WO2002006443A2 - Lipoxygenase-inducible promoter, expression cassettes comprising same and transformed plants - Google Patents

Abstract

The invention concerns novel polynucleotides comprising a novel regulating sequence promoting a tobacco lipoxygenase gene inducible by an inducer and in particular in response to a stress by a pathogen. The invention also concerns an expression cassette comprising said regulating promoter sequence. The invention further concerns plant cells or plants transformed with said inventive polynucleotide or expression cassette.

Description

 Inducible lipoxy enase promoter, expression cassettes comprising it and transformed plants

The present invention relates to polynucleotides comprising a novel promoter regulatory sequence inducible in response to aggression by a pathogen or in response to another inducing agent. The present invention also relates to expression cassettes and vectors comprising a promoter regulatory sequence according to the invention which controls the expression of a coding sequence and in particular of a heterologous coding sequence, heterologous meaning here a coding sequence different from the native coding sequence. The invention also relates to a host organism and in particular plant cells and plants transformed with a polynucleotide, an expression cassette or a vector according to the invention.

Plant lipoxygenases (LOXs) are involved in various physiological processes and these functions can be performed by different isoenzymes. This diversity in biological functions also results in regulatory mechanisms as well as different tissue and subcellular locations depending on the isoforms. An important role is attributed to LOXs in stress responses, in particular injury, and parasitic attacks (Blée, Prog. Lipid Res., 1: 33-37, 1998). A strong induction of expression of LOX genes is thus measured in many monocotyledonous or dicotyledonous plants interacting with bacteria, viruses or fungi, as well as following mechanical injuries or caused by insects on the leaves. A cDNA and a genomic fragment encoding a tobacco LOX has been cloned (Rancé, Doctoral Thesis of the University of Paris XI, 1997; Véronési et al., Plant Physiol., 108: 1342, 1995; GenbankX84040). DNA / RNA hybridization experiments (northern hybridization) have shown that this LOX gene is expressed in tobacco cells in culture following the application of elicitor and in tobacco plants inoculated with the fungus Phytophthora parasitica nicotianae, Ppn (Véronési et al., Plant Physiol., 112: 997-1004, 1996). In Arabidopsis thaliana, there are at least two genes encoding LOXs. The _4tLOXl gene is constitutively expressed in all plant organs with higher expression levels in the roots and in young germinations (Melan et al, Plant Physiol., 101: 441-450, 1993). Strong induction of ^ tLOXl activity is detected following infection with Pseudomonas syringae. A differential accumulation of LOX transcripts is highlighted between a compatible or incompatible interaction. The induction of gene expression is indeed greater and earlier after inoculation with an avirulent strain. Furthermore, it has been shown that the expression of the _4tLOXl gene was inducible after treatment with MeJa (methyl jasmonate) or with abscissic acid. Another _4tLOX2 gene, divergent in terms of its peptide sequence (approximately 40% similarity with the protein sequences of Arabidopsis _4tLOXl and of tobacco NtLOXl) and having a sequence of addressing to chloroplasts, is preferentially involved in the synthesis of acid. jasmonic in response to the injury (Bell et al, PΝAS, 92: 8675-8679, 1995). Complementary DNA coding for LOXs has been isolated in particular from potatoes, tomatoes, rice, barley, soybeans, peas and beans.

However, a limited number of LOXs genes specifically involved in the resistance mechanisms of plants have been isolated. Consequently, the number of promoters of LOX genes from characterized plants is limited compared with the numerous clones of cDNA isolated. The promoter of the _4tLOX1 gene induced by the pathogenesis has not been isolated and characterized and the promoters of LOX genes isolated so far relate to LOXs normally expressed in the seeds, during germination or ripening of the fruits. Rouster et al. (Plant J., 11: 513-523, 1997) have thus isolated the LOXl promoter from barley, normally active in germination and in leaves treated with MeJa. The pea LOX2 promoter has also been cloned and used for expression of the reporter gene uidA in transgenic tobacco plants (Forster et al., Plant Mol. Biol., 26: 235-248, 1994). The results show high levels of expression in seeds, leaves and stems. The cloned promoter therefore does not have any marked tissue specificity whereas the pea LOX2 gene is preferentially expressed in the seeds. Promoters of two LOX genes from tomatoes which are normally expressed during fruit ripening have also been isolated (Beaudoin and Rothstein, Plant Mol. Biol., 33: 835-846, 1997). LOX promoters with specific activity in plant defense and resistance mechanisms have not yet been isolated and characterized.

The present invention relates to a tobacco lipoxygenase 1 promoter. This promoter has a specific expression profile of the defense mechanisms of plants, it is activated in response to an inducing agent and in particular in response to the aggression of a plant by a pathogenic agent.

Description of the sequence list

SEQ ID NO.l: Promoter of tobacco lipoxy genase 1

SEQ ID NO. 2-3: PCR primers

Description of the Invention Polynucleotides

The present invention relates to novel polynucleotides comprising a promoter regulatory sequence functional in plants.

By promoter regulatory sequence is meant according to the invention the non-coding region of a gene involved in the binding with RNA polymerase and with other factors which are responsible for the initiation and regulation of the transcription leading to to the production of an RNA transcript.

According to the present invention, the term “polynucleotide” means a single-stranded nucleotide chain or its complement or a double-stranded nucleotide chain which may be of DNA or RNA type. Preferably, the polynucleotides of the invention are of the DNA type, in particular double stranded DNA. The term

"polynucleotide" also refers to the modified oligonucleotides and polynucleotides.

The polynucleotides of the present invention are isolated or purified from their natural environment. Preferably, the polynucleotides of the present invention can be prepared by standard molecular biology techniques as described by Sambrook et al. (see Sambrook et al., Molecular Cloning: A Labratory Manual, 1989) or by biochemical synthesis.

In a first embodiment, the invention relates to a polynucleotide comprising a plant promoter regulatory sequence of SEQ ID No. 1. In another embodiment, the invention also relates to polynucleotides comprising a polynucleotide chosen from the following polynucleotides: a) a polynucleotide capable of hybridizing selectively to the polynucleotide of SEQ ID No. 1; b) a polynucleotide homologous to the polynucleotide of SEQ ID No. 1.

By "homologous" is meant according to the invention a polynucleotide having one or more sequence modifications compared to the reference sequence. These modifications can be deletions, additions or substitutions of one or more nucleotides of the reference sequence. Advantageously, the percentage of homology will be at least 70%, 75%, 80%, 85%, 90%, 95% and preferably at least 98% and more preferably at least 99% by compared to the reference sequence. The methods for measuring and identifying the homologies between the nucleic acid sequences are well known to those skilled in the art. One can use for example the PILEUP or BLAST programs (in particular Altschul et al., J. Mol.Evol. 36: 290-300, 1993; Altschul et al., J.Mol.Biol. 215: 403-10, 1990) . Preferably we will use the default settings. The invention therefore relates to polynucleotides comprising polynucleotides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% and preferably at least 98% and more preferably at least 99% homology with the polynucleotide of SEQ ID No. 1. Preferably the invention relates to a polynucleotide comprising a polynucleotide of at least 50, 100, 200, 300, 400, 500, 1000 nucleotides having at least 70%, 75%, 80 %, 85%, 90%, 95%, 98% and preferably at least 98% and more preferably at least 99% of homology with the polynucleotide of SEQ ID No. 1. Preferably, polynucleotides homologous to a reference polynucleotide retain the function of the reference sequence.

By "sequence capable of selective hybridization" is meant according to the invention the sequences which hybridize with the reference sequence at a level above the background noise significantly. The level of the signal generated by the interaction between the sequence capable of selective hybridization and the reference sequences is generally 10 times, preferably 100 times more intense than that of the interaction of the other DNA sequences generating the background noise. The stringent hybridization conditions allowing selective hybridization are well known to those skilled in the art. In general, the hybridization and washing temperature is at least 5 ° C. lower than the Tm of the reference sequence at a given pH and for a given ionic strength. Typically the hybridization temperature is at least 30 ° C. for a polynucleotide of 15 to 50 nucleotides and at least 60 ° C for a polynucleotide of more than 50 nucleotides. As an example, hybridization is carried out in the following buffer: 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, 500 μg / ml denatured salmon sperm DNA. The washes are for example carried out successively at low stringency in a 2X SSC buffer, 0.1% SDS, at medium stringency in a 0.5X SSC buffer, 01% SDS and at high stringency in a 0.1X SSC buffer, 0, 1% SDS. Hybridization can of course be carried out according to other usual methods well known to those skilled in the art (see in particular Sambrook et al., Molecular Cloning: A Labratory Manual, 1989). The invention therefore relates to polynucleotides comprising a polynucleotide capable of selectively hybridizing with the polynucleotide of SEQ ID No. 1. Preferably, the invention relates to a polynucleotide comprising a polynucleotide of at least 50, 100, 200 , 300, 400, 500, 1000 nucleotides capable of selectively hybridizing with the polynucleotide of SEQ ID No. 1. Preferably, the polynucleotides selectively hybridizing to a reference polynucleotide retain the function of the sequence reference.

In a third embodiment, the invention relates to a biologically active fragment of the polynucleotide according to SEQ ID No. 1.

By “biologically active fragment” is meant above a polynucleotide having a promoter activity or a transcription regulation activity in particular in response to attacks by a pathogenic agent or by another agent inducing a defense reaction of plants.

In a preferred embodiment, the polynucleotides of the present invention comprise at least one CAAT box (positions 2055-2059 of SEQ ID No. 1) and at least one TATA box (positions 2178-2185 of SEQ ID No. 1 ). Preferably, the polynucleotides according to the invention also comprise regulatory elements, in particular at least one G box (positions 1989-1992, 1847-1850, 1636-1639, 1162-1165, 1038-1041, 189-192, 67- 70 of SEQ ID No. 1) and / or at least one ERE box. Preferably, the polynucleotides of the present invention comprise at least one polynucleotide chosen from the following polynucleotides: a) a polynucleotide whose sequence is between position 1938 and position 1945 of SEQ ID No. 1; and b) a polynucleotide whose sequence is between position 576 and position 584 of SEQ ID No. 1; and c) a polynucleotide whose sequence is between position 513 and position 520 of SEQ ID No. 1. In a preferred embodiment, the polynucleotides of the present invention comprise a polynucleotide whose sequence is between position 821 and position 875 of SEQ ID No.l.

In a preferred embodiment, the polynucleotides of the present invention have promoter activity in plants. Advantageously, the promoter activity is not detected in healthy plants.

In one embodiment, the polynucleotides of the present invention have promoter activity in certain healthy plant organs important for plant survival and persistence. In particular, the promoter activity of the polynucleotides of the present invention is detected in the cotyledons of the embryo, in the hypocotyle and the cotyledonary leaves during germination, in the region of the collar and in the petioles of the leaves of growing plants and adult plants, as well as in the flower's reproductive organs and in the flower's petals. This tissue expression profile in organs essential for the survival and persistence of the plant is consistent with a role of defense and protection. In particular, activity is detected in the abscission zones of the leaves and flowers.

Preferably, the polynucleotides of the present invention have an activity which promotes and regulates expression in relation to the defense mechanisms of plants and to pathogenesis. Preferably, this promoter activity is induced in response to an inducing agent and in particular in response to an attack by a pathogen or by a chemical agent. By "inducing agent" is meant any chemical agent as well as any agent produced by a pathogen capable of causing a defense reaction of plants. Mention will in particular be made of chemical elicitors as well as elicitors of pathogenic agents such as bacteria, viruses and fungi. By inducing agent is also meant any pathogenic agent capable of causing a defense reaction of the plant.

Advantageously, the promoter activity of the polynucleotides of the present invention is induced following eliciting treatments, to aggression by pathogenic agents, in particular bacterial, fungal or viral, and in particular by infection with Phytophthora. Preferably, this induction is very early and is measurable before the onset of necrosis or symptoms. Preferably, the induction amplitude is high, at the quantitative level, an induction of a factor of at least 15, 25, 50, 75 and preferably a factor of 100 is measured after 12 hours. Preferably, the promoter activity of the polynucleotides of the present invention is induced by inducing agents, in particular by treatment with MeJa (methyl jasmonate), ACC (1-aminocyclopropane-1 carboxylic acid) and with heavy metals.

Advantageously, the promoter activity of the polynucleotides according to the present invention is induced by λ-carrageenan, a polysaccharide extracted from red algae of the genus Gigartina (Mercier et al., New Phytologist 149: 43-51, 2001). Advantageously, this induction is obtained by treatment of the leaf tissue by infiltration.

The techniques for evaluating the promoter activity of a polynucleotide are well known to those skilled in the art. These techniques conventionally involve the use of an expression vector comprising, in the direction of transcription, the polynucleotide to be tested and a reporter gene (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989). Expression tapes

According to one embodiment of the invention, the polynucleotides having a promoter or regulatory activity according to the invention are inserted into an expression cassette using cloning techniques well known to those skilled in the art. This expression cassette comprises the elements necessary for the transcription and translation of the sequences coding for a reporter gene or a polypeptide of interest. The polynucleotides of the present invention having promoter and / or expression regulating activity are therefore advantageously used to express a heterologous gene in a host organism and in particular in plant cells or in plants. Advantageously, this expression cassette comprises both elements making it possible to produce a polypeptide by a host cell and elements necessary for the regulation of this expression. The present invention therefore also relates to functional expression cassettes in plant cells or plants comprising, in the direction of transcription, a 5 ′ promoter regulatory sequence, a coding sequence and a 3 ′ regulatory sequence, in which the 5 'promoter regulatory sequence comprises a polynucleotide chosen from the following polynucleotides: a) a polynucleotide comprising a plant promoter regulatory sequence characterized in that it comprises the polynucleotide of SEQ ID No. 1; and b) a polynucleotide capable of selectively hybridizing to the polynucleotide of SEQ ID No. 1; and c) a polynucleotide homologous to the polynucleotide of SEQ ID No. 1; and d) a polynucleotide according to b) or c) having promoter activity in plant cells and plants; e) a polynucleotide according to b), c) or d), the promoter activity of which is induced in response to an inducing agent and in particular in response to aggression by a pathogen.

Any gene of interest can be expressed in a host organism under the control of a promoter according to the invention. Preferably, the polynucleotides according to the present invention having promoter activity are used for the expression of a heterologous gene in plant cells or in a plant. Advantageously, the coding sequence comprises a sequence coding for a reporter gene or a sequence coding for a protein or a polypeptide of interest. Among the reporter genes, mention will be made in particular of the reporter genes GUS (β-glucuronidase), GFP (green fluorescent protein), LUC (luciferase), CAT (chloramphenicol transferase) or β-galactosidase (lacZ). Preferably, the protein of interest is a protein which confers on plants properties of resistance to diseases or insects.

In a preferred embodiment, the protein of interest is chosen from fungal elicitors or lytic peptides.

With regard to the mode of induction of the promoter according to the invention, the protein of interest is advantageously a protein which confers on plants properties of resistance to diseases and pathogens.

Among the proteins or peptides of interest which confer new disease resistance properties, mention will be made in particular of chitinases, glucanases, oxalate oxidase, all these proteins and their coding sequences being widely described in the literature, or even antibacterial peptides and / or antifungals, in particular peptides of less than 100 amino acids rich in cysteines such as thionines or plant defensins, and more particularly lytic peptides of all origins comprising one or more disulfide bridges between cysteines and regions comprising basic amino acids, in particular the following lytic peptides: androctonine (WO 97/30082 and PCT / FR98 / 01814, deposited on August 18, 1998) or drosomicine (PCT / FR98 / 01462, deposited on July 8, 1998).

According to a preferred embodiment of the invention, the protein or peptide of interest is chosen from fungal eliciting peptides, in particular elicitins (Kamoun et al., Mol. Plant microb. Internet, 6: 15-25, 1993 ; Panabières et al., Mol. Plant Microb. Internet, 8, 1995).

Among the proteins of interest conferring new insect resistance properties, there will be mentioned more particularly the Bt proteins widely described in the literature and well known to those skilled in the art. Mention will also be made of proteins extracted from bacteria such as Photorabdus (WO 97/17432 & WO 98/08932).

The expression cassettes according to the present invention can also include any other sequence necessary for the expression of the gene of interest, such as for example regulatory elements or signal sequences allowing the addressing of the polypeptide of interest.

The construction techniques of these expression cassettes are widely described in the literature (see in particular Sambrook et al., Molecular Cloning: A Labratory Manual, 1989).

Certain elements of the expression cassettes according to the invention are illustrated below without implied limitation.

As a 5 ′ regulatory sequence, the polynucleotides according to the invention can be used alone or associated with at least part of a promoter of a gene expressing itself naturally in plants, in particular a promoter expressing in particular in plant leaves, such as promoters known as constitutive of bacterial, viral or plant origin or also light dependent promoters such as that of a gene for the small subunit of plant ribulose- biscarboxylase / oxygenase (RuBisCO) or any known suitable promoter that can be used. Among the promoters of plant origin, mention will be made of the histone promoters as described in application EP 0 507 698, or the rice actin promoter (US Pat. No. 5,641,876). Among the promoters of a plant virus gene, there may be mentioned that of the cauliflower mosaic (CAMV 19S or 35S), or the circovirus promoter (AU 689 311).

The polynucleotides according to the invention can also be used in combination with at least part of a promoter specific for regions or specific tissues of plants, and more particularly promoters specific for seeds (Datla, R. et al., Biotechnology Ann. Rev., 3: 269-296, 1997), in particular the promoters of napine (EP 255 378), of phaseoline, of glutenin, of heliantinin (WO 92/17580), of albumin (WO 98/45460) or of oelosin (WO 98 / 45461).

In the expression cassettes of the present invention, any regulatory sequence can be used which makes it possible to increase the level of expression of the coding sequence inserted in said expression cassette. According to the invention, it is possible in particular to use, in association with the promoter regulatory sequence, other regulatory sequences which are located between the promoter and the coding sequence, such as transcription activators ("enhancer"). Among the leader sequences derived from viruses, mention will be made, for example, of the activator of the tobacco mosaic virus (TMV) described in application WO 87/07644, or the activator of the tobacco etch virus (TEV). Different sequences derived from plant introns can also be used to increase the level of expression of the gene of interest, especially in monocotyledonous plants. Mention will be made, for example, of the intron I of the corn gene AdhI (Callis et al., Genes Develop., 1: 1183-1200, 1987).

A wide variety of terminator sequences can be used in the expression cassettes according to the invention. These sequences allow the termination of transcription and polyadenylation of the mRNA. Any terminator sequence functional in the selected host organism can be used. For expression in plants, it is possible in particular to use the terminator nos of Agrobacterium tumefaciens, or alternatively terminator sequences of plant origin, such as for example the histone terminator (see EP 0 633 317), the terminator CaMV 35 S and the tml terminator. These terminator sequences are usable in monocotyledonous and dicotyledonous plants.

The present invention also relates to a polynucleotide comprising an expression cassette according to the invention and in particular a vector comprising an expression cassette according to the invention. Advantageously, the expression cassettes according to the present invention are inserted into a vector for their replication or for the transformation of a host organism.

vectors The present invention also relates to transformation or expression vectors comprising at least one polynucleotide or an expression cassette according to the present invention. The vectors of the present invention are used in particular for transforming a host organism and for expressing a polypeptide of interest in said host organism. The host organism is for example a bacterium, a yeast, a fungus, a plant cell or a plant. This vector can in particular consist of a plasmid, a cosmid, a bacteriophage or a virus in which a polynucleotide or an expression cassette according to the invention is inserted. In general, any vector capable of being maintained, self-replicating or propagating in a host cell in order to induce the expression of a polynucleotide or a polypeptide can be used.

The techniques for constructing these vectors and the techniques for inserting an appropriate sequence into these vectors are widely described in the literature (see in particular Sambrook et al., Molecular Cloning: A Labratory Manual, 1989). Advantageously, the vectors according to the invention comprise at least one origin of replication. Preferably, the vectors of the invention also comprise at least one selection marker and preferably a selection marker usable in plant cells or in plants. Among the selection markers, there may be mentioned antibiotic resistance genes such as the npt11 gene for resistance to kanamycin (Bevan et al., Nature 304: 184-187, 1983) and the hph gene for resistance to hygromycin (Gritz et al., Gene 25: 179-188, 1983). We will also mention the herbicide tolerance genes such as the bar gene (White et al., NAR 18: 1062, 1990) for bialaphos tolerance, the EPSPS gene (US 5,188,642) for glyophosate tolerance or the HPPD gene. (WO 96/38567) for tolerance to isoxazoles. It will also be possible to use the genes coding for easily identifiable reporter enzymes such as the enzyme GUS or genes coding for pigments and enzymes regulating the production of pigments in the transformed cells. Such selection marker genes are described in particular in patent applications EP 242 236, EP 242246, GB 2 197 653, WO 91/02071, WO 95/06128, WO 96/38567 or WO 97/04103.

Advantageously, these vectors are used for the transformation of a host organism. Those skilled in the art will choose the appropriate transformation vectors in particular according to the host organism to be transformed and according to the transformation technique implemented.

For the transformation of plant cells or plants, it will in particular be a virus which can be used for the transformation of developed plants and which additionally contains its own elements of replication and expression. Preferably, the vector for transforming plant cells or plants according to the invention is a plasmid.

Many vectors have been developed for the transformation of plants with Agrobacterium tumefaciens. Other vectors are used for transformation techniques not based on the use of Agrobacterium. These vectors are well known to those skilled in the art and widely described in the literature. Transformation

The subject of the invention is also a method of transforming host organisms, in particular plant cells with a polynucleotide, an expression cassette or a transformation or expression vector according to the invention.

The invention also relates to a process for transforming plant cells in which at least one polynucleotide, an expression cassette and / or a vector according to the invention is integrated into the genome of said plant cells. According to the present invention, the transformation of the host organism can be obtained by any suitable known means, the techniques of transformation and in particular of transformation of plants are amply described in the specialized literature. For the processes of transformation of plant cells and regeneration of plants, the following patents and patent applications may be mentioned in particular: 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 5,204,253,175 EP 486 233, EP 486 234, EP 539 563, EP 674725, WO 91/02071 and WO 95/06128.

Certain techniques use Agrobacterium in particular for the transformation of dicotyledons. A series of methods consists in using as a means of transfer into the plant a chimeric gene inserted into a Ti plasmid of Agrobacterium tumefaciens or Ri of Agrobacterium rhizogenes. Other methods include bombarding cells, protoplasts, or tissue with particles to which DNA sequences are hung. Other methods can also 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 nature of the host organism, in particular the plant cell or the plant.

The present invention also relates to plant cells and plants obtained by the transformation methods described above. Host organizations

The present invention also relates to a host organism transformed with a polynucleotide, an expression cassette or a vector according to the invention.

By host organism is meant in particular according to the invention any mono or multicellular organism, lower or higher, in particular chosen from bacteria, yeasts, fungi or plant cells and plants. Advantageously, the bacteria are chosen from Escherichia coli, the yeasts are chosen from Pichia pastoris and Saccharomyces cerevisae, the fungi are chosen from Aspergillus niger. Preferably, the host organism is a plant cell or a plant.

In a preferred embodiment, the invention relates to transformed plant cells or transformed plants comprising at least one polynucleotide, an expression cassette and / or a vector according to the invention.

By "plant cell" is meant according to the invention any cell originating from a plant and which can constitute undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of plants, plants or seeds. The term "plant" according to the invention means any differentiated multicellular organism capable of photosynthesis, in particular monocotyledons or dicotyledons, more particularly crop plants intended or not for animal or human food, such as corn, wheat, l barley, sorghum, rapeseed, soybeans, rice, sugar cane, beet, tobacco, cotton, sunflower, clover etc.

According to a particular embodiment of the invention, the host organism comprises at least one other heterologous gene coding for a peptide, a polypeptide or a protein of interest. The polynucleotide comprising a polynucleotide according to the invention and the other heterologous gene (s) may have been introduced into the host organism simultaneously by means of the same vector comprising them, or using several vectors, or sequentially using several vectors, or alternatively by crossing several host organisms, each comprising a heterologous gene.

By heterologous gene is meant according to the invention any gene introduced artificially into the host organism, and more particularly artificially integrated into its genome, the methods allowing this introduction or integration possibly being those described above.

The plants transformed according to the invention can thus contain other genes of interest, conferring on plants new agronomic properties. Among the genes conferring new agronomic properties on transformed plants, there may be mentioned the genes conferring tolerance to certain herbicides, those conferring tolerance to certain insects, those conferring tolerance to certain diseases. Such genes are described in particular in patent applications WO 91/02071 and WO 95/06128. Mention may also be made of the genes modifying the constitution of the modified plants, in particular the content and quality of certain essential fatty acids (EP 666 918) or also the content and quality of proteins, in particular in the leaves and / or seeds. of said plants. Mention will in particular be made of the genes coding for proteins enriched in sulfur amino acids (WO 98/20133; WO 97/41239; WO 95/31554; WO 94/20828; WO 92/14822 These other genes of interest can be combined with chimeric gene according to the invention either by conventional crossing of two plants each containing one of the genes (the first the chimeric gene according to the invention and the second the gene coding for the protein of interest) or by carrying out the transformation of cells plants of a plant containing the gene coding for the protein of interest with the chimeric gene according to the invention.

The present invention also relates to plants containing transformed cells as defined above, in particular plants regenerated from the transformed cells and their progeny. The regeneration is obtained by any suitable process which depends on the nature of the species, as for example described in the references above.

The present invention also relates to genetically modified plants in the genome of which a polynucleotide or an expression cassette according to the invention are stably integrated and transmissible by sexual reproduction. The present invention also relates to plants obtained by crossing the regenerated plants above with other plants. It also relates to the seeds of transformed plants.

The examples below illustrate the invention, without however seeking to limit its scope.

All the methods or operations described below in these examples are given by way of examples and correspond to a choice made from among the different methods available to achieve the same result. This choice does not affect the quality of the result and therefore, any suitable method can be used by those skilled in the art to achieve the same result. Most of the methods for engineering DNA fragments are described in "Current Protocols in Molecular Biology" Volumes 1 and 2, Ausubel F.M. et al. or in Sambrook et al 1989.

Description of the figures

Figure 1: Glucuronidase activity of PILOX plants after infiltration of Ppn elicitor, in the infiltrated area. A: The enzymatic activity was assayed (in pmol MU / h / μg prot) in leaf discs taken from areas infiltrated by the Ppn elicitor, at the times indicated (4, 8, 12, 24 hours). The measurements were carried out for 3 plants of the same transgenic line (1, 2, 3). Two controls were carried out on plants after water infiltration (4) and on control plants without infiltration (5). B: The enzymatic activity was assayed (in pmol MU / h / μg prot) in leaf discs taken from areas infiltrated by the Ppn elicitor, at the times indicated (4, 8, 12, 24 hours). The measurements were carried out for 3 different transgenic lines (l-.line 4, 2: line 5, 3: line3).

Figure 2: Glucuronidase activity of PILOX plants after infiltration of Ppn elicitor, away from the infiltrated area.

The enzymatic activity was assayed (in pmol MU / h / μg prot) in leaf discs taken from the non-infiltrated half of leaves treated with Ppn elicitor, at the times indicated (0, 3, 7 days). The cross indicates the death of the tissue in the infiltrated area. Leaf discs were taken from the infiltrated area (1), the near area (2), the far area (3) and from an upper leaf (4).

Figure 3: Glucuronidase activity in PILOX plants inoculated with Ppn race 0.

The glucuronidase activity was measured (in pmol MU / h / μg prot) in the roots of inoculated plants, at the times indicated (5, 12, 24, 48 hours and 3 days). Line 5 was inoculated with water (2) and with Ppn race 0 (1). Line 21 was inoculated with water (4) and with Ppn race 0 (3).

Figure 4: Effect of MeJa (methyl jasmonate) on the glucuronidase activity of PILOX plants.

The glucuronidase activity was measured in plants 24 hours after treatment with MeJa (250 μM). For each measurement, the specific activity of the treated plants was compared to that of control plants treated with ethanol, controls to which the value 1 was assigned. The histograms represent the specific activity compared to that of the controls. The measurements carried out on three lines of PILOX plants are shown (lines 5, 9 and 13). Two tests (1) and (2) were carried out for each line.

EXAMPLES

Example 1; Obtaining a tobacco genomic clone containing the promoter region of the pathogenesis-induced LOX gene Description of the probes used TL-J2 is a complementary DNA of 2888 bp, corresponding to a pathogen-induced tobacco LOX gene. Obtaining this complementary DNA is described by Véronési et al. (Plant Physiol., 108: 1342, 1995), its sequence is deposited in GenBank under the accession number X84040. An EcoRI fragment of 1409 bp, corresponding to nucleotides 1222 to 2630 of TL-J2, called TL-35, was also used. Description of the tobacco genomic bank used

The tobacco genomic bank was acquired from the company Clontech (4030 Fabian Way, Palo Alto, CA 94303-4607, USA, product reference FL1071d). This is a library produced by partial digestion with the DNA enzyme Mbol isolated from plants tobacco Nicotiana tabacum, cv. Xanthi-nc, harvested 30 days after emergence, then selection of fragments of a size between 8 and 22 kb and insertion of the fragments obtained at the BamUL site of the EMBL3 vector. Tobacco LOX genomic clones Screening of the tobacco library using complementary DNA TL-35 led to the isolation and purification of a bacteriophage clone named Al, exhibiting strong probe hybridization TL-35. A step of subcloning the phage DNA made it possible to isolate and sequence fragments of the clone A1 which hybridized to the LOX probe. Analysis of the sequences obtained has shown that the insert A1 contains the 3 'terminal part of the NtLOX1 gene, and in particular the nucleotides 260 to 2888 of the ADΝC TL-J2 divided into 8 exons separated by 7 introns. The obtaining of a second genomic tobacco clone overlapping with the clone Al and containing the 5 'terminal region of the NtLOXl gene, is described in the doctoral thesis of the University of Paris XI-Orsay by Iann Rancé (1997, pages 46 to 48). This clone was named 2A5. Partial sequencing of phage DNA showed that clone 2A5 contained nucleotides 1 to 259 of TL-J2 cDNA, the untranslated region of the 5 'terminal end of the gene and all of exon 1 The insert 2A5 overlaps with the clone Al on 2557 nt, which correspond to nucleotides 297 to 2853 of the sequence of intron 1 (with a total size of 3798 bp). A 2330 bp region containing the 5 'terminus of the LOX gene and the upstream regions has been sequenced and designated as PILOX. The sequence of this fragment is presented in SEQ ID No. 1. Isolation of a promoter fragment

The 2330 bp PILOX DNA fragment containing the nucleotides 728 to 3047 of the genomic clone 2A5 was isolated by chain amplification (PCR). This fragment contains the putative TATA box and approximately 2.2 kb of upstream sequence which potentially includes the majority of the cz ^' s regulatory elements. The 3 ′ terminal region of the fragment contains the 88 nucleotides of the leader region of the LOX RNA, the ATG initiator codon for the translation of the LOX gene as well as the nucleotides coding for the first 6 amino acids of LOX from tobacco. The sequences of the two primers used in the PCR reaction are indicated below and note the insertion of the Xbal site in the sense primer and of the Smal site in the antisense primer (underlined characters). Sense primer: 5 * CCC TTT CTA GAC CAT TTA TTT CCC 3 'Anti-Sense primer: 5' CTG TGA TTC CCG GGA CAA TCT TCT 3 ' The amplification reaction contained 300 ng of phage DNA from clone 2A5, 25 picomoles of each of the two primers, 200 μM dNTP and 1 unit of Vent polymerase (New England) and its reaction buffer. The amplification was carried out over 20 cycles and the sequence of the fragment obtained was verified. Clone fragment sequence analysis

A sequence comparison between the tobacco PILOX promoter and the GenBank nucleic acid database does not reveal significant homologies with other promoters. In particular, a sequence alignment between the tobacco LOX promoter and a 700 bp fragment upstream of the LOX1 transcribed sequence from Arabidopsis thaliana, (Melan et al, Biochem. Biophys. Actophys, 1210: 377-380, 1994) or the LOXl promoter of barley, inducible by MeJa, does not reveal conserved regions. However, this analysis makes it possible to detect in the PILOX promoter a 50 bp sequence (position 821-875 of SEQ ID No. 1), upstream of the TATA box, which has 90% homology with an intron region. 2 of a tobacco gene coding for a phenylalanine ammonia-lyase as well as with an intron region 5 of the tobacco rbvmfl gene coding for methyl-transferase of the large Rubisco subunit.

Potential regulatory elements, involved in regulation by methyl jasmonate or by elicitors, were also sought in the sequence of the tobacco PILOX promoter.

Many promoters induced by methyl jasmonate (MeJa) such as the parsley chalcone synthase promoter (Schulze-Lefert et a, EMBO J., 8: 651-656, 1989), the promoter of the gene coding for the protein reserve (VSP) soybeans (Mason et al, Plant Cell, 5: 241-251, 1993) or the promoter of the proteinase inhibitor II gene from potato (Kim et al, Plant Physiol., 99 : 571-576, 1992) contain in their sequence a G box (CACGTG) essential for induction. A TGACG motif analogous to the asl element identified in the CaMV 35S promoter is also involved in the response to the MeJa of the LOX 1 lipoxygenase promoter from barley (Rouster et al, Plant J., 11: 513-523, 1997). Analysis of the tobacco LOX promoter sequence does not reveal TGACG or G box consensus motifs. Several ACGT motifs which can be assimilated to poorly preserved G boxes are however identified in the promoter. Several sequence elements involved in the response to elicitors have also been identified in different plant promoters. ERE boxes (AATTGACC) are mainly found for Elicitor Responsive Element, identified in particular in the PRl promoter of the PRm gene of maize (Raventos et al, Plant J., 7: 147-155, 1995) and in the PRl promoter of parsley (Rushton et al, EMBO J., 15: 5690-5700, 1996). E1RE boxes (GTCAG) for Elicitor Responsive Element are also involved in activation by elicitors as shown in particular for the promoter of the tobacco gene encoding a class I chitinase (Fukuda et al, Plant Mol. Biol., 24: 485-493, 1994). Recently, a GCC box also called JERE element (Jasmonic acid and Elicitor-Responsive Element) involved in both activation by elicitors and jasmonic acid was characterized in the promoter of the gene encoding strictosidine synthase from Catharanthus roseus ( Menke et al, EMBO J., 18: 4445-4463 1999). The search for these elements in the tobacco LOX promoter sequence did not identify any GCC or E1RE boxes. Three patterns similar to the ERE box can however be located upstream of the TATA box. Example 2: Obtaining the PILOX fusion construct:; GUS

After a digestion reaction with the enzymes Xbal and Smal, the amplified promoter fragment was cloned at the same sites in the vector pBHO1 (Clontech). The cloning is carried out so that the ATG codon of the LOX gene is in the same reading frame as the coding sequence of the reporter gene uidA. The chimeric construction thus obtained is called PlLOX :: GUS. EXAMPLE 3 Genetic Transformation of Tobacco

The PlLOX :: GUS construct was transferred by electroporation into Agrobacteria of strain LBA4404. The recombinant bacteria obtained were then used for the infection of tobacco leaf discs, Nicotiana tabacum, lines 46.8 and 49.10 according to standard procedures (Horsch et al, Plant Molecular Biology Manual, Kluwer Académie Publishers, 1988). The tobacco lines used are quasi-isogenic and are differentiated by the presence in line 46.8 of a locus of resistance to race 0 of Ppn. The plants regenerated on a Murashige and Skoog (MS) medium containing 150 μg / ml of Kanamycin are placed in a culture chamber and then in a greenhouse to obtain the Tl seeds. The presence of the LOX-uidA promoter expression cassette as well as the number of copies of the transgene in the genome of the regenerated plants were determined by DNA / DNA hybridization experiments (Southern). The results show that the primary transformants of line 46.8 contain from 1 to 5 copies of the transgene. Plants 46.8 having a low number of copies (single copy or two copies) were selected for the study of the LOX promoter. For these lines, the Tl seeds resulting from the self-fertilization of the primary transformants were put to germinate on selective medium and the plants obtained were used for the study of the activity of the promoter.

The activity of the PILOX promoter was characterized from three types of experiments involving: - infiltration of leaves with preparations of elicitors

- root inoculations with Phytophthora parasitica nicotianae (Ppn).

- induction tests on young plants to analyze the influence of various molecules on the regulation of promoter activity.

The experiments were carried out with transgenic plants, of generation Tl, originating from independent lines and containing one or two copies of the transgene per genome.

Example 4 Detection and Measurement of Glucuronidase Activity

Histo-chemical detection of GUS activity is carried out essentially according to the method described by Jefferson et al. (EMBO J., 6: 3901-3907, 1987). Tissues are removed from the transgenic plants and directly incubated at 37 ° C. in a 50 mM phosphate buffer, pH 7 containing 0.5 mM of ferro and ferri cyanide potassium salts, 1 mM of X-GLUC (Eurogentec); and 0.1% Triton X100.

After coloring, the samples are placed in a 70% ethanol solution, making it possible to remove the chlorophyll pigments, then observed for analysis. The GUS activity assay is carried out according to the standard protocol (Jefferson et a,

EMBO J., 6: 3901-3907, 1987). The collected samples are ground in an extraction buffer to obtain a protein extract on which the glucuronidase activity is measured. The enzymatic reaction is carried out for 50 μL of extract in the presence of the substrate MUG (Sigma) at 1 mM. The appearance of methyl umbelliferone (MU) is measured by fluorometric determination and the activity values are expressed in picomoles of MU produced per hour and per μg of proteins. The amount of protein is evaluated by the method of Bradford (Biorad). Example 5 Activity of the PILOX Promoter in Transgenic Plants During Development

This study was carried out essentially on Tl plants, derived from independent transgenic lines, containing one or two copies of the PILOX :: GUS construct. Analysis of the activity of the reporter gene in the floral parts was only carried out on the primary transformants.

We can distinguish three distinct expression profiles during development.

Germination

The β-glucuronidase activity was analyzed in young germinations harvested at different stages of development.

Stage 1: 5 days after sowing.

Germination starts with the exit of the radicle through the integuments. At this early stage, there is an intense limited coloration in a circular area around the radicle where it emerges from the seed coat. No coloration is detected elsewhere on the radicle. The expression GUS is localized at the level of the collar but the internal tissues of the integuments, around the zone of exit of the radicle, also seem strongly colored.

Stage 2: 7 days after sowing.

At this stage, an elongation of the radicle and the hypocotyle is observed. The cotyledons are generally outside the integuments, which for their part still remain attached to the seedling at the level of the collar. The expression GUS is detected at the level of the neck and the elongated hypocotyle. A strong expression is also visible on the cotyledons. The staining seems to be localized on the epidermal cells but histological sections will be necessary to confirm this result. No activity is detected in the roots and there is a clear delimitation at the level of the collar between colored and non-colored zone.

Stage 3: 10 days after sowing.

There is a general development of the seedlings with a root, an elongated hypocotyle and two developed cotyledons. A residual GUS expression persists in the cotyledons. The intensity of this coloring is variable depending on the individual and decreases during development.

Vegetative stage

Analysis of reporter gene expression in different plant tissues in growing and adult plants showed β-glucuronidase activity limited to the collar region as well as to the leaf petioles. GUS expression was detected at the collar at all stages of development analyzed. Analysis of sections on fresh material has shown that this expression is localized at the level of the conductive elements. A GUS coloration is visible in the central cylinder of the upper part of the root and in the vessels of the internal phloem in the lower part of the stem. GUS expression was also observed at the base of the leaf petioles. Microscopic observation of cross sections at the nodes of the stem has shown that the cells which develop a blue color belong to the cortical parenchyma. These indications suggest that the colored zone constitutes the future abscission zone of the leaf. With the exception of these tissues, no GUS activity was detected in the vegetative organs (leaves, stems, roots). Flowering plant

Flowers at different stages as well as still immature capsids were collected for the histochemical analysis of the expression GUS. The results show that the green parts of the flower such as the sepals and the receptacle do not show GUS staining. GUS expression is however detectable in the petals but limited to the upper pigmented area. The stamens do not seem to express the gene while a strong expression is detected in the pollen. There is also a blue color at the stigma (may be due to the deposition of pollen) as well as at the base of the embryonic sac. Inside the embryo sac, we observe that the ova also develop a GUS expression. This activity is moderate and seems mainly localized in surface cells. The abscission zone between the style and the embryonic sac is also strongly colored. At the level of the capsids, a GUS expression limited to the seeds is observed while the placenta and the vascular system are not colored. Cross sections made from seeds collected in the green capsules show that the expression GUS is essentially localized at the level of the cotyledons of the embryo whereas no expression is detected at the level of the endosperm. Histological analysis of glucuronidase activity made it possible to precisely locate the tissue profile of the activity of the PILOX promoter. The expression GUS is detected in the cotyledons of the embryo, in the hypocotyle and the cotyledonary leaves during germination. GUS activity is also detected in the reproductive organs of the flower as well as in the petals. Many LOXs are expressed during germination as well as in flowers or fruits. These LOXs are generally involved specifically in development and are involved in the growth and cell senescence processes. The expression of certain defense-related genes (Henning et al, Plant J., 4: 481-493, 1993) can however be detected in the flowers and in the seeds. In addition, Melan et al. (Plant Physiol., 105: 385-393, 1994) have shown that the _4tLOXl gene induced during pathogenesis is also transiently activated in the early stages of the germination of _4 seeds. thaliana. We can therefore think that the expression GUS visualized in organs essential for the survival and persistence of the plant is also in agreement with a role of defense and protection. This is also underlined by the expression GUS detected in the collar and in the abscission zones, two types of tissue corresponding to zones of communication between organs. This type of tissue-specific expression has also been detected for other defense-related genes. In particular, it has been shown that the promoter of the PRB-1b gene of tobacco expresses itself specifically in the abscission zones of the leaves and flowers (Eyal et al, Plant J., 4: 225-234, 1993). The constitutive expression of LOX in these compartmentalization zones could constitute an obstacle to the colonization of the plant by a pathogenic agent. Example 6; Elicitor infiltration

Elicitor infiltrations are carried out using a syringe without a needle on the underside of adult leaves of 8-week-old plants and cultivated in a culture chamber (photoperiod: 16 h day / 8 h night; temperature 25 ° C day / 22 ° C night; humidity 80%). The GUS assays carried out from the elicited plants are carried out on samples composed of three leaf discs of 1 cm in diameter taken from the treated areas. Several samples are taken for each treatment. The experiments were carried out with transgenic plants, of generation Tl, originating from independent lines and containing one or two copies of the transgene per genome. Infiltration of Ppn elicitor (Phytophthora parasitica nicotianae)

A preparation of Ppn walls at 30 μg / ml is infiltrated on the abaxial surface of leaves of adult transgenic plants. Areas of approximately 10 cm ² are infiltrated except for the search for systemic activation of the promoter for which half-leaves have been fully infiltrated. Total or partial necrosis of the infiltrated tissue is generally visible 24 to 48 hours after infiltration. The histochemical study of the expression of the reporter gene 6 to 8 hours after infiltration shows that a strong GUS activity is visible in the infiltrated tissues. Analyzes carried out 12 and 24 hours after infiltration show that at this stage, the GUS coloration is no longer visible except around the periphery of the necrotic area. No induction of the reporter gene is detected in areas infiltrated with water or non-infiltrated.

A fluorometric assay of the β-glucuronidase activity was carried out from leaf discs taken from the infiltrated areas. The results obtained relate to three independent lines of PlLOX :: GUS transgenic plants. An early induction of the reporter gene is measured as early as 4 hours after treatment. The maximum level of expression is reached between 12 and 24 hours after infiltration and corresponds to approximately 100 times the level measured in areas infiltrated with water. The results obtained for plants belonging to the same line or to independent lines are different in terms of amplitude but all have the same general profile.

Infiltrations of the Ppn elicitor solution were also carried out on half of adult leaves. The purpose of these experiments is to search for the remote activation of the PILOX promoter in the part of the untreated leaf as well as in the leaves of higher or lower rank. The GUS activity assays were carried out 3 days and 7 days after treatment. An increase in GUS activity is detected 3 days after infiltration into untreated tissue close to the infiltrated area. The activity levels measured remain moderate and represent around 20% of the values generally measured in areas infiltrated by the elicitor. This activity in the proximal tissues decreases at 7 days while at the same stage a slight increase in GUS activity is measured in tissues distant from the infiltrated area but located on the same sheet. No induction of activity was detected in the untreated leaves of higher or lower rank.

These results are shown in Figures 1-2. Heavy metal infiltration Lead nitrate (PbNO ₃ ) infiltration was carried out to analyze the activity of the promoter following treatment with heavy metals. These act as abiotic elicitors and cause cell necrosis of the treated tissues. Our experiments demonstrate an induction of gene activity GUS after infiltration of PbNO ₃ at 10 mM. The revelation of β-glucuronidase, 20 hours after treatment, reveals a strong blue coloration in the infiltrated areas during necrosis. Example 7: Realization of wounds Wounds are carried out on plants at the same stage of development as those used for the infiltration of elicitors. Developed leaves are fully injured using a needleless syringe. The end of the syringe is pressed against the underside of the sheet until an impression is obtained. These traces become necrotic after the second or third day after treatment. The GUS assays carried out on injured plants are carried out on samples composed of three leaf discs of 1 cm in diameter taken from the treated areas. Several samples are taken for each treatment. The GUS activity assays are carried out using leaf discs taken from injured leaves 24 hours and 7 days after treatment. The data obtained show that under the conditions of the experiment the injury does not induce an increase in glucuronidase activity, although the injured areas appear necrotic. EXAMPLE 8 Root Infection with Zoospores of Race 0 of Ppn Obtaining Zoospores

A pastille of Ppn race 0 mycelium previously cultivated on a synthetic medium (Keen, Science, 187: 74-75, 1975) is placed on an Agryl P17 canvas (Sodoca, France) placed in a V8 liquid medium, 5% (juice Campbell). After 4 days of culture at 25 ° C. the cloth on which the mycelium has grown is transferred to 1.5% agar. This transfer leads to a nutritional deficiency which will induce the formation of sporocysts in the fungus. After 4 days of culture at 25 ° C, the mycelium is covered with 10 ml of distilled water and transferred to 4 ° C for 30 minutes. This thermal shock causes the release of the zoospores which are recovered with the liquid and counted in a Thoma cell. Plant inoculation

Transgenic Tl generation plants grown in a culture chamber and 5 weeks old are removed and the roots are cleared of vermiculite. The roots are then placed in a 2.5 ml tube containing a suspension of zoospores of race 0 of the fungus at 400,000 spores / ml. The plants are placed in a culture chamber for the duration of the experiment. Control plants, not inoculated, are placed in distilled water. The GUS activity in infected plants is measured independently on the leaf tissues collected above the collar and on the root-collar part. Measurement of GUS activity The histo-chemical revelation of β-glucuronidase shows a strong induction of the reporter gene in the roots of inoculated plants. GUS activity is visible in the roots from 5 hours after inoculation but the tissue coloration is more intense after 12 hours and 24 hours. Four to five days after inoculation, the GUS activity in the roots appears much lower. The results are shown in Figure 3. In the entire root bundle, only a few main roots are strongly colored. Several profiles are observed: certain roots develop a coloration over their entire length which seems to concern all cells. On other roots, intense activity is observed at the level of the root primordia as well as in the central cylinder. Intense GUS activity is also visible in the collar. The non-inoculated plants have an activity in the collar which is less intense than in the case of inoculation. The roots of uninoculated plants do not develop GUS staining. During the interaction, the LOX promoter is not very active in the aerial organs. Glucuronidase expression was however detected on the old cotyledonary leaves, close to the collar and in the petioles of the first leaves. The fluorometric assay of GUS activity in inoculated plant roots shows rapid induction, detectable 5 hours after inoculation with maximum expression between 12 hours and 24 hours. The expression then gradually decreases until 3 ^ιeme day post-inoculation. In leaf tissue, taken above the collar, there is a smaller induction of GUS expression (about 10 times less than in root tissue) with maximum activity measured 48 hours after inoculation.

The PILOX promoter is induced more strongly during an incompatible interaction than during a compatible interaction

The induction of the promoter was quantified during a compatible or incompatible interaction.

46.8 transgenic plants (lines 5 and 9) were infected either with zoospores of race 0 of Ppn, (incompatible interaction), or with zoospores of race 1 (compatible interaction). At 3, 8 or 24 hours after inoculation, plants from both batches are harvested and used for fluorimetry measurements. From 3 hrs, a GUS activity is detected for the two types of interactions, greater than that detected in the controls not inoculated in plants of the line 5. The time 8 h discriminates the two types of interactions: the specific activity measured in resistant plants is more important than that measured in sensitive plants. At 24 h it is twice as high. The specific activity in uninoculated plants remains almost zero. Similar results are obtained in plants of line 9.

These results seem to show that lipoxygenase is induced much more strongly during an incompatible interaction than during a compatible interaction. Example 9: Treatments with Secondary Messengers and Phytosanitary Products

The purpose of these experiments is to analyze the influence of molecules regulating the activity of defense genes on the activity of the PILOX promoter. There are typically three ways of transducing signals triggering defense reactions: one involving methyl jasmonate, another involving salicylic acid and a third involving ethylene. These three molecules were therefore tested on the induction of the promoter to characterize which signaling pathway is mobilized to regulate the activation of the PILOX promoter during pathogenesis. It should be noted that we used ACC (1-aminocyclopropane-1 carboxylic acid) rather than ethylene gas. ACC, the substrate for ACC oxidase, is the direct precursor of ethylene.

In addition, two products from the phytosanitary industry, Bion ^® (Novartis) and Aliette ^® (Rhône-Poulenc) were also tested. Aliette is the commercial formulation of fosetyl-Al, a product used as a systemic fungicide, in particular against oomycete fungi.

Bion is the commercial formulation of BTH (benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester), a chemical analogue of salicylic acid which induces the acquisition of systemic resistance (SAR) in treated plants.

The Tl transgenic seeds are sterilized and germinated on an MS agar medium containing 150 μg / ml of kanamycin. The plants are harvested at 6 weeks after sowing and placed in Petri dishes containing an MS 0.1X liquid medium, pH 5.8, without sucrose and containing 0.001% of Triton X100. Concentrated solutions different molecules or products tested are then added to the medium to reach the following concentrations:

Methyl jasmonate (Aldrich): 200 μM, 500 μM and 1 mM

ACC (Sigma) ImM Salicylic acid (Sigma) 50 μM, 200 μM and ImM.

BION ^® 500 mg / 1 or 1.2 mM active substance

ALIETTE ^® 125mg / l or 282 μM active substance

The dishes are sealed with Parafilm and placed in culture chambers for 24 hours before analysis of the GUS activity. The GUS activity assays are carried out on samples corresponding to 3 plants collected. Treatment with ACC (1-aminocyclopropane-1 carboxylic acid)

The experiments carried out with ACC ImM made it possible to measure moderate inductions of glucuronidase activity, which vary according to the lines by a factor 1.5 to 2. Treatment with MeJa (methyl jasmonate)

Plants treated with methyl jasmonate made it possible to demonstrate a high induction of GUS activity. The PILOX plants were treated with 250 μM or 500 μM of MeJa. The induction of the promoter is revealed by measurement in fluorimetry of the GUS activity on 3 batches of 2 plants. All the treated plants have a GUS activity of 2 to 8 times greater than that of the controls after treatment with 250 μM of MeJA. The results are shown in FIG. 4. The same results are obtained with a concentration of 500 μM. However, an experiment has made it possible to reach a maximum induction value of a factor of 15. The histochemical analysis of the glucuronidase activity has shown that the induction of activity seems to take place preferentially in the leaves. Note that residual GUS activity is detected in the cotyledon leaves of untreated plants. Treatment with SA (salicylic acid)

The GUS activity assays after application of salicylic acid did not reveal activation of the GUS expression in the treated plants. The coloring of plants treated with AS is similar to that of untreated plants and does not demonstrate an inducing effect of SA on the PILOX promoter. These results are confirmed by fluorimetry measurements which also do not show an inducing effect of the SA on the PILOX promoter at the doses tested. It has previously been shown on cell cultures that SA inhibits a Ppn-induced signaling pathway: cells treated with an SA + Ppn elicitor combination have a lower ethylene production than plants treated with the elicitor alone (Rickauer et al., Planta, 202: 155-162, 1997). It was interesting to study the effect of SA on the inducing activity of the Ppn elicitor on the LOX signaling pathway in our transgenic plants. Plants treated with the elicitor show a significant blue coloration, both in the leaves and in the roots. When the plants are treated with the elicitor and the SA, this coloration is less, suggesting that the SA also has an antagonistic effect on the induction of LOX by the elicitor. Treatment with H ₂ O2

Hydrogen peroxide was used to test the effect of a molecule involved in oxidative shock on the PILOX promoter. The transgenic plants were treated with three concentrations of hydrogen peroxide (200 μM, 2 and 20 mM), with or without the presence of catalase (which detoxifies H ₂ O ₂ in planta). GUS activity is detected histochemically after 1 h or 4 h of treatment. Under these conditions, no inducing effect of hydrogen peroxide on the PILOX promoter has been demonstrated. We also produced hydrogen peroxide directly in our treatment media using glucose oxidase and glucose (6mM). These tests also do not demonstrate an inducing effect of H O ₂ on the PILOX promoter. Treatment with phytosanitary products

The assay of GUS activity in the tissues of plants treated with a Aliette solution at 125 mg / 1 made it possible to demonstrate a marked increase in activity. On two independent transgenic lines, we measure activation levels between 2 and 8 with an average of 4.5. These results are confirmed by the histochemical analysis of the glucuronidase which shows the appearance of a blue coloration in the treated plants. The induction seems to concern the leaf tissue as well as the roots.

The LOX promoter is not induced by BION. Indeed, all of the experiments carried out on two transgenic lines indicate an average induction of activity by a factor of 1.5.

Claims

 claims

1) Polynucleotide comprising a plant promoter regulatory sequence characterized in that it comprises the polynucleotide of SEQ ID No. 1.

2) Polynucleotide characterized in that it comprises a polynucleotide chosen from the following polynucleotides: a) a polynucleotide capable of hybridizing selectively to the polynucleotide of SEQ ID No. 1; and b) a polynucleotide homologous at least 80% to the polynucleotide of SEQ ID No. 1.

3) Polynucleotide according to claim 2 characterized in that it has a promoter activity in plant cells and plants.

4) Polynucleotide according to one of claims 2 or 3 characterized in that said promoter activity is induced in response to aggression by a pathogen.

5) Cassette of functional expression in plant cells or plants comprising, in the direction of transcription, a promoter regulatory sequence in 5 ′, a coding sequence and a regulatory sequence in 3 ′, characterized in that the sequence of 5 'promoter regulation comprises a polynucleotide according to one of claims 1 to 4.

6) Expression cassette according to claim 5 characterized in that the coding sequence comprises a sequence coding for a reporter gene or a sequence coding for a protein of interest.

7) Expression cassette according to claim 6 characterized in that the protein of interest is a protein conferring on plants resistance properties to diseases or insects.

8) Expression cassette according to claim 7 characterized in that the protein of interest is chosen from fungal elicitors or lytic peptides.

9) Vector comprising a polynucleotide according to one of claims 1-8.

10) Process for transforming plant cells, characterized in that it consists in integrating into the genome of said plant cells at least one polynucleotide according to one of claims 1 to 4, at least one expression cassette according to one of claims 5 to 8 or a vector according to claim 9.

11) Transformed plant cells characterized in that they comprise a polynucleotide according to one of claims 1 to 4, an expression cassette according to one of claims 5 to 8 or a vector according to claim 9.

12) Process for obtaining transformed plants characterized in that: a) plant cells are transformed by integration into the genome of said plant cells of at least one polynucleotide according to one of claims 1 to 4, at least an expression cassette according to one of claims 5 to 8 or a vector according to claim 9; and b) a plant is regenerated from the transformed plant cells.

13) Process for obtaining transformed plants characterized in that: a) plant cells are transformed by integration into the genome of said plant cells of at least one polynucleotide according to one of claims 1 to 4, at least an expression cassette according to one of claims 5 to 8 or a vector according to claim 9; and b) a plant is regenerated from the transformed plant cells, and c) the regenerated plant is crossed with other plants.

14) Transformed plants capable of being obtained by a process according to claims 12 or 13.

15) Transformed plants characterized in that they comprise a polynucleotide according to one of claims 1 to 4, an expression cassette according to one of claims 5 to 8 or a vector according to claim 9.

16) Transformed plants characterized in that they contain transformed cells according to claim 11.

17) Plants transformed according to one of claims 14 to 16, characterized in that they are chosen from the following plants: tobacco, wheat, barley, sorghum rice, corn, soybeans, rapeseed , cotton, sunflower, beet, clover.

18) Seeds of the plants transformed according to one of claims 14 to 17.