial, viral or plant origin, such as, for example, 0* that of a gene of the small subunit of ribulose biscarboxylase (RuBisCO) or that of a gene of a-tubulin (European Application EP No. 0 652 286), or alternatively of a plant virus gene such as, for example, that of o cauliflower mosaic virus (CAMV 19S or 35S), but any suitable promoter can be used. Preferably, recourse is made to a promoter regulation sequence which favours the 30 overexpression of the coding sequence, such as, for example, that comprising at least one histone promoter such as described in European Application EP 0507698.

According to the invention, it is equally possible to use, in association with the promoter regulation sequence, other regulation sequences which are situated between the promoter and the coding sequence, such as "enhancer" transcription activators, such as, for example, tobacco etch virus (TEV) translation activator described in the Application W087/07644, or of transit peptides, either single, or double, and in this case possibly separated by an intermediate sequence, that is to say comprising, in the transcription direction, a sequence coding for a transit peptide of a plant gene coding for a plastid 15 localization enzyme, a part of the sequence of the N-terminal mature part of a plant gene coding for a plastid localization enzyme, then a sequence coding for a second transit peptide of a plant gene coding for a plastid localization enzyme, formed by a part of the 20 sequence of the N-terminal mature part of a plant gene coding for a plastid localization enzyme, such as o- described in European Application No. 0 508 909.

It is possible to use as terminator or polyadenylation regulation sequence any corresponding sequence of bacterial origin, such as, for example, the nos terminator of Agrobacterium tumefaciens, or even of plant origin, such as, for example, a histone terminator such as described in European Application EP No. 0 633 317.

An object of the present invention is also plant cells, of monocotyledonous or dicotyledonous plants, especially of crops, transformed according to one of the processes described above and comprising in their genome an efficacious quantity of a gene expressing hydroxyphenylpyruvate dioxygenase (HPPD). The invention also provides a plant regenerated from such transformed plant cells. It has been observed that plants transformed in this way have a significant tolerance to certain novel herbicides such as the isoxazoles described especially in French Patent Applications 9506800 and 95 13570 and especially of 4-[4-CF3-2-(methylsulphonyl)benzoyl]-5cyclopropylisoxazole, and especially isoxaflutole, a selective maize herbicide, the diketonitriles such as those described in European Applications 0 496 630, 0 496 631, in particular 2-cyano-3-cyclopropyl-l-(2-

SO

2

CH

3 -4-CF 3 -phenyl)propane-l,3-dione and 2-cyano-3- 20 cyclopropyl-1-(2-S0 2

CH

3 -4-2,3-Cl 2 -phenyl)propane-1,3dione, the triketones described in European Applications 0 625 505 and 0 625 508, in particular sulcotrione.

Finally, an object of the invention is a method of weeding plants, especially crops, with the aid of a herbicide of this type, characterized in that S. this herbicide is applied to plants transformed according to the invention, both pre-sowing, preemergence and post-emergence of the crop.

1: An object of the invention is also the use of the HPPD gene as a marker gene in the course of the "transformation-regeneration" cycle of a plant species and selection on the above herbicide.

The different aspects of the invention will be better understood with the aid of the experimental examples below.

Example 1: Isolation of the HPPD gene of P. fluorescens A32 Starting from the amino-acid sequence of HPPD of Pseudomonas sp. P.J. 874 (published by Rietschi U.

et al., 1992, Eur. J. Biochem. 205: 459-466), the sequence of different oligonucleotides is deduced in order to amplify by.PCR a part of the coding sequence of HPPD of P. fluorescens A 32 (isolated by McKellar, 15 R.C. 1982, J. Appl. Bacteriol., 53: 305-316). An amplification fragment of the gene of this HPPD has been used to screen a partial genomic bank of fluorescens A32 and thus to isolate the gene coding for this enzyme.

20 A) Preparation of genomic DNA of P. fluorescens A32 The bacteria was cultivated in 40 ml of M63 minimum medium (KH 2

PO

4 13.6 g/l, (NH 4 2

SO

4 2 g/l, MgSO 4 0.2 g/l, FeSO 4 0.005 g/l, pH-7 plus L-tyrosine 10 mM as the sole carbon source) at 28°C for 48 hours.

After washing, the cells are taken up in 1 ml of lysis buffer (100 mM tris HC1, pH 8.3, 1.4 M NaCl and 10 mM EDTA) and incubated for 10 minutes at 65 0

C.

8 After a phenol/chloroform treatment (24:1) and a chloroform treatment, the nucleic acids are precipitated by addition of one volume of isopropanol, then taken up in 300 Al of sterile water and treated with final 10 Ag/ml RNAse. The DNA is treated afresh with phenol/chloroform, chloroform and reprecipitated by addition of 1/10 of the volume of 3 M sodium acetate, pH 5 and 2 volumes of ethanol. The DNA is then taken up in sterile water and determined.

B) Choice of the oligonucleotides and syntheses Starting from the amino-acid sequence of HPPD of Pseudomonas sp. P.J. 874, five oligonucleotides are chosen, two directed in the terminal NH 2 direction of the protein towards the COOH terminal of the protein and three directed in the opposite direction (see Fig. The choice was dictated by the two following rules: a stable 3' end of the 20 oligonucleotide, that is to say at least two bases without ambiguity.

the smallest degeneracy possible.

The oligonucleotides chosen have the following sequences: P1: 5'TA(C/T)GA(G/A)AA(C/T)CCIATGGG3' P2: 5'GA(G/A)ACIGGICCIATGGA3' P3: 5'AA(C/T)TGCATIA(G/A)(G/A)AA(C/T)TC(C/T)TC3' P4: 5'AAIGCIAC(G/A)TG(C/T)TG(T/G/A)ATICC3' 5'GC(C/T)TT(A/G)AA(A/G)TTICC(C/T)TCIC3' They were synthesized on a Cyclone plus DNA synthesizer of the make MILLIPORE.

With these five oligonucleotides, the amplification fragments which must be obtained theoretically by PCR starting from the sequence SEQ ID No. 1 have the following sizes: with the primers P1 and P3 r r r r r approximately 690 bp with the primers approximately 720 bp with the primers approximately 1000 bp with the primers approximately 390 bp with the primers approximately 420 bp with the primers approximately 700 bp C) Amplification P. fluorescens A32.

P1 and P4 P1 and P5 P2 and P3 P2 and P4 P2 and P5 of a coding part of HPPD of 9 The amplifications were carried out on a PERKIN ELMER 9600 PCR apparatus and with PERKIN ELMER Taq polymerase with its buffer under standard conditions, that is to say for 50 il of reaction mixture there are dNTP at 200 jiM, primers at 20 pM, units of Taq polymerase and 2.5 .g of DNA of P. fluorescens A32.

The amplification programme used is 5 min at and then 35 <45 sec 95°C, 45 sec 49°C, 1 min 720C> cycles followed by 5 min at 72 0

C.

Under these conditions, all the amplification fragments obtained have a size compatible with the theoretical sizes given above, which is a good indication of the specificity of the amplifications.

The amplification fragments obtained with the sets of primers P1/P4, P1/P5 and P2/P4 are ligated into pBSII after digestion of this plasmid by Eco RV and treatment with the terminal transferase in the presence of ddTTP as described in HOLTON T.A. and GRAHAM M.W. 1991, Vol. 19, No. 5, p. 1156.

A clone of each of the three types is 15 partially sequenced; this allows it to be confirmed that a part of the coding region of the HPPD of P. fluorescens A32 has indeed been amplified in the three cases. The P1/P4 fragment is retained as probe in order to screen a partial genomic bank of 20 P. fluorescens A32 and to isolate the complete gene of the HPPD.

D) Isolation of the gene By Southern it is shown that a 7 Kbp fragment hybridizes, after digestion of the DNA of P.

fluorescens A32 by the restriction enzyme BamHI, with the probe HPPD P1/P4. 400 g of DNA of P. fluorescens A32 are thus digested with the restriction enzyme BamHI and the DNA fragments making up approximately 7 Kbp are 11 purified on agarose gel.

These fragments are ligated into pBSII the latter is digested with BamHI and dephosphorylated by treatment with alkaline phosphatase. After transformation in E. coli DHlOb, the partial genomic bank is screened with the probe HPPD P1/P4.

A positive clone was isolated and called pRP A. Its simplified map is given in Figure 2. On this map is indicated the position of the coding part of the HPPD gene. It is composed of 1077 nucleotides which code for 358 amino acids (see SEQ ID No. The HPPD of P. fluorescens A32 has a good amino-acid homology with that of Pseudomonas sp. strain P.J. 874, in fact there is 92 agreement between these two proteins (see Fig. 3).

Example 2: Construction of two chimeric genes To confer plant tolerance to herbicides inhibiting HPPD, two chimeric genes are constructed: The first consists in putting the coding part 20 of the HPPD gene of P. fluorescens A32 under the control of the double histone promoter (European Patent No. 0 507 698) followed by tobacco etch virus translational enhancer (TEV) (pRTL-GUS (Carrington and Freed, 1990; J. Virol. 64: 1590-1597)) with the terminator of the nopaline synthase gene. The HPPD will then be localized in the cytoplasm.

The second will be identical to the first, except that the optimized transit peptide (OTP) is 12 intercalated between the TEV transcription activator and the coding part of the HPPD (European Application EP No. 0 508 909). The HPPD will then be localized in the chloroplast.

A) Construction of the vector pRPA-RD-153: pRDA-RD-11 A derivative of pBS-II SK(-) (Stratagene catalog #212206) containing the polyadenylation site of nopaline synthase (NOS polyA) (European Application EP No. 0 652 286) is cloned between the KpnI and Sall sites. The KpmI site is transformed into a NotI site by treatment with T4 DNA polymerase I in the presence of 150 pM of 3 deoxynucleotide triphosphates and then ligation with an NotI linker (Stratagene catalog #1029). An NOS polyA 15 cloning cassette is thus obtained.

pRPA-RD-127: A derivative of pRPA-BL-466 (European Application EP No. 0 337 899) cloned in 33 pRPA-RD-11 creating an expression cassette of the oxy gene and containing the promoter of the small subunit 20 of ribulose biscarboxylase: "promoter (SSU) oxy gene NOS polyA" To create this plasmid, pRPA-BL-488 was digested with XbaI and HindIII to isolate a fragment of 1.9 kbp comprising the SSU promoter and the oxy gene which was ligated into the plasmid pRPA-RD-11, digested with compatible enzymes.

pRPA-RD-132: This is a derivative of pRPA- BL-488 (European Application EP No. 0 507 698) cloned into pRPA-RD-127 with creation of an expression cassette of the oxy gene with the double histone promoter: "double histone promoter oxy gene NOS polyA" To produce this plasmid, pRPA-BL-466 is digested with HindIII, treated with Klenow and then redigested with NcoI. The purified fragment of 1.35 kbp containing the histone double promoter H3A748 is ligated with the plasmid pRPA-RD-127 which had been digested with XbaI, treated with Klenow and redigested with NcoI.

pRPA-RD-153: This is a derivative of pRPA-RD-132 containing the translation activator of the tobacco etch virus (TEV). pRTL-GUS (Carrington and o 15 Freed, 1990; J. Virol. 64: 1590-1597) is digested with Ncol and EcoRI and the 150 bp fragment is ligated into pRPA-RD-132 digested with the same enzymes. An expression cassette containing the promoter "double histone promoter TEV oxy gene NOS polyA" S 20 is thus created.

B) Construction of the vector pRPA-RD-185: pUC19/GECA: A derivative of pUC-19 (Gibco catalog #15364-011) containing numerous cloning sites.

pUC-19 is digested with EcoRI and ligated with the oligonucleotide linker 1: Linker 1: AATTGGGCCA GTCAGGCCGT TTAAACCCTA GGGGGCCCG CCCGGT CAGTCCGGCA AATTTGGGAT CCCCCGGGC TTAA The selected clone contains an EcoRI site followed by the polylinker which contains the following sites: EcoRI, Apal, AvrII, PmeI, Sfil, SacI, KpnI, Smal, BamHI, XbaI, Sall, PstI, SphI and HindIII.

pRPA-RD-185: this is a derivative of pUC19/GECA containing a modified polylinker. pUC19/GECA is digested with HindIII and ligated with the oligonucleotide linker 2: Linker 2: AGCTTTTAAT TAAGGCGCGC CCTCGAGCCT GGTTCAGGG AAATTA ATTCCGCGCG GGAGCTCGGA CCAAGTCCC TCGA The selected clone contains a HindIII site in the centre of the polylinker which now contains the following sites: EcoRI, Apal, AvrII, Pmel, Sfil, SacI, KpnI, Smal, BamHI, XbaI, Sall, PstI, SphI, HindIII, PacI, AscI, XhoI and EcoNI.

15 C) Construction of the vector pRP T: pRP 0: a derivative of pRPA-RD-153 containing an expression cassette of HPPD, double histone promoter TEV HPPD gene terminator Nos. To produce pRP O, pRPA-RD153 is digested with HindIII, 20 treated with Klenow and then redigested with NcoI to remove the oxy gene and replace it by the HPPD gene coming from the pRP A plasmid by BstEII digestion, Klenow treatment and redigestion with NcoI.

pRP R: to obtain the plasmid, pRP 0 was digested with PvuII and SacI, the chimeric gene was purified and then ligated into pRPA-RD-185 and the latter was digested with PvuII and SacI.

pRP T: was obtained by ligation of the chimeric gene coming from pRP R after digestion with SacI and HindIII in the plasmid pRPA-BL 150 alpha2 digested with the same enzymes (European Application EP No. 0 508 909).

The chimeric gene of the pRP T vector thus has the following structure: Double histone TEV Coding region nos promoter of HPPD terminator D) Construction of the pRP V vector pRP P: this is a derivative of pRPA-RD-7 (European Application EP No. 0 652 286) containing the optimized transit peptide followed by the HPPD gene. It p.

was obtained by ligation of the coding part of HPPD coming from pRP A by BstEII and NcoI digestion, Klenow treatment and from the plasmid pRPA-RD-7, the latter digested with SphI and AccI and treated with DNAse polymerase T4.

pRP Q: a derivative of pRPA-RD-153 e*0* containing an expression cassette of HPPD, double histone promoter TEV OTP HPPD gene Nos e..

terminator. To construct it, the plasmid pRPA-RD-153 is digested with SalI, treated with Klenow and then redigested with NcoI to remove the oxy gene and replace it by the HPPD gene released from the pRP P plasmid by BstEII digestion, Klenow treatment and redigestion with NcoI.

pRP S: to obtain it, the plasmid pRP Q was digested with PvuII and SacI to release the chimeric gene, which was ligated into pRPA-RD-185, the latter digested with PvuII and SacI.

pRP V: it was obtained by ligation of the chimeric gene released from pRP S, after digestion with SacI and HindIII, into the plasmid pRPA-BL 150 alpha2 (European Application EP No. 0 508 909).

The chimeric gene of the pRP Q vector thus has the following structure: Double histone TEV OTP Coding region nos promoter of HPPD terminator Example 3: Transformation of the industrial *o tobacco PBD6 In order to determine the efficacy of these two chimeric genes, these were transferred to industrial tobacco PBD6 according to the transformation and regeneration procedures already described in European Application EP No. 0 508 909.

*oo* 1) Transformation: The vector is introduced into the nononcogenic strain of Agrobacterium EHA 101 (Hood et al., 1987) which carries the cosmid pTVK 291 (Komari et al., 1986). The transformation technique is based on the procedure of Horsh R. et al. (1985), Science, 227, 1229-1231.

2) Regeneration: The regeneration of the tobacco PBD6 (origin 17 SEITA France) from foliar explants is carried out on a Murashige and Skoog (MS) base medium comprising 30 g/l of sucrose as well as 200 ug/ml of kanamycin. The foliar explants are selected on plants in the greenhouse or in vitro and transformed according to the foliar discs technique (Science 1985, Vol. 227, p.

1229-1231) in three successive steps: the first comprises the induction of shoots on an MS medium to which is added 30 g/l of sucrose containing 0.05 mg/1 of naphthylacetic acid (NAA) and 2 mg/l of benzylaminopurine (BAP) for 15 days. The shoots formed during this step are then developed by culture on an MS medium to which is added 30 g/l of sucrose, but not containing any hormone, for 10 days. Developed shoots 15 are then selected and cultured on an MS rooting medium of half salts, vitamins and sugars content and not containing any hormone. At the end of approximately days, the rooted shoots are placed in earth.

Example 4: Measurement of the tolerance of the tobacco to 4-[4-CF 3 cyclopropylisoxazole: post-emergence treatment On leaving in-vitro culture, the transformed tobacco plantlets were acclimatized in a greenhouse relative humidity; temperature: 20°C during the night and 23 0 C during the day) for five weeks and then treated with 4-[4-CF 3 cyclopropylisoxazole.

The control tobacco, non-transformed and treated with 4-[4-CF 3 cyclopropylisoxazole in doses ranging from 50 to 400 g/ha, develops chloroses in approximately 72 hours, which intensify to develop into very pronounced necroses in a week (covering approximately 80 of the terminal leaves).

After transformation, this same tobacco, which overexpresses the HPPD of P. fluorescens, is very well protected against treatment with 4-[4-CF 3 -2- (methylsulphonyl)benzoyl]-5-cyclopropylisoxazole at a dose of 400 g/ha.

If the overexpressed enzyme is in the cytoplasm, that is to say if the transformation was carried out with the gene carried by the vector pRP T, 15 then the plant shows very slight chloroses which are all localized on the intermediate leaves.

If the overexpressed enzyme is in the chloroplast, that is to say if the transformation was carried out with the gene carried by the vector pRP V, 20 then the plant is perfectly protected and does not show any symptoms.

Example 5: Measurement of the tolerance of the tobacco to 4-[4-CF3-2-(methylsulphoyl)benzoyl]-5cyclopropylisoxazole: pre-emergence treatment a) in vitro test: Tobacco seeds harvested from plants from the "transformation-regeneration" cycle and resistant to isoxaflutole foliar treatment are used at a dose of *9 19 400 g/h described in Examples 1 to 3.

These seeds were sown in boxes containing plant agar at 10 g/l and isoxaflutole at different concentrations ranging from 0 to 1 mg/l. Germination was then carried out at 25*C with a photoperiod of 12 hours of light/12 hours of darkness.

According to this protocol, wild tobacco seeds were germinated as well as seeds of the two types of transgenic tobacco, that is to say CY tobaccos, with localization of the HPPD in the cytoplasm, and the CO tobaccos with localization of the HPPD in chloroplast.

Resistance measurements are carried out visually between 2 and 3 weeks after sowing.

The results are recorded in the table below.

15 isoxaflutole Wild tobacco CY tobacco CO tobacco concentration 0 mg/1 100 of the 100 of the 100 of the seeds ger- seeds seeds minate germinate germinate without without without symptomso symptoms 0 symptoms 0.05 mg/l 20 of the 75 of the 75 of the seeds seeds seeds germinate germinate* germinate* and show without without symptomso symptomso symptomso 1 mg/1 no 75 of the 75 of the germination seeds seeds germinate* germinate* with slight without symptoms 0 symptoms 0 o the symptoms which the plantlets show in the course 5 of germination are more or less significant deformations of the cotyledons and above all a bleaching of the tissues which are normally photosynthetic and thus green.

75 of the seeds germinate because seeds from the self-fertilization of single-locus plants coming from the "transformation-regeneration" cycle and thus only carrying the tolerance gene on one chromosome were sown.

Working in the same way with the following products, Product No. 51 of American Patent 4 780 127, the same results are obtained at a concentration of 0 mg/l and 0.1 mg/l on wild tobacco and CO tobacco.

b) greenhouse test: Measurement is carried out as in Example 4, apart from the treatment being carried out preemergence, 24 hours before sowing. Wild sowing is carried out normally. Under these conditions, it is observed that, for the non-treated control sowings, there is no germination for any dose of herbicide at least equal to 10 g/ha. On the contrary, the CY tobaccos do not show any symptoms, such as defined in paragraph up to and including 100 g/ha. Similarly, the CO tobaccos do not show any symptoms, such as defined in paragraph up to and including 200 g/ha.

.a These results show clearly that the HPPD gene of P. fluorescens confers a tolerance to the tobacco against pre-emergence treatments with isoxaflutole.

This tolerance is better if the protein is localized in the chloroplast in place of the cytoplasm.

Example 6: With the aim of studying whether the HPPD gene of Pseudomonas fluorescens can be used as a marker gene in the course of the "transformation-regeneration" cycle of a plant species, tobacco was transformed with the HPPD gene and transformed plants were obtained after selection on isoxaflutole.

Material and methods and results The chimeric gene pRP V described below is transferred into industrial tobacco PBD6 according to the transformation and regeneration procedures already described in European Application EP No. 0 508 909.

The chimeric gene of the vector pRP V has the following structure: Double histone TEV OTP Coding region nos promoter of HPPD terminator 1) Transformation: 10 The vector is introduced into the Agrobacterium EHA 101 non-oncogenic strain (Hood et al., 1987) which carries the cosmid pTVK 291 (Komari et al., 1986). The transformation technique is based on the procedure of Horsh et al. (1985).

15 2) Regeneration: The regeneration of the tobacco PBD6 (origin SEITA France) from foliar explants is carried out on a Murashige and Skoog (MS) base medium comprising 30 g/l of sucrose as well as 350 mg/1 of cefotaxime and 1 mg/l 20 of isoxaflutole. The foliar explants are selected on plants in a greenhouse or in vitro and transformed according to the foliar discs technique (Science 1985, Vol. 227, p. 1229-1231) in three successive steps: the first comprises the induction of shoots on an MS medium to which is added 30 g/l of sucrose containing 0.05 mg/l of naphthylacetic acid (NAA) and 2 mg/l of benzylaminopurine (BAP) for 15 days and 1 mg/l of isoxaflutole. The green shoots formed in the course of this step are then developed by culture on an MS medium to which are added 30 g/l of sucrose and 1 mg/l of isoxaflutole, but not containing hormone, for 10 days.

Developed shoots are then selected and are cultured on an MS rooting medium of half salts, vitamins and sugars content and 1 mg/l of isoxaflutole and not containing any hormone. At the end of approximately 15 days, the rooted shoots are placed in earth.

All the plantlets obtained according to this protocol are analysed by PCR with specific primers of the HPPD of P. fluorescens. This PCR analysis has enabled it to be confirmed that all the plantlets thus obtained have indeed integrated the HPPD gene.

15 In conclusion, this assay confirms that the HPPD gene can be used as marker gene and that, associated with this gene, isoxaflutole can be a good selection agent.

Examples 7 and 8: Isolation of the HPPD gene of Arabidopsis thaliana and of the HPPD gene of carrot (Daucus carotta) a) Construction of cDNA banks mRNAs extracted from young plantlets of Arabidopsis thaliana and mRNAs extracted from carrot cells in culture served to construct two cDNA banks in the vector Uni Zap T XR marketed by the company Stratagen, following the protocol recommended by this company.

b) Screening of the cDNA banks These two banks were screened with the aid of a probe corresponding to a cDNA of Arabidopsis thaliana of partial length, obtained via the Arabidopsis Biological Resource Center (Ohio, USA) and indexed: EST clone No.

91B13T7. This clone is formed of approximately 500 base pairs of which only 228 had been sequenced by the MSU- DOE Plant Research Laboratory in the context of random sequencing of cDNA of Arabidopsis thaliana. We completely sequenced the 500 base pairs before using this clone to screen our cDNA banks of Arabidopsis thaliana and of carrot with the aid of the classical technique of hybridization of lysis regions (reference c) A cDNA of Arabidopsis thaliana (SEQ ID No. 2) corresponding to 1338 base pairs was obtained. This cDNA has a translation initiation start codon in position 25 and a translation end codon in position 1336. This cDNA is thus complete and codes for a protein of 445 amino acids.

20 d) A cDNA of carrot (Daucus carotta) (SEQ ID No. 3) corresponding to 1329 base pairs was obtained. This cDNA has a translation initiation start codon in position 1 and a translation finish codon in position 1329. This cDNA is thus complete and codes for a protein of 442 amino acids.

The sequences illustrated are the following: SEQ ID No. 1 Sequence of the HPPD gene of Pseudomonas fluorescens A32 SEQ ID No. 2 cDNA sequence of HPPD of Arabidopsis thaliana SEQ ID No. 3 cDNA sequence of HPPD of Daucus carotta The figures below are given by way of indication to illustrate the invention.

Ficure 1 represents the protein sequence of the HPPD of Pseudomonas sp. strain P.J 874 and the theoretical nucleotide sequence of the corresponding coding part; the five oligonucleotides chosen to carry out the amplification of a part of this coding region are symbolized by the five arrows.

Figure 2 represents the mapping of the plasmid with the genomic DNA fragment of 7 kb comprising the gene of the HPPD of P. fluorescens A32.

Fiqure 3 gives the comparison of the amino-acid sequences of the HPPD of P fluorescens A32 and of the HPPD of Pseudomonas sp. strain P.J.874 (only the divergent amino acids between the two sequences are indicated) as well as the concensus sequence.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

••go 25A The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

o* 0, Sequence listing GENERAL INFORMATION APPLICANT: Sailland. Alain Rolland. Anne Matninge, Michel Palle". Kenneth F TITLE OF INVENTION: DNA SEQUENCE OF A H-YDROXYPHIENYLPYRUVATE

DIOXYGENASE

GENE AND OBTAINMENT OF PLANTS COMPRISING THIS HYDROXYPHIENYLPYRUVATE DIOXYGENASE GENE, WHICH ARE RESISTANT TO CERTAIN HERBICIDES (iii) NUM1BER OF SEQUENCES: 3 CORRESPONDENCE

ADDRESS:

ADDRESSEE. Francos Chruten AB STREET: 14-20 rue Pierre BAIZET CITY: Lyon Cedex 09 (E COUNTRY: France ZIP: 69263 COMPUTER READABLE FORM: MEDIUM TYPE. Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOMIAS-DOS; SOFTWARE: Patentlin Releise Version #1.25 (vj) CURRENT APPUCATION DATA..

APPLICATION NUMBER. FIR PH95033 FILING DATE. 02-JUN-195

CLASSIFICATION:

(viii) ATTORNEY/AGEhrr

INFORMATION:

NAME: Chreien. Francois (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 72-29-26-42 TELEFAXC 72-29-25-43 INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

LENGTH: 1077 base pairs TYPE. nucleic acid STRANDEDNESS: double TOPOLOGY: linear (11) MOLECULE TYPE: DNA (genomic) (00 HYPOTHETICAL:

NO

ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE ORGANISM: Pseudomonas fiuorescefls (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i: ATGGCAGATC TATACGAAAA CCCAATGOGC CTGATGGGCT TTGAATTCAT CGAATTAGCG TCrGCCGACGC CGGTACCCT GGCCCATC TTCGAGATCA TC-GGCTTCAC CAAAkGTCGCG ACCCACCGTT CCAAGAACGT GCACCTGTAZ CGCCAGGGCG AGATCAACCT GA7CCTCAAC AACGAGCCCA ACAGCATCGC CTCCTACTTT GCGGCCGAAC ACGGCCCGTC rGGTGTGCGGC ATGGCGTTCC GCGTGAAGGA CTCG-CAAAAG GCCTACAACc GCGCCCTGGA ACTCGGCGCC CAGCCGATCC- ATATTGACAC CGGGCCGATG GAATTGAACC TGCCGGCGAT CAAGGGCATC GGCGGCGCGC CGTTGTACCT GATCGACCGT TTCGGCGAAG GCAGCTCGAT CTACGACATC GACTTCGTGT ACCTCGAAGG TGTGGAGCGC AATCCGGTCG GTG6A=GCT CAAAGTCATC GACCACCTGA CCCAkCAACGT CTATCGCGGC CGCATZGGTCT ACTGGGCCAA CTTCTACGAG AAATTGTTCA ACTTCCGTGA AGCGCGTTAC TTCGATATCA AWCGAGTA CACCGGCCTG ACTTCCAAGG CCATGAGTGC GCCGGACGGC ATGATCCGCA TCCCGCTGAA CGAAGAGTCG TCCAAGGGCG CGGGGCGAT CGAAGAGTTC CTGATFGCAGT TCAACGGCGA AGGCATCCAG CACGTGGCGT TCCTCACCGA CGACCTGGTC AA GAccTGGG ACGCGTTGAA GAAAATCGGC ATGCGCTTCA TGALCCGCGCC GCCAGACACT TAT~TACGAA6A TGCTCGAAGG CCGCCTGCCT GACCACGGCG AGCCGGTGGA TCAACTGCAG GCPLCGCGGTA TCCTGCTGGA CGGATCTTCC GTGGAAGGCG ACAAACGCCT GCTGCTGCAG ATCTTCTCGG AAACCCTGAT GGGCCCGGTG TTCTTCGAAT TCALTCCAGCG CAAGGGCGAC GATGGGTTTG GCGAGGGCAA CTTCAAGGCG CTGTTCGAGT CCATCGAACG TGACCAGGTG CGTCGTGGTG TATTGACCGC CGATTAA INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1338 base pairs (B TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear Chi) MOLECULE TYPE: cDNA (ii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Arabidopsis thaliana STRAIN: Columbia DEVELOPMENTAL STAGE: Young green plant (vii) IMMEDIATE SOURCE: LIBRARY: Uni zap XR STRATAGENE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ATGGGCcAcc AAAACGCCGC CGTTTCAGAG AATCAAAACC ATGATGACGG CGCTGCGTCG TCG;CCGGGAT TCAAGCTCGT CGGATTTTCC AAGTTCGTAA GAAAGAATCC AAAGTCTGAT 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1077 120 AAATTCAAGG TTAArGCcTT CCATCACATC G;AGTTCTGGT GCG;GCG-ACGC AACCAACG;TC GCTCGTCGCT TCTCCTGGGG TCTGGGGATG AGATTCTCCG CCAAATCC-GA TCTTTCCACC GWAACAIGG TTCACGCCTC TTACCTACc ACCTCCGGT6 ACCTCCGATT

CCTT.TTCACT

GCTCCTTACT CTCCGTfZTCT CTCCGCCGGA GAGATTAC CGACAACCAC AGCTTCTATC CCAAGTTTCG ATCACGGCTC GCCGTTGCGA TTGAAGTAG;A GCTATTCCTT CGTCrCCTcC C-,AtACGr.Cr ATGTTGTTCT GAATTCTTGC CAGGGTTCGA ACCGGCGGC TTGACCACGC GTAGCGGGGT

TCACTGGTTT

GCCGAGAGCG

GTTTAAATTC

TTGTCGTCC TTCTTCTCTT

CACATGGTCT

AGACGCAGAG TCAGCTTTCT CCATCAGTGT TATCGTCCTC AATGAA.GCAG TTACGATCGC CCGATATGTT .AGTTACWAG CAGAAGATAc GCGTGTAGAG GATGCGTCGT

CGTTCCCATT

CGTGGGAAAC GTTCCTGAGC TTGGTCCGGC TCACCAATTC GCAGAGTTCA CAGCAGACGA6 AGCGGTCCTG GCTAGCAATG A2TGAAATGGT

CGGTGTTAGA

AGCTAATGGC

TGAGGTTAAA

CGAAAAATCC

GGATTATGGT

TTTAACTTAT

CGTTGGAAC

0 XZTAACGAGC CAGTG-CACr- AACAAAGAGG AhAGTCAGA6 TTCAGACGTA TTTGGAACAT AACGAAGGCr- CAGGGCTACA AC-ATCTGGCT CTGATGAGTG AAGACATAXT CAGGACCCTG AGAGAGATGA GGAAGAGGG CAGTATTGGA GGATTCGACT TCATGCCTTC

TCCTCCGCCT

ACTTACTACC AGAATCTCAA GAAACGGGTC GGCGACGTGC TCAG<CGATGA TCAGATCAAG GAkGTGTGAGG AATTAGGGA.T TCTTGTAGAC AGAGATrGATC AAGGGACGTT

GCTTCAAATC

TTCACAAAAC CACTAGGTG, CGCCGACG ATATTTATAG AGATAATCCA

GAGAGTAGGA

TGCA.TGATrQA AAGATGAGGA AGGGAAW=C TACCAGAGTC; GAGGATG;TGG. TGGTTTTGGC AAAGGCAATT TCTCTGAr.CT CTTCAAGTCC ATTGAAGAAT ACGAAAAGAC TCTTGAAGCC AAACAGT'rAG

TGGGATGA

INFORMATION FOR SEQ ID NO:3: SEQUENCE

CHARACTERISTICS:

LENGTH: 1329 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Daucus carota DEVELOPMENTAL STAGE: Suspension cells (vii) IMMEDIATE SOURCE: LIBRARY: Uni zap XR STRATAGENE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGGAAA AAcAATCGGA AGCTGAAATT CTCTCAAGCA ATTCATCAAA CACCTC'rCCr 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1338 GcAACATTCA AGCTGGTCGG TTTCAA-ZAAC TTCGTCCGCG CCAACCCCAA G-TCCC;ATCAC TTCGCCGTGA AGCGGTTCCA CCACATTGAG TTCTGCGTGCG GCGACGCCAC CAACACGTCG CGGCGGTTCT CGTGGGGCCT CGGCATGCCT TTGGTGGCGA AATCGGATCT CTCTACTGGA AACTCTGTTC ACGCTTCTTA TCTTGTTCGC TCGGCGAATC TCAGTT.TCGT CTTCACCGCT CCTTACTCTC CGTCCArGAC CACTTCCTCT GGTTCAGCTG CCATCCCGTC TTTTTCGGCA TCGGGTTTTC ACTCTTTTGC GGCCAAACAC GGCCTTGCTG TTCGGGCTAT TGCTCTTGAA GTTGCTGACG TGGCTGCTGC GTTTGAGGCC AGTGT2'GCGC GTGGGGCCAG GCCGGCTTCG GCTCCTGTTG AA2TTGGACGA CCAGGCGTGG TTGGCTGAGG TGGAGTTGTA CGGAGATGTG GTCTT.GAGGT TTGTTAGTTT TGGGAGGGAG GAGGGTTTGT TTTTGCCTGG ATTCGAGGCG GTGGAGGGGA CGGCGTCGTT TCCGGATTTG GATTATGGAA TTAGAAGACT TGATCATGCG GTGGGGAATG TTACCGAGTT GGGGCCTGTG GTGGAGTATA TTAAAGGGTT TACGGGGTTT CATGAMTTTG CGGAGTTTAC ADCGGAGGATr GTGGGGACTT TGGAGAGTGG GTTGAATTCG GTGGTGTTGG CGAATAATGA GGAGATGGTT CTGTTGCCCT TGAATGAGCC TGTGTATGGG ACCAAGAGGA AGAGTCAGAT ACAGACTTAC TTGGAGCACA ATGAAGGGGC TGGAGTGCAG CATTTGGCTT TA~rGAGTGA GGATATTTTT AGGACTTTAA GGGAGATGAG GAAGAGGAGT TGCCTTGGTG GTTTTGAGTT TATGCCTTCG CCACCGCCTA CGTATTACAA GAATTTGAAG AATAGGGTCG GGG.ATGTG;TT GAGTGATGAA cAGA~cAArG ArTGTGAAGA TTTGGGGATT TTGGTGGATA GGGATGATCA GGGTACATTG CTTCAAATCT TTACCAAGCC TGTAGGTGAC 12C 9 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1329 AGGCCTACCT TATPTCATAGA GATCATTCAG GGGCAGATGT ACCAGAAGGG CGGGTGCGGA TTCAAGTCCA TCGAAGAATA TGAAAAAACA

GCTGCATGA

AGGGTAGGGT GCATGCTCAA GGACGATGCA GGATTTGGGA AGGGGAACTT CTCAGAGCTG CTTGAAGCTA AACAAATCAC TGGATCTGCT

Claims (27)

1. Isolated sequence of a gene of plant origin which encodes hydroxyphenylpyruvate dioxygenase (HPPD) and which hybridizes with the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

2. Sequence according to Claim 1, characterized in that it is from Arabidopsis.

3. Sequence according to Claim 1, characterized in that is from an umbelliferous plant.

4. Sequence according to Claim 3, wherein the umbelliferous plant is a carrot. Chimeric gene for the genetic transformation of plants comprising, in the transcription direction: at least one promoter regulation sequence which is operable in plants, a heterologous coding sequence of plant origin which encodes hydroxyphenylpyruvate dioxygenase (HPPD) and which hybridizes with the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3, at least one polyadenylation sequence.

6. Chimeric gene according to Claim characterized in that the coding sequence is a sequence such as defined in any one of Claims 2 to 4.

7. Chimeric gene according to Claim 5 or Claim 6, characterized in that the promoter regulation sequence favours the overexpression of the coding sequence.

8. Chimeric gene according to Claim 7, characterized in that the promoter regulation sequence comprises at least one histone promoter.

9. Chimeric gene according to one of Claims R* to 8, characterized in that it comprises, between the promoter regulation sequence and the coding sequence, a ^74 Q:\Opr\VpaW\FcbaryWeb 2003\2302734 ameded claims avmic crop.49.doc-18 Februry 2003 31 transit peptide. Chimeric gene according to any one of Claims 5 to 9, characterized in that it comprises, between the promoter regulation sequence and the coding sequence, an optimized transit peptide comprising, in the transcription direction, a sequence coding for a transit peptide of a plant gene coding for a plastid localization enzyme, a part sequence of the N-terminal mature part of a plant gene coding for a plastid localization enzyme,then a sequence coding for a second transit peptide of a plant gene coding for a plastid localization enzyme.

11. Chimeric gene according to one of Claims to 10, characterized in that it comprises, between the promoter regulation sequence and the coding sequence, a sequence of a transcription activator (enhancer).

12. Vector utilizable for the genetic transformation of plants, characterized in that it comprises a chimeric gene according to one of Claims 5 to 20 11.

13. Plant cell, characterized in that it comprises a chimeric gene according to one of Claims 5 to 11.

14. Plant, characterized in that it is regenerated from cells according to Claim 13.

15. Plant according to Claim 14, characterized in that it belongs to the dicotyledonous plant family.

16. Plant according to Claim 15, characterized o*o* in that it is a tobacco plant.

17. Plant according to Claim 16, characterized inthat it is a tobacco plant PBD6.

18. Process of transformation of plants to make QAOp\Vp\FbmrykFcb 2003\2302734 -cl.dd claims avmdc crop.49.do-18 Fcbmary 2003 32 them tolerant to HPPD inhibitors, characterized in that a gene expressing an exogenous HPPD, which hybridizes with the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3, is introduced into a vegetable cell.

19. Process according to Claim 18, characterized in that the transfer is carried out using Agrobacterium tumefaciens or Agrobacterium rhizogenes. Process according to Claim 18, characterized in that the transfer is carried out by delivery by bombardment with the aid of charged particles of the DNA.

21. Process of transformation of plants, characterised in that a gene expressing an exogenous HPPD of plant origin, which hybridizes with the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3, is introduced into a vegetable cell as a selection marker.

22. Process according to any one of Claims 18 to 21, characterized in that a chimeric gene according to any one of Claims 5 to 11 is introduced. S 20 23. Process of selective herbicidal treatment of plants, characterized in that an HPPD inhibitor is applied to a transformed plant comprising cells according to Claim 13.

24. Process according to Claim 23, characterized in that the plants are the plants according to any one of Claims 14 to 17. Process according to Claim 23 or Claim 24, characterized in that the plants are obtained by the process according to any one of Claims 18 to 22. 30 26. Process according to any one of Claims 23 o to 25, characterized in that the HPPD inhibitor is an |r isoxazole, a diketonitrile, a triketone or sukotrione. Q:\Oplc \Vpa\FebnaryFeb 2003\2302734 amnded claims avidc crop.49.doc-18 Febrary 2003 d 0 33

27. Process according to Claim 28, characterized in that the HPPD inhibitor is 4-[4-CF3-2- isoxazole.

28. Sequence according to any one of Claims 1 to 4, substantially as herein before described with reference to the figures and/or examples.

29. Chimeric gene according to any one of Claims 5 to 11, substantially as herein before described with reference to the figures and/or examples.

30. Vector according to Claim 12, substantially as herein before described with reference to the figures and/or examples.

31. Plant cell according to Claim 13, substantially as herein before described with reference to the figures and/or examples.

32. Plant according to any one of Claims 14 to 17, substantially as herein before described with reference to the figures and/or examples.

33. Process according to any one of Claims 18 S 20 to 27, substantially as herein before described with 0 reference to the figures and/or examples. 0oo DATED this 18th day of February 2003 Aventis CropScience S.A. DAVIES COLLISON CAVE Patent Attorneys for the applicant ees *Joe o f *oo* *0000 oo ooeot C