FR2825578A1 - Reducing sensitivity of plants to diseases and pathogens, by overexpressing a lipoxygenase, also vectors and cassettes for the process and transformed plants - Google Patents

Abstract

Reducing the sensitivity of plants to diseases and attack by pathogens comprising overexpressing a lipoxygenase (I) in the plants, is new. Independent claims are also included for the following: (1) expression cassette (EC) functional in plant cells and plants, comprising a constitutive plant promoter that controls expression of a sequence (II) encoding a (I) at least 90 % homologous with a 862 residue amino acid sequence (S1), given in the specification; (2) vector containing EC; (3) plant cells transformed to contain EC and/or the vector of (2); and (4) transformed plants containing EC, the vector of (2) and/or the cells of (3).

Description

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Over-expression of a lipoxygenase in plants and reduction in the sensitivity of plants to diseases and attacks by pathogenic organisms
The present invention relates to methods for decreasing the susceptibility of plants to disease and attack by pathogenic organisms. The methods according to the invention consist in over-expressing a lipoxygenase in plants to reduce their sensitivity to diseases and aggressions. The invention also relates to expression cassettes, vectors and transformed plants used in the methods according to the invention.

 Lipoxygenases (LOXs) are ubiquitous enzymes in higher plants and mammals. They catalyze the oxygenation of polyunsaturated fatty acids containing a (Z, Z) -1,4-pentadiene motif. In plants, the enzyme's substrates are linoleic (C18: 2) and linolenic (C18: 3), two major constituents of cell membranes. These polyunsaturated fatty acids are generally complexed in the form of membrane phosphoglycerolipids and are only accessible to LOXs after the action of a phospholipase of type A2 or lipolytic acyl hydrolases. Recent work, however, suggests that certain LOXs could, under certain circumstances, oxidize esterified fatty acids and membrane lipids (1-4). Arachidonic acid, which is not detectable in plants, but is one of the membrane constituents in Oomycetes, is also a substrate for plant LOXs (5).

 LOXs are classified according to the position of the carbon on which molecular oxygen is preferentially inserted. In plants, there are 13-LOXs and 9-LOXs; the same enzyme can however use either position, either indifferently or with a preference for a position. This specificity can be modified as a function of the pH and O2 concentration conditions of the medium (4). The position specificity of a LOX is not directly predictable from its primary sequence, even if certain structural elements linked to this property are now known (6,7).

 The products formed in the reaction are hydroperoxides of fatty acids, very reactive and capable of causing, via radical reactions, the degradation of the major constituents of the cell (lipids, proteins, nucleic acids) (8). Fatty acid hydroperoxides are quickly converted to a series of compounds with diverse biological activities. All products derived from polyunsaturated fatty acids via oxygenation are collectively called oxylipins.

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 Plant LOXs have been associated with various physiological processes, based on gene expression profiles and enzyme activity. Thus, it has been proposed that LOXs be involved in the regulation of seed ripening, germination, fruit ripening, senescence of leaves and flowers. The precise participation of LOXs in these processes remains to be determined, however. An important role is also attributed to LOXs in stress responses, in particular injury, and parasitic attacks (4,5, 9). A strong induction of LOX gene expression is thus measured in many monocotyledonous or dicotyledonous plants during their interaction with bacteria, viruses or fungi, as well as after mechanical injury or caused by insects on the leaves.

 This diversity in biological functions could be ensured due to the presence of different isoenzymes, presenting regulatory mechanisms as well as very varied tissue and subcellular locations, depending on the species and isoforms considered (5). Added to this is the diversity of oxylipins generated from LOX products, which is modulated according to the type of hydroperoxide formed and the nature of the enzymes which metabolize them.

 The hydroperoxides of fatty acids generated by LOX are in fact converted according to several distinct enzymatic pathways (4). The hydroperoxide lyase (HPL) path catalyzes the cleavage of 13-or 9-hydroperoxides of fatty acids to result in the synthesis of volatile aldehydes in C6 or C9 and short chain acids in C12-or in C9, like 12-oxo-trans-9-dodecenoic acid, precursor of traumatic acid. C6 aldehydes play an important role in the fragrance of plants, but some such as trans-2hexenal also have anti-microbial properties (10). Recently, it has been shown that trans-2-hexenal can also act as a signal molecule enabling the activation of defense genes (11). Traumatic acid, also called the wound hormone, is believed to play a role in tissue healing by promoting cell division at injury sites (5).

A second enzymatic pathway involved in the metabolism of the fatty acid hydroperoxides produced by LOX concerns the allene oxide synthase (AOS). This enzyme catalyzes the dehydration of 13-hydroperoxylinolenic acid and forms an oxide allene which is the precursor of jasmonic acid. Jasmonic acid is a key molecule in the plant's signaling mechanisms enabling the activation of numerous defense genes (12) including the genes coding for protease inhibitors, active against insects, and also many PR proteins. (chitinase, defensin, thionine, glucanase).

 The role of jasmonates as a signal molecule has been recently emphasized by various

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 experiments showing that mutants of Arabidopsis or tomato affected in the biosynthesis of these molecules or their perception, showed a deficiency in the induction of defense genes (13), a greater sensitivity to organisms normally non-pathogenic for them wild plants (14) or reduced resistance against insects (15). A third enzymatic way of converting hydroperoxides concerns the formation of divinyl ethers of fatty acids. These compounds, which can be formed from 13-or 9-hydroperoxides, have been isolated from various plants. The first divinylether synthase (DES) was recently cloned in tomatoes (16). The fatty acid divinylether produced from 9-hydroperoxides, colneleic acid and colnelenic acid, have antifungal properties, in particular with regard to Phytophthora infestans (17).

 Other modifications of the products of the reaction catalyzed by LOX relate to epoxidation by epoxygenases and peroxygenases. This metabolic pathway allows the synthesis of molecules with antimicrobial activity and could intervene in the synthesis of cutin monomers which, in addition to their structural function, can also induce the activity of defense genes (18).

 The hydroperoxides of fatty acids produced by LOX can finally generate the formation of free radicals involved in mechanisms of degradation of membranes linked to cell death (5).

 All of these data therefore show that the initial activity of LOX is essential for the synthesis of a set of molecules, some of which exhibit antimicrobial activities or are involved in signaling leading to defense.

 LOX activity increases markedly in tobacco in response to elicitors (19, 20). The isolation of a LOX complementary DNA clone, called pTL-J2 (21), allowed the characterization of the corresponding tobacco gene, called LOX. DNA / RNA hybridization experiments (northern hybridization) have shown that LOTI is expressed in tobacco cells in culture following the application of elicitor and in tobacco plants inoculated with the oomycete Phytophthora parasitica nicotianae, Ppn (22).

 Transcripts corresponding to this gene are not detectable in healthy plants or untreated cells. A biochemical study shows that in vitro the LOX of elicited tobacco cells allows the production of 9-and 13-hydroperoxides of fatty acids with preferential insertion of molecular oxygen in position 9 (20).

 Following the root inoculation of tobacco with Ppn, a strong induction of gene expression is measured. In the case of an incompatible interaction (plant

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 resistant / avirulent microorganism race), an early induction of gene activity is measured, with a maximum of accumulation of transcripts at 24 hours (22). The induction of the gene is later and of smaller amplitude in the case of a compatible interaction (susceptible plant / virulent race), as has also been observed in other gene-for-gene models, such as interactions between tomatoes and Pseudomonas syringae (23), rice and Magnaporthe grisea (24), and potatoes and Phytophthora infestans (25).

The expression of the LOX1 gene is not detected in the tissues of healthy tobacco plants, with the exception of flowering plants for which LOX transcripts are detected in small quantities in the petals and sepals, and young germinations (22 , 26). In the latter case, a transient expression of the LOX1 gene is detected between the second and fourth day after the start of germination.

maximum à 12 heures. Un polysaccharide issu d'algues marines, le -carraghénane, est également inducteur du gène LOX1 lorsqu'il est appliqué à des plantes de tabac par infiltration dans le mésophylle foliaire (28). Villalba et al. (27) have shown that an elicitor glycoprotein isolated from Ppn walls, called CBEL for Cellulose Binding Elicitor Lectin, induces the LOX1 gene when it is infiltrated into the leaf mesophyll of tobacco plants. LOX1 transcripts appear during the first four hours after infiltration and their level is

maximum at 12 o'clock. A polysaccharide from marine algae, carrageenan, is also an inducer of the LOX1 gene when applied to tobacco plants by infiltration into the leaf mesophyll (28).

 In tobacco cells, expression of the LOX1 gene is detected following elicitor treatments. In the case of Ppn elicitor (wall extract), induction of expression of the LOX1 gene is detected within the first two hours after treatment, with a maximum accumulation at 24 hours (22). Cryptogeine, an elicitor peptide of Phytophthora cryptogea, oomycete for which tobacco is not a host, as well as the endopolygalacturonase of Colletotrichum lindemuthianum also allow the induction of the LOX1 gene (26). It has also been shown that the induction of the LOX1 gene was earlier than that of defense genes such as those encoding the PR proteins which are chitinases and ssl-3 glucanases, suggesting the potential role of the LOX pathway in transduction of the signal triggering the defense reactions (26). LOX1 expression in cells is also inducible by methyl-jasmonate (22,29), thus indicating a possible self-amplification of this pathway, when no accumulation of LOX transcripts is detected after the application of acid salicylic (22).

 Analysis of the expression of the LOX1 gene shows that the induction of LOX constitutes an early response of the plant to infection by Ppn, suggesting a role in the establishment of resistance. This potential role has been confirmed by obtaining plants

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 transgenics expressing the DNA complementary to the gene in antisense orientation. Indeed, these plants, originating from the line 46-8 normally resistant to race 0 of Ppn, exhibit greatly reduced LOX activity levels and have lost their capacity to trigger the incompatible reaction (30, 31). This experiment clearly shows that the expression of the LOX gene is necessary for establishing the state of resistance in the gene-for-gene interaction between tobacco and Ppn. LOX nonsense plants are also more sensitive to another pathogenic tobacco fungus, Rhizoctonia solani (30, 31). In parallel, Rustérucci and collaborators (32), have shown that 9hydroperoxides accumulate during the hypersensitive type response of tobacco to cryptogeine, and that this accumulation is necessary for the development of this reaction.

In another solanacea, the potato, oxylipins from the 9-LOX pathway, in particular colneleic and colnelenic acids, preferentially accumulate in cultured cells in response to a P. infestans elicitor (33). All of these data suggest a major role for the 9-LOX pathway in nightshade, including tobacco, in the response to pathogens, in particular Oomycetes. In these plants, the 13-LOX pathway participates in the response to pathogens, as shown by the early synthesis of jasmonates (29); it is also involved in the response to injury and insects.

Thus, potato plants expressing a 13-LOX antisense construct have been shown to be more susceptible to attack by insects (34).

 Few attempts to over-express LOX in plants have been reported in the literature. Indeed, the expression of a LOX poses feasibility questions because of the potential toxic properties of LOXs, if they directly oxidize biomembranes, and of the products they form.

In tobacco, the introduction of soy LOX2 under the control of a chimeric promoter, formed by fusion of the cauliflower mosaic virus (CaMV) 35S promoter and an "enhancer" isolated from the alfalfa mosaic, allowed the production of transgenic calluses having a LOX activity approximately 2 times stronger than that of calluses transformed with the vector devoid of LOX sequence. These calluses are capable of forming 3 to 6 times more volatile aldehydes, produced from the 13-LOX pathway via a 13-
HPL, which the calluses control. Transgenic plants have been regenerated from these calluses but although having a strong accumulation of the heterologous protein, these plants do not have a LOX activity different from that of control plants. However, they form larger amounts of volatile aldehydes than the control plants (35). More recently, a 13-LOX specific lipid body from cucumber seeds has been

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 expressed in tobacco (36). The heterologous protein accumulates throughout the plant, in transgenic tobacco, and in particular in seeds where it is localized mainly in lipid bodies such as in cucumber. Its presence results in a qualitative modification of the LOX activity of transgenic plants, both in vitro and in vivo. A 13-LOX chloroplastic from Arabidopsis, AtLOX2, was used in a direction-oriented construction under the control of the CaMV 35S promoter to transform arabettes. However, the authors focused on the transformation events that led to a decrease in LOX activity by co-suppression (37). Finally, in the lens, the transient transformation of protoplasts with a construction containing a lens LOX under the control of the CaMV 35S promoter led to a 20% increase in the LOX activity of these protoplasts (38). None of the transformed plants or cultures described above have been tested for response to attack by pathogens. In addition, the experiments carried out mainly concern 13-LOXs.

 In addition, the hypotheses concerning the participation of lipoxygenases in the defense mechanisms of plants are essentially based on the biological activities of certain oxylipins (39). However, the biosynthesis of a majority of oxylipins, and in particular jasmonic acid and volatile aldehydes from the 13-LOX pathway, and the divinyl ethers from the 9-LOX pathway, requires the activity of enzymes metabolizing the LOX products, such as AOS, HPL, or DES, beyond LOX itself. The expression of genes corresponding to these enzymes is often inducible. In tomatoes and Arabidopsis thaliana, the expression of genes encoding OSA and HPL is inducible by injury (40-42). Consequently, the action of lipoxygenases alone does not therefore seem sufficient to trigger the resistance mechanisms of plants.

 Cultivated plants are attacked by many pathogenic organisms such as viruses, bacteria and fungi, but also by pests such as insects. These attacks weaken the plants and decrease the yields of their crop. There is therefore an important need to increase the resistance mechanisms of plants and to decrease their sensitivity to diseases and attacks by parasitic or pathogenic organisms. The response mechanisms of plants to attack by pathogens have been the subject of numerous studies. It is now commonly accepted that the lipoxygenase pathway in plants participates in their defense system and in the establishment of a state of resistance through the pathway of oxylipins in particular.

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However, the knowledge of these metabolic pathways and lipoxygenases has not made it possible to develop methods allowing the plant resistance to be directly increased.

 This problem is solved by the present invention since it has now been found that the over-expression of a lipoxygenase in plants directly reduces the susceptibility of plants to diseases and to attacks by pathogens. Unexpectedly, and although lipoxygenase integrates into complex metabolic pathways, the overexpression of a lipoxygenase in plants is sufficient to improve the response of plants to attack by pathogens. In addition, the overexpression of lipoxygenase does not substantially affect the phenotype of the transformed plants apart from the new acquired properties.

 The present invention therefore consists in over-expressing a lipoxygenase in plants to reduce the susceptibility of plants to diseases and attacks by pathogenic or pest organisms. The invention also relates to preferred expression cassettes for the overexpression of lipoxygenase in plants as well as plant cells and transformed plants.

 Description of the list of sequences SEQ ID No. 1: Tobacco lipoxygenase (LOX1).

SEQ ID No. 2: Expression cassette CaMV 35S promoter-Coding sequence of the LOXl tobacco gene - Terminator nos.

SEQ ID No. 3-6: Primers for PCR.

Description of the invention
The present invention relates to a method for reducing the susceptibility of plants to disease and attack by pathogenic organisms. This process involves overexpressing a lipoxygenase in these plants.

By "lipoxygenase" is meant an enzyme catalyzing the dioxygenation of polyunsaturated fatty acids containing a (Z, Z) -1,4-pentadiene motif. In plants, the enzyme's substrates are linoleic (C18: 2) and linolenic (C18: 3), two major constituents of cell membranes.

 "Over-expression" means that the lipoxygenase is expressed at a level higher than the level of expression observed in a reference plant (not induced). This over-expression results in a greater accumulation of transcripts of the lipoxygenase gene,

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 lipoxygenase itself and by increased specific lipoxygenase activity in plant tissues. To reduce the sensitivity of plants to diseases and attacks by pathogenic organisms, it is important that the level of expression of lipoxygenase is higher than that of a reference plant when aggression by the pathogenic organism occurs.

 The overexpression of lipoxygenase makes it possible to decrease the susceptibility of plants to diseases and to attacks by pathogenic organisms. By "attacks by pathogenic organisms" is meant in particular attacks on plants by viruses, bacteria, fungi, oomycetes or even insects.

 In a preferred embodiment, the method includes constitutively over-expressing a lipoxygenase in plants. The term "constitutively" designates the temporal and spatial expression of the lipoxygenase in plants in the methods according to the invention. By "constitutively" means the expression of a lipoxygenase in the tissues of the plant throughout the life of the plant and in particular during the whole of its vegetative cycle. In a first embodiment, the lipoxygenase is expressed constitutively in all plant tissues. In a second embodiment, the lipoxygenase is expressed constitutively in the roots, the leaves, the stems, the flowers and / or the fruits. In another embodiment of the invention, the lipoxygenase is expressed constitutively in the roots, the leaves and / or the stems.

 In a particular embodiment of the invention, the lipoxygenase is an "inducible" lipoxygenase in a reference plant. In the present invention, the term "inducible" lipoxygenase means a lipoxygenase which is not expressed or expressed at very low levels and whose expression is strongly induced in response to elicitors in the response to stress, injuries and in particular diseases and attacks by pathogenic organisms.

 In a preferred embodiment of the methods according to the invention, the lipoxygenase preferably has 9-lipoxygenase activity. Lipoxygenases (LOX) are classified according to the position of the carbon on which molecular oxygen is preferentially inserted.

In plants, 13-LOX and 9-LOX are distinguished. Methods for determining the specificity of lipoxygenase activity are described in the literature (Fournier et al., Plant 1. 3: 63-70,1993; Homung et al., PNAS 96: 4192-4197.1999; Rustérucci and al., 1. Biol. Biochem. 274: 36446-36455,1999).

 Any lipoxygenase whose over-expression in plants makes it possible to reduce the susceptibility of plants to diseases and attacks by pathogenic organisms is

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utilisable dans les procédés selon l'invention. Les lipoxygénases sont connues de l'homme du métier et d'autres lipoxygénases peuvent être identifiées en utilisant des techniques connues. On citera notamment à titre d'exemple les lipoxygénases de pomme de terre (Kolomoiets M. V. et al., Plant Physio. 124 : 1121-1130, 2000), de tomate (Genbank AY008278) de tubercule de pomme de terre (Royo et al., JBiol. Chem., 271 : 21012-21019, 1996 ; Casey, R., Plant Physiol., 107 : 265-266, 1995), la lipoxygénase de graine d'amande (Mita et al., Eur. J Biochem., 268 : 1500-1507, 2001) et la lipoxygénase de grain d'orge (Van Mechelem et al., Biochem. Biophys. Acta, 1254 : 221-225, 1995).

usable in the methods according to the invention. Lipoxygenases are known to those skilled in the art and other lipoxygenases can be identified using known techniques. Mention may in particular be made, for example, of potato lipoxygenases (Kolomoiets MV et al., Plant Physio. 124: 1121-1130, 2000), of tomato (Genbank AY008278) of potato tuber (Royo et al. , JBiol. Chem., 271: 21012-21019, 1996; Casey, R., Plant Physiol., 107: 265-266, 1995), almond seed lipoxygenase (Mita et al., Eur. J Biochem. , 268: 1500-1507, 2001) and barley grain lipoxygenase (Van Mechelem et al., Biochem. Biophys. Acta, 1254: 221-225, 1995).

Preferably, the lipoxygenase is a plant lipoxygenase.

In a particular embodiment of the invention, it is a solanaceous plant lipoxygenase. Among the solanaceae plants, mention will be made in particular of tobacco, tomatoes, potatoes and even chilli peppers.

In another preferred embodiment of the invention, the lipoxygenase has at least 80% homology with tobacco lipoxygenase 1 (LOX1) of SEQ ID No. 1. Advantageously, the percentage of homology will be d '' at least 80%, 85%, 90%, 95% and preferably at least 98% and more preferably at least 99% relative to SEQ ID No. 1. The term "homologous" denotes a polypeptide which may have a deletion, an addition or a substitution of at least one amino acid. The methods for measuring and identifying homologies between polypeptides or proteins are known to those skilled in the art. One can use for example the UWGCG package and the BESTFITT program to calculate homologies (Devereux et al., Nucleic Acid Res. 12, 387-395, 1984). Preferably, these homologous lipoxygenases retain the same biological activity as the tobacco lipoxygenase (LOX1) of SEQ ID No. 1. Preferentially, these polypeptides therefore have lipoxygenase activity and even more preferably lipoxygenase activity.

In an even more preferred embodiment, the methods according to the present invention use the lipoxygenase of SEQ ID No. 1.

The overexpression of lipoxygenase in plants is achieved by transforming the plants or by applying to plants a molecule stimulating the synthesis of lipoxygenase in the plant.

In a preferred embodiment of the invention, the lipoxygenase is over-expressed by integration into the genome of plants of an expression cassette comprising a sequence coding for a lipoxygenase under the control of a promoter functional in plants.

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 By "promoter" 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 production of an RNA transcript. Plant promoters, which can be used in the methods according to the present invention, are widely described in the literature.

 Preferably, the promoter is a constitutive promoter in plants. The constitutive promoters which can be used in the methods according to the invention are also well known to those skilled in the art.

 As promoter regulatory sequence in plants, any promoter sequence of a gene expressing itself naturally in plants can be used, for example promoters known as constitutive of bacterial, viral or plant origin. Bacterial promoters such as that of the octopine synthase gene or the nopaline synthase gene, viral promoters, such as the cauliflower mosaic virus 35S promoter or the CSVMV promoter (WO 97/48819) will be cited and promoters of plant origin such as the promoter of the histone gene (EP0507698) or the promoter of a rice actin gene (US 5,641,876). 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").

 In a preferred embodiment of the invention, the constitutive promoter is the 35S promoter of the cauliflower mosaic virus.

 In another embodiment of the invention, the constitutive expression or the overexpression of the lipoxygenase is obtained by transforming the plants so as to place a constitutive promoter or an "enhancer" sequence upstream or close to the lipoxygenase gene in plants. One can in particular use any regulatory sequence making it possible to increase the level of expression of the lipoxygenase in plants.

 Preferably, the lipoxygenase is over-expressed in the stems, leaves and / or roots of plants.

 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, beets, tobacco, cotton, etc.

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 The overexpression of lipoxygenase can be obtained in any plant according to methods known to those skilled in the art.

 In a particular embodiment of the invention, the plants are chosen from solanaceous plants. Among the solanaceae plants, mention will be made in particular of tobacco, tomatoes, potatoes and even chilli peppers.

 In a preferred embodiment of the invention, the lipoxygenase is over-expressed by integration into the genome of plants of an expression cassette comprising a sequence coding for a lipoxygenase under the control of a promoter functional in plants. A polynucleotide encoding a lipoxygenase is inserted into an expression cassette using cloning techniques well known to those skilled in the art.

This expression cassette includes the elements necessary for the transcription and translation of the sequences coding for lipoxygenase in plants. Advantageously, this expression cassette comprises both elements making it possible to produce lipoxygenase by the transformed plants and elements necessary for the regulation of this expression. The present invention also relates to preferred expression cassettes which can be used in the methods according to the invention.

 In one embodiment, the present invention relates to functional expression cassettes in plant cells and plants comprising a promoter having constitutive activity in plants controlling the expression of a polynucleotide encoding a lipoxygenase homologous to at least 90 % with lipoxygenase of SEQ ID No. 1. Advantageously, the percentage of homology will be at least 80%, 85%, 90%, 95% and preferably at least 98% and more preferably d '' at least 99% compared to SEQ ID No. l. Preferably, this polynucleotide codes for a lipoxygenase having 9-lipoxygenase activity. More preferably, this polynucleotide codes for the lipoxygenase of SEQ ID No. 1. In a particular embodiment of the invention, the promoter is the 35S promoter of the cauliflower mosaic virus.

 The expression cassettes according to the invention preferably comprise a terminator sequence. These sequences allow the termination of transcription and polyadenylation of the mRNA. Any functional terminator sequence in plants 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 (EP 0 633 317), the terminator CaMV 35 S and the tml terminator. These terminator sequences are usable in monocotyledonous and dicotyledonous plants.

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

 Advantageously, the expression cassettes according to the present invention are inserted into a vector for their replication or for the transformation of plants.

 The present invention also relates to vectors for the transformation of plants comprising at least one expression cassette according to the present invention. This vector can in particular consist of a plasmid or a virus into which an expression cassette according to the invention is inserted. 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. Preferably, the vectors of the invention also comprise at least one selection marker. Among the selection markers, there may be mentioned antibiotic resistance genes such as the nptII 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 HPPD gene (WO 96/38567) for tolerance to isoxazoles.

 The present invention therefore relates to vectors comprising an expression cassette according to the invention.

 The invention also relates to a process for transforming plants with an expression cassette or a vector according to the invention.

 According to the present invention, the transformation of plants can be obtained by any suitable known means, the techniques for transforming plants are amply described in the specialized literature.

 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 tissues with particles to which the DNA sequences are attached. Other methods can also be used such as micro-injection or electroporation, or even direct precipitation using PEG.

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 Those skilled in the art will choose the appropriate method depending on the nature of the plant cell or the plant.

 The present invention relates to transformed plant cells comprising 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 present invention also relates to the transformed plants comprising an expression cassette, a vector and / or cells transformed according to the invention.

 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, beets, tobacco, cotton, etc.

 FIGURES Figure 1 Figure 1 shows the 35S-9-LOX construct used for tobacco processing. The coding sequence 9-LOX (LOX /) was obtained by PCR amplification and then inserted between the promoter 35S of the cauliflower mosaic virus (35S) and the terminator of the nopaline synthase of Agrobacterium tumefasciens (tnos). This vector also includes the neomycin phosphotransferase gene (NPTII) which confers resistance to kanamycin in bacteria and plants. "F" ("35S sense") designates the sense primer and "R" ("reverse LOX1") denotes the reverse primer.

Figure 2 Figure 2 is a histogram representing the measurement of specific activity
LOX in the stems of tobacco plants in nKAT / mg protein. 46-8 WT and
49-10 WT designates the parental lines. S46-21, S46-26, S49-18 designate the transgenic lines. Indeed, prior to inoculation tests by
Ppn, the specific LOX activity as well as the level of corresponding transcripts were analyzed for stems of 12-week-old tobacco plants. We observe that the level of specific LOX activity in the three

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 Transgenic lines retained is significantly higher than that measured in the corresponding parental lines.

Figure 3 Figure 3 is a histogram representing the measurement of the length of the lesions in mm. These measurements were taken 48 hours (48 hours) or 72 hours (72 hours) after inoculation. The numbers in parentheses correspond to the independent repetitions performed. The two lines S46-21 and S46-26, inoculated with Ppn 1 have lesion lengths significantly shorter than those obtained with the same pathogen in the wild parent, 46-8 WT. Similarly, the S49-18 line has shorter lesions than those measured in its wild relative 49-10 WT during the inoculation of these two lines with Ppn 0. These differences are significant at 48 hours and 72 hours after l 'inoculation.

 EXAMPLES Example 1: Biological material Wild tobacco plants (Nicotiana tabacum L.) from the two quasi-isogenic lines 46-8 (46-8 WT) and 49-10 (49-10 WT) were used (Helgeson et al. , Phytopath. 62, 1439-1443, 1972). These lines are distinguished by the presence in the 46-8 WT line of a locus of resistance to race 0 of Ppn. Thus, the 46-8 WT line is resistant to race 0 of Ppn and sensitive to race 1 of this pathogen while the line 49-10 WT is sensitive to both races of Ppn.

2-1 (hygrométrie 40%, température constante 25 C, lumière 60 Ilmol. m-2. s : 16h, obscurité : 8h), les plantes sauvages ou transgéniques résistantes à la kanamycine, sont transférées sur vermiculite en salle à culture (hygrométrie 80%, lumière 125 umol. m s : 16h, 25 C, obscurité : 8h, 18 C). The seeds of wild or transgenic lines are sterilized (Rancé et al., Plant Cell Report, 13,647-651, 1994) and sown on solid MS medium, added in the case of transgenic lines, kanamycin (50 ug. Mr1). After 4 weeks of growth in vitro

2-1 (humidity 40%, constant temperature 25 C, light 60 Ilmol. M-2. S: 16h, darkness: 8h), wild or transgenic plants resistant to kanamycin, are transferred to vermiculite in the culture room (hygrometry 80%, light 125 umol. Ms: 16h, 25 C, darkness: 8h, 18 C).

 The Ppn strains used correspond to isolates 1156 (race 0) and 1452 (race 1) (Hendrix, J. W. & Apple, J. L., Tobacco Science 11, 148-150,1967). The Ppn mycelium is grown in the dark on a solid synthetic medium (Keen, N. T., Science 187,74-75, 1975).

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Example 2: Obtaining the CaMV 35S promoter cassette-coding sequence LOX1terminator nos (p35S-LOXl)
TL-J2 is a complementary DNA of 2888 bp, corresponding to the LOX1 gene from tobacco induced by pathogenesis. Obtaining this complementary DNA is described by Véronési et al. (Véronési et al., Plant Physiol. 108,1342, 1995), its sequence is deposited in GenBank under accession number X84040. This cDNA was used as a template for the PCR amplification of the LOX coding sequence.

 Primers were synthesized to amplify a 2.6 kb DNA fragment, covering positions 49 to 2667 of the cDNA: - Sense primer = 5'-GTTATCAAACAGTTTAAAATGTTTCTGGAG-3 '- Reverse primer = 5'-TGATTTAAAGTTCTATATTGAC-3 '' These primers also allow the introduction of DraI sites (underlined in the primer sequence) upstream of the translation initiation codon and downstream of the stop codon (indicated in bold in the primer sequence) of the sequence LOX.

de 20 cycles, incluant chacun 1 min de dénaturation à 94 C, 1 min d'hybridation à 50 C et 6 min d'extension à 72 C, suivis d'une étape finale de 40 min d'extension à 72 C. The PCR reaction was carried out in a total volume of 25 μl, containing 50 ng of plasmid pTL-J2.50 pmol of each of the sense primers and reverse above, 2.5 units of DNA polymerase Pfu (Stratagene Cloning Systems) and adjusted to 200 µM of each dNTP and 2 mM MgCL. After 5 min of denaturation at 94 C, the thermal cycler program was composed

20 cycles, each including 1 min of denaturation at 94 C, 1 min of hybridization at 50 C and 6 min of extension at 72 C, followed by a final step of 40 min of extension at 72 C.

The DNA of this reaction was digested with DraI, and separated on 0.8% agarose gel. The 2.6 kb blunt-end fragment was purified from the gel (Kit QiaEx II, Qiagen) and cloned at the SmaI site of the vector pIPMO (Rancé et al., PNAS 6554-6559,1998) between the CaMV 35S promoter and the 3 ′ untranslated region of the nopaline synthase gene of Agrobacterium tumefaciens (terminator nos). This vector also includes two copies of the neomycin phosphotransferase (NPTII) gene which confers resistance to kanamycin in bacteria and plants. The ligation mixture was used to transform competent Escherichia coli XL1Blue bacteria, and colonies resistant to kanamycin were selected and then screened for the presence of LOX sequence using the molecular probe TL-J2. Positive colonies were cultured and the corresponding plasmids purified. The orientation of the LOX1 sequence was examined for each of the plasmids by PCR using the following primers:

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 - Primer F, sense 35S: 5'-GGCCATGGAGTCAAAGATTC-3 'targeting nucleotides 6906-6925 of the CaMV 35S promoter (sequence available in Genbank under accession number J02048).

- Primer R, reverse LOX7: 5'-GCTCTGGCATGAAATTTCG-3 'targeting nucleotides 2290-2272 (non-coding strand) of TL-J2.

The amplification reactions were carried out in a volume of 50 μl and included 100 ng of plasmid to be tested, 10 pmol of each primer and 1 unit of Taq DNA polymerase in a medium adjusted to 200 μM of each dNTP and 1.5 mM MgCl. The thermal cycler program included an initial denaturation step of 5 min at 94 C, then 40 cycles each consisting of 1 min denaturation at 94 C, 1 min hybridization at 65 C and 2 min extension at 72 C, followed a final elongation step of 10 min at 72 C. The reaction products were separated on a 0.8% agarose gel. A plasmid for which the presence of an amplification product of the expected size (2.8 kb) indicated the sense orientation of the LOX sequence with respect to the CaMV 35S promoter in the construction, was selected.

The LOX sequence and its junctions with the promoter and the terminator have been fully sequenced. The plasmid thus verified is named p35S-LOXl. The sequence of the CaMV 35S-LOX1 construction is described in SEQ ID No. 2.

EXAMPLE 3 Genetic Transformation of Tobacco
The plasmid p35S-LOX1 was mobilized in the LBA4404 strain of Agrobacterium tumefaciens by thermal shock (Holsters et al., Mol. Gen. Genet. 163,181-187, 1978). A colony resistant to kanamycin was isolated, the plasmid purified, and the integrity of the construction verified by PCR with the primers F and R and under the conditions described above for the determination of the relative orientation of the LOX sequence. The recombinant bacteria obtained were then used for the infection of tobacco leaf discs, Nicotiana tabacum, lines 46-8 WT and 49-10 WT according to protocols already described (Horsch et al., Science 227,1229-1231, 1985 ).

u. g. ml'' de Kanamycine ont été placées en chambre de culture puis en serre pour l'obtention des graines Tl, par auto-fécondation. Les lignées transgéniques régénérées à partir des lignées parentales 46-8 WT et 49-10 WT sont nommées plantes S46-x et S49-x, respectivement. Plants regenerated on a Murashige and Skoog (MS) medium containing 150

ug ml '' of Kanamycin were placed in a culture chamber and then in a greenhouse to obtain the Tl seeds, by self-fertilization. The transgenic lines regenerated from the parental lines 46-8 WT and 49-10 WT are called plants S46-x and S49-x, respectively.

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Dellaporta et collaborateurs (Dellaporta et al., Plant Mol. Biol. Rep. 1, 19-21, 1983). Example 4: Characterization of the primary transformants
The presence of the 35S-LOX1 expression cassette and the number of copies of the transgene in the genome of the regenerated plants were determined by PCR and DNA / DNA hybridization experiments (Southern). The genomic DNA of wild plants or of regenerated plants resistant to kanamycin was prepared according to the method described by

Dellaporta et al. (Dellaporta et al., Plant Mol. Biol. Rep. 1, 19-21, 1983).

The integrity of the transferred T-DNA was verified by PCR amplification using the primers F sense 35S and R reverse LOXl)), described above. The reaction conditions were as described above, but the amount of template DNA in this case was 800 ng of genomic DNA. The number of copies of T-DNA inserted was estimated by Southern blot (50). The genomic DNA (15 μg) was digested with BamH1 and the digestion products were separated on agarose gel, then transferred to a nylon membrane. A CaMV 35S probe (nucleotides 6909 to 7440 of the Genbank sequence J02048) was used after labeling with [a-32p] dCTP. The number of bands hybridizing this probe, after revelation by autoradiography, is a good indication of the number of insertion sites of the construction.

ira). Cette construction, appelée p35S-LOXl contient également un gène de résistance à la kanamycine (NPTII) permettant de sélectionner les cellules végétales transformées. Les lignées de tabac 46-8 WT et 49-10 WT ont été transformées par Agrobacterium tumefaciens LBA 4404 dans laquelle la construction p35S-LOXI a été introduite. A partir des cals sélectionnés sur un milieu contenant de la kanamycine, 15 transformants primaires indépendants S46-x dérivant de la lignée 46-8 WT ont été régénérés. La lettre x désigne le numéro de la plante obtenue. De même, 25 transformants primaires indépendants S49-x ont été régénérés à partir de la lignée 49-10 WT. Ces plantes ont été acclimatées en chambre de culture puis transférées en serre jusqu'à floraison. Les graines correspondant aux plantes Tl ont été obtenues par auto-fécondation des transformants primaires. Example 5 Transformation of Tobacco by the Sequence Coding for Tobacco LOX1 Under the Control of the Constitutive Promoter CaMV 35 S
In order to constitutively express tobacco LOX1 in transgenic tobacco, the corresponding coding sequence was introduced in sense orientation into the transfer DNA of the binary vector pIPMO, downstream of the constitutive promoter CaMV 35S (p35S) (FIG.

go). This construct, called p35S-LOXl also contains a kanamycin resistance gene (NPTII) used to select the transformed plant cells. Tobacco lines 46-8 WT and 49-10 WT were transformed with Agrobacterium tumefaciens LBA 4404 in which the construction p35S-LOXI was introduced. From the calli selected on a medium containing kanamycin, 15 independent primary transformants S46-x derived from the line 46-8 WT were regenerated. The letter x indicates the number of the plant obtained. Likewise, 25 independent primary transformants S49-x were regenerated from the 49-10 WT line. These plants were acclimatized in the growing room and then transferred to the greenhouse until flowering. The seeds corresponding to the Tl plants were obtained by self-fertilization of the primary transformants.

The integrity of the construct introduced into the genome of transgenic plants was verified by PCR amplification from a preparation of genomic DNA from the primary transformants cultivated on kanamycin and from a pair of primers, one specific

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 of the 5 ′ region of the CaMV 35S promoter (F) and the other of the 3 ′ region of the LOXI coding sequence (R). The amplification products were separated on an agarose gel and revealed with ethidium bromide. For 10 of the 11 primary transformants analyzed, the profile obtained corresponds to a single strip whose size (2.8 kb) corresponds to the estimated size of the product. In addition, this profile is identical to that obtained with the binary vector p35S-LOXi, which suggests that at least one copy has been integrated into the genome of these transformants.

In contrast, a primary transformant does not have such a profile although it is resistant to kanamycin, indicating incomplete integration of the construct. The parental lines 46-8 WT and 49-10 WT, analyzed as negative controls do not show a signal corresponding to the construction.

The number of copies inserted into each of the regenerated lines was estimated by Southern type hybridization from genomic DNA digested with BamHI and from a probe homologous to the CaMV 35S promoter. The transfer DNA has two BamHI sites: the first is located between the CaMV 35S promoter and the LOXI sequence and a second in the LOTI sequence. The BamHI fragments hybridizing the radiolabelled CaMV 35S probe therefore result from a first cut between the CaMV 35S promoter and LOXI and from a second cut in the plant genome, upstream of the left border of the transfer DNA. The insertion of the transfer DNA into the genome of the plants being random, the BamHI fragments hybridizing the radiolabelled CaMV 35S probe, obtained in the case of multiple insertions, will have sizes depending on the position of the BamH1 site in the DNA. genomics and therefore probably different. This is why the number of these fragments makes it possible to evaluate the number of insertion sites in the genome. The profiles obtained indicate that the primary transformants S46-3, S46-4, S46-26, S49-8, and S49-13 contain a copy of the transgene, while two copies have been inserted into the genome of lines S49-18 and S49-28, and three copies in the genome of lines S46-21, S49-14 and S49-30. The CaMV 35S radiolabelled probe did not hybridize with the genomic DNA corresponding to the S49-24 lines, nor with that of the parental lines 46-8 WT and 49-10WT.

EXAMPLE 6 Extraction and Analysis of RNAs
Total RNA was isolated from frozen samples of T1 generation or wild plants. The plant material was ground in liquid nitrogen and the RNA extracted using the Extract-all Kit (Eurobio). The nucleic acid concentration was estimated by spectrophotometry. The northern blot experiments were performed as

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précédemment décrit (Rickauer et al., Planta 202, 155-162, 1997). Les filtres ont été hybridés avec la sonde TL-J2 radiomarquée.

previously described (Rickauer et al., Planta 202, 155-162, 1997). The filters were hybridized with the radiolabelled TL-J2 probe.

Example 7 Analysis of the Accumulation of LOX Transcripts in the T1 Transgenic Lines The level of LOX expression was evaluated in the various T1 transgenic lines by measuring the accumulation of the LOX transcripts by northern blot. The total RNA samples were prepared from young 4-week-old transgenic tobacco plants selected in vitro on a medium containing kanamycin. The evaluation of the respective levels of transgene expression in these lines was carried out by comparing the profiles obtained with the level of transcripts detected in wild plants, as well as in a control tobacco cell suspension (negative controls), or in a tobacco cell suspension treated with Ppn elicitors (positive control). The results obtained indicate that the level of transcripts is low, or even undetectable, in the transgenic lines S46-3, S46-4, S49-8, S49-13, S49-24, S49-28 and S49-30. On the other hand, the lines S46-21, S46-26, S49-14 and S49-18 show a significant accumulation of LOX transcripts reaching, after quantification, from 30 to 66% of the level detected in elicited tobacco cells. No accumulation of LOX transcript is detected in the wild line or in the control tobacco cells. The introduction of the promoter construct 35S-LOX1 in tobacco is therefore accompanied by an important constitutive expression in the transgenic lines S46-21, S46-26, S49-14 and S49-18.

Example 8 Immunodetection of LOX Obtaining a polyclonal rabbit serum anti-LOX1 from tobacco: Rabbits were immunized with a fusion protein expressed in Escherichia coli and comprising the 244 C-terminal residues of LOX1 from tobacco fused to glutathione S-transferase (GST) from Schistosomajaponicum. An XhoI fragment of pTL-J2, corresponding to nucleotides 1921 to 2888, was inserted at the site XhoI of the vector pGEX-5X-3 (Pharmacia, sequence available in Genbank under the accession number U13858) which made it possible to obtain d 'a translational fusion with the coding sequence of GST. A colony of bacteria containing the recombinant plasmid was selected and cultured. These bacteria were treated with isopropylthio-p-galactoside at 4 mM for 16 hours at 37 ° C. in order to induce the production of the fusion protein. The bacteria were harvested by centrifugation at 6000 x g for 10 min and then the proteins were extracted by

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suspension du culot bactérien dans une solution tamponnée ajustée à 140 mM NaCl, 2, 7 mM KCI, 10 mM Na2HP04, 1, 8 mM KH2P04, pH 7, 3, à raison de 40 ul de solution par ml de culture, puis soniquation du mélange en 3 cycles de 1 min chacun, sur la glace. Le soniquat a été centrifugé à 10000 x g pendant 5 min et les protéines insolubles contenues dans le culot de centrifugation ont été récupérées et extraites dans le tampon de charge SDSPAGE IX (50) à 100 C pendant 10 min. Après une nouvelle centrifugation à 10000 xg pendant 5 min, l'extrait protéique a été chargé sur un gel dénaturant de polyacrylamide à 8%. Après électrophorèse et brève coloration au bleu de Coomassie, le gel a été décoloré et la bande correspondant à la protéine de fusion (55 kDa) a été excisée du gel et utilisée pour l'immunisation des animaux (Eurogentec). L'un des sérums, qui présentait le meilleur titre par rapport à la protéine de fusion et à la LOX1 de tabac a été retenu comme sérum anti- LOX1. Analyse western : Des extraits enzymatiques dialysés et concentrés, préparés comme décrit ci-dessus, ont été séparés en SDS-PAGE sur un gel à 10% à raison de 100 ig de protéines par piste, et après électrophorèse, les protéines séparées ont été transférées sur membrane de nitrocellulose par electro-transfert. Les analyses western ont été réalisées selon des protocoles standards. Le sérum anti-LOX1 à la dilution de 1 : 1000 a été utilisé comme anticorps primaire, et des IgGs de chèvre anti-IgGs de lapin, couplées à la phosphatase alcaline (Sigma), ont été utilisées comme anticorps secondaire. L'activité enzymatique phosphatase alcaline a été détectée par la méthode au NBT-BCIP.

suspension of the bacterial pellet in a buffered solution adjusted to 140 mM NaCl, 2.7 mM KCI, 10 mM Na2HP04, 1.8 mM KH2P04, pH 7.3, at a rate of 40 μl of solution per ml of culture, then sonication of the mix in 3 cycles of 1 min each, on ice. The soniquat was centrifuged at 10,000 xg for 5 min and the insoluble proteins contained in the centrifugation pellet were collected and extracted into the loading buffer SDSPAGE IX (50) at 100 C for 10 min. After a further centrifugation at 10,000 xg for 5 min, the protein extract was loaded onto a denaturing 8% polyacrylamide gel. After electrophoresis and brief staining with Coomassie blue, the gel was discolored and the band corresponding to the fusion protein (55 kDa) was excised from the gel and used for animal immunization (Eurogentec). One of the sera, which had the best titer compared to the fusion protein and to LOX1 from tobacco, was selected as anti-LOX1 serum. Western analysis: Dialysed and concentrated enzymatic extracts, prepared as described above, were separated on SDS-PAGE on a 10% gel at the rate of 100 μg of proteins per lane, and after electrophoresis, the separated proteins were transferred on nitrocellulose membrane by electro-transfer. Western analyzes were performed according to standard protocols. Anti-LOX1 serum at a dilution of 1: 1000 was used as the primary antibody, and goat anti-rabbit IgG IgGs, coupled with alkaline phosphatase (Sigma), were used as a secondary antibody. The alkaline phosphatase enzyme activity was detected by the NBT-BCIP method.

EXAMPLE 9 Detection of the LOX1 Protein in the Tl Transgenic Lines
Using transgenic lines constitutively expressing the LOXI transgene, the search for the LOX1 protein was undertaken by western analysis. Soluble protein extracts, prepared from the aerial parts of 8 week old plants, were separated by SDS-PAGE. The detection of the LOX1 protein was carried out using a polyclonal rabbit serum directed against the C-terminal part of the LOX1 protein. The immunochemical revelation shows the presence of a single band in the lanes corresponding to the transgenic lines S46-26 and S49-18. The size of the corresponding product, between 79 and 101 kDa, is consistent with the size calculated from the primary sequence of the LOX1 protein (92kDa). In contrast, the LOX1 protein is not detected in extracts prepared from the parental lines 46-8 WT and 49-10 WT. The constitutive expression of the LOXI transgene is therefore accompanied by an accumulation of the corresponding protein in the transgenic lines S46-26 and S49-18.

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Example 10: Measurement of LOX activity
The samples of wild or transgenic plants were frozen and then ground in liquid nitrogen and homogenized in 0.25 M sodium phosphate buffer, pH 6.5, containing 5% polyvinylpolypirrolidone, at a rate of 1 ml of buffer per g of fresh material. . After thawing, the extracts were mixed with a vortex and centrifuged for 5 min at 12,000 x g. The centrifugation supernatant constitutes the crude enzyme extract.

Two methods of measuring LOX activity were used.

mesure de la formation de diènes conjugués à , 234 nm (s=27000 M-l. cm'l) pendant 4 min à 30 C, dans du tampon phosphate de sodium 0, 25 M, pH 6, 5, saturé en air. L'acide linoléique a été utilisé comme substrat à la concentration finale de 820 uM. Les résultats sont exprimés en nanokatal. mg-l protéines. La teneur en protéines des aliquotes testés a été déterminé par la méthode de Bradford (Bradford, Anal. Biochem. 72, 248-254, 1976). Chromatographic method (CCM): A protocol was adapted from a method described by Caldelari and Farmer (Caldelari, D. & Farmer, EE, Phytochemistry 47,599-604, 1998). The LOX test was carried out with an aliquot of the crude enzyme extract corresponding to 50 μg of proteins, in a total volume of 0.4 ml of 0.25 M sodium phosphate buffer, pH 6.5, saturated with air. and containing linoleic acid labeled with 14C on carbon 1, at a final concentration of 1.2 u. M, for 30 min at 30 C. The reaction mixture was then extracted 2 times with a methanol-chloroform mixture (2: 1) and the organic phases were concentrated under nitrogen flow. The extracts were separated by thin layer chromatography on silica plates, in an ether-hexaneacidic mixture (70: 30: 1). The radio-labeled products resulting from the metabolism of linoleic acid as well as the remaining substrate were revealed by phosphorimaging. The amount of substrate remaining in each reaction was estimated by comparison with a control reaction without enzyme extract (ImageQuaNT software) Spectrophotometric method: The crude enzyme extract was dialyzed and concentrated by centrifugation on an Ultrafree-4 unit (Millipore) equipped with '' a Biomax 1 OkDa NMWL membrane, for 30 min at 3500 xg and at 4 ° C., then underwent three washing steps by adding 0.5 ml of 0.25 M sodium phosphate buffer, pH 6.5 and centrifugation in the same unit. LOX activity was determined in a test with a total volume of 475 Ill, by

measurement of the formation of conjugated dienes at 234 nm (s = 27000 Ml. cm'l) for 4 min at 30 ° C. in 0.25 M sodium phosphate buffer, pH 6.5, saturated with air. Linoleic acid was used as a substrate at the final concentration of 820 µM. The results are expressed in nanokatal. mg-l proteins. The protein content of the aliquots tested was determined by the method of Bradford (Bradford, Anal. Biochem. 72, 248-254, 1976).

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Exemple 11 : Transformation in vitro de l'acide linoléique par les plantes transgéniques Tl
Le niveau d'activité LOX des plantes transgéniques a été comparé à celui des lignées parentales 46-8 WT et 49-10 WT en mesurant, in vitro, la capacité de différents extraits enzymatiques à transformer un substrat naturel de cette enzyme, l'acide linoléique. Ces extraits, préparés à partir des parties aériennes de plantes âgées de 8 semaines, ont été incubés in vitro avec de l'acide linoléique marqué au 14C. Les produits radiomarqués, extraits puis séparés par chromatographie en couche mince (CCM), ont été révélés à l'aide d'un phosphorimager. A partir de l'image digitalisée de la CCM, l'acide linoléique non métabolisé à la fin de la réaction a été quantifié pour chaque piste et exprimé en pourcentage de l'acide linoléique mesuré dans une réaction témoin ne comportant pas d'extrait enzymatique. Ces pourcentages correspondent à la moyenne de trois répétitions indépendantes. Dans les essais, l'acide linoléique disparaît presque complètement dans les pistes correspondant aux plantes transgéniques S46-26 et S49-18 avec seulement 5 et 10 % de substrat restant en fin de réaction alors que dans le cas des lignées parentales 46-8 WT et 49-10 WT, environ 50 % du substrat n'est pas métabolisé. Afin de vérifier que cette différence entre les lignées WT et transgéniques est bien due à l'expression constitutive du transgène introduit, la même réaction a été réalisée en pré-incubant les extraits enzymatiques avec de l'ETYA (acide 5,8, 11, 14-eicosatétraynoïque), un inhibiteur spécifique des LOXs. Dans ce cas, environ 50 % de l'acide linoléique est détecté en fin de réaction aussi bien pour les lignées parentales que pour les lignées transgéniques. Ceci suggère que l'ensemble des lignées possède une activité indépendante de la LOX, capable de métaboliser une partie de l'acide gras introduit. Dans le cas où les extraits enzymatiques sont bouillis avant d'être incubés avec le substrat, entre 80 et 90% de l'acide gras est extrait en fin de réaction, montrant qu'il s'agit bien d'une réaction enzymatique et suggérant soit qu'une partie du substrat (entre 10 et 20%) est dégradée chimiquement, soit qu'elle n'est pas extractible dans les conditions utilisées. Cette expérience montre donc que les lignées transgéniques S46-26 et S49-18 présentent une activité de conversion de l'acide linoléique sensible à l'ETYA, ce qui n'est pas le cas des lignées parentales 46-8 WT et 49-10 WT.

EXAMPLE 11 In Vitro Transformation of Linoleic Acid by Tl Transgenic Plants
The LOX activity level of the transgenic plants was compared to that of the parental lines 46-8 WT and 49-10 WT by measuring, in vitro, the capacity of different enzymatic extracts to transform a natural substrate of this enzyme, the acid linoleic. These extracts, prepared from the aerial parts of 8-week-old plants, were incubated in vitro with 14C-labeled linoleic acid. The radiolabelled products, extracted and then separated by thin layer chromatography (TLC), were revealed using a phosphorimager. From the digitalized image of the TLC, the non-metabolized linoleic acid at the end of the reaction was quantified for each lane and expressed as a percentage of the linoleic acid measured in a control reaction not containing an enzymatic extract. . These percentages correspond to the average of three independent repetitions. In the tests, the linoleic acid disappears almost completely in the tracks corresponding to the transgenic plants S46-26 and S49-18 with only 5 and 10% of substrate remaining at the end of the reaction whereas in the case of the parental lines 46-8 WT and 49-10 WT, approximately 50% of the substrate is not metabolized. In order to verify that this difference between the WT and transgenic lines is indeed due to the constitutive expression of the transgene introduced, the same reaction was carried out by pre-incubating the enzymatic extracts with ETYA (acid 5,8, 11, 14-eicosatetraynoic), a specific LOX inhibitor. In this case, approximately 50% of the linoleic acid is detected at the end of the reaction both for the parental lines and for the transgenic lines. This suggests that all of the lines have an activity independent of LOX, capable of metabolizing part of the fatty acid introduced. In the case where the enzymatic extracts are boiled before being incubated with the substrate, between 80 and 90% of the fatty acid is extracted at the end of the reaction, showing that it is indeed an enzymatic reaction and suggesting either that part of the substrate (between 10 and 20%) is chemically degraded, or that it is not extractable under the conditions used. This experiment therefore shows that the transgenic lines S46-26 and S49-18 exhibit activity of conversion of linoleic acid sensitive to ETYA, which is not the case for the parental lines 46-8 WT and 49-10 WT.

This shows that the constitutive expression of the LOXI transgene, as well as the presence of the LOX1 protein in the transgenic lines also results in an increase in LOX activity in these plants. This increase in activity was also measured in the S46-21 line.

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EXAMPLE 12 Inoculation of Tobacco Plants with Ppn pn A method of stem tobacco inoculation with Ppn was used. Wild tobacco plants (lines 46-8 WT and 49-10 WT) or transgenic, 12 weeks old, were inoculated by application of a mycelium tablet on the stem after section of the apical part thereof ( about a third of the top) with a razor blade. The mycelium pellets came from cultures in agar medium 7 days old. Control plants were treated identically, with the exception of the application of the mycelium tablet, replaced by a tablet of sterile medium. The control and inoculated stems were covered with an aluminum film to preserve the plant tissues and the mycelium from drying out.

Example 13: Observation and measurement of symptoms
Symptoms were observed and quantified 48 hours or 72 hours after inoculation. The stems were cut longitudinally and the length of the lesions was measured for each half-stem at five equidistant points, distributed over the entire width of the section. The length of lesion used for each individual corresponds to the average of these 10 measurements.

Example 14 Measurement of the Accumulation of LOXI Transcripts and of the Specific LOX Activity in the Stems of the Tl Transgenic Lines
The method used to test the interaction between tobacco and the pathogenic microorganism, Ppn, consists in inoculating the stem with Ppn mycelium, after section of the plant apex. As a preamble to this experiment, the level of expression of the transgene as well as the specific LOX activity of the stems of the transgenic lines S46-21, S46-26 and S49-18 were compared with those observed in the parental lines 46-8 WT and 49-10 WT. For each line, total RNAs were prepared from a pool of 3 pieces of stem, each from an independent plant. The result of the hybridization with a radiolabeled LOI probe confirms the accumulation of LOX transcripts in the stems of the transgenic lines S46-21, S46-26 and S49-18 while no LOX transcript is detected in the parental lines 46 -8 WT and 49-10 WT. The specific LOX activity was also measured in this organ from concentrated and dialyzed enzyme extracts. The analysis was carried out with a spectrophotometer by measuring, at 234 nm, the appearance of the fatty acid hydroperoxides. For each line studied, 3 independent measurements were carried out. The

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results obtained, gathered in a histogram (Fig. 2), indicate that the specific LOX activity measured in the transgenic lines S46-21, S46-26 and S49-18 is 1.8 to 5 times greater than the level d activity measured in parental lines 46-8 WT and 49-10 WT. In addition, these activity levels reach 25% (S46-21) and 70% (S49-18) of the LOX activity level measured in 24 hour elicited tobacco cells (353.8 nkat. Mg 'protein, data not shown). This analysis therefore confirms that the constitutive expression of the LOX transgene as well as the increase in LOX activity measured in the transgenic plants also characterize the stems.

Example 15 Analysis of the Interaction between Ppn and the Tl Transgenic Lines Before PN and the T Lines constitutive LOX Activity
In order to examine the consequences of the constitutive expression LOXI in the transgenic lines S46-21, S46-26 and S49-18 on their interaction with Ppn, plants of these lines, 12 weeks old, were inoculated with this agent pathogenic to the stems. The symptoms obtained after inoculation with a virulent Ppn race were compared to those observed during a compatible interaction involving the corresponding parental line. Thus, the lines S46-21, S46-26 and 46-8 WT were inoculated by race 1 of Ppn while the lines S49-18 and 49-10 WT were inoculated by race 0 of Ppn. An incompatibility control was carried out by inoculating the line 46-8 WT with race 0 of Ppn. Symptoms obtained 48 hours or 72 hours after inoculation were observed on longitudinal sections of the stems and lesions were measured (Figure 3).

The symptoms observed on the parental lines 46-8 WT and 49-10 WT are typical of tobacco / Ppn interactions; line 46-8 WT, inoculated with race 0 of Ppn, presents dry and localized lesions characteristic of an incompatible interaction. On the other hand, the long macerated brown lesions observed in the 46-8 WT / Ppn 1 and 49-10 WT / Ppn 0 interactions reflect the colonization of the stem by the pathogen and are typical of compatible interactions. In comparison with the latter, the lesions measured in the transgenic lines, inoculated by the same virulent race as that used with the corresponding parental line, are markedly reduced. In the two transgenic lines selected, S46-21 and S46-26, inoculation by race 1 of the fungus does not cause the formation of these long macerated lesions. The lesions are much smaller than in the compatible case but also much less macerated. This difference is also observed when the compatible interaction 49-1 OWT / Ppn 0 (wild line sensitive to Ppn 0) and the interaction between the

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lignée transgénique S49-18, qui est issue de la lignée 49-1 OWT, et Ppn 0. On observe que les lésions provoquées lors de l'interaction S46-26/Ppn 1 ressemblent davantage aux nécroses apparaissant lors d'une interaction incompatible (46-8 WT I Ppn 0), qu'aux lésions accompagnant la colonisation des tissus de la plante par le champignon dans le cas d'une interaction compatible (46-8 WT/Ppn 1). Par exemple, les lésions obtenues 48 heures après l'inoculation dans les interactions S46-21/Ppn 1 et S46-26/Ppn 1 sont respectivement 3,4 et 2,4 fois plus courtes que celles mesurées dans l'interaction compatible 46-8 WT/Ppn 1. Pour la lignée S49-18, l'inoculation par la race 0 de Ppn provoque des lésions 2 fois plus courtes que celles observées pour la lignée parentale 49-10 WT inoculée par la même race du champignon. L'ensemble de ces résultats montre que l'expression constitutive LOX dans les lignées transgéniques s'accompagne d'une limitation nette de la progression du champignon.

transgenic line S49-18, which comes from the line 49-1 OWT, and Ppn 0. It is observed that the lesions caused during the interaction S46-26 / Ppn 1 more closely resemble the necroses appearing during an incompatible interaction ( 46-8 WT I Ppn 0), only to lesions accompanying colonization of plant tissues by the fungus in the case of a compatible interaction (46-8 WT / Ppn 1). For example, the lesions obtained 48 hours after inoculation in the S46-21 / Ppn 1 and S46-26 / Ppn 1 interactions are 3.4 and 2.4 times shorter respectively than those measured in the compatible interaction 46- 8 WT / Ppn 1. For the S49-18 line, inoculation with race 0 of Ppn causes lesions 2 times shorter than those observed for the parental line 49-10 WT inoculated by the same race of the fungus. All of these results show that the constitutive expression LOX in the transgenic lines is accompanied by a clear limitation of the progression of the fungus.

 Beyond the reduction in size of the lesions observed in the transgenic lines inoculated by a virulent race of Ppn, the nature of the latter is also modified. The lesions obtained in the S46-26 / Ppn 1 interaction are not macerated as in the 46-8 WT / Ppn 1 compatible interaction but rather dry as in the 46-8 WT / Ppn 0 incompatible interaction. This shows that the constitutive LOX activity measured in the transgenic lines S46-21, S46-26 and S49-18 actively participates in the resistance of tobacco to Ppn.

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Claims (18)

 Claims 1) Method for reducing the susceptibility of plants to diseases and attack by pathogenic organisms, characterized in that it consists in over-expressing a lipoxygenase in said plants. 2) Method according to claim 1 characterized in that it consists in constitutively over-expressing a lipoxygenase in said plants. 3) Method according to one of claims 1-2 wherein said lipoxygenase has a 9-lipoxygenase activity. 4) Method according to one of claims 1-3 wherein said lipoxygenase is a plant lipoxygenase. 5) Method according to one of claims 1-4 wherein said lipoxygenase is a solanaceous plant lipoxygenase. 6) Method according to one of claims 1-5 wherein said lipoxygenase is at least 80% homologous to the lipoxygenase of SEQ ID No. 1. 7) The method of claim 6 wherein said lipoxygenase is shown in the SEQ ID No. 1. 8) Method according to one of claims 1-7 wherein said lipoxygenase is over-expressed by integration into the genome of said plant of an expression cassette comprising a sequence coding for said lipoxygenase under the control of a functional promoter in plants. 9) The method of claim 8 wherein said promoter is a constitutive promoter in plants. <Desc / Clms Page number 31> 10) The method of claim 9 wherein said constitutive promoter is the promoter 35S of the cauliflower mosaic virus. 11) Method according to one of claims 1-10 wherein said lipoxygenase is over-expressed in the stems, leaves and roots of said plants. 12) Cassette of functional expression in plant cells and plants characterized in that it comprises a promoter having constitutive activity in plants controlling the expression of a polynucleotide encoding a lipoxygenase homologous at least 90% to the lipoxygenase of SEQ ID No. 1. 13) Expression cassette according to claim 12, in which said polynucleotide codes for a lipoxygenase having 9-lipoxygenase activity. 14) Expression cassette according to one of claims 12 or 13 in which said polynucleotide codes for the lipoxygenase of SEQ ID No. 1. 15) Expression cassette according to one of claims 12-14 in which said promoter is the 35S promoter of the cauliflower mosaic virus. 16) Vector characterized in that it comprises an expression cassette according to one of claims 12-15. 17) Transformed plant cells characterized in that they comprise an expression cassette according to one of claims 12-15 and / or a vector according to claim 16. 18) Transformed plants characterized in that they comprise an expression cassette according to one of claims 12-15, a vector according to claim 16 and / or transformed plant cells according to claim 17.