CA2309880A1 - Chimera gene having a light dependent promoter providing tolerance to hppd inhibitors - Google Patents

Abstract

The invention concerns a chimera gene comprising at least an elementary chimera gene including, in the transcriptional direction, elements regulating in 5' necessary for its transcription into plants, at least one heterologous coding portion comprising a sequence coding for an enzyme providing plants with tolerance to herbicides inhibitors of HPPD and at least one terminator or polyadenylation regulating sequence, wherein the elements regulating in 5' ensure the transcription of the elementary chimera gene at the level of chlorophyllian tissues. The invention also concerns the transformation of plants and the plants transformed with said chimera gene. It further concerns a method for growing transformed plants which consists in applying a HPPD inhibitor for controlling weeds.

Description

Chimeric gene before a dependent light promoter conferring tolerance to HPPD inhibitors The subject of the present invention is a chimeric gene having a light promoter dependent operably linked to a gene coding for HPPD {hydroxy phenyl pyruvate dioxygenase), providing improved tolerance to herbicides fHPPD inhibitors to a plant cell and a plant normally sensitive.
The invention also relates to transformed plant cells and palntes.
with the chimeric gene according to the invention, a process for transforming cells plants and plants and a method for growing transformed plants in which apply a HPPD inhibitor herbicide for weed control.
Herbicides targeting HPPD are in particular isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, US 5 424 276) in in particular isoxaflutole, a selective herbicide for corn, diketonitriles (EP
496 630, EP
496 631), in particular 2-cyano-3-cyclopropyl-1- (2-S02 CH3-4-CF3 phenyl) propane-1,3-dione and 2-cyano-3-cyclopropyl-1- (2-S02 CH3-4-2,3 Cl2 phenyl) propane-1, 3-dione, triketones (EP 625 505, EP 625 508, US 5,506,195), in especially sulcotrione or pyrazolinates. Genes encoding an HPPD
imparting tolerance to these herbicides and transgenic plants containing this gene show significant tolerance to said herbicides are described in the request WO 96/38567 and in patent application FR 97 14264 filed on 7 November 1997, the content of which is included here by reference.
Until now all the tests carried out to confer a good tolerance to fHPPD inhibitors have been using "constitutive" promoters or allowing expression in all kinds of plant tissue, tissue root, tissue leaves, areas growing more or less quickly. To do this plants dicots like tobacco, Arabidopsis thaliana, rapeseed and soybeans have summer transformed either with:
- the pRP T chimeric gene Double histone promoter I TEV I Coding region of HPPD I Terminator nos - the chimney supper pRP V
Double histone promoter I TEV I OTP I Coding meeting of HPPD I Terminator our These tests allowed us to confirm that this type of expression allows in the broadleaf weeds get very good tolerance.

2 Monocotyledonous plants such as mace have also been transformed with the chimeric gene pRPA-RD-1004 (patent application PCT / FR97 / 01256 filed on July 1997):
histoneIntron promoter OTP Terminator coding region 1 of the HPPD nos H3C4 from mas Adhl We have now found that we could confer on a plant cell and a 5 plants improved herbicide tolerance by over-expression of fHPPD by through a "light dependent" promoter (hereinafter called LD promoter).
The use of these LD promoters leads to the expression of the chimeric gene in the chlorophyll tissues or "green tissues" without any expression in the fabrics no chlorophyll or very weak expression in non-tissue chlorophyllians like 10 root tissue, such expression being sufficient to enhance tolerance of plants transformed with herbicides that inhibit HPPD.
The present invention therefore firstly relates to a chimeric gene comprising at least minus one elementary chimeric gene containing, in the sense of transcription, S 'regulatory elements necessary for its transcription in plants, at least a heterologous coding part comprising a coding sequence coding for a enzyme conferring on plants tolerance to HPPD inhibitor herbicides and at minus a terminating regulatory or polyadenylation sequence, the elements of 5 'regulation ensuring the transcription of the elementary chimeric gene at level of chlorophyll tissues, the so-called 5 'regulatory elements comprising preference at least one promoter regulatory sequence of the LD promoter type.
By "LD promoter" is meant according to the invention any gene promoter coding for peptides whose transcription is induced by light which East functional as a promoter in plant cells. These LD promoters can be of bacterial, viral or vegetable origin. Such promoters are notably described by Terzaghi & coll. (Light-regulated transcription, Annu. Rev. Plant Physiol.
Plant Mol. Biol., 1995, 46: 445-474) the content of which is incorporated herein by reference.
As useful LD promoter according to the invention, mention will be made more particularly of promoter of a gene for the small plant, ribulose-biscarboxylase (rbcs) subunit protein "Light-harvesting chlrorophyll a / b binding" (LHCP), plastocyanine (pet E ~ and phenylalanine ammonia lyase (pal). Preferably we use a sequence of promoter regulation which promotes overexpression of the coding sequence in the chlorophyll tissues, such as, for example, that comprising at least one fragment promoter of the small rbcs subunit (SSU) of a plant plus particularly isolated from Heliauthus annuus as described in the US patent 5,559,024.

3 ~ VO 99/25842 PCT / FR98 / 02414 Among the SSU promoters, we can also cite the SSU promoter of petunia describes in US patent 4,962,028.
By functional fragment is meant according to the invention any DNA sequence from the SSU promoter sequence reproducing the function of the sequence where she is from.
According to a preferred embodiment of the invention, the sequence of the SSU promoter of Heliauthus annuus comprises the DNA sequence represented by sequence identifier no.1 (SEQ ID NO: 1) or a sequence counterpart of said sequence. More preferably, the sequence of the SSU promoter consists of the DNA sequence represented by sequence identifier # 1.
By "homologous" is meant according to the invention a DNA sequence having one or more sequence changes from the DNA sequence of reference described by sequence identifier No. 1, and reproducing the function of this sequence. These modifications can be obtained according to the techniques usual of mutation, or by choosing the synthetic oligonucleotides that can to be employed in the preparation of said sequence by hybridization. So advantageous, the degree of homology will be at least 70% compared to the sequence of reference, preferably at least 80%, more preferably at least 90%.
By "plant cell" is meant according to the invention any cell originating from a monocotyledonous plant and which can constitute undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of plants monocotyledons, monocotyledonous plants or seeds.
The term "plant" according to the invention means any multicellular organism differentiated capable of photosynthesis, especially plants monocotyledons or dicotyledons, preferably cultured or not intended for animal or human food, such as wheat, barley, oats, rice, corn, sorghum, sugar cane, soy, rapeseed, cotton, tobacco, beet or still plants of vegetable or floral cultures.
According to the invention, it is also possible to use, in combination with the sequence of LD type promoter regulation, other regulatory sequences, which are located between the promoter and the coding sequence, such as activators of trancription "enhancer", such as the translation activator for the etch virus tobacco (VTE) described in the article by Carrington and Freed, 1990; J. Virol. 64: 1590-1597, or some sequences encoding transit peptides, either single or double, and in that case possibly separated by an intermediate sequence, i.e.
including, in the sense of transcription, a sequence coding for a transit peptide of a uncomfortable plant coding for a plastid localized enzyme, part of sequence of the mature N-terminal part of a plant gene encoding an enzyme to location

4 plastidiale, then a sequence coding for a second transit peptide of a uncomfortable plant coding for a plastid localization enzyme, consisting of part of sequence of the mature N terminal part of a plant gene coding for a enzyme to plastid localization, as described in the patent application EP
508 909.
As coding sequence for an enzyme which confers tolerance on plants with herbicides that inhibit HPPD, it is possible in particular to use all those known to confer plant tolerance to certain HPPD inhibitors such as that sequences coding for an HPPD described in patent application WO 96/38567 and in patent application FR 9714264 filed on November 7, 1997. This HPPD
can be of any kind.
More particularly, this sequence can be of bacterial origin, such than in particular the genus Pseudomonas or of plant origin, such as especially from monocotyledonous or dicotyledonous plant, in particular Arabidopsis or umbelliferae such as the carrot (Daucus carota). She can be native or wild or possibly mutated while basically keeping a property of tolerance herbicide against fHPPD inhibitors, such as family of isoxazoles or that of triketones or pyrazinolates.
As a regulatory or polyadenylation sequence, it is possible to use any corresponding sequence of bacterial origin, such as example the terminator nos of Agrobacterium tumefaciens, or of plant origin, like example a histone terminator as described in the European application EP

317.
The present invention also relates to a cloning or expression vector for the transformation of a plant cell or a monocotyledonous plant or dicotyledonous. The vector according to the invention comprises, in addition to the chimeric gene ci-above, at minus an origin of replication. This vector can consist of a plasmid, a cosmid, a bacteriophage or a virus, transformed by the introduction of the gene chimera according to the invention. Such plant cell transformation vectors and of plants monocotyledons are well known to those skilled in the art and widely described in the literature. Preferably, the transformation vector of plant cells or plants according to the invention is a plasmid.
The invention also relates to a method for transforming cells.
plants by integration of at least one nucleic acid fragment or a gene chimera as defined above, transformation which can be obtained by any known means suitable with the vector according to the invention.
A series of methods involves bombarding cells or protoplasts with particles to which the DNA sequences are attached. Another series of methods consists in using a gene as a means of transferring into the plant chimera inserted into a Ti plasmid of Agrobacterium tumefaciens or Ri of Agrobacterium rhizogenes. Other methods can be used such as micro-injection or electroporation, or even direct precipitation using PEG.
Those skilled in the art will choose the appropriate method depending on the

5 nature of the plant cell or plant.
The present invention also relates to plant cells or plants, herbicide tolerant transformants targeting HPPD and containing less a chimeric gene according to the invention defined above.
The present invention also relates to plants containing cells transformed, especially plants regenerated from cells transformed. The regeneration is obtained by any suitable process which depends on the nature of the species.
For plant cell transformation and regeneration processes of plants, in particular the following patents and patent applications: US
4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267,159, EP 604 662, EP 672,752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US
5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US
5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442 174, EP
486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 and WO 95/06128.
The present invention also relates to the transformed plants derived from the cultivation and / or crossing of the above regenerated plants, as well as seeds transformed plants.
The present invention also relates to a method for controlling bad grasses in a surface of a field including seeds or plants transformed with the chimeric gene according to the invention, which method consists in apply in the said field surface a toxic dose for the said bad herbs of a herbicide targeting HPPD, without however affecting way substantial seeds or plants transformed with the so-called chimeric gene according to the invention.
The present invention also relates to a method for cultivating plants.
transformed according to the invention with a chimeric gene according to the invention which process consists in planting the seeds of said transformed plants in a surface of a field suitable for growing said plants, to be applied to said said surface field a weed toxic dose of a herbicide with the target the HPPD defined above in the presence of weeds, without affecting of substantially said seeds or said transformed plants, then to harvest the plants cultivated when they reach the desired maturity and possibly at separate the seeds from the harvested plants.

6 In the above two processes, the application of the herbicide having as its target HPPD can be made according to the invention, both in pre-planting, pre-emergence and post-emergence of the culture.
By herbicide within the meaning of the present invention means an active material herbicide alone or combined with an additive which modifies its effectiveness as for example an agent increasing (activity (synergistic) or limiting activity (in English safener).
Of course, for their practical application, the above herbicides are associate of manner known per se to the adjuvants of formulations usually used in agrochemical The various aspects of the invention will be better understood using the examples below.
Example 1: Construction of a chimeric gene with an HPPD sequence and a LD promoter.
To confer plant tolerance to herbicides that inhibit HPPD, builds a chimeric gene called pRPA-RD-2005:
It consists in putting the coding part of the fHPPD gene of P. fluorescens A32 under the control of the promoter of the small, isolated RuBisCO subunit of heliauthus annuus (US 5,559,024), of the optimized transit peptide (OTP) (EP 508,909), even monitoring of the fHPPD coding region of Pseudomonas fluorescens (WO 96/38567) at its turn followed by the terminator nos of Agrobacterium tumefaciens. The HPPD will so localized in the chloroplast and the gene is mainly expressed in fabrics chlorophyllians.
PRPA-RD-2005 is a binary vector of pRPA-BL-150Aa2 type (EP 508 909) containing an fHPPD expression cassette: promoter of small sub unit of ribulose discarboxylase-OTP-HPPD gene-terminator nos -To build it, we used pRPA-RD 2004 which contains the cassette fHPPD expression Construction of pRPA-RD-2004 - pRD-207 is a pBluescript SK (-) (stratagene catalog # 21 2206) containing the nopaline synthase gene (nos terminator). PRD-207 is used as vector for the construction of pRPA-RD-2004 - pRD-208 contains the OTP / HPPD cassette: nos. It is obtained from plasmid pRPA S, described in WO 96/38567, digested with XbaI / CIaI, treatment with polymerase pfu type. The cassette is introduced into the open pRD-207 by digestion SaII, klenow treatment.
- The promoter of the small subunit of ribulose biscarboxylase of heliauthus annuus (SSU HA; SEQ ID NO: 1) comes from the plasmid pRD-127 described in WO

7 96/38567 digested NcoI / XbaI is introduced into pRD-208. This construction constitutes pRPA-RD-2004 Construction of PDR-2005 - To build pRPA-RD-2005, we open the vector pRPA-BL-150Aa2 by XbaI / HindIII restriction enzymes to which the cassette is inserted expression of the HPPD of pRPA-RD-2004 (described above) by the same enzymes Its structure is therefore:
Promoter SSU OTP fHPPD coding region Terminator nos Example 2: Processing of PBD6 industrial tobacco.
In order to determine the effectiveness of this chimeric gene of Example 1, it was transferred to PBD6 industrial tobacco according to processing procedures and of regeneration already described in patent application EP 508 909.
1) Transformation:
The vector is introduced into the non-oncogenic strain of Agrobacterium EHA 101 or LBA 4404 (flood and A1,1987) carrying the cosmid pTVK 291 (Komari and a1.1986). The transformation technique is based on the procedure of Horsh R. et al.
(1985) Science, 227, 1229-1231.
2} D; ~ generation:
The regeneration of PBD6 tobacco (from SEITA France) from explants foliar is carried out on a Murashige and Skoog (MS) base medium including 30g / 1 sucrose as well as 350 mg / 1 cefotaxime and 1 mg / 1 diketonitrile deriving from fisoxaflutole or 10 mg / 1 of 1- [4- (trifluoromethyl) -2- (methylsulfonyl) phenyl] -2-cyano-3- (1-methylcyclopropyl) -propan-1,3-dione, another inhibitor of HPPD. The explants leaves are taken from plants in the greenhouse or in vitro and transformed according to technique of leaf discs (Science 1985, Vo1 227, p.1229-1231.) in three steps successive: the first includes the induction of shoots on an MS medium added 30g / 1 sucrose containing O.OSmg / 1 naphthylacetic acid (ANA) and 2 mg / 1 of benzylaminopurine (BAP) for 15 days and 1 mg / 1 of isoxaflutole. The shoots green formed during this stage are then developed by culture on a middle MS
supplemented with 30 g / 1 of sucrose and 1 mg / 1 of isoxaflutole or 10 mg / 1 of 1- [4-(trifluoromethyl) -2- (methylsulfonyl) phenyl] -2-cyano-3- (1-methylcyclopropyl) -propan-1,3-dione but not containing hormone, for 10 days. Then we take of sprouts developed and cultivated on a medium of MS rooting content half 3 S in salts, vitamins and sugars and 1 mg / 1 of isoxaflutole or 10 mg / I of 1- [4-(trifluoromethyl) -

8 2- (methylsulfonyl) phenyl] -2-cyano-3- (I-methylcyclopropyl) -propan-1,3-dione and born containing no hormone. After about 15 days, the rooted shoots are passed into the earth.
EXAMPLE 3 Tolerance of Tobacco Transformed with the Chimeric Gene pItPA-RD-2005 Leaving the in vitro step of Example 2, the tobacco seedlings transformed have been acclimatized in the greenhouse (60% relative humidity; temperature:
20 ° C at night and 23 ° C the day) for three weeks then treated with 4- [4-(trifluoromethyl) -2-(methylsulfonyl) benzoyl] -5-cyclopropylisoxazole (isoxafutole).
IO Control tobacco, unprocessed and treated with isoxafutole at doses of 400 g / ha, develops chloroses in about 72 hours, which intensify to evolve towards very pronounced necrosis in a week (covering about 80% of the leaves final).
The corresponding processed tobacco, hereafter tobacco 2005, which overexpresses HPPD
of P. fluorescens, is very well protected against treatment with isoxafutole at the dose of 400 g / ha.
Example 4: Greenhouse tolerance of tobacco 2005 In this example we compare the greenhouse tolerance of 2005 tobacco to that of COl I tobacco, transformed with the pRPA-V chimeric gene described in the application for patent WO 96/38567 and that of unprocessed tobacco.
Description of tobacco trials.
Seeds of each of these types of tobacco are sown in the ground and the day even a treatment with isoxaflutole doses of 0/30/200/400 g / ha applied so in pre-emergence are carried out.
Buildings tested Coll: Double histone ~ TEV ~ OTP ~ HPPD Pseudomonas Nos 2005: SSU ~ OTP ~ HPPD Pseudomonas Nos PBD6: not transformed.
Balance sheet For PBD6 tobacco, germination is normally at 0 g / ha and no germination takes place from the dose of 30 g / ha. If by chance a seedling gets up she is white and dies very quickly.
For seeds from a CO I 1 tobacco, the emergence is normal and without symptoms phytotoxicity for doses ranging from 0 to 200 g / ha. At 400 g / ha, the seeds germinate, emergence takes place normally but there is stunting or "stunting ", fairly clear compared to untreated plants.

9 For 2005 tobacco seeds, emergence is normal and without symptoms phytotoxicity for doses ranging from 0 to 400 g / ha. None of the three doses of isoxaflutole, in a greenhouse, does not cause symptoms of phytotoxicity or "
stunting ".
Example 5: Tolerance in the field of tobacco tobacco 2005 The comparative tolerance tests of Example 4 are reproduced in the field for the same tobacco 2005, CO11 and PBD6.
Description of tobacco trials.
Young seedlings were transplanted individually in minimottes to be transplanted a second time in the field. Processing post was a week later with doses of isoxaflutole 0/100/200/300/400/500/600 g / ha.
Balance sheet The diagram below comparing CO11 and 2005 tobacco, shows clearly that the promoter SSU confers better tolerance than double histone associated with TEV "enhancer".
~ 2005 ~ COl 1 ~ Wild type I

, ~ 90 '~~ 70 ~, 30 oh 10 Thus, 13 days after treatment at 400g / ha, 15% phytotoxicity is observed sure CO11 tobacco while 2005 tobacco has little or no phytotoxicity.
Similarly, 20 days after a 400g / ha treatment, more than 20% of 20 phytotoxicity on CO11 tobacco whereas 2005 tobacco presents less than 10% of phytotoxicity.
Under field culture conditions, plants transformed with the uncomfortable pRPA-RD-2005 including the promoter SSU have better tolerance to fHPPD inhibitors than those transformed with the pRPA-V gene including double histone promoter combined with TEV "enhancer" of the state of technical.

LIST OF SEQUENCES
(1) GENERAL INFORMATION:
(i) DEPOSITOR:
(A) NAME: RHONE-POULENC AGROCHEMISTRY
(B) STREET: 14-20 Rue Pierre BAIZET
(C) CITY: LYON
(E) COUNTRY: France (F) POSTAL CODE: 69009 (ii) TITLE OF THE INVENTION: Chimeric gene having a light promoter dependent conferring tolerance to HPPD inhibitors (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER-DETACHABLE FORM:
(A) TYPE OF SUPPORT: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 766 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomics) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

Claims (20)

Claims 1. Chimeric gene comprising at least one elementary chimeric gene comprising, in the direction of transcription, 5' regulatory elements required to its transcription in plants, at least one heterologous coding part including a coding sequence encoding an enzyme conferring tolerance to plants to the HPPD inhibitor herbicides and at least one regulatory sequence terminator or for polyadenylation, characterized in that the regulatory elements at 5' ensure the transcription of the elementary chimeric gene at the tissue level chlorophyllians. 2. chimeric gene according to claim 1, characterized in that said 5' regulatory elements include at least one regulatory sequence LD promoter-like promoter. 3. chimeric gene according to claim 2, characterized in that the LD promoter is of bacterial, viral or plant origin. 4. chimeric gene according to claim 3, characterized in that the LD promoter is selected from a functional sequence of the promoter of a gene small subunit of plant RuBisCO, light-harvesting chlrorophyll a/b binding protein (LHCP), plastocyanin (pet E) and phenylalanine ammonia lyase (pal). 5. chimeric gene according to claim 4, characterized in that the LD promoter comprises a functional fragment of the promoter of the small sub unity of the RuBisCO of a plant. 6. chimeric gene according to claim 5, characterized in that the LD promoter comprises a functional functional fragment of the promoter of the small RuBisCO subunit of Helianthus annuus. 7. chimeric gene according to claim 6, characterized in that the LD promoter comprises the DNA sequence represented by the identifier of sequence No. 1 (SEQ ID NO:1) or a homologous sequence of said sequence. 8. chimeric gene according to one of claims 1 to 7, characterized in that the elementary chimeric gene comprises in association with the elements of regulation in 5' at least one regulatory sequence, located between the promoter and the sequence coding, chosen from transcription activators and/or sequences coding for transit peptides. 9. Chimeric gene according to one of claims 1 to 8, characterized in that the coding sequence for an enzyme conferring on plants tolerance to herbicides HPPD inhibitors is chosen from sequences encoding an HPPD
original bacterial or vegetable origin. 10. Cloning or expression vector for transformation of a cell plant or of a monocotyledonous or dicotyledonous plant, characterized in that that he further comprises the chimeric gene according to one of claims 1 to 9, at least a origin of replication. 11. Vector according to claim 10, characterized in that it is a plasmid. 12. Process for transforming plant cells, characterized in that integrating into said plant cell at least one chimeric gene according to one from claims 1 to 9. 13. Transformed Herbicide Tolerant Plant Cell Targeting HPPD, characterized in that it comprises at least one chimeric gene according to one of claims 1 to 9. 14. Transformed plant tolerant to herbicides targeting HPPD, characterized in that it comprises cells transformed according to the claim 13. 15. Transformed plant according to claim 14, characterized in that it is regenerated regenerated from transformed cellsaccording to the claim 14. 16. Transformed plant tolerant to herbicides targeting HPPD, characterized in that it is derived from the cultivation and/or crossing of plants regenerated according to claim 15. 17. Seeds of transformed plants according to one of claims 14 to 16. 18. Method for controlling weeds in an area of a field comprising seeds or plants transformed according to one of claims 14 to 17, characterized in that one applies to the said surface of the field a dose toxic for said weeds of a herbicide targeting HPPD, without Nevertheless substantially affect said seeds or transformed plants. 19. Process for growing plants transformed according to one of claims 14 to 16, characterized in that it consists in planting the seeds according to claim 17 in an area of a field suitable for the cultivation of say transformed plants, to be applied to said surface of said field a dose toxic to weeds of an HPPD-targeted herbicide if present of weeds, without substantially affecting said seeds or say them transformed plants, then harvesting the cultivated transformed plants when they reach the desired maturity and eventually separate the seeds from the plants transformed harvested. 20. Method according to one of claims 18 or 19, characterized in that the HPPD-targeted herbicide is applied pre-plant and/or withdrawn and/or post-emergence of the crop.