EP1392834A2 - Überexpression einer lipoxygenase in pflanzen und verminderung der empfänglichlichkeit von pflanzen gegenüber pathogenbefall - Google Patents

Überexpression einer lipoxygenase in pflanzen und verminderung der empfänglichlichkeit von pflanzen gegenüber pathogenbefall

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Publication number
EP1392834A2
EP1392834A2 EP02748941A EP02748941A EP1392834A2 EP 1392834 A2 EP1392834 A2 EP 1392834A2 EP 02748941 A EP02748941 A EP 02748941A EP 02748941 A EP02748941 A EP 02748941A EP 1392834 A2 EP1392834 A2 EP 1392834A2
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EP
European Patent Office
Prior art keywords
plants
lipoxygenase
plant
lox
promoter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP02748941A
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English (en)
French (fr)
Inventor
Laurent Mene-Saffrane
Marie-Thérèse ESQUERRE-TUGAYE
Jo[Lle Fournier
Roland Beffa
Marie-Claire Le Trève Collonge GROSJEAN-COURNOYER
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Biogemma SAS
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Rhobio SA
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Publication date
Priority claimed from FR0107470A external-priority patent/FR2825578A1/fr
Application filed by Rhobio SA filed Critical Rhobio SA
Publication of EP1392834A2 publication Critical patent/EP1392834A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • 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 attacks.
  • the invention also relates to expression cassettes, vectors and transformed plants used in the methods according to the invention.
  • Lipoxygenases are ubiquitous enzymes in higher plants and mammals. They catalyze the dioxygenation of polyunsaturated fatty acids containing a (Z, Z) -1,4-pentadiene motif.
  • the enzyme's substrates are linoleic (Cl 8: 2) and lmolenic acid (Cl 8: 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 type A 2 or lipolytic acyl hydrolases.
  • 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 O 2 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).
  • LOXs 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). Strong induction of gene expression
  • LOX 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.
  • 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 lead to the synthesis of volatile aldehydes in C6 or C9 and short chain acids, in Cl 2- or C9, such as 12-oxo-t? v y-9-dodecenoic acid, precursor of traumatic acid.
  • C6 aldehydes play an important role in the fragrance of plants, but some such as trans-2-hexenal also have anti-microbial properties (10).
  • 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 alien oxide synthase (AOS). This enzyme catalyzes the dehydration of 13-hydroperoxylinolenic acid and forms an alien oxide 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 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 exhibit antifungal properties, in particular with regard to Phytophthora infestons (17).
  • LOX 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 LOX1.
  • LOX1 DNA / RNA hybridization experiments (northern hybridization) have shown that LOX1, 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.
  • LOXl gene is not detected in the tissues of healthy tobacco plants except for 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.
  • LOX1 gene In tobacco cells, expression of the LOX1 gene is detected following elicitor treatments. In the case of Ppn elicitor (wall extract), an 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 a peptide elicitor 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 LOXl gene (26).
  • LOXl expression in cells is also inducible by methyl-jasmonate (22, 29), thus indicating a possible self-amplification of this pathway, while no accumulation of LOX transcripts is detected after the application of d salicylic acid (22).
  • 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.
  • 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.
  • 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 transgemal 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 route via a 13-HPL, than the control calluses.
  • 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.
  • 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 oxylipin pathway in particular. However, the knowledge of these metabolic pathways and of lipoxygenases has not made it possible to develop methods making it possible to directly increase the resistance of plants.
  • the present invention therefore consists in over-expressing a lipoxygenase in plants to reduce the sensitivity 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.
  • SEQ ID No. 1 Tobacco lipoxygenase (LOXl).
  • SEQ ID No.2 Cassette of Expression Promoter CaMV 35S - Coding sequence of the LOXl gene of tobacco - Terminator nos.
  • SEQ ID No. 3-6 Primers for PCR.
  • 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.
  • lipoxygenase is meant an enzyme catalyzing the dioxygenation of polyunsaturated fatty acids containing a (Z, Z) -1,4-pentadiene motif.
  • the enzyme's substrates are linoleic (C18: 2) and linolenic (C18: 3), two major constituents of cell membranes.
  • 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, of lipoxygenase itself and by increased specific lipoxygenase activity in plant tissues. To reduce the susceptibility of plants to disease and attack 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.
  • attacks by pathogenic organisms is meant in particular attacks on plants by viruses, bacteria, fungi, oomycetes or even insects.
  • 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.
  • 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.
  • lipoxygenase is expressed constitutively in all plant tissues.
  • the lipoxygenase is expressed constitutively in the roots, the leaves, the stems, the flowers and / or the fruits.
  • the lipoxygenase is expressed constitutively in the roots, the leaves and or the stems.
  • the lipoxygenase is an "inducible" lipoxygenase in a reference plant.
  • inducible lipoxygenase is understood to mean 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.
  • the lipoxygenase preferably has 9-lipoxygenase activity.
  • Lipoxygenases LOX
  • LOX Lipoxygenases
  • 13-LOX and 9-LOX are distinguished.
  • Methods for determining the specificity of lipoxygenase activity are described in the literature (Foumier et al., Plant J. 3: 63-70, 1993; Hornung et al., PNAS 96: 4192-4197, 1999; Rustérucci and al., J. Biol. Biochem. 274: 36446-36455, 1999).
  • 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 Physiol. 124: 1121-1130, 2000), of tomato (Genbank AY008278) of potato tuber (Royo et al. , J. Biol.
  • the lipoxygenase is a plant lipoxygenase.
  • it is a soloxygenase lipoxygenase.
  • solanaceae plants mention will be made in particular of tobacco, tomatoes, potatoes and even chilli peppers.
  • the lipoxygenase has at least 80% homology with the tobacco lipoxygenase 1 (LOXl) of SEQ ID No. 1.
  • the percentage of homology will be at least 80%, 85%, 90%, 95% and preferably at least 98% and more preferably at least 99% compared to SEQ ID No .l.
  • 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.
  • these homologous lipoxygenases retain the same biological activity as the tobacco lipoxygenase (LOXl) of SEQ ID No. 1.
  • these polypeptides therefore have lipoxygenase activity and even more preferably 9-lipoxygenase.
  • 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 plants or by applying to plants a molecule stimulating the synthesis of lipoxygenase in the plant.
  • 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 functional promoter in plants.
  • 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.
  • 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.
  • 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).
  • the constitutive promoter is the 35S promoter of the cauliflower mosaic virus.
  • 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.
  • a constitutive promoter or an "enhancer" sequence upstream or close to the lipoxygenase gene in plants.
  • lipoxygenase is over-expressed in the stems, leaves and / or roots of plants.
  • plant 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.
  • the overexpression of lipoxygenase can be obtained in any plant according to methods known to those skilled in the art.
  • the plants are chosen from solanaceous plants.
  • solanaceae plants mention will be made in particular of tobacco, tomatoes, potatoes and even chilli peppers.
  • 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 functional promoter 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.
  • this expression cassette comprises both elements making it possible to cause lipoxygenase to be produced 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.
  • 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.
  • 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.
  • this polynucleotide codes for a lipoxygenase having 9-lipoxygenase activity. More preferably, this polynucleotide codes for the lipoxygenase of SEQ ID No.1.
  • 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. The construction techniques of these expression cassettes are widely described in the literature (see in particular Sambrook et al., Molecular Cloning: A Laboratory
  • 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.
  • the vectors of the invention also comprise at least one selection marker.
  • antibiotic resistance genes such as the nptllpoxa gene: 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).
  • 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.
  • 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.
  • the transformation of plants can be obtained by any suitable known means, the techniques for transforming plants are amply described in the specialized literature.
  • 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. 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.
  • 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.
  • 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.
  • Figure 1 shows the 35S-9-LOX construct used for tobacco processing.
  • the coding sequence 9-LOX (LOXl) was obtained by PCR amplification and then inserted between the promoter 35S of the cauliflower mosaic virus (35 S) and the terminator of the nopaline synthase of Agrobacterium tumefasciens (tnos).
  • This vector also includes the neomycin phosphotransferase (NPTII) gene which confers resistance to kanamycin in bacteria and plants.
  • NPTII neomycin phosphotransferase
  • F 35S sense
  • R reverse LOXl
  • Figure 2 is a histogram representing the measurement of the specific LOX activity in the stems of tobacco plants in nKAT / mg protein. 46-8 WT and 49-10 WT denote the parental lines. S46-21, S46-26, S49-18 designate the transgenic lines. Indeed, prior to inoculation tests by
  • Figure 3 is a histogram representing the measurement of the length of the lesions in mm. These measurements were performed 48 hours (48 hours) or 72 hours
  • 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., Phytopoth. 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.
  • the Ppn strains used correspond to isolates 1156 (race 0) and 1452 (race 1) (Hendrix, JW & Apple, JL, Tobacco Science 11, 148-150, 1967). The Ppn mycelium is grown in the dark on a solid synthetic medium (Keen, NT, Science 187, 74-75, 1975).
  • Example 2 Obtaining the CaMV 35S promoter cassette-LOX1 coding sequence-nos terminator (p35S-LOX!
  • 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 Véroocci et al. (Véroocci 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 LOX1 coding sequence.
  • - Sense primer 5'-GTTATCAAACAGTTTAAAATGTTTCTGGAG-3 '
  • These primers also allow the introduction of Dral sites (underlined in the primer sequence) upstream of the translation initiation codon and downstream of the stop codon (indicated in bold characters in the primer sequence) of the LOXL sequence.
  • the PCR reaction was carried out in a total volume of 25 ⁇ l, 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 ⁇ M of each dNTP and 2 mM MgCl 2 .
  • the thermal cycler program consisted of 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 40 min extension step at 72 ° C.
  • the DNA of this reaction was digested with Dral, 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 from Agrobacterium tumefaciens (terminator nos).
  • This vector also includes two copies of the neomycin phosphotransferase gene (NPT1I) which confers resistance to kanamycin in bacteria and plants.
  • NPT1I neomycin phosphotransferase gene
  • the ligation mixture was used to transform competent Escherichia coli XLIBlue 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 LOXl sequence was examined for each of the plasmids by PCR using the following primers: - Primer F, "35S sense”: 5'-GGCCATGGAGTCAAAGATTC-3 'targeting nucleotides 6906-6925 of the CaMV 35S promoter (sequence available in Genbank under accession number J02048).
  • the amplification reactions were carried out in a volume of 50 ⁇ l 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 ⁇ M of each dNTP and 1.5 mM MgCl 2 .
  • 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 to 72 ° C, followed by a final elongation step of 10 min at 72 ° C.
  • the reaction products were separated on a 0.8% agarose gel.
  • 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-ZOX7 was mobilized in the LBA4404 strain of Agrobacterium tumefaciens by thermal shock (Holsters et al., Mol. Gen. Genêt. 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 tabocum, lines 46-8 WT and 49-10 WT according to protocols already described (Horsch et al., Science 227, 1229-1231, 1985 ).
  • the plants regenerated on a Murashige and Skoog (MS) medium containing 150 ⁇ g.ml "1 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 at from the parental lines 46-8 WT and 49-10 WT are named plants S46-x and S49-x, respectively.
  • genomic DNA of wild plants or of regenerated plants resistant to kanamycin was prepared according to the method described by
  • the integrity of the transferred T-DNA was verified by PCR amplification using the primers
  • p35S-ZO Z also contains a kanamycin resistance gene (NPTIL) used to select the transformed plant cells.
  • NPTIL kanamycin resistance gene
  • the letter x indicates the number of the plant obtained.
  • 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 from the 5 'region of the CaMV 35S promoter (F) and the other from the 3' region of the coding sequence LOXl (R).
  • the amplification products were separated on an agarose gel and revealed with ethidium bromide.
  • the profile obtained corresponds to a single strip whose size (2.8 kb) corresponds to the estimated size of the product.
  • this profile is identical to that obtained with the binary vector p35S-O i, which suggests that at least one copy has been integrated into the genome of these transformants.
  • 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 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 LOXl sequence and a second in the LOXl sequence.
  • the BamHI fragments hybridizing the radiolabelled CaMV 35S probe therefore result from a first cut between the CaMV 35S promoter and LOX1 and from a second cut in the plant genome, upstream from the left border of the transfer DNA.
  • 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.
  • the level of LOX expression was evaluated in the different Tl transgenic lines by measuring the accumulation of 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.
  • 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.
  • Rabbits were immunized with a fusion protein expressed in Escherichia coli and comprising the 244 C-terminal residues of tobacco LOXl fused with glutathione S-transferase (GST) ) of Schistosomajaponicum.
  • GST glutathione S-transferase
  • a colony of bacteria containing the recombinant plasmid was selected and cultured. These bacteria were treated with 4 mM isopropylthio- ⁇ -galactoside for 16 hours at 37 ° C. in order to induce the production of the fusion protein.
  • the bacteria were harvested by centrifugation at 6000 xg for 10 min and then the proteins were extracted by suspension of the bacterial pellet in a buffered solution adjusted to 140 mM NaCl, 2.7 mM KC1, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.3, at a rate of 40 ⁇ l of solution per ml of culture, then sonication of the mixture 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 SDS-PAGE 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.
  • the samples of wild or transgenic plants were frozen 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 material.
  • the centrifugation supernatant constitutes the crude enzyme extract.
  • Chromatographic method fCCM A protocol was adapted from a method described by Caldelari and Farmer (Caldelari, D. & Farmer, E.E., Phytochemistry 47, 599-604,
  • the LOX test was carried out with an aliquot of the crude enzyme extract corresponding to 50 ⁇ g 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 14 C on carbon 1, at a final concentration of 1.2 ⁇ M, for 30 min at 30 ° C.
  • the reaction mixture was then extracted twice with a methanol-chloroform mixture (2: 1) and the
  • Spectrophotometric method The crude enzyme extract was dialyzed and concentrated by centrifugation on an Ultrafree-4 unit (Millipore) equipped with a Biomax lOkDa NMWL membrane, for 30 min at 3500 xg and at 4 ° C, then underwent three stages. of washing by adding 0.5 ml of 0.25 M sodium phosphate buffer, pH 6.5 and centrifugation in the
  • 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 14 C-labeled linoleic acid.
  • 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.
  • 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.
  • a stem injection method of tobacco using 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 were 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.
  • 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.
  • 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.
  • 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.
  • total RNAs were prepared from a pool of 3 pieces of stem, each from an independent plant.
  • the result of hybridization with a radiolabelled LOXl 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 enzymatic extracts and dialysis. 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 results obtained, gathered in a histogram (Fig.
  • 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.
  • 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.
  • 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 49-10WT / Ppn 0 interaction (wild line sensitive to Ppn 0) and the interaction between the transgenic line S49-18, which comes from the line 49-10WT, and Ppn 0.
  • the lesions caused during the interaction S46-26 / Ppn 1 more closely resemble the necroses appearing during an incompatible interaction (46 -8 WT / 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).
  • the lesions obtained 48 hours after inoculation in the S46-21 1 Ppn 1 and S46-26 / Ppn 1 interactions are 3.4 and 2.4 times shorter respectively than those measured in the compatible interaction.
  • Plants constitutively expressing the LOX1 of tobacco were previously obtained. In a stem inoculation test, these plants showed reduced susceptibility to Phytophthora parasitica var. nicotianae (Ppn) compared to the wild line from which they come. The behavior of these plants in a root inoculation test has been studied.
  • the seeds of wild or transgenic lines were sterilized and sown in Petri dishes, on solid MS medium at a rate of approximately 30 seeds per dish, by inserting a disc of synthetic cloth between the medium and the seeds.
  • the boxes were placed in an inclined position to orient the growth of the roots.
  • a method of root inoculation of tobacco with a suspension of Ppn zoospores was used.
  • a colony of Ppn mycelium obtained on V8 medium was placed in a deficiency on agar water for 4 days, then the zoospores were released by cold shock (30 min at 15 ° C. then 30 min at room temperature) in 10 ml of water.
  • the zoospore suspension is adjusted to 4000 spores / ml.
  • the liquid MS medium is removed and replaced by the spore suspension. Plants with no symptoms of disease are counted after 6-11 days, and the percentage of survival (symptomless plants / total plants) is calculated for each combination.
  • plants of the transgenic line S46-21 were inoculated at the roots by the virulent race 1 of this pathogen.
  • the fate of the inoculated plants was compared to that of wild plants of the corresponding parental line 46-8 WT inoculated by this same breed (compatible interaction).
  • An incompatibility control was carried out by inoculating the line 46-8 WT with race 0 of Ppn under the same conditions as well as a non-inoculated control.
  • the plants were observed 6 or 11 days after inoculation in a first experiment, and 7 days after inoculation in a second experiment. Plants free of disease symptoms were counted and survival percentages were calculated (Table 1).
  • the 46-8 WT line inoculated by race 0 of Ppn, does not show any symptoms and the percentage of survival is very close to 100%.
  • colonization of plants by the pathogen results in significant mortality and a low percentage of survival (20 to 24%).
  • the plants of the transgenic sense line LOX S46-21 inoculated with the same virulent race as that used with the original line 46-8 WT, have a much higher percentage of survival, and this in two independent experiments (survival rate 80 to 88%). All of these results confirm that the expression constitutive LOX in tobacco is accompanied by a remarkable decrease in sensitivity to Ppn.
  • lipid-body lipoxygenase is
  • Jasmonic acid carboxyl methyltransferase A key enzyme for 20 jasmonate-regulated plant responses, Proc. Natl. Acad. Sci. U. S. A. 98, 4788-4793.

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EP02748941A 2001-06-07 2002-06-06 Überexpression einer lipoxygenase in pflanzen und verminderung der empfänglichlichkeit von pflanzen gegenüber pathogenbefall Withdrawn EP1392834A2 (de)

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FR0107470A FR2825578A1 (fr) 2001-06-07 2001-06-07 Sur-expression d'une lipoxygenase dans les plantes et diminution de la sensibilite des plantes aux maladies et aux agressions par des organismes pathogenes
FR0107470 2001-06-07
FR0114358A FR2825580B1 (fr) 2001-06-07 2001-11-07 Sur-expression d'une lipoxygenase dans les plantes et diminution de la sensibilite des plantes aux maladies et aux agressions par des organismes pathogenes
FR0114358 2001-11-07
PCT/FR2002/001943 WO2002099112A2 (fr) 2001-06-07 2002-06-06 Sur-expression d'une lipoxygenase dans les plantes et diminution de la sensibilite des plantes aux maladies et aux agressions par des organismes pathogenes

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