EP1163348A1 - Inuzierbarer comtii promoter, diesen enthaltendes chimäres gen sowie transgene pflanzen - Google Patents

Inuzierbarer comtii promoter, diesen enthaltendes chimäres gen sowie transgene pflanzen

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Publication number
EP1163348A1
EP1163348A1 EP00912723A EP00912723A EP1163348A1 EP 1163348 A1 EP1163348 A1 EP 1163348A1 EP 00912723 A EP00912723 A EP 00912723A EP 00912723 A EP00912723 A EP 00912723A EP 1163348 A1 EP1163348 A1 EP 1163348A1
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EP
European Patent Office
Prior art keywords
promoter
plants
sequence
plant
nucleic acid
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.)
Withdrawn
Application number
EP00912723A
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English (en)
French (fr)
Inventor
Bernard Fritig
Valérie TOQUIN
Pierrette Geoffroy
Michel Legrand
Serge Kauffmann
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Rhobio SA
Original Assignee
Rhobio SA
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Filing date
Publication date
Priority claimed from FR9903700A external-priority patent/FR2791359A1/fr
Application filed by Rhobio SA filed Critical Rhobio SA
Publication of EP1163348A1 publication Critical patent/EP1163348A1/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Withdrawn legal-status Critical Current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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C12N9/1011—Catechol O-methyltransferase (2.1.1.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8239—Externally regulated expression systems pathogen inducible
    • 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
    • C12N15/8283—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 virus resistance
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)

Definitions

  • COMTII inducible promoter chimeric gene comprising it and transformed plants
  • the present invention relates to a new promoter regulatory sequence inducible in response to an injury, mechanical or chemical, or in response to aggression by a pathogenic agent, in particular bacterial, fungal or viral, or by an insect or a nematode.
  • the present invention also relates to a chimeric gene (or expression cassette) comprising the promoter regulatory sequence according to the invention which controls the expression of a heterologous coding sequence, heterologous meaning here a coding sequence different from the native coding sequence.
  • the present invention also relates to a host organism comprising said chimeric gene, the transformed plants comprising it and the seeds (seeds) of said transformed plants.
  • PAL phenylalanine ammonia lyase
  • HMG HMG-CoA reductase
  • PI proteinase inhibitors
  • 5,670,349 is also known, as the promoter of apple beta-galactosidase (ABG1) or the promoter of apple amino cyclopropane carboxylate syntase (ACC synthase) described in patent application WO 98/45445.
  • the present invention relates to a new nucleic acid fragment, in particular isolated, comprising an inducible plant promoter (or promoter regulatory sequence), said inducible promoter consisting of the promoter of a plant O-methyltransferase class II gene (hereinafter COMT II).
  • Class II O-methyltransferase genes including the class II caffeic acid-O- methyltransferase gene, from plants are silent (inactive) in the absence of any aggression since non-aggressed plants do not express it , or at least at levels undetectable by the usual methods of analysis.
  • the COMT II messenger is undetectable by the "Northern blot" technique in different tissues of a healthy untreated plant, such as tobacco (Pellegrini & al., 1993).
  • This COMTII and its promoter are activated (or induced) by injuries, viral infections, aggressions to UV rays, or to chemical aggressions by different products like benzothiazole (BTH), methyl jasmonate or elicitors of vegetable origin like pectin.
  • BTH benzothiazole
  • methyl jasmonate or elicitors of vegetable origin like pectin.
  • the isolated nucleic acid fragment according to the invention consists of a plant COMTII promoter.
  • COMTII of plant is meant according to the invention any OMT of plant which is not expressed in healthy untreated plants, but which is it following a mechanical, chemical attack by a pathogen, an insect or a nematode.
  • COMTII plant of origin is meant according to the invention any differentiated multicellular organism capable of photosynthesis, whether it is monocotyledon or dicotyledonous, such as for example rice, wheat, barley, sunflower, corn, tobacco, rapeseed, soybeans or Arabidopsis thaliana.
  • COMT II is a COMT of a dicotyledonous plant, preferably tobacco.
  • promoter is meant according to the invention the non-coding region of a gene involved in the binding with AR polymerase and other factors which are responsible for the initiation and modulation of transcription leading to the production of 'an RNA transcript. It is more particularly any sequence 5 ′ to the translation initiation site or “start” codon (ATG) of the COMTII coding sequence allowing the transcription and expression of said coding sequence.
  • the promoter according to the invention advantageously comprises a sequence of more than 600 nucleotides upstream of the ATG of COMT II, preferably of more than 700, of more than 800 or even more than 900 nucleotides upstream of ATG, more preferably more than 1000 nucleotides upstream of the ATG, even more preferably more than 1200 nucleotides upstream of the ATG. Promoters comprising more than 1500 nucleotides upstream of the COMTII ATG are also part of the present invention.
  • the promoter includes a transcription initiation site.
  • the transcription initiation site is generally located less than 100 nucleotides upstream of the ATG, advantageously approximately 90 nucleotides upstream.
  • the 3 ′ end of the COMTII promoter according to the invention is located between the transcription initiation site and the ATG.
  • the 3 ′ end of the COMTII promoter is located between 10 and 50 nucleotides downstream from the transcription initiation site, more preferably between 20 and 40 nucleotides downstream, even more preferably between 20 and 30 nucleotides downstream.
  • the COMTII promoter according to the invention also comprises at least one TATA box and at least one CAT box.
  • the TATA box is generally located less than 50 nucleotides upstream from the transcription initiation site, about 40 nucleotides upstream.
  • the CAT box is generally located less than 100 nucleotides upstream from the transcription initiation site, preferably about 100 nucleotides and / or 80 nucleotides upstream.
  • the promoter comprises two CAT boxes.
  • the promoter according to the invention also comprises regulatory elements involved in the expression of genes for the metabolism of phenylpropanoids and defense-associated genes, in particular at least one A box and / or at least one L box and / or at least an inverted L box and / or at least one P box and / or at least one inverted W box.
  • Box A includes the following sequence CCGTCC. It is generally located less than 410 nucleotides from the site of initiation of transcription.
  • Box L includes the following sequence CTTCAACAACCAACC. It is generally located less than 180 nucleotides from the site of initiation of transcription.
  • the first reverse L box includes the following sequence GTTAGGTGAAG. It is generally located less than 1000 nucleotides upstream from the transcription initiation site.
  • the promoter according to the invention comprises two inverted L boxes, one at approximately 970 nucleotides upstream from the transcription initiation site and the other at approximately 440 nucleotides upstream.
  • the second inverted L box comprises the following sequence TGTTAGGTGTGTTT.
  • the P box includes the following sequence CACACCAACTCCCA. It is generally located less than 750 nucleotides upstream from the transcription initiation site.
  • the reverse W box includes the following sequence GGTCAA. It is generally located less than 1200 nucleotides upstream from the transcription initiation site.
  • the promoter according to the invention comprises two inverted W boxes, one at approximately 1110 nucleotides upstream and the other at approximately 210 nucleotides upstream.
  • the promoter according to the invention also comprises at least one E box and / or at least one G box and / or at least one GT box.
  • Box E includes the following sequence TTCCATC AAG. It is generally located less than 110 nucleotides upstream from the transcription initiation site.
  • Box G includes the following sequence CCACGT. It is generally located less than 600 nucleotides upstream from the transcription initiation site.
  • the GT box includes the following sequence GGTTAA. It is generally located less than 450 nucleotides upstream from the transcription initiation site.
  • the promoter according to the invention comprises two GT boxes, one at approximately 400 nucleotides upstream and the other at approximately 280 nucleotides upstream.
  • the promoter according to the invention consists of the tobacco COMTII promoter defined by the nucleotide sequence upstream of the ATG represented by the sequence identifier 1 (SEQ ID NO 1) .
  • the tobacco COMTII promoter comprises the sequence between nucleotides 557 and 1795 of SEQ ID NO 1, the sequences capable of hybridizing selectively to said sequences and their homologous sequences.
  • the COMTII tobacco promoter comprises the sequence between nucleotides 557 and 889 of SEQ ID NO 1, the sequences capable of hybridizing selectively to said sequences and their homologous sequences.
  • the COMTII inducible tobacco promoter is activated by wounds, viral infections, attacks by UV rays, or chemical attacks by various products such as benzothiazole (BTH), methyl jasmonate or eliciters of plant origin such as pectin. .
  • BTH benzothiazole
  • methyl jasmonate methyl jasmonate
  • eliciters of plant origin such as pectin.
  • the functional study of the promoter made it possible to identify the regions involved in the induction of the promoter under different conditions.
  • the fragment comprising the sequence between nucleotides 698 to 1365 is necessary and sufficient to confer sensitivity and induction by injury, by VMT, megaspermine, pectin and chitin.
  • the COMTII tobacco promoter therefore comprises the sequence between nucleotides 698 and 1365 of SEQ ID NO 1, the sequences capable of hybridizing selectively to said sequences and their homologous sequences.
  • This fragment can be associated with another promoter to induce the expression of a gene by injury, by VMT, megaspermine, pectin or chitin.
  • This fragment can thus be associated with the minimum promoter of the RNA 35S of CaMV.
  • the present invention therefore therefore also relates to a chimeric promoter comprising the sequence between nucleotides 698 and 1365 of SEQ ID NO 1, the sequences capable of hybridizing selectively to said sequences and their homologous sequences.
  • the tobacco COMTII promoter therefore comprises the sequence between nucleotides 815 and 1795 of SEQ ID NO 1, the sequences capable of hybridizing selectively to said sequences and their homologous sequences.
  • sequence capable of hybridizing selectively is meant according to the invention the sequences which hybridize with the above sequences at a level higher than the background noise significantly.
  • the background noise can be linked to the hybridization of other DNA sequences present, in particular other cDNAs present in a cDNA library.
  • the level of the signal generated by the interaction between the sequence capable of hybridizing selectively and the sequences defined by the above sequence ID according to the invention is generally 10 times, preferably 100 times more intense than that of l interaction of other DNA sequences generating background noise.
  • the level of interaction can be measured, for example, by labeling the probe with radioactive elements, such as 32P.
  • Selective hybridization is generally obtained by using very severe environmental conditions (for example 0.03 M NaCl and 0.03 M sodium citrate at about 50 ° C-60 ° C).
  • the hybridization can of course be carried out according to the usual methods of the prior art (in particular Sambrook & al., 1989, Molecular Cloning: A Labratory Manual).
  • homologous sequence is meant according to the invention any sequence comprising more than 70% homology, preferably more than 80% homology, even more preferably more than 90% homology, and which retains the functional elements of COMTII giving it its properties as an inducible promoter.
  • the methods for measuring and identifying the homologies between the nucleic acid sequences are well known to those skilled in the art.
  • the isolation and cloning of a COMT II gene is done by analysis of a genomic library prepared from the DNA of the plant of interest.
  • the genomic DNA is cut by one or more appropriate restriction enzymes and introduced into a suitable vector to constitute, by methods known to those skilled in the art, a library containing all of the genomic DNA of the plant (Ausubel et al ., 1998; Sambrook et al., 1989).
  • the clone (s) containing a COMT II gene is (are) isolated with a nucleotide probe.
  • the probe sequence is. either deduced from the protein sequence if the enzyme has been purified (by monitoring its activity, for example), or prepared from a cDNA clone from a library.
  • This cDNA library is prepared from mRNA extracted from treated tissues so as to induce expression of the COMT II gene (by injury, infection or chemical treatment as described in the examples or Figures 1-5).
  • the cDNA library is then screened by antibodies directed against a COMT II protein (for example from tobacco) or by a nucleotide probe deduced from the COMT II protein of the plant in question or deduced from the sequences conserved in the COMTs of plants.
  • the cDNA thus isolated is characterized by its nucleotide sequence or by the enzymatic activity of the recombinant protein obtained after expression of the cDNA in a prokaryotic or eukaryotic organism.
  • the non-coding cDNA sequences (3 ′ and / or 5 ′) are used to select, by PCR, under very selective conditions, the genomic clone (s) containing the COMT II gene expressed during the processing used to construct the cDNA library.
  • the Promoter sequences are then isolated by PCR or any other suitable method well known to those skilled in the art.
  • the present invention also relates to a chimeric gene (or expression cassette) functional in plant cells and plants comprising, in the direction of transcription, a 5 'regulatory sequence, a coding sequence and a 3' regulatory sequence, the 5 ′ regulatory sequence comprising a COMT II promoter according to the invention defined above.
  • plant cell 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.
  • 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, rapeseed, soy, rice, sugarcane, beet, tobacco, cotton, etc.
  • the COMTII promoter according to the invention can be used alone or associated with at least part of a promoter of a gene expressing itself naturally in plants, in particular a promoter expressing in particular in the leaves of plants, such as, for example, so-called promoters of bacterial, viral or plant origin or also so-called light-dependent promoters such as that of a gene for the small ribulose-biscarboxylase / oxygenase (RuBisCO) subunit of plant or any known suitable promoter that can be used.
  • promoters of plant origin mention will be made of the histone promoters as described in application EP 0 507 698, or the rice actin promoter (US Pat. No. 5,641,876).
  • the promoters of a plant virus gene mention will be made of that of the cauliflower mosaic (CAMV 19S or 35S), or the circovirus promoter (AU 689 31 1).
  • the COMTII promoter according to the invention can also be used in combination with at least part of a promoter specific for particular regions or tissues plants, and more particularly promoters specific to seeds ([22] Datla, R. & al., Biotechnology Ann. Rev. (1997) 3, 269-296), in particular promoters of napine (EP 255 378), phaseoline, glutenin, heliantinin (WO 92/17580), albumin (WO 98/45460) or oelosin (WO 98/45461). According to the invention, it is also possible to use, in association with the promoter
  • transcription activators such as for example the translational activator of the mosaic virus of the tobacco (VMT) described in application WO 87/07644, or the tobacco etch virus (VET) described by Carrington & Freed.
  • any corresponding sequence of bacterial origin such as for example the nos terminator of Agrobacterium tumefaciens, or of plant origin, such as for example a histone terminator as described in EP 0 633 317.
  • the coding sequence of the chimeric gene according to the invention comprises a coding sequence for a reporter gene, such as the GUS coding sequence, or a coding sequence for a protein of interest.
  • the protein of interest is advantageously a protein which confers on plants resistance properties to diseases or insects.
  • chitinases glucanases, oxalate oxidase
  • all these proteins and their coding sequences being widely described in the literature, or even antibacterial peptides and / or antifungals, in particular peptides of less than 100 amino acids rich in cysteines such as thionines or plant defensins, and more particularly lytic peptides of all origins comprising one or more disulfide bridges between cysteines and regions comprising amino acids basic, in particular the following lytic peptides: androctonine (WO 97/30082 and PCT / FR98 / 01814, deposited on August 18, 1998) or drosomicine (PCT / FR98 / 01462, deposited on July 8, 1998).
  • the protein or peptide of interest is chosen from fungal eliciting peptides, in particular elicitins (Kamoun & al., 1993; Panab restaurants & al., 1995).
  • the fungal elicitor peptide is megaspermine. Megaspermine and its coding sequence is represented by sequence identifier No. 13 (SEQ ID 13).
  • the chimeric gene according to the invention comprising, in the direction of transcription, a 5 ′ regulatory sequence comprising a COMT II promoter and a coding sequence for megaspermine comprises the DNA sequence represented by the identifier of sequence n ° 14 (SEQ ID 14).
  • the present invention also relates to a chimeric gene (or expression cassette) functional in plant cells and plants comprising, in the direction of transcription, a 5 'regulatory sequence, a coding sequence for an elicitor and a regulatory sequence in 3 ', the 5' regulatory sequence comprising an inducible promoter.
  • the elicitor is an elicitin, more preferably megaspermine as defined above.
  • the inducible promoter is advantageously chosen from the promoters of phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG), chitinases, glucanases, proteinase inhibitors (PI), genes of the PR1 family, nopaline synthase (nos) or the vspB gene (US 5,670,349, Table 3), the HMG2 promoter (US 5,670,349), the apple beta-galactosidase (ABG1) promoter or the amino cyclopropane promoter apple carboxylate syntase (ACC synthase) (WO 98/45445).
  • PAL phenylalanine ammonia lyase
  • HMG HMG-CoA reductase
  • PI proteinase inhibitors
  • genes of the PR1 family nopaline synthase (nos) or the vspB gene
  • the HMG2 promoter US 5,670,
  • the present invention also relates to a cloning and / or expression vector for the transformation of plant cells or plants containing at least one chimeric gene as defined above.
  • This vector comprises, in addition to the above chimeric gene, at least one origin of replication.
  • This vector can consist of a plasmid, a cosmid, a bacteriophage or a virus, transformed by the introduction of the chimeric gene according to the invention.
  • transformation vectors are well known to those skilled in the art and widely described in the literature.
  • it will in particular be a virus which can be used for the transformation of developed plants and containing in addition its own elements of replication and expression.
  • the vector for transforming plant cells or plants according to the invention is a plasmid.
  • the chimeric gene according to the invention can be used in association with a selection marker gene, either in the same vector, the two genes being associated in a convergent, divergent or collinear manner, or alternatively in two vectors used simultaneously for the transformation of the host organism.
  • selection marker gene either in the same vector, the two genes being associated in a convergent, divergent or collinear manner, or alternatively in two vectors used simultaneously for the transformation of the host organism.
  • antibiotic resistance genes there may be mentioned antibiotic resistance genes, herbicide tolerance genes (bialaphos, glyphosate or isoxazoles), genes coding for easily identifiable reporter enzymes such as the enzyme GUS, genes coding for pigments or enzymes regulating the production of pigments in transformed cells.
  • herbicide tolerance genes biashos, glyphosate or isoxazoles
  • genes coding for easily identifiable reporter enzymes such as the enzyme GUS
  • genes coding for pigments or enzymes regulating the production of pigments in transformed cells are described in particular in patent applications EP 242 236, EP 242 246, GB 2 197 653, WO 91/02071, WO 95/06128, WO 96/38567 or WO 97/04103.
  • the subject of the invention is also a method of transforming plant cells by integrating into the genome of said plant cells at least one chimeric gene as defined above, transformation which can be obtained by any suitable known means, fully described in the specialized literature and in particular the references cited in the present application, more particularly by the vector according to the invention.
  • a series of methods involves bombarding cells, protoplasts or tissues with particles to which the DNA sequences are attached.
  • Another 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 can be used such as micro injection or electroporation, or even direct precipitation using PEG.
  • Those skilled in the art will choose the appropriate method depending on the nature of the host organism, in particular the plant cell or the plant.
  • the present invention also relates to plant cells or transformed plants containing a chimeric gene defined above.
  • the present invention also relates to plants containing transformed cells, in particular plants regenerated from transformed cells.
  • the regeneration is obtained by any suitable process which depends on the nature of the species, as for example described in the references above.
  • the following patents and patent applications may be mentioned: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267,159, EP 604 662, EP 672 752 , US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253,175 EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 and WO 95/06128.
  • the present invention may be mentioned: US
  • the transformed plants which can be obtained according to the invention can be of the monocotyledon type such as for example cereals, sugar cane, rice and corn or dicotyledons such as for example tobacco, soybeans, rapeseed, cotton, sunflower, beet, clover, etc.
  • the plants transformed according to the invention may contain other genes of interest, conferring on plants new agronomic properties.
  • genes conferring new agronomic properties on transformed plants there may be mentioned the genes conferring tolerance to certain herbicides, those conferring tolerance to certain insects, those conferring tolerance to certain diseases.
  • Such genes are described in particular in patent applications WO 91/02071 and WO 95/06128.
  • Mention may also be made of the genes modifying the constitution of the modified plants, in particular the content and quality of certain essential fatty acids (EP 666 918) or also the content and quality of proteins, in particular in the leaves and / or seeds. of said plants.
  • genes of interest can be combined with the chimeric gene according to the invention either by conventional crossing of two plants each containing one of the genes (the first the chimeric gene according to the invention and the second the gene coding for the protein d 'interest) or by transforming plant cells of a plant containing the gene coding for the protein of interest with the chimeric gene according to the invention.
  • Example 1 Isolation of the COMT class II gene Screening of a tobacco genomic library made it possible to isolate 6 different clones containing COMT class II genes (COMTII). The latter were first characterized by their restriction profiles which revealed a certain heterogeneity among the different clones.
  • COMTIIs form a multigene family composed of six to seven genes, only one of which is transcribed in defense reactions since only one type of cDNA has been characterized in a library made from leaves inoculated with the tobacco mosaic virus. (VMT) (Pellegrini & al. 1993).
  • VMT tobacco mosaic virus
  • PCR reactions were carried out using primers derived from the 3 'non-coding region of the cDNA. Under conditions of high selectivity, a single clone is then amplified. The amplification products were sequenced. The sequences obtained showed perfect homology with those of the 3 'non-coding regions of the cDNA.
  • the selected genomic clone was subcloned into a bacterial vector (pue 18) and represents a 14 kb insert of which 9 kb are located upstream of the ATTI of the COMTII gene.
  • the transcription initiation site was determined by the primer extension technique and is located 90 nucleotides from the translation initiation site.
  • the promoter was sequenced over a length of 1771 kilobases. Non-specific elements common to the eukaryotic genes involved in the initiation of transcription such as the TATA box and the CAT box were found in the promoter
  • COMTII (SEQ ID NO 1). Regulation sites have been identified by comparison of the promoter sequences of the COMTII gene with those of genes involved in defense mechanisms.
  • the COMTII promoter contains specific elements of the phenylpropanoid metabolism genes involved in the stress response, such as the three boxes P, A, L (initially identified in the gene
  • PAL parsley (SEQ ID NO 1). These three boxes are involved in the response to elicitors and the P and L boxes are also involved in the response to UV.
  • the COMTII promoter also has elements playing an important role in the induction of PR genes by elicitors such as the W box (SEQ ID NO 1).
  • General regulatory elements are found in the COMTII promoter such as the G, GT boxes and the activating element of the simian virus SV4O (SEQ ID NO 1).
  • Box G is an element present in a wide variety of plant promoters.
  • the G box associated with specific cis elements, is involved in the regulation of many genes responding to different physiological and environmental signals.
  • the promoter region responsible for the regulation of genes by methyl jasmonate, consists of a G box associated with sequences rich in nucleotides C.
  • a similar organization is found at the level of the promoter of genes specifically induced during the injury.
  • the L boxes, present in the COMTII promoter could be involved in this kind of interactions because they are motifs rich in nucleotides C.
  • GT boxes represented several times in promoters, seem to play a role in modulating the expression of certain plant genes, either as an activator or as a repressor.
  • the functional analysis of the COMTII promoter was carried out by transgenesis in stable expression.
  • the transgene was obtained by transcriptional fusion between the promoter and a reporter gene, the GUS gene ( ⁇ -glucuronidase).
  • GUS gene ⁇ -glucuronidase
  • the GUS activity was tested in these plants under different conditions of induction of defense reactions, by a fungal elicitor injected into the leaves (megaspermine), or after exposure to UV. The results obtained are reported in Tables 1 and 2 below.
  • the GUS activity is expressed in pmol MU / min. mg.
  • the control (T) consists of the infiltration of water into the leaves of the transformed plants.
  • Control plants transformed with an empty vector had an activity of about 10-30 pmol / min.mg.
  • the control (T) corresponds to the basal GUS activity in the untreated plants.
  • the size of the promoter must be greater than 600 bp, in this case 1239 bp to allow induction and high expression of the GUS gene.
  • the deletions of the promoter corresponding to the constructs (-420 to +24), of (-313 to +24) and of (-121 to +24) cause a complete loss of the expression of the GUS gene under all the conditions tested.
  • the activity of the GUS gene under the control of the 1239 bp promoter is 1000 times greater than that observed for the other constructions. Activation of the COMTII promoter by the wound and methyl jasmonate and by elicitors of various origins and types.
  • Transgenic plants with the COMTII (- 1215 to +24) / GUS construction have been treated with various chemicals, regulators of defense reactions, with salicylic acid (SA) and methyl-2-6-dichloroisonicotinic (INA) , by fungal elicitors such as glucans or chitin fragments and a plant-based elicitor such as pectin.
  • SA salicylic acid
  • INA methyl-2-6-dichloroisonicotinic
  • the GUS activity was measured in the sheets 16 h after infiltration of these compounds and the results reported in Table 3 below.
  • the GUS activity is expressed in pmole MU / min.mg.
  • the control (T) corresponds to the basal GUS activity in the untreated transformed plants.
  • GUS activity (of the order of 3) is obtained in plants infiltrated with pectic fragments compared to the control.
  • the induction of the 1239 bp promoter has been studied during injury or after treatment with methyl jasmonate (a molecule which plays a role in signaling defense responses during injury) and with benzothiadiazole (BTH) (chemical inducer of RAD).
  • BTH benzothiadiazole
  • the GUS activity is measured 16 hours after treatment. The results are reported in Table 4 below. Table 4 - Induction of GUS activity by different compounds and stress
  • the control (T) corresponds to the GUS activity of the untreated plants.
  • the promoter is activated by the various treatments.
  • Induction factors range from 2.5 (BTH) to 14.5 (methyl jasmonate).
  • Activation of the COMTII promoter during viral infection The inoculation of VMT in tobacco requires the production of micro-wounds on the leaves allowing the entry of the virus and its multiplication.
  • GUS activity and COMTII activity were measured in injured leaves and in leaves inoculated by the virus.
  • the results show that the GUS gene under the control of the COMTII promoter has an induction kinetics identical to that of the endogenous COMTII gene, followed by the measurement of the catalytic activity of the corresponding proteins.
  • the COMTII promoter is induced early by the injury and exhibits maximum activity at 4 p.m.
  • the same peak of activity is observed at 4 p.m. during the viral infection and is due to the leaf injury caused by the inoculation of the virus.
  • the GUS activity is strongly stimulated from the 3rd day and progresses until the 7th day.
  • the local and systemic induction of the promoter during viral infection was measured at 3 and 7 days after inoculation of VMT.
  • the GUS activities, expressed in pmol MU / min.mg, are reported in Table 5 and represent an average of the values obtained in 9 transformants.
  • the control T corresponds to the GUS activity of the untreated plants.
  • the histochemical test carried out on leaves from 35S / GUS transgenic plants is the positive control of the experiment. Cross sections of injured leaves also show that all cell types are induced by the injury.
  • the functional analysis of these different constructions was carried out on transgenic tobacco in stable expression.
  • the studies were first carried out on a population of around 20 vitroplants, allowing the selection of 3 to 7 lines per construction which analyzed at the age of 20 to 45 days after transfer to the greenhouse.
  • Table 7 Induction by injury, methyl jasmonate, UV and VMT of GUS activity in tobacco vitroplants transformed by the various COMTII / GUS constructs.
  • vitroplant leaves (4 to 6 weeks old after transplanting) were injured with forceps and the GUS histochemical activity was revealed 16 hours after injury.
  • the fragment (- 1073 to - 406) allows the induction of the reporter gene by the different treatments mentioned above, the proximal part of the promoter between -406 and +24 is therefore not essential for the promoter activity.
  • the fragment -1071 to - 406 bp therefore appears to be necessary and sufficient to confer sensitivity to the inducers studied.
  • elicitin genes such as those of parasiticin and cryptogein have shown that these genes code for a preprotein (Kamoun et al., 1993; Panab restaurants et al., 1995).
  • the coding sequence of these elicitins comprises a signal peptide of 20 amino acids, allowing their excretion in the extracellular medium followed by a sequence of 98 amino acids corresponding to the mature protein.
  • Amplification reactions were carried out on reverse transcripts of Phytophthora megasperma using a sense primer derived from the nucleotide sequence of cryptogeine and oligodT as antisense primer. An amplified fragment of approximately 450 nucleotides was obtained. This fragment was cloned into a bacterial vector in order to be sequenced.
  • the nucleotide sequence corresponding to the signal peptide also has 100% identity with that of cryptogeine.
  • the native cDNA of ⁇ megaspermine was fused on the one hand to the COMT II promoter and on the other hand to the 35S promoter.
  • the COMT II promoter of 1239 bp was used because it has all the regulatory elements necessary for its induction.
  • transgenic tobacco Nicotiana tabacum Samsun NN tobacco plants were transformed with the two constructs via Agrobacterium tumefaciens. Six primary transformants for each construction were regenerated and self-fertilized. Plants from the second generation have a normal phenotype except certain individuals with the ⁇ -megaspermine gene under the control of the 35S promoter. These plants have a growth delay and also have a poorly developed root system. However, no tissue necrosis reminiscent of that developed by leaf infiltration and which can be linked to the expression of elicitin is observed in transgenic plants.
  • ⁇ megaspermine was analyzed in the leaves of transgenic plants having the cDNA of elicitin under the control of the 35S promoter by Western-Blot (FIG. 2A) and by Northern-Blot (FIG. 2B). Surprisingly, ⁇ megaspermine is undetectable in all of the transformants analyzed ( Figure 2A). The transcription level of elicitin was therefore examined in these plants by Northern-Blot ( Figure 2B). Transcripts could be detected and the level of expression varies from one transformant to another. It appears that in 2 types of plants (E and F) the size of the transcripts is smaller than the size of the complete messenger. In this case, the absence of protein could be explained by the fact that the cDNA is truncated.
  • the size of the transcripts corresponds to that of the elicitin expressed by the fungus and the rate of transcripts is not negligible, except for plant B showing a very small proportion of transcripts. Plants A which have the highest transcript levels also show growth retardation. Subsequently, only the transformants having the complete transcripts of ⁇ megaspermine were tested for their resistance.
  • the expression of ⁇ -megaspermine under the control of the COMT II promoter was analyzed by Western-Blot in the leaves of transgenic tobacco (FIG. 3).
  • the expression was studied in healthy untreated plants to determine the basic level of expression of elicitin and after injury to analyze the level of induction obtained.
  • untreated tissue the level of ⁇ megaspermine is undetectable.
  • elicitin is detected in very small quantity in injured tissues. This is due to activation of the COMT II promoter by the injury.
  • the accumulation of COMT II was also examined in the same transgenic plants using anti-COMT II antibodies.
  • COMT II is detected at a non-negligible level in untreated tissue, whereas normally only a very weak COMT II activity is detected in healthy plants not transformed or transformed with the chimeric COMT II :: GUS gene (Collendavelloo et al, 1981; Pellegrini et al., 1993). COMT II is also produced in larger amounts in injured tissue compared to similarly injured control plants.
  • the COMT II promoter allows an induced expression of elicitin in transgenic tobacco. This was verified for the 6 transformants. However, the amount of elicitin detected in plants after induction is very low. The presence of COMT II in untreated plants suggests that a very small amount of elicitin (not detected by the method used) is produced in the healthy plant and that this synthesis is sufficient to induce the endogenous COMT II gene. In addition, it would appear that an induced expression of elicitin during the injury also allows greater induction of the endogenous COMT II gene.
  • VMT Tobacco Mosaic Virus
  • Processed tobacco has the N resistance gene and therefore reacts with HR during inoculation with VMT.
  • resistance to the virus is quantified by measuring the size of the lesions 7 days after infection, when they have reached their almost final size. The greater the resistance developed by the plant, the smaller the size of the lesions, thus illustrating a greater confinement of the pathogen at the site of infection.
  • the promoter of the COMT II gene being strongly induced around lesions during HR at VMT, it should allow a strong expression of ⁇ megaspermine at the site of infection.
  • transgenic plants a, b, c, d, e and f expressing elicitin under the control of the COMT II promoter and the transformants A, B, C, D having the cDNA of ⁇ -megaspermine under the control of the 35S promoter were inoculated by VMT.
  • the control plants are transgenic plants for the chimeric gene "COMT II promoter :: GUS”.
  • FIG. 4 represents the distribution of the size of the lesions measured on the control plants and for a line expressing elicitin under the control of the promoter 35S (plants A) and a line expressing elicitin under the control of the promoter COMT II (plants b). This distribution follows a Gaussian curve which allows the statistical analysis of the results.
  • the control plants have a size of lesions, the average of which is 3.3 + 0.5 mm.
  • the average lesion size obtained for plants A was not significantly different from that of the controls (3.1 ⁇ 0.6 mm), on the other hand, that obtained for plants b was significantly smaller than the controls (1.4 ⁇ 0.6 mm).
  • the results obtained for each independent line are grouped in FIG. 5 and show that all of the transformants (plants A, B, C and D) having the cDNA for ⁇ -megaspermine under the control of the 35S promoter exhibit a size of lesions which does not differ not that of the witnesses.
  • all the plants expressing elicitin under the control of the COMT II promoter have a size mean lesions significantly lower than that of control plants. This reduction is more or less significant depending on the transformants.
  • the plants expressing ⁇ -megaspermine under the control of the COMT II promoter chosen for this test correspond to line b which has a high level of resistance to VMT.
  • Plants A possessing the cDNA of ⁇ -megaspermine under the control of the 35S promoter were also inoculated as were control plants COMT II :: GUS.
  • For each transgenic line 5 plants were inoculated.
  • Transgenic plants A show a mosaic similar to the control plants. On the other hand, a reduction in these symptoms is observed in the transgenic plants b.
  • the viral load was examined in the systemic leaves of the same level (3 rd leaf above the inoculated leaf). This analysis was carried out by Western-Blot using antibodies directed against the protein shell of the virus (FIG. 6). The amount of virus present in the different transgenic plants is evaluated relative to the amount of virus present in the control plants. The results obtained show that the plants expressing ⁇ megaspermine under the control of the promoter 35S have 10 to 15 times less virus than the control plants. This reduction in the amount of virus may seem significant. Be aware, however, that the amounts of virus produced in wild plants can vary by the same amount. In contrast, the transgenic plants expressing megaspermine under the control of the COMT II promoter contain 1,000 to 10,000 times less virus than the control plants. This represents a considerable and very significant reduction in viral load.
  • Antifungal resistance We examined whether elicitin production in planta induced resistance to a soil fungus, Phytophthora parasitica vav.nicotianae. This fungus infects tobacco plants through the roots invading the root and then the vascular system, thus causing sclerosis of the conducting vessels. Symptoms of infection result in black rot at the neck. This mode of infection is difficult to implement because it requires an adequate concentration of zoospores and meets strict conditions of temperature and humidity. An "artificial" mode of inoculation consists in applying, after decapitation of tobacco, the mycelium of the fungus at the level of the severed stem. After 7 days the stems are removed and the symptoms of infection are examined inside the stems.
  • the inoculation was carried out on 7 COMT II :: GUS control plants.
  • 7 COMT II GUS control plants.
  • Plants A, B, C and D having the "35S-megaspermine promoter" construct were tested.
  • the lines a and b having a high increased resistance to VMT were chosen as well as the line e having a lower level of resistance.
  • the progression of symptoms measured in the stems of control plants reached an average length of 5.3 cm. A slowing down of the symptoms of infection is observed in the stems of transgenic plants expressing ⁇ -megaspermine under the 35S promoter control.
  • the progression of the fungus is also greatly slowed down in the stems of transgenic plants expressing ⁇ megaspermine under the control of the COMT II promoter.
  • the stems of the COMT II were induced during infection with P. parasitica.
  • the stems of the COMT II : meg transformants a, b and e are infected over a length of 0.7 cm, 1 cm and 3.5 cm respectively.
  • Plants a and b are the most resistant to infection by P. nicotianae and also correspond to the plants with the highest resistance to VMT.
  • the resistance levels induced in these plants are related to the expression rate of elicitin.
  • An analysis of the levels of transcripts by Northern-Blot could perhaps confirm this hypothesis, knowing the difficulty of detecting the protein by Western-Blot.
  • a tobacco genomic DNA library (Nicotiana tabacum var. Xanthi) constructed in ⁇ - ⁇ MBL3 (Clontech) was screened with a radioactive COMT II cDNA probe (Pellegrini & al., 1993).
  • Six positive genomic clones were isolated after four rounds of purification. These purified clones were tested by PCR to identify the one which comprises the COMT gene expressed during the hypersensitivity (HR) response of tobacco leaves to VMT.
  • the 5 ′ and 3 ′ primers for the PCR analysis are represented by the oligonucleotides 1 and 2 below (S ⁇ Q ID NO 4 and S ⁇ Q ID NO 5 respectively):
  • Olig ⁇ l 5 'CGTTTCGCAA TGTGATTTGA TC 3'
  • Oligo2 5 'CTCAAAATGA CATCCTTTCA TAC 3'
  • the clone COMT II genome was purified on Quiagen tip according to the protocol described by the manufacturer and subcloned into the SalI restriction site of the pue 18 plasmid vector. DNA sequencing.
  • DNA sequencing was carried out on denatured double-stranded DNA according to the method of Sallger & al. (1977) using the “rhodamin dye terminator cycle ready” kit with ampliPaq FS DNA polymerase (Perkin-Elmer, P / N402078) and an Applied Biosystems 373 sequencer (Perkin-Elmer). The sequence was determined on both strands with overlaps using primers synthesized from already determined sequences. Analysis of primer extension products.
  • the primer extension reaction was carried out on total RNA according to the method described in Current Protocols in Molecular Biology (Trienzenberg, 1992).
  • RNA isolated from tobacco leaves infected with VMT and from uninfected tobacco leaves was hybridized to the following oligonucleotide (SEQ ID NO 6), complementary to COMT II mRNA and labeled with 1 'end 5':
  • Oligo3 5 'CTGAAGATGT CAATAGTTGC ATGGC 3'
  • the extension product was separated on a 6% polyacrylamide gel.
  • the location of the transcription initiation site was determined on the basis of the comigration of the extension products with the sequence scale of the obtained from the corresponding region of the gene (Sallger & al., 1977). Construction of plasmids.
  • the truncated versions of the COMTII promoter were amplified by PCR using the primers PAS1 in 3 'and PSI, PS2, PS3 and PS4 in 5' represented below (SEQ ID NO 7 to 11 respectively), leading respectively to amplification nucleotide fragments of the following lengths: -1215 / + 24, (new constructs greater than 600 bp) -420 / + 24, -313 / + 24 and -121 / + 24 (numbering relating to the site of initiation of transcription) .
  • PAS1 5 GGTCTAGAGG GCCTTTTAGA GTGTTTTTGT TAG 3'
  • PS4 5 'TTTAAGCTTA AAGAGAACCA GACAATATT 3' The built -1728 / + 24, -1471 / + 24, -956 / + 24, -937 / + 24, -882 / + 24, -746 / + 24, - 676 / + 24, -560 / + 24, -435 / + 24, were obtained with the primers PAS2 in 3 'and respectively the primers PS5, PS6, PS7, PS8, PS9, PS10, PSI 1, PS12 and PS13 in 5' presented below (SEQ ID NO 15 to 24 respectively): PAS2: 5 'CGCGGATCCC CTTTTAGAGT GTTTTTGTTA GGC 3'
  • PS5 5 'ACGCGTCGAC GTTAGGGACA ATCTATAGTG TCAC 3'
  • PS ⁇ 5 'ACGCGTCGAC GCTCCGAGGA TTTGGCTGTC GCGG 3'
  • PS7 5 'ACGCGTCGAC GCTGGTTAGG TGAAGTAAAG CATG 3'
  • PS 8 5 'ACGCGTCAGATAG CGC 3 '
  • PS 10 5 'ACGCGTCGAC GACTTTAACA CACCAACTCC C 3' PS11: 5 'ACGCGTCGAC CGGATCTAGA ATTTGGGTTC ATTC 3' PS 12: 5 'ACGCGTCGAC GTGTATACTC CACGTCTCCG GATAC 3' PS 13: 5 'ACGCGTCGTTTGTGT3GTGT3 which was placed upstream of the minimum 35S promoter was obtained by using the 3 'primer PAS3 and the 5' primer PS14 (SEQ ID NO 25 and 26):
  • PAS3 5 'CGCGGATCCG CTTAACACCA AACACACCTA ACATTG 3'
  • PS 14 5 'ACGCGTCGAC CAGTGGTGAG TTTAGCTGTC 3'
  • the amplification was carried out for 30 cycles with an initial step of 4 min at 95 ° C and a final step of 5 min at 72 ° C, using the genomic clone as template. Each cycle consists of 1 min at 95 ° C followed by 1 min of hybridization and then
  • the hybridization step is carried out at 54 ° C, 59 ° C, 55 ° C, and 50 ° C to amplify, respectively, the fragments -1215 / + 24, - 420 / + 24, -313 / + 24 and -121 / + 24.
  • the hybridization temperature is
  • the promoter fragment -1215 / + 24 is digested with Sali and Xbal, the corresponding sites being present in the primers PSI and PSA1 respectively.
  • the promoter fragments of 420, 313 and 121 base pairs are digested with HindIII and Xbal (the site for HindIII is present in the primers PS2, PS3 and PS4, and the site Xbal in the primer PAS1). / + 24, -1471 / + 24, -956 / + 24, -937 / + 24, -
  • the construct consisting of the CaMV 35S promoter upstream of the megaspermine gene was obtained by replacing the Gus gene with the megaspermine gene (SEQ ID NO 12) in the plasmid pBil 21 (Clontech).
  • RNA 10 mg is used as a matrix for the synthesis of the first strand.
  • the total RNAs are heated for 3 min at 65 ° C, cooled on ice, and incubated for 2 h at 42 ° C in 50 ml of reverse transcription buffer (50 mM Tris-HCL pH 8.3 - 15 mM MgCl2 - 75 mM KCL - 1 mM DTT) containing 1 mM of each dNTP, 40 pmol of antisense primer corresponding to oligodT, and 20 U of reverse transcriptase of AMV (avian myeloblastosis virus). The mixture is heated to 94 ° C to stop the reaction.
  • reverse transcription buffer 50 mM Tris-HCL pH 8.3 - 15 mM MgCl2 - 75 mM KCL - 1 mM DTT
  • AMV avian myeloblastosis virus
  • the pellet After precipitation of the reaction mixture with ethanol, the pellet is dissolved in sterile distilled water. The synthesis of the second strand is initiated by Taq DNA polymerase. 1/20 of the reverse transcript is used for PCR amplification in 50 ml of buffer (10 mM Tris-HCL pH 8.3 - 50 mM KCL - 1.5 mM MgCl2 - 0.01% BSA), 200 mM dNTP, 0.1 mM sense and antisense primers and 1 unit of Taq.
  • the primer sense (5 '
  • the antisense primer corresponds to the oligodT having the restriction sites
  • reaction mixture is heated for 3 min at 94 ° C, then is subjected to 30 reaction cycles each comprising 3 steps: 1 min at 94 ° C, 1 min at 49 ° C and 1 min at 72 ° C. After the last cycle, the elongation is continued for 10 min at 72 ° C.
  • the amplification product obtained is then cloned into the vector pGEM (Promega) in order to be sequenced.
  • the different COMTII-GUS promoter constructs obtained previously in the plasmid pBHO1 are introduced into a strain of Agrobacterium tumefaciens GV3101 (pPM6000) (Rossi & al., 1993) by electroporation (Nagel & al., 1990).
  • Tobacco plants (Nicotiana tabacum cv Samsun NN) are transformed by infiltration of Agrobacterium on 10-day plants (Rossi & al., 1993).
  • the plants are regenerated on a Murashige & Skoog (MS) medium (GIBCO BRL) supplemented with sucrose (30 g / 1 for stem formation and 15 g / 1 for root formation), 6-benzylaminopurine (Serva) (2 mg / ml), and naphthalene acetic acid (Serva) (0 , 05 mg / ml).
  • MS Murashige & Skoog
  • Serva 6-benzylaminopurine
  • Serva naphthalene acetic acid
  • Kanamycin 150 mg / ml
  • Control plants were prepared by transformation with the empty vector pBHO1 or the vector p min GUS (containing the reporter gene GUS under control of the minimum promoter of the RNA 35 S of CaMV). Seven to 20 independent transformants are regenerated for each construct. The primary transformants are self-fertilized and the FI seeds are germinated on an MS medium comprising 300 mg / l of kanamycin
  • the histochemical localization of GUS in transgenic plants is carried out according to the method described by Jefferson & al. (1987).
  • the histochemical reaction is incubated in the dark at 37 ° C for 12 hours.
  • the tissues are rinsed, first with a 50 mM phosphate buffer to terminate the reaction, then several times with 70% to 90% ethanol to remove the pigmentation from the tissues.
  • the tissues are rinsed in 70% ethanol> and are included in a fixing historesin (Jung) for cross sections of leaves.
  • the blocks of historesins are cut with a microtome and photographs are taken at a magnification of 10 to 40 times by a binocular microscope.
  • the COMTII and GUS activities were assayed on 100 mg of tissue.
  • the tissue is homogenized in a 100 mM sodium phosphate buffer at pH 7.5 and 10 mM DTT after addition of polyclar AT (Serva) and quartz.
  • the crude extracts are clarified by centrifugation and by filtration on glass wool.
  • GUS and COMTII activities are measured on the same raw extracts.
  • an aliquot of the protein extract is added to 1 ml of phosphate buffer comprising 100 ⁇ M of catechol, 50 ⁇ M of tritiated S-adenosyl-L-methionine (1.5 ⁇ 10 5 cpm / ml) and incubated for 3 hours at 37 ° C.
  • the reaction is stopped by adding 100 ⁇ l of 9M sulfuric acid.
  • the radioactive product of the reaction ferulic acid
  • an NA scintillation solution Beckman
  • the fluorimetric measurement of the GUS activity is carried out on the same samples according to the procedure of Jefferson & al. (1987). Protein content is determined by the method of Bradford (1976) using the Biorad reagents.
  • the vector pGEM Promega not having the cloning sites compatible with those of pBHO1, a step of subcloning in the vector Bluescript (pSK) was necessary.
  • the vector pBil01 CO T // - G £ / S previously constructed and corresponding to the promoter -1215 / + 24 fused to the reporter gene GUS was used to obtain the chimeric gene comt II-meg.
  • the GUS gene is excised by digestion of the binary plasmid with BamHI and Sstl. A SnabI site present in the GUS gene makes it possible to cut the latter and prevents it from religating with the binary plasmid since no step of purification of the vector is carried out. Restriction enzymes are inactivated by heat.
  • the digested DNA is extracted with a phenol: chloroform (1: 1) mixture, then chloroform: isoamilic alcohol (24: 1) and precipitated with ethanol.
  • the digested DNA is resuspended in sterile water and ligated with the insert.
  • the vector pBil21 (Clontech) carrying the 35S-GUS gene was used to obtain the 35S-meg chimeric gene. Cloning is carried out according to the method described above.
  • Transgenic tobacco plants are cultivated in vitro under a 12h (24 ° C) / 12 (20 ° C) light cycle for 5 weeks after germination. They are propagated on an MS medium with the addition of kanamycin
  • the tissues are finely ground in liquid nitrogen, then in 1 to 2 volumes of extraction buffer (0.2 M sodium borate pH 9 - 30 mM EGTA - SDS 1% - 5 mM DTT).
  • extraction buffer 0.2 M sodium borate pH 9 - 30 mM EGTA - SDS 1% - 5 mM DTT.
  • the mixture is poured into a tube containing 1 volume of phenol / chloroform (1: 1), vortexed, then centrifuged for 10 min at 5000 g.
  • a second Phenol / chloroform extraction (1: 1) is performed, followed by a third with chloroform / isoamyl alcohol (24: 1).
  • the RNAs present in the supernatant are specifically precipitated by adding 1 volume of a solution of LiCl (4 M) EDTA (10 mM) overnight at 0 ° C.
  • RNAs are assayed by measuring the absorbance of a solution diluted to 260 (1 unit DO260 ⁇ 40 ⁇ g / ml of RNA), and their integrity is checked by migration on a non-denaturing agarose gel stained with BET. Electrophoresis
  • RNAs are separated on a denaturing agarose gel prepared in a MOPS xl buffer containing 16% formaldehyde. 1.2% agarose (w / v) gels were used.
  • the RNA samples are denatured for 15 min at 65 ° C. in the presence of 3 volumes of denaturation solution (MOPS ⁇ 5 lO ⁇ l, formamide 50 ⁇ l, formaldehyde 16 ⁇ l) per volume of RNA, and quickly cooled in ice.
  • Load buffer is added (MOPSxl, glycerol 50%, bromophenol blue 0.05%>) at a rate of 1/10 of the volume.
  • MOPSxl glycerol 50%, bromophenol blue 0.05%>
  • the gel can be stained with BET (0.5 ⁇ g / ml) for 1 to 2 min, then washed thoroughly with sterile water. The RNAs are then visualized under UV light.
  • RNA size marker (Promega) deposited on the edge of the gel.
  • the gel is rinsed with sterile water in order to remove the excess formaldehyde.
  • the migration band corresponding to the size marker is cut, colored with BET and photographed.
  • the RNAs are transferred by capillary action for 5 h on a positively charged nylon membrane (Hybond N + , Amersham) with SSC x20 buffer. After transfer, the membrane is rinsed in SSC x2 buffer and the RNAs are fixed so covalent on the nylon membrane by exposure to ultraviolet light (1200 J, UV Stratalinker 2400, Stratagene). The membrane is hybridized to a radioactive probe and treated as described in Sambrook et al.
  • Extraction can be done immediately after harvest or on frozen samples.
  • 150 g of leaf tissue are ground in a mortar in 0.5 M sodium acetate buffer pH 5.2 containing 2- ⁇ -mercaptoethanol (15 mM) and activated carbon. The volume of buffer is adjusted so as to obtain a fine and homogeneous ground material. This is then filtered on gauze and then centrifuged for 30 min at 15,000 g. The supernatant constituting the crude extract is then analyzed. Protein determination
  • the protein concentration of the crude extracts is determined by the method of Bradford (1976) in microtitration plates: to 10 ⁇ l of an extract to be tested are added 200 ⁇ l of Bradford reagent xl (Biorad). 2 ⁇ l of raw leaf extract are supplemented to 10 ⁇ l with buffer. Each sample is tested three times. After 5 to 10 min, the plate is read with a spectrophotometer MR 700 (Dynatech) with filter 5 (660 nm). The blank consists of 10 ⁇ l of buffer and the values are compared to a standard range produced with bovine serum albumin (SAB, Sigma).
  • SAB bovine serum albumin
  • the gel analysis makes it possible to determine the molecular mass of the proteins by comparing their relative mobility with those of proteins of known molecular mass.
  • 20% (v / v) loading buffer 60 mM Tris-HCl pH 6.8, 5% (v / v) 2- ⁇ -mercaptoethanol, 10% (v / v) glycerol, 0.01 % (v / v) bromophenol blue,
  • the samples are heated at 100 ° C for 1 min before being deposited.
  • the gels (0.75 mm x 7 cm x 9 cm) are poured between a glass plate and an alumina plate.
  • the electrophoresis buffer is composed of 192 mM glycine, 25 mM Tris and
  • Protein transfer and immunodetection on nitrocellulose After electrophoretic migration on acrylamide gel, the proteins can be transferred, in a liquid medium in the transfer buffer (0.16 M Tris - 1.20 M glycine), onto a nitrocellulose or nylon PVDF type membrane (Immobilon, Millipore) . Before the transfer, the gel is balanced in the transfer buffer. The PVDF membrane is activated for 1 min in methanol and then equilibrated in the transfer buffer. The electrotransfer takes place for 90 min at 150 mA.
  • Immunodetection was carried out using specific antibodies directed against megaspermine or the VML shell protein. The revelation is carried out by chemiluminescence with the ECL kit (Amersham).
  • Plant treatment Before treatment, the plants are conditioned for a few days in an air-conditioned cubicle at 22 ° C ⁇ 1 ° C, under a brightness of 5000 lux and a photoperiod D / N of 16h / 8h. These conditions are maintained during infection, with the exception of infection with Erwinia carotovora.
  • VMT Tobacco Mosaic Virus
  • the leaves of tobacco plants, 6 weeks old, are rubbed using a glass spatula soaked in a buffer solution containing viral RNA (1 to 5 ⁇ g / ml) and an abrasive, celite (10 mg / ml).
  • the inoculum per leaf (for a plant) corresponds to
  • the mycelium of P. p. nicotianae is cultivated on a Petri dish containing a solid medium (oat medium: 100 g of crushed oat seeds are suspended in 1 l of distilled water. The medium is filtered through gauze. The medium is autoclaved after addition of 15 g of agar).
  • the inoculation was carried out after decapitation of the tobacco plants.
  • the stem of 10-week-old tobacco plants is cut under the apical bud.
  • a pellet of mycelium from P. p. nicotianae is taken at the periphery of a 7-day culture on oat medium and is deposited on the section of the stem.
  • the rod brought into contact with the mycelium pellet is encapsulated in aluminum foil to avoid too rapid drying of the tissues at the inoculation site.
  • the Erwinia carotovora strain is cultured overnight at 28 ° C in LB medium. After centrifugation, the bacterial pellet is taken up in a solution of
  • Treatment by an elicitor Solutions are infiltrated by a syringe fitted with a fine needle into the mesophyle of the leaves. The infiltrated areas are delimited with a "felt-tip” marker. The infiltrated areas are harvested 16 hours after treatment.
  • the leaves are treated with a solution of ⁇ -megaspermine, a protein elicitor purified from a culture medium of Phytophthora megasperma (Kauffmann & al., 1993), or of oligosaccharides such as chitin oligomers (100 ⁇ g / ml), fragments of glucan (200 ⁇ g / ml), and fragments of pectins (200 ⁇ g / ml). Control plants are infiltrated with water.

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EP00912723A 1999-03-22 2000-03-22 Inuzierbarer comtii promoter, diesen enthaltendes chimäres gen sowie transgene pflanzen Withdrawn EP1163348A1 (de)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
FR9903700 1999-03-22
FR9903700A FR2791359A1 (fr) 1999-03-22 1999-03-22 Promoteur inductible comtii, gene chimere le comprenant et plantes transformees
FR9907646 1999-06-11
FR9907646A FR2791360B1 (fr) 1999-03-22 1999-06-11 Promoteur inductible, comtii, gene chimere le comprenant et plantes transformees
PCT/FR2000/000714 WO2000056897A1 (fr) 1999-03-22 2000-03-22 Promoteur inductible comtii, gene chimere le comprenant et plantes transformees

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US7122718B2 (en) 2000-07-28 2006-10-17 Dairy Australia Limited Modification of plant resistance to diseases and/or pests
EP2557174B1 (de) * 2004-04-06 2016-03-23 Fibria Celulose S/A Promoter mit Cambium/Xylem-bevorzugter Expression und ihre Verwendung
EP2714901B1 (de) 2011-05-31 2024-09-18 Keygene N.V. Schädlingsresistente pflanzen
IN2014MN00853A (de) 2011-10-19 2015-04-17 Keygene Nv
WO2013095125A1 (en) 2011-12-16 2013-06-27 Keygene N.V. Method for producing a plant having enhanced disease resistance to nematodes
WO2014142647A1 (en) 2013-03-14 2014-09-18 Wageningen Universiteit Fungals strains with improved citric acid and itaconic acid production
EP3344774A1 (de) 2015-09-04 2018-07-11 Keygene N.V. Diplospory-gen
WO2020239984A1 (en) 2019-05-29 2020-12-03 Keygene N.V. Gene for parthenogenesis

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NL9000773A (nl) * 1990-04-02 1991-11-01 Rijkslandbouwhogeschool Werkwijze voor het beschermen van planten tegen pathogenen.
GB9119279D0 (en) * 1991-09-10 1991-10-23 Ici Plc Modification of lignin synthesis in plants
US5670349A (en) * 1993-08-02 1997-09-23 Virginia Tech Intellectual Properties, Inc. HMG2 promoter expression system and post-harvest production of gene products in plants and plant cell cultures
US6100451A (en) * 1995-05-18 2000-08-08 Board Of Trustees Of The University Of Kentucky Pathogen-inducible regulatory element
AU9072298A (en) * 1997-08-13 1999-03-08 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Tissue-specific poplar promoters

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WO2000056897A1 (fr) 2000-09-28
CA2366217A1 (fr) 2000-09-28

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