AU1161499A - Chimeric hydroxyphenylpyruvate dioxygenase, DNA sequence, and herbicide-tolerant plants containing such a gene - Google Patents

Description

WO 99/24586 1 PCT/FR98/02391 Chimeric hydroxyphenylpyruvate dioxygenase, DNA sequence, and herbicide-tolerant plants containing such a gene. 5 The present invention relates to a nucleic acid sequence encoding a chimeric hydroxyphenylpyruvate dioxygenase (HPPD), a chimeric gene comprising this sequence as encoding sequence, and its use for obtaining plants which are resistant to certain 10 herbicides. The hydroxyphenylpyruvate dioxygenases are enzymes which catalyse the transformation reaction of parahydroxyphenylpyruvate (HPP) into homogentisate. This reaction takes place in the presence of iron (FE 2+ 15 and in the presence of oxygen (Crouch N.P. et al., Tetrahedron, 53, 20, 6993-7010, 1997). We may put forward the hypothesis that the HPPDs contain an active site which is suitable for catalysing this reaction, in which the iron, the substrate and the water molecule 20 combine, even though such an active site has never been described to date. Moreover, there are also known certain molecules which inhibit this enzyme and which attach themselves competitively to the enzyme to inhibit the 25 transformation of HPP into homogentisate. It has been found that some of these molecules can be used as herbicides, insofar as the inhibition of the reaction WO 99/24586 2 PCT/FR98/02391 in the plants leads to bleaching of the leaves of the treated plants and to the death of these plants (Pallett K.E. et al., 1997, Pestic. Sci. 50 83-84). Such herbicides of the prior art which target HPPD are, 5 in particular, the isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, US 5 424 276), in particular isoxaflutole, which is a selective maize herbicide, the diketonitriles (EP 496 630, EP 496 631) in particular 2-cyano-3 10 cyclopropyl-1- (2-SO 2

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3 -4-CF 3 -phenyl) -propane-1, 3-dione and 2-cyano-3-cyclopropyl-1-(2-SO 2

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3 -4-2,3-Cl 2 -phenyl) propane-1,3-dione, the triketones (EP 625 505, EP 625 508, US 5,506,195), in particular sulcotrione, or else the pyrazolinates. 15 To make the plants herbicide-tolerant, three principal strategies are available, viz. (1) making the herbicide non-toxic using an enzyme which transforms the herbicide or its active metabolite into non-toxic degradation products, such as, for example, the enzymes 20 for tolerance to bromoxynil or to Basta (EP 242 236, EP 337 899); (2) conversion of the target enzyme into a functional enzyme which is less sensitive to the herbicide or its active metabolite, such as, for example, the enzymes for tolerance to glyphosate 25 (EP 293 356, Padgette S.R. et al., J. Biol. Chem., 266, 33, 1991); or (3) overexpression of the sensitive enzyme, in such a way that the plant produces high Ij Irl~l1_" WO 99/24586 3 PCT/FR98/02391 enough quantities of the target enzyme with regard to the kinetic constants of this enzyme relative to the herbicide in such a way that it has enough functional enzyme despite the presence of its inhibitor. 5 With this third strategy, it has been described that plants which are tolerant to HPPD inhibitors (WO 96/38567) were successfully obtained, it being understood that a simple strategy of overexpressing the sensitive (unaltered) target enzyme 10 was successfully employed for the first time to impart to the plants a herbicide tolerance which is at an agronomical level. Despite the success obtained with this simple strategy of overexpressing the target enzyme, the 15 system for HPPD inhibitor tolerance must be varied to obtain tolerance whatever the culture conditions of the tolerant plants or the commercial doses of herbicide application in the field. It is known from the prior art (WO 96/38567) that enzymes of different origin 20 (plants, bacteria, fungi) have primary protein sequences which differ substantially and that these enzymes have an identical function and essentially similar or related kinetic characteristics. It has now been found that all these HPPDs 25 have, on the one hand, many sequence homologies in their C-terminal portion (Fig. 1) and, on the other hand, an essentially similar tertiary (three- WO 99/24586 4 PCT/FR98/02391 dimensional) structure (Figure 2). Asregards the competitive inhibition characteristic, the hypothesis is put forward that the HPPD inhibitors attach themselves to the enzyme at the active site of the 5 latter, or in its vicinity, in such a way that the access of HPP to this active site is blocked and its conversion in the presence of iron and of water is prevented. It has now been found that, by mutating the enzyme in its C-terminal portion, it was possible to 10 obtain functional HPPDs which are less sensitive to HPPD inhibitors, so that their expression in the plants allows an improved HPPD inhibitor tolerance. As regards these elements, it can thus be concluded that the active site of the enzyme is located in its C-terminal 15 portion, while its N-terminal portion essentially ensures its stability and its oligomerization (Pseudomonas HPPD is a tetramer, plant HPPDs are dimers). It has now been found that it was possible to 20 generate a chimeric enzyme by combining the N-terminal portion of a first enzyme with the C-terminal portion of a second enzyme so as to obtain a novel functional chimeric HPPD, which allows each portion to be selected for its particular properties, such as, for example, to 25 select the N-terminal portion of a first enzyme for its stability properties in a given cell (plant, bacterium and the like) and the C-terminal portion of a second RA1 WO 99/24586 5 PCT/FR98/02391 enzyme for its kinetic properties (activity, inhibitor tolerance, and the like). The present invention therefore primarily relates to a chimeric HPPD which, while being 5 functional, that is to say retaining its properties of catalysing the conversion of HPP into homogentisate, comprises the N-terminal portion of a first HPPD in combination with the C-terminal portion of a second HPPD. 10 Each portion of the chimeric HPPD according to the invention is derived from an HPPD of any origin and is, in particular, selected from amongst plant, bacterial or fungal HPPDs. According to a preferred embodiment of the 15 invention, the N-terminal portion of the chimeric HPPD is derived from a plant HPPD, this plant preferably being chosen from dicots, in particular Arabidopsis thaliana or Daucus carota, or else from monocots, such as maize or wheat. 20 According to another preferred embodiment of the invention, the C-terminal portion of the chimeric HPPD is derived from a plant HPPD as defined above or from an HPPD of a microorganism, especially a bacterium, in particular Pseudomonas, more particularly 25 Pseudomonas fluorescens, or of a fungus, it being possible for this C-terminal portion to be natural or mutated by substituting one or more amino acids of the

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WO 99/24586 6 PCT/FR98/02391 C-terminal portion of the original HPPD, especially for making it less sensitive to HPPD inhibitors. A number of HPPDs and their primary sequence have been described in the prior art, especially HPPDs 5 from bacteria such as Pseudomonas (Riietschi et al., Eur. J. Biochem., 205, 459-466, 1992, WO 96/38567), plants such as Arabidopsis (WO 96/38567, Genebank AF047834) or carrot (WO 96/38567, Genebank 87257), from Coccicoides (Genebank COITRP), or mammals such as mouse 10 or pig. Since the alignment of these sequences is known through the usual methods of the art, for example the method described by Thompson, J.D. et al. (CLUSTAL W: Improving the sensitivity of progressive multiple 15 sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice, Nucleic Acids Research, 22:4673-4680, 1994) and through access to these computer programs for sequence alignment, for example via internet, the person skilled 20 in the art can define sequence homologies relative to a reference sequence and recognize the key amino acids, or else define the regions which they have in common, which especially allows a C-terminal region and an N-terminal region to be defined on the basis of this 25 reference sequence. The reference sequence for the present invention is the sequence from Pseudomonas, and all WO 99/24586 7 PCT/FR98/02391 definitions and indications of specific amino acid positions relate to the primary sequence of Pseudomonas HPPD. The appended Figure 1 shows an alignment of a plurality of HPPD sequences which are described in the 5 prior art and which are aligned relative to the Pseudomonas HPPD sequence by way of reference; they include the HPPD sequence of Streptomyces avermitilis (Genebank SAV11864), of Daucus carota (Genebank DCU 87257), of Arabidopsis thaliana (Genebank AF047834), of 10 Zea mays, of Hordeum vulgare (Genebank HVAJ693), of Mycosphaerella graminicola (Genebank AF038152), of Coccicoides immitis (Genebank COITRP), and of Mus musculus (Genebank MU54HD). This figure shows the numbering of the amino acids of the Pseudomonas 15 sequence, and the amino acids which they have in common are designated by an asterisk. Based on such an alignment, and with the definition of the Pseudomonas amino acid, it is easy to identify the position of the corresponding amino acid in a different HPPD sequence 20 by its position and its nature (the alignment of sequences of different origin, viz. plants, mammals and bacteria, demonstrate that this alignment method, which is well known to those skilled in the art, can be applied generally to any other sequence). An alignment 25 of different HPPD sequences is also described in- the Patent Application WO 97/49816. R9 WO 99/24586 8 PCT/FR98/02391 The C-terminal portion of the HPPDs, which is where the active site of the enzyme is located, differs from its N-terminal portion by a linkage peptide, as shown by the schematic diagram of the tertiary 5 structure of the Pseudomonas HPPD monomer given in Figure 2. This structure was obtained by the usual methods of crystal X-ray diffraction analysis. The linkage peptide will allow the N-terminal end of the C-terminal portion of the enzyme to be defined, this 10 peptide being located between amino acids 145 and 157 in the case of Pseudomonas (cf. Figure 1). The C-terminal portion can thus be defined as being composed of the sequence which is delimited, on the one hand, by the linkage peptide and, on the other 15 hand, by the C-terminal end of the enzyme. The sequence alignment shown in the appended Figure 1 demonstrates for all sequences two amino acids in positions 161 and 162 in the case of the Pseudomonas sequence (D = Asp161 and H = His162). Relative to the Pseudomonas HPPD, it 20 can thus be defined that the linkage peptide which represents the N-terminal end of the C-terminal portion of the HPPD is located approximately between 5 and 15 amino acids upstream of the amino acid Asp161. The present invention equally relates to a 25 nucleic acid sequence encoding an above-described chimeric HPPD. According to the present invention, "nucleic acid sequence" is to be understood as being a ) -7 R4 WO 99/24586 9 PCT/FR98/02391 nucleotide sequence which can be of the DNA or the RNA type, preferably of the DNA type, especially double stranded, whether it is of natural or synthetic origin, especially a DNA sequence for which the codons encoding 5 the chimeric HPPD according to the invention will have been optimized according to the host organism in which it will be expressed, these optimization methods being well known to those skilled in the art. The sequences encoding each HPPD derived from 10 the chimeric HPPD according to the invention can be of any origin. In particular, it can be of bacterial origin. Bacteria of the following types can be mentioned as specific examples: Pseudomonas sp., for example Pseudomonas fluorescens or else cyanobacteria 15 of the type Synechocystis. The origin of the sequence can also be a plant, especially it may be derived from dicots such as tobacco, Arabidopsis, Umbelliferae such as Daucus carota, or else monocots such as Zea mays or wheat. The encoding sequences and the means of 20 isolating and cloning them are described in the abovementioned references, whose contents are incorporated herein by reference. The encoding sequences of the N-terminal and C-terminal portions of the chimeric HPPD according to 25 the invention can be assembled by any usual method for constructing and assembling nucleic acid fragments TRA, which are well known to those skilled in the art and K~. 441 WO 99/24586 10 PCT/FR98/02391 widely described in the literature and illustrated especially by the use examples of the invention. The present invention therefore also relates to a process of generating a nucleic acid sequence 5 encoding a chimeric HPPD according to the invention, this process being defined hereinabove. Another subject of the invention is the use of a nucleic acid sequence encoding a chimeric HPPD according to the invention in a process for the 10 transformation of plants, as marker gene or as encoding sequence which allows a tolerance to HPPD inhibitor herbicides to be imparted to the plant. It is well understood that this sequence can also be used in combination with another marker gene, or marker genes, 15 and/or a sequence, or sequences, encoding one or more agronomic properties. The present invention also relates to a chimeric gene (or expression cassette) comprising an encoding sequence as well as heterologous regulatory 20 elements in positions 5' and 3' which can function in a host organism, in particular plant cells or plants, the encoding sequence comprising at least one nucleic acid sequence encoding a chimeric HPPD as defined above. By host organism there is to be understood 25 any single-celled or lower or higher multi-celled organism into which the chimeric gene according to the invention can be introduced so as to produce chimeric 44 kiA-7 WO 99/24586 11 PCT/FR98/02391 HPPD. In particular, these are bacteria, for example E. coli, yeasts, in particular of the genera Saccharomyces or Kluyveromyces, Pichia, fungi, in particular Aspergillus, a baculovirus, or, preferably, plant cells 5 and plants. By plant cell, there is to be understood according to the invention any plant-derived cell which can constitute indifferentiated tissues, such as calli, differentiated tissues such as embryos, parts of 10 plants, plants or seeds. By plant there is to be understood according to the invention any differentiated multi-celled organism which can photosynthesize, in particular monocots or dicots, more particularly crop plants for 15 animal or human nutrition or non-food crop plants, such as maize, wheat, oilseed rape, soya, rice, sugar cane, beet, tobacco, cotton and the like. The regulatory elements required for expressing the nucleic acid sequence encoding an HPPD 20 are well known to those skilled in the art and depend on the host organism. They comprise, in particular, promoter sequences, transcription activators, terminator sequences, inclusive of start and stop codons. The means and methods for identifying and 25 choosing the regulatory elements are well known to those skilled in the art and widely described in the literature.

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WO 99/24586 12 PCT/FR98/02391 More particularly, the invention relates to the transformation of plants. Any regulatory promoter sequence of a gene which is expressed naturally in plants, in particular a promoter which is expressed 5 particularly in the leaves of plants, such as, for example, so-called constitutive promoters derived from bacteria, viruses or plants such as, for example, a histone promoter as described in Application EP 0 507 698, or a rice actin promoter, of a plant 10 virus gene, such as, for example, that of cauliflower mosaic virus (CAMV 19S or 35S), or else so-called light-dependent promoters such as that of a gene of the small subunit of ribulose biscarboxylase/oxygenase (Rubisco) of a plant, or any other suitable known 15 promoter can be used as regulatory promoter sequence in plants. In combination with the regulatory promoter sequence, it is also possible to use, in accordance with the invention, other regulatory sequences which 20 are located between the promoter and the encoding sequence, such as transcription activators (enhancers), such as, for example, the tobacco mosaic virus (TMV) translation activator described in Application WO 87/07644 or the tobacco etch virus (TEV) translation 25 activator described by Carrington & Freed. As regulatory terminator or polyadenylation sequence, there can be used any corresponding sequence 4 WO 99/24586 13 PCT/FR98/02391 derived from bacteria, such as, for example, the Agrobacterium tumefaciens nos terminator, or else derived from plants, such as, for example, a histone terminator as described in Application EP 0 633 317. 5 According to a particular embodiment of the invention, at the 5'-position of the nucleic acid sequence encoding a chimeric HPPD there is employed a nucleic acid sequence encoding a leader peptide, this sequence being located between the promoter region and 10 the sequence encoding the chimeric HPPD in such a manner as to allow expression of a fusion protein of leader peptide/chimeric HPPD, the latter being as defined above. The leader peptide allows the chimeric HPPD to be sent to the plastids, more particularly the 15 chloroplasts, the fusion protein being cleaved between the leader peptide and the chimeric HPPD as it passes through the plastid membrane. The leader peptide can be simple, such as an EPSPS leader peptide (described in US Patent 5,188,642) or a leader peptide of that of the 20 small ribulose biscarboxylase/oxygenase subunit (ssu Rubisco) of a plant, which may comprise some amino acids of the N-terminal portion of the mature ssu Rubisco (EP 189 707) or else a multiple leader peptide comprising a first plant leader peptide fused with a 25 portion of the N-terminal sequence of a mature protein localized in the plastids which is fused to a second -RAL/ plant leader peptide as described in Patent EP 508 909, 3 I O

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WO 99/24586 14 PCT/FR98/02391 and more particularly the optimized leader peptide comprising a sunflower ssu Rubisco leader peptide fused to 22 amino acids of the N-terminal end of maize ssu Rubisco fused to the leader peptide of maize ssu 5 Rubisco as described together with the encoding sequence in Patent EP 508 909. The present invention also relates to the fusion protein of leader peptide/chimeric HPPD, the two elements of this fusion protein being defined further 10 above. The present invention also relates to a cloning and/or expression vector for transforming a host organism containing at least one chimeric gene as defined hereinabove. This vector comprises, in addition 15 to the above chimeric gene, at least one replication origin. This vector can be constituted by a plasmid, a cosmid, a bacteriophage or a virus which is transformed by introducing the chimeric gene according to the invention. Such transformation vectors according to the 20 host organism to be transformed are well known to those skilled in the art and widely described in the literature. To transform plant cells or plants, this will be, in particular, a virus which may be employed for transforming developed plants and furthermore 25 contains its own replication and expression elements. The transformation vector for plant cells or plants according to the invention is preferably a plasmid. -4J WO 99/24586 15 PCT/FR98/02391 A further subject of the invention is a process for the transformation of host organisms, in particular plant cells, by integrating at least one nucleic acid sequence or chimeric gene as defined 5 hereinabove, which transformation may be carried out by any suitable known means which have been widely described in the specialist literature and in particular in the references cited in the present application, more particularly by the vector according 10 to the invention. One series of methods consists of bombarding cells, protoplasts or tissues with particles to which the DNA sequences have been attached. Another series of methods consists in using a chimeric gene inserted into 15 an Agrobacterium tumefaciens Ti plasmid or Agribacterium rhizogenes Ri plasmid as transfer means into the plant. Other methods can be used, such as microinjection or electroporation, or else direct precipitation with PEG. The person skilled in the art 20 will choose the appropriate method according to the nature of the host organism, in particular of the plant cell or the plant. A further subject of the present invention are host organisms, in particular plant cells or 25 plants, which are transformed and contain a chimeric gene comprising a sequence encoding a chimeric HPPD as defined hereinabove.

WO 99/24586 16 PCT/FR98/02391 A further subject of the present invention are the plants which comprise transformed cells, in particular the plants regenerated from transformed cells. Regeneration is effected by any suitable 5 process, which depends on the nature of the species as described, for example, in the references hereinabove. Patents and patent applications which are cited in particular for the processes for transforming plant cells and regenerating plants are the following: 10 US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267,159, EP 604 662, EP 672 752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, 15 US 5,510,318, US 5,204,253, US 5,405,765, EP 442 174, EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 and WO 95/06128. The present invention also relates to the transformed plants obtained from growing and/or 20 hybridizing regenerated plants hereinabove as well as to the seeds of transformed plants. The transformed plants which can be obtained according to the invention can be of the monocotyledonous type such as, for example, cereals, 25 sugar cane, rice and maize, or of the dicotyledonous type such as, for example, tobacco, soya, oilseed rape, cotton, beet, clover and the like.

WO 99/24586 17 PCT/FR98/02391 Another subject of the invention is a method of selectively controlling weeds in plants, in particular crops, with the aid of an HPPD inhibitor, in particular a herbicide defined hereinabove, 5 characterized in that this herbicide is applied to transformed plants according to the invention, either pre-planting, pre-emergence or post-emergence of the crop. The present invention also relates to a 10 method of controlling weeds at the surface of a field with seeds or plants transformed with the chimeric gene according to the invention, the process consisting in applying, to this surface of the field, a dose of an HPPD inhibitor herbicide which is toxic to these weeds, 15 without, however, substantially affecting the seeds or plants transformed with this chimeric gene according to the invention. The present invention also relates to a method of growing plants which have been transformed 20 according to the invention with a chimeric gene according to the invention, the method consisting in planting the seeds of these transformed plants at a surface of a field which is suitable for growing these plants, in applying, if weeds are present, to this 25 surface of this field a dose of a herbicide which targets the HPPD defined hereinabove and which is toxic to the weeds, without substantially affecting these

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WO 99/24586 18 PCT/FR98/02391 seeds or these transformed plants, and then in harvesting the grown plants when they achieve the desired maturity and, finally, in separating the seeds from the harvested plants. 5 In the two above methods, the herbicide which targets HPPD can be applied according to the invention, either pre-planting, pre-emergence or post-emergence of the crop. Herbicide is to be understood as meaning for 10 the purposes of the present invention a herbicidally active substance alone or in combination with an additive which modifies its efficacy such as, for example, an agent which increases the activity (synergist) or limits the activity (safener). The HPPD 15 inhibitor herbicides are in particular previously defined. Of course, the above herbicides are combined in a manner known per se with the formulation auxiliaries conventionally used in agrochemistry for their practical application. 20 When the transformed plant according to the invention comprises another tolerance gene for another herbicide (such as, for example, a gene encoding a chimeric or non-chimeric EPSPS which imparts glyphosate tolerance to the plant), or when the transformed plant 25 is naturally insensitive to another herbicide, the process according to the invention may comprise the simultaneous or staggered application of an HPPD

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WO 99/24586 19 PCT/FR98/02391 inhibitor in combination with said herbicide, for example glyphosate. Another object of the invention is the use of the chimeric gene encoding a chimeric HPPD as marker 5 gene during the transformation regeneration cycle of a plant species and selection based on the above herbicide. The different aspects of the invention will be understood better with reference to the experimental 10 examples which follow. All the methods or operations described hereinbelow in these examples are given by way of example and represent a choice between the various methods which are available for arriving at the same 15 result. This choice has no effect on the quality of the result and, as a consequence, any suitable method may be used by the person skilled in the art to arrive at the same result. Most of the DNA fragment engineering methods are described in "Current Protocols in 20 Molecular Biology" Volumes 1 and 2, Ausubel F.M. et al., which are published by Greene Publishing Associates and Wiley-Interscience (1989), or in Molecular cloning, T. Maniatis, E.F. Fritsch, J. Sambrook, 1982. -0 A4 WO 99/24586 20 PCT/FR98/02391 Example 1: Colorimetric screening test for mutants which are tolerant to 2-cyano-3-cyclopropyl-1 (2-SO 2

-CH

3 -4-CF 3 -phenyl) -propane-1, 3-dione pRP C: The vector pRP A (which is described 5 in the application WO 96/38567), which contains a genomic DNA fragment and the encoding region of the Pseudomonas fluorescens A32 HPPD gene, was digested with NcoI, purified and then ligated into the expression vector pKK233-2 (Clontech) which itself had 10 been digested with NcoI, the only cloning site of this vector. The orientation of the gene in the resulting vector pRP C, which allows expression under the control of the trc promoter, was checked. A YT-broth-type culture medium with 1% 15 agarose (Gibco BRL ultra pure) and 5 mM L-tyrosine (Sigma), which contains the selection agent for the above vector pRP C is poured into 96-well plates at 100 pl per well. 10 pl of an E. coli culture in the exponential growth phase which contains the vector pRP 20 C is applied to each well. After 16 hours at 370C, the wells which do not contain the culture medium or those which have been seeded with an E. coli culture containing the vector PKK233-2 are transparent, while the wells seeded with an E. coli culture containing the 25 vector pRP C are brown. A series was established with identical culture medium containing different concentrations WO 99/24586 21 PCT/FR98/02391 (0 mM to 14 mM) of 2-cyano-3-cyclopropyl-1-(2 methylsulphonyl-4-trifluoromethylphenyl)-propane-i,3 dione (EP 0 496 631) which was solubilized in water and brought to pH 7.5. This molecule is a diketonitrile, 5 known as an efficient inhibitor of HPPD activity (Pallett, K.E. et al. 1997. Pestic. Sci. 50, 83-84). In the bacterial culture containing the vector pRP C, a total absence of staining is observed for 7 mM of the above compound. 10 Identical results were obtained when 2-cyano 3-cyclopropyl-1-(2-methyl-4-trifluoromethylphenyl)pro pane-1,3-dione was substituted by 2-cyano-3 cyclopropyl-l-(2-methylsulphonyl-4-(methylthio)phenyl) propane-1,3-dione and 2-(2-chloro-3-ethoxy-4-(ethyl 15 sulphonyl)benzoyl)-5-methyl-1,3-cyclohexadione, (WO 93/03009). The end concentrations of the two molecules in DMSO solution are 3.5 mM and 7 mM, respectively. These results confirm that a test based on 20 the HPPD activity, whatever the origin of this activity, allows the identification of HPPD activities which present a tolerance to HPPD activity inhibitors from the family of the isoxazoles as well as the triketones. RA nRA1 -1344 WO 99/24586 22 PCT/FR98/02391 Example 2: Preparation and evaluation of the activity of chimeric HPPDs The purpose is to generate fusions between HPPDs of phylogenetically different organisms, such as 5 monocotyledonous and dicotyledonous plants or else such as plants [lacuna] bacteria, with the following advantages: - HPPDs are obtained whose characteristics in terms of codon usage are more advantageous 10 for the expression in hosts where a tolerance to HPPD inhibitors is desirable, but difficult to obtain with a non-chimeric HPPD, - HPPDs are obtained whose characteristics in terms of weak affinity to the inhibitors are 15 more advantageous, - an HPPD activity is obtained in vitro or in vivo with a gene which is interesting, but of which no partial clone is available. The fusions of N- and C-terminal domains of 20 plants were tested. 1) Construction Fusions were carried out between the N-terminal regions of dicots and the C-terminal region of maize. These regions were introduced into dicots, 25 which are phylogenetically related. The search for the fusion zone was based on comparisons of protein sequences of different HPPDs RAi/ WO 99/24586 23 PCT/FR98/02391 (Figure 1). By homology with the Pseudomonas fluorescens HPPD, the N-terminal portions of Arabidopsis thaliana and of Daucus carota were identified; they terminate with tyrosine 219 and 5 tyrosine 212, respectively. The partial gene of Zea mays thus corresponds to 80% of the C-terminal portion of the protein. The PINEP region, which is highly conserved in plants, was chosen as the exchange zone. Upstream 10 there is the N-terminal portion, and downstream there is the C-terminal portion. By studying nucleotide sequences, it has been attempted to introduce a restriction site, if possible a single restriction site, which modifies the protein sequence in this 15 region little or not at all. The AgeI restriction site, which meets these criteria, can be obtained by modifying the codon usage for the adjacent amino acids E and P. The AgeI site was introduced by directed 20 mutagenesis using the USE method for the Arabidopsis thaliana and Daucus carota HPPD, with the aid of the degenerated oligonucleotides AgeAra and AgeCar, and with the USE selection oligonucleotide AlwNI, which can be used with vector pTRC. 25 AgeAra 5'pCCGATTAACGAACCGGTGCACGGAAC 3' AgeCar 5'pCCCTTGAATGAACCGGTGTATGGGACC 3' USE AlwNI 5'pCTAATCCTGTTACCGTTGGCTGCTGCC 3' RA1 rw I WO 99/24586 24 PCT/FR98/02391 PCR mutagenesis was used to introduce simultaneously the SalI site at the end of the gene and the AgeI site in Zea mays so as to facilitate subcloning of all the fusions in the same vector at the 5 same sites. The primers used are AgeMais and SalMaisRev. AgeMais 5'CCGCTCAACGAACCGGTGCACGGCACC 3' SalMaisRev 10 5'GCAGTTGCTCGTCGACAAGCTCTGTCC 3' After replacing the AgeI/SalI fragment of the Arabidopsis thaliana HPPD clone with the Zea mays or Daucus carota AgeI/SalI fragment, clones are obtained which encode chimeric Arabidopsis thaliana/Zea mays and 15 Arabidopsis thaliana/Daucus carota HPPDs, which are termed pFAM and pFAC, respectively (which stands for fusion of the N-terminal of Arabidopsis thaliana with a C-terminal of maize or of carrot). Similarly, clones pFCM and pFCA were obtained with the Daucus carota HPPD 20 clone. 2) in vivo activity The brown staining of the isolated colonies that reflects the HPPD activity was observed for the fusions FAM, FAC, FCA and FCM. The brown staining was 25 scored on a scale from 1 to 10, 10 being the most intense (as described in Example 1), by comparison with R4 WO 99/24586 25 PCT/FR98/02391 HPPDs derived from Arabidopsis thaliana (FAA) and Daucus carota (FCC). N-terminal C-terminal fragment fragment **C **M **A FC* 5 2 2 FA* 9 9 10 5 In general, the activity drops for each fusion in comparison with the HPPD which provides the complete N-terminal domain at the beginning of the C-terminal domain. Where the N-terminal originates from 10 Arabidopsis thaliana, the drop in activity is slight, but where the N-terminal originates from Daucus carota, the activity measured drops more markedly. However, these experiments demonstrate that the chimeric HPPDs are active enzymes and that it is 15 currently quite possible to express these chimeras in plants and, for example, to express an HPPD with an N-terminal derived from dicots and a C-terminal derived from monocots in the plants. I 4, '"

Claims (26)

1. Chimeric HPPD comprising the N-terminal portion of a first HPPD in combination with the C terminal portion of a second HPPD. 5

2. Chimeric HPPD according to Claim 1, characterized in that each portion of the chimeric HPPD is derived from an HPPD of any origin, in particular selected from amongst plant, bacterial or fungal HPPDs.

3. Chimeric HPPD according to either of 10 Claims 1 and 2, characterized in that the N-terminal portion of the chimeric HPPD is derived from a plant HPPD, this plant preferably being chosen from dicots, in particular Arabidopsis thaliana or Daucus carota, or from monocots, such as maize or wheat. 15

4. Chimeric HPPD according to one of Claims 1 to 3, characterized in that the C-terminal portion of the chimeric HPPD is derived from a plant HPPD or an HPPD of a microorganism.

5. Chimeric HPPD according to Claim 4, 20 characterized in that the plant HPPD is selected from amongst the HPPDs of dicots, in particular Arabidopsis thaliana or Daucus carota or of monocots, such as maize or wheat.

6. Chimeric HPPD according to Claim 4, 25 characterized in that the HPPD of a microorganism is a bacterial HPPD, in particular Pseudomonas, more particularly Pseudomonas fluorescens. WO 99/24586 27 PCT/FR98/02391

7. Nucleic acid sequence encoding a chimeric HPPD according to one of Claims 1 to 6.

8. Chimeric gene comprising an encoding sequence as well as heterologous regulatory elements in 5 positions 5' and 3' which can function in a host organism, in particular plant cells or plants, characterized in that the encoding sequence comprises at least one nucleic acid sequence encoding a chimeric HPPD according to Claim 7. 10

9. Chimeric gene according to Claim 8, characterized in that the host organism is selected from amongst bacteria, for example E. coli, yeasts, in particular of the genera Saccharomyces or Kluyveromyces, Pichia, fungi, in particular 15 Aspergillus, baculoviruses, or plant cells and plants.

10. Chimeric gene according to Claim 8, characterized in that the host organism is a plant cell or a plant.

11. Chimeric gene according to Claim 10, 20 characterized in that it comprises, at the 5'-position of the nucleic acid sequence encoding a chimeric HPPD, a nucleic acid sequence encoding a plant leader peptide, this sequence being located between the promoter region and the sequence encoding the chimeric 25 HPPD in such a manner as to allow expression of a fusion protein of leader peptide/chimeric HPPD. WO 99/24586 28 PCT/FR98/02391

12. Fusion protein of leader peptide/chimeric HPPD, the chimeric HPPD being defined according to one of Claims 1 to 6.

13. Cloning and/or expression vector for 5 transforming a host organism, characterized in that it contains at least one chimeric gene according to one of. Claims 8 to 11.

14. Process for the transformation of a host organism, characterized in that at least one nucleic 10 acid sequence according to Claim 7 or a chimeric gene according to one of Claims 8 to 11 is stably integrated into this host organism.

15. Process according to Claim 14, characterized in that the host organism is a plant 15 cell.

16. Process according to Claim 15, characterized in that a plant is regenerated from the transformed plant cell.

17. Transformed host organism, in particular 20 a plant cell or plant, characterized in that it contains a nucleic acid sequence according to Claim 7 or a chimeric gene according to one of Claims 8 to 11.

18. Plant cell, characterized in that it contains a nucleic acid sequence according to Claim 7 25 or a chimeric gene according to one of Claims 8 to 11.

19. Transformed plant, characterized in that it contains transformed cells according to Claim 18. WO 99/24586 29 PCT/FR98/02391

20. Plant according to Claim 19, characterized in that it is regenerated from transformed cells according to Claim 18.

21. Transformed plant, characterized in that 5 it is obtained from growing and/or hybridizing regenerated plants according to Claim 20.

22. Transformed plants according to one of Claims 19 to 21, characterized in that they are selected from amongst the monocots, in particular 10 cereals, sugar cane, rice and maize, or the dicots, in particular tobacco, soya, oilseed rape, cotton, beet and clover.

23. Seeds of transformed plants according to one of Claims 19 to 22. 15

24. Method of controlling weeds at the surface of a field with seeds according to Claim 23 or transformed plants according to one of Claims 19 to 22, the process consisting in applying, to this surface of the field, a dose of an HPPD inhibitor herbicide which 20 is toxic to these weeds, without, however, substantially affecting the seeds or plants transformed with this chimeric gene according to the invention.

25. Method of growing transformed plants according to one of Claims 19 to 22, the process 25 consisting in planting the seeds of these transformed plants according to Claim 23 at the surface of a field which is suitable for growing these plants, in WO 99/24586 30 PCT/FR98/02391 applying, if weeds are present, to this surface of this field a dose of a herbicide which targets HPPD and which is toxic to the weeds, without substantially affecting these seeds or these transformed plants, and 5 then in harvesting the grown plants when they achieve the desired maturity and, finally, in separating the seeds of the harvested plants.

26. Process according to either of Claims 24 and 25, characterized in that the HPPD inhibitor is 10 selected from amongst the isoxazoles, in particular isoxaflutole, the diketonitriles, in particular 2 cyano-3-cyclopropyl-1- (2-SO 2 CH 3 -4-CF 3 -phenyl) -propane 1,3-dione and 2-cyano-3-cyclopropyl-1- (2-SO 2 CH 3 -4-2,3 C1 2 -phenyl)-propane-1,3-dione, the triketones, in 15 particular sulcotrione, or the pyrazolinates. WO 99/24586 PCT/FR98/02391 Ii I~O I b Li . 04I ali m 1ai -t o~~t A) 0 )0a0 V)0) 4- 0 a -0) 440 0) 0 4-0 )0)H ) -~1 0 ()H )to10 0 C H 0) '4) t~4O -H0) U ) -~40)u0 0 4 -H 00) 00 )4)Q )~ u - W00 u) S a 0 ) 4-)0 4a) O)HO -0 a 0~ )- 01H0 0 O)-H.0S-H 0 -ri 0 0 ). -J -H (t0 0)0 .4 0 0 -,I )O)-J 0 -100 4- 00 0 4-4 001 (a0) 4-4 0 0 4- ) 0 4-4 0))004.4U)0)4-40U) U) to -1 0) )04 U)) 0)0044))4-4) (D w )4 0 w w 0) 00i) (1)4-V ) 00w>w,00)4w 4w0 W MO 4 -1 4 (1 Cf) A 4 1 /5U M U) - 0 r - WO 99/24586 PCT/FR98/02391 W.~ 1% :0 ;m 14 va c a "I ~0 C I 1444 a d 0 ~~O a 40)~ba 0 Ai A U 4A CA at-S 49 -4 (0 o 06 V4 0 0 20 0 0 - ) 4-4 (1) IL H H 4 (12- (0 -H-~ U) (-H 4- U) rq z -H () .,-J M li 1 z4 ara : 41 (a (a0 ~ 5(1 (L)( 0 rl() ) ( ) 0 C -I ( -1ir 4 0o 'H 4 (0 (0 4 0o 'HI (0 (0 ro 01 020 'H0 (o 4-10 ) 02044 -1 ( 4-1 ) (a 44r--I0 (o 4- 0 (a 44 l :j W (1 - -A HU) 0 Q ) u(0) )rq U a ) u :3 (12 -H1A UH0( 02 'H)~ H 0(1 r-HG)D> U (a >- . a) 03--HG() > 02(0Z)-, a) 03 -HGW) W 0(3>1r a) 02-H.5-H 0 0 E 0 0-H.05rz-HO0 0 l 0 U) -H.05- -H 0 0 E 0 0 04 M 0 U)4-'0 0 : 0 Q0 (0 0 U) J0 0 :: : 0 Q 0 0 0 U) 4-)0 :: u00-0 4 u0()0 '4-4 0 0 -Q0G0 '4-4 U00"o .00 w):J L4-4 :50 >1 0 W P(0 4- U 2 00>,0G) 0 M J W2: 0 >10 W 4 4-) 2/5 WO 99/24586 PCT/FR98/02391 a~> Ii IN~~iiaa - 4M 4M411 auaaa C . 3::% x~fhU a 00 5l rd W a~ ~ lc 0000000 m Wa W Cc t 0 a) 01 0a -H a a 0)) 0 00 )0 '-4 ( 4o i U $- 4 c r 0 4 ( - l U)U 10 J U ) U0 r- y P U ) U) - 0 ) - U) (0 -)0 a) U ) )A U 0 4)U H )(1) U 0 0 *H)U) 0 -H -H H- 0 (a HU -H 4 l -- 4 0 ( - '0-H4 -H 4 U 0 -H u 0 (aH0 0, r:- 0 *Hu0m U)0 g0 4 0 -HU)O4F) U) -H : 0 -0 r4 0 U -,A-H 0 0 ri 0 U) -H 0 0 r: 0 05 *HQ4U) *H -00 : 0 *HU 4) 0 0 * 5 3 0 4 0 HU)0 -4,0 U 0)50 -H U) 1) 0)4-H : 0) -0 14 r= -1 ) 0 4 0 -H o4- 0: 4-4u _ -H u 4)0j440 00 j 4 'H 0) 0i 0) 0 'H M) -0W j0 > 4m4 : i0 Q 4M4 C -Z g0 0 )' U) 04- 0 )4 U) -H0) 10 )4 U) L -40 4J0) 4- 0-40 -0) 4-4 0 40 43/54 WO 99/24586 PCT/FR98/02391 00 4-)g sE U) *I o 4r C i) U) ) -1 0 1 4-J -) 0 (L 0 a)U) -i c E -H 0 o E 0 -0 0 , U) 4- 0 7$ 0 4 -0 (0 (1) 4- U ZJi U04)L 0WW~-1 O~nI4/5