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WO2001011036A1 - Augmentation de la resistance de vegetaux a des pathogenes - Google Patents

Augmentation de la resistance de vegetaux a des pathogenes Download PDF

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Publication number
WO2001011036A1
WO2001011036A1 PCT/US2000/021624 US0021624W WO0111036A1 WO 2001011036 A1 WO2001011036 A1 WO 2001011036A1 US 0021624 W US0021624 W US 0021624W WO 0111036 A1 WO0111036 A1 WO 0111036A1
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snf
plant
kinase
promoter
transformed
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David Bisaro
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Ohio State University Research Foundation
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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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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

Definitions

  • Plant pathogens are of great economic importance, as plant disease accounts for a significant fraction of crop losses.
  • the present invention provides a method of making plants with enhanced resistance to infection with plant pathogens, including viral pathogens, bacterial pathogens, and fungal pathogens.
  • the present invention provides a method of preparing plants with enhanced resistance to infection with plant pathogens.
  • the method comprises transforming a plant cell with a DNA construct which comprises an exogenous SNF-1 transgene, i.e., a DNA which encodes an SNF-1 protein kinase or the catalytic domain of such kinase.
  • the transgene also comprises a promoter which regulates expression of the SNF-1 kinase or the catalytic domain.
  • the promoter is operably linked to the DNA sequence which encodes the SNF-1 kinase or catalytic domain.
  • the method further comprises the step of generating a transformed plant from the transformed plant cell.
  • the transformed plant expresses the SNF-1 kinase or the catalytic domain and, thus, contains an SNF-1 kinase or catalytic domain that is encoded by the SNF-1 transgene as well as the SNF-1 kinase that is encoded by the plants own SNF-1 gene.
  • Such plants are referred to as "overexpressors .
  • the present method is especially useful for producing plants with enhanced resistance to plant pathogens, particularly viral pathogens, more particularly Geminiviruses . It is expected that the present method is also useful for producing plants with enhanced resistance to abiotic stress. Examples of abiotic stress are ozone, heat stress, and salt stress.
  • the present invention also provides a plant cell having a SNF-1 transgene stably integrated into its genome.
  • the transgene comprises a DNA sequence encoding a SNF-1 kinase or the catalytic domain of such kinase and a promoter which controls expression of the DNA coding sequence in the plant cell.
  • the present invention also relates to cell cultures consisting of such transformed cells, plants regenerated from such transformed cells and seeds of such transformed plants.
  • FIGURES Figure 1 shows the nucleotide sequence, SEQ ID NO: 1, and amino acid sequence, SEQ ID NO:2, of SNF1 kinase from Arabidopsis thaliana .
  • the cDNA was obtained from a two-hybrid screen and sequenced by standard methods. The sequence is identical to a previously reported SNF1 cDNA from the same species (Le Guen, L., Thomas, M. , Bianchi, M., Halford, N.G., and Kreis, M. (1992) Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase. Gene 120: 249- 254) .
  • Figure 2 shows an amino acid sequence alignment of SNF1 proteins from yeast , SEQ ID NO: 3, Arabidopsis, SEQ ID NO: 2, and tobacco, SEQ ID NO : 4.
  • the sequences shown were obtained from GenBank and aligned using the ClustalW algorithm.
  • FIG. 3 Mean latent period following Beet Curly Top Virus (BCTV) inoculation of transgenic antisense SNFl plants.
  • BCTV Beet Curly Top Virus
  • the mean latent period (days post-inoculation) is indicated, and the number of infected versus inoculated plants for each treatment is given in parenthesis .
  • ID50 for BCTV on non- transgenic plants is reached at approximately 18-fold dilution of the inoculum, whereas the ID50 is reached at 1, 150-fold dilution in line AS-4, and following 6,250- fold dilution in line AS-12.
  • FIG. 5 is a graph showing BCTV ID 50 values on non- transgenic and a sense (overexpressing) SNFl line.
  • the present method provides a method of transforming a plant cell which is useful for preparing a plant with enhanced resistance to plant pathogens, particularly viral pathogens, and to abiotic stress.
  • the method of transforming the cell comprises the steps of introducing into a plant sample an exogenous DNA fragment which comprises a transgene comprising a sequence which encodes a SNF-1 kinase protein or the catalytic domain thereof and a promoter which is operably linked to SNF-1 kinase encoding sequence, i.e., the promoter controls expression of the SNF-1 kinase or catalytic domain.
  • the cells are then grown under conditions that allow for expression of the SNF-1 kinase or SNF-1 catalytic domain, and, preferably, expression of a selectable or screenable marker gene that, preferably, is co- introduced into the plant sample with the SNFl transgene.
  • the marker gene may be on the same DNA fragment as the SNF-1 transgene or different DNA fragment.
  • transgenic plants which contain and express the SNF- 1 transgene are selected and used to generate pathogen resistant transgenic plants.
  • Expression of the transgene preferably, is assayed by conventional techniques such as for example Northern analysis or RT- PCR.
  • the transgenic plants produced in accordance with the present method contain the transgene within the genome of their cells, i.e., the transgenic plants are stably transformed. It has been determined that such transgenic plants are resistant to infection with geminiviruses, particularly Beet Curly Top Virus (BCTV) .
  • BCTV Beet Curly Top Virus
  • resistant means a significant increase in the amount of geminivirus required to produce disease symptoms as compared to a similar non-transgenic plant which does not contain the transgene.
  • TGMV TGMV
  • TGMV TGMV
  • SNFl is a serine/threonine kinase that plays a key role in glucose sensing and signal transduction pathways 5 in yeast and plant cells.
  • a similar role is ascribed to the homologous AMP-activated protein kinase (AMPK) in mammalian cells (for review see Johnston, M. (1999) Feasting, fasting, and fermenting: glucose sensing in yeast and other cells. Trends in Genetics 15: 29-33).
  • AMPK homologous AMP-activated protein kinase
  • SNFl kinase is required for the expression of glucose-repressed genes (e.g. SUC2, which encodes invertase, an enzyme that hydrolyzes sucrose to glucose and fructose) .
  • SUC2 glucose-repressed genes
  • invertase an enzyme that hydrolyzes sucrose to glucose and fructose
  • Plant homologues have been cloned from Arabidopsis, tobacco, potato, barley, and rye. The amino acid sequences of the Arabidopsis and tobacco SNF-1 kinase are shown in Figure
  • a SNF-1 transgene is a polynucleotide having a sequence which encodes a protein whose amino acid sequence is at least 90% identical, preferably 95% identical, more preferably at least 97%
  • the coding sequence encode the N terminal portion of the plant or yeast SNF-1 kinase .
  • the preferred N terminal portion comprises from about amino acid 1 to about amino acid 350.
  • the preferred N-terminal portion comprises from about amino acid 1 to about amino acid 400.
  • the SNF-1 encoding sequence may be a heterologous SNF-1 encoding sequence, i.e., an SNF-1 gene from yeast or a different plant species.
  • a tobacco plant may be transformed with an SNF-1 gene from Arabidopsis .
  • the encoding sequence may be a homologous SNF-1 kinase encoding sequence, i.e., an SNF-1 gene from the same plant species.
  • a tobacco plant may be transformed with an SNF-1 gene from another tobacco plant .
  • the protein encoded by the SNF-1 transgene need not have an amino acid sequence which is 100% identical to a known amino acid sequence, referred to hereinafter as a "reference sequence" .
  • Such protein may have an altered sequence in which one or more of the amino acids in the reference sequence is deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence.
  • the altered protein has an amino acid sequence which is at least 90% identical to the reference sequence, preferably at least 95% identical, more preferably at least 97% identical, most preferably at least 99% identical to the reference sequence.
  • Altered sequences which are at least 95% identical have no more than 5 alterations, i.e.
  • Percent identity is determined by comparing the amino acid sequence of the variant with the reference sequence using MEGALIGN project in the DNA STAR program. Sequences are aligned for identity calculations using the method of the software basic local alignment search tool in the BLAST network service (the National Center for Biotechnology Information, Bethesda, MD) which employs the method of Altschul, S. F., Gish, W. , Miller, W. , Myers, E. W. & Lipman, D. J. (1990) J. Mol . Biol . 215, 403-410.
  • Identities are calculated by the Align program (DNAstar, Inc.) In all cases, internal gaps and amino acid insertions in the candidate sequence as aligned are not ignored when making the identity calculation. The alterations are designed not to abolish the kinase activity of the altered protein or polypeptide.
  • conservative amino acid substitutions involve substitution of one aliphatic or hydrophobic amino acids, e.g. alanine, valine, leucine and isoleucine, with another; substitution of one hydroxyl-containing amino acid, e.g. serine and threonine, with another; substitution of one acidic residue, e.g. glutamic acid or aspartic acid, with another; replacement of one amide- containing residue, e.g.
  • asparagine and glutamine with another; replacement of one aromatic residue, e.g. phenylalanine and tyrosine, with another; replacement of one basic residue, e.g. lysine, arginine and histidine, with another; and replacement of one small amino acid, e.g., alanine, serine, threonine, methionine, and glycine, with another.
  • one aromatic residue e.g. phenylalanine and tyrosine
  • basic residue e.g. lysine, arginine and histidine
  • replacement of one small amino acid e.g., alanine, serine, threonine, methionine, and glycine
  • the transgene further comprises a promoter which is operably linked to the SNF-1 coding sequence for expression of the coding sequence.
  • the transgene further comprises a polyadenylation signal.
  • the promoter preferably, is a plant promoter, for example the 35S cauliflower mosaic virus (CaMV) promoter or a nopaline synthase or octopine synthase promoter. Examples of other constitutive promoters used in plants are the 19 S promoter, and promoters from genes encoding actin or ubiquitin.
  • the promoter is a regulatable or inducible promoter.
  • an inducible promoter is the chemically inducible promoter known as the tobacco PR-la promoter.
  • an inducible promoter is one which is wound inducible.
  • Such promoters are described by Stanford et al . , Mol. Gen. Genet. 215: 200-208 (1989); Xu et al . , Plant Molec. Biol . 22: 573-588 (1993), Logemann et al . , Plant Cell 1: 151- 158 (1989); Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993); Firek et al . , Plant Molec. Biol. 22: 129- 142 (1993); and Warner et al . , Plant J. 3: 191-201
  • tissue specific promoters include tissue specific promoters. Examples of such promoters are green tissue specific promoters, root specific promoters, stem specific promoters, and flower specific promoters such as those described by Hudspeth & Gurla, Plant Molec. Biol. 12: 579-589 (1989) and de Framond, FEBS 290: 103-106 (1991) .
  • control SNFl expression in transgenic plants may prove toxic.
  • Such control preferably, is achieved by using promoters less active than the normally strong and constitutive 35S CaMV promoter, or by selecting 35S lines that are low level expressors. Additional control is also achieved by placing the transgene under the control of tissue specific and/or developmentally regulated promoters, or by using inducible promoters (e.g. a glucocorticoid-inducible promoter) .
  • the exogenous DNA fragment preferably, also comprises other appropriate regulatory signals, such as a leader sequence, transcription terminator, and polyadenylation site , which direct expression of the operably linked SNF-1 coding sequence in the plant cell .
  • regulatory signals are readily available in the art.
  • Suitable plant cells are from monocotyledonous or dicotyledonous plant .
  • Suitable monocotyledous species are, by way of example, barley, wheat, maize and rice.
  • Suitable dicotyledonous species include, but are not limited to, tobacco, tomato, sunflower, petunia, cotton, sugarbeet, potato, lettuce, melon, soybean, canola and pepper.
  • the method is useful for conferring enhanced pathogen resistance to a wide variety of plants .
  • Agricultural crop plants are of particular importance.
  • Any type or source of plant cells which serve as target for transformation by one or more delivery methods can serve as the host cells for transformation.
  • Such sources include, by way of example, immature and mature embryos, pollen, protoplasts, suspension. Delivery of the DNA fragment in to the host plant cells may be accomplished by a variety of techniques available in the art. Such techniques include non- biological mechanisms such as microprojectile bombardment, electroporation, microinjection, induced uptake, and aerosol beam injection.
  • the DNA construct comprising the exogenous SNF-1 transgene may be subcloned into a vector effective for introducing the DNA construct into the plant.
  • Ti plasmid vectors effective for this purpose are pMON 530, pBI221, pGMVNEO pCMCHOO, and pDG208.
  • the DNA construct is subcloned into a binary Ti plasmid plant vector and mobilized into Agrobacterium . Tumefaciens, and the A . Tumefaciens transformant is then used for infection and transformation plant cells or tissues.
  • Binary plant transformation vectors are known in the art
  • the SNFl gene cloned in a Ti plasmid vector is introduced into the plant sample using an Agrobacterium transformant.
  • the Agrobacterium transformant is cocultivated with plant cells or plant tissues.
  • the Agrrojbacterium binds to the plant cell walls and transfers the plasmid or a portion thereof into the plant cell.
  • transformation results from the transfer of a specific portion of the plasmid, referred to hereinafter as "T-DNA" , into the genome of plant cells.
  • the T-DNA is transferred and integrated into the plant genome as a discrete unit .
  • the T-DNA contains the exogenous SNF-1 gene or the catalytic domain thereof, which, preferably, is flanked by a promoter and polyadenylation signals.
  • the T-DNA also contains a screenable marker gene, or or selectable marker resistance gene, such as Tn5 neomycin phosphotransferase II, which confers resistance to kanamycin.
  • the transformed plant cells are selected by growth in selection medium. Thereafter, transformed plants are regenerated from the cells using conventional techniques and analyzed to ensure that the transformed plant contains the exogenous gene and is expressing the exogenous gene.
  • Leaf discs or tissue cultures of transformed plant cells are propagated to generate transformed whole plants.
  • the transformed leaf discs or plant cell are cultured on a suitable medium, preferably, a selectable growth medium. Plants may be regenerated from the resulting callus.
  • Transgenic plants are those whose cells stably integrate the exogenous transgene into their genome, the exogenous gene being expressible in the cells.
  • Resistance or sensitivity of the transgenic plant to a pathogen is assessed by the ability of the plants to grow, grow faster, or avoid disease symptoms in the presence of a predetermined dose or inoculum of the pathogen as compared to plants of the same species which have not been transformed in accordance with the present method.
  • TrAP geminiviral proteins
  • L2 protein from beet curly top virus
  • transgenic plants show ES not only to the DNA-containing geminiviruses TGMV and BCTV, but also to the RNA virus tobacco mosaic virus, indicating that the ES phenotype is quite general and may extend to all viruses, bacterial pathogens, fungal pathogens, and abiotic stress.
  • TrAP and L2 proteins during the geminiviral infection process is to inhibit the activity of SNFl kinase, thereby disabling a general host defense.
  • We attempted to reproduce the ES phenotype by expressing an antisense SNFl kinase construct (driven by the CaMV 35S promoter) in transgenic plants.
  • jbent amiana plants comprising the exogenous Arabidopsis S ⁇ F-1 gene in antisense orientation relative to the 35S promoter were tested by challenge inoculation with BCTV.
  • Viruses were delivered to plants by the agroinoculation procedure described in Elmer et al . (1988) Agrobacterium-media ed inoculation of plants wi th tomato golden mosaic virus DNAS . Plant Mol. Biol. 10:225-234.
  • the data shown in Figures 3 and 4 show that the ES phenotype does in fact result following expression of antisense SNFl kinase in transgenic N. benthamiana plants.
  • the ES phenotype is characterized by a reduction in mean latent period of from 5-7 days (Fig. 3) , and a reduction in viral ID50 from 60- to 330- fold (Fig. 4) , depending on the transgenic line.
  • infection levels comparable to those seen with non-transgenic plants can be achieved with much less virus inoculum in the case of the transgenic antisense S ⁇ F1 lines.
  • SEQ ID NO: 1 The nucleotide sequence, SEQ ID NO: 1, and amino acid sequence, SEQ ID NO:2, of SNFl kinase from Arabidopsis thaliana are shown in Figure 1.
  • the SNFl gene was obtained in a yeast two-hybrid screen using a truncated TGMV TrAP protein as bait, and an Arabidopsis cDNA library as prey.
  • the cDNA was full- length, and was recognized as encoding SNFl by virtue of its homology to yeast SNFl and to the tobacco SNFl proteins (Fig. 2) , and by its identity to previously cloned Arabidopsis SNFl (Le Guen, L., Thomas, M. , Bianchi, M. , Halford, N.G., and Kreis, M. (1992) Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNFl protein kinase. Gene 120: 249-254). (Fig. 1 and 2).
  • the SNFl gene was cloned from the yeast two-hybrid vector by PCR using
  • SNFl cDNA obtained by RT-PCR from any species in which the SNFl sequence is known such as for example, tobacco, and potato, barley and rye.
  • the sequences of the known SNFl genes are generally available on a publically available database such as GenBank.
  • the Ti plasmid vector pMON530 was cut with Smal and treated with calf intestinal alkaline phosphatase to prevent re-ligation.
  • the SNFl sequence was then removed from pET by cutting with Ndel (from the pET3 polylinker) and BamHI, and rendered flush ended by treatment with T4 DNA polymerase.
  • the flush-ended pMON530 and SNFl DNA fragments were mixed and ligated, and used to transform E. coli .
  • Clones containing the SNFl gene in the sense and antisense orientation relative to the 35S promoter in pMON530 were selected and mated into A . tumefaciens for transformation of plants.
  • the binary system consists of two components: 1) a disarmed A . tumefaciens Ti plasmid, which provides functions required for excision of the T-DNA from the Ti plasmid, and for its transfer and integration into the plant genome and 2) a second, smaller binary plasmid vector of about 6 - 10 kb that contains the T-DNA to be transferred, i.e. the Arabadopsis SNF-1 gene and a drug resistance gene.
  • binary vectors also include a streptomycin/spectinomycin resistance gene (outside the T-DNA) for selection in E. coli and A . tumefaciens .
  • the binary vector employed was pMON530 prepared as described in (Rogers et al . (1987) Improved vectors for plant transformation : Expression cassette vectors and new selectable markers . Methods in Enzymology 153: 253-277).
  • Non-transformed A . tumef ciens containing the resident, disarmed Ti plasmid was obtained from Monsanto. Cultures of the non-transformed A .
  • tumefaciens were grown overnight in LB broth containing 25 ⁇ g/ml chloramphenicol and 50 ⁇ g/ml kanamycin.
  • Cultures of E. coli containing the mobilization plasmid (e.g. pRK2013) were grown overnight in LB broth containing 50 ⁇ g/ml kanamycin.
  • Cultures of E. coli containing the binary plasmid were grown overnight in LB broth containing 50 ⁇ g/ml spectinomycin.
  • the plates were incubated at 28°C until colonies were detected on the plates .
  • Plasmid DNA was isolated from A . tumefaciens transformants according to the method of Dhaese et al . (1979) Nucleic Acids Research 7: 1837 and used to verify the presence and integrity of Arabidopsis SNF-1 gene in T-DNA by Southern blot analysis or PCR.
  • a . tumefaciens transformants were also used to transform leaf discs from Nicotiana benthamiana plants. This step varies depending on the species to be transformed. For petunia and tobacco leaf discs are also used. For tomato, cotyledon pieces are used. For Arabidopsis thaliana , sterile root pieces are used.
  • the plant tissues Prior to transformation, the plant tissues are sterilized in a solution of 20% Clorox, 0.5% Tween 20 for 15 min.
  • the leaf discs were placed upside down on MS104 plates and preincubated for 48 hours at room temperature in continuous light to increase the transformation efficiency Thereafter, the sterilized leaf discs were soaked in a liquid culture of an A. tumefaciens transformant. This is done by placing 10-20 discs in a sterilin tube (with a loose cap) and adding 1 ml of an overnight culture. The discs were then removed, blotted dry with sterile filter paper, and placed upside down on an MS104 plate with no selection.
  • the discs were transferred to MS104 plates containing selection medium (750 ⁇ g/ml carbenicillin to kill the Agrobacterium and 300 ⁇ g/ml kanamycin to select for the desired T-DNA marker) . After about 1-2 weeks, the discs form callus around the edges of the disc. This is followed by the appearance of shoots. Shoots were removed at regular intervals from the callus and transferred to rooting media (MSO plates containing the antibiotics present in the MS104 plates) . Shoots with roots were transferred to sterile soil in pots and covered with clear plastic to retain humidity. After 2-4 days, plastic can be removed and transgenic plants treated as normal plats.
  • selection medium 750 ⁇ g/ml carbenicillin to kill the Agrobacterium and 300 ⁇ g/ml kanamycin to select for the desired T-DNA marker
  • Transformed leaf discs are harvested and analyzed for presence of SNFl transgene and mRNA after 2-6 days. Alternatively transformed regenerants are obtained and analyzed in the same manner.
  • DNA, RNA or protein is isolated from the leaf discs or regenerated plants by conventional methods.
  • the presence of an integrated SNF- 1 transgene in the genome of the plant is examined by restriction endonuclease digestion followed by Southern blot analysis, or by PCR using primers designed to recognize T-DNA border sequences or by PCR using primers designed to amplify a region within the transgene.
  • Expression of RNA encoding the SNF-1 transgene is examined by Northern blot analysis or by RT-PCR. Expression of protein is examined by Western blotting.
  • Transgenic lines are established by selfing transformed plants to homozygosity using conventional techniques.
  • Transgenic N. benthamiana plants made as described above and comprising the exogenous Arabadopsis S ⁇ F-1 gene in sense orientation relative to the 35S promoter were tested by challenge inoculation with BCTV.
  • Viruses were delivered to plants by the agroinoculation procedure described in Elmer et al . (1988) Agro acterium-mediated inoculation of plants wi th tomato golden mosaic virus DNASs Plant Mol. Biol. 10:225-234.

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Abstract

La présente invention concerne un procédé visant à l'obtention de végétaux présentant une résistance accrue à l'infection par des phytopathogènes. A cet effet, on transforme une cellule végétale au moyen d'une construction d'ADN comprenant un transgène SNF-1 exogène, c'est à dire un ADN codant une protéine kinase SNF-1 ou le domaine catalytique d'une telle kinase. Ce transgène comprend également un promoteur régulant l'expression de la kinase SNF-1 ou du domaine catalytique SNF-1. Ce promoteur est fonctionnellement relié à la séquence d'ADN codant la kinase SNF-1 ou le domaine catalytique SNF-1. Ce procédé comporte également une phase de génération de végétal transformé à partir de la cellule végétale transformée. Le végétal transformé exprime la kinase SNF-1 ou le domaine catalytique SNF-1, et par tant, contient une kinase SNF-1 ou un domaine catalytique SNF-1 qui est codé par le transgène SNF-1, de même que la kinase SNF-1 qui est codée par le gène SNF-1 propre des végétaux. La présente invention concerne une cellule végétale dans le génome de laquelle est intégré de façon stable un transgène SNF-1. Ce transgène comprend également une séquence d'ADN codant une kinase SNF-1 ou le domaine catalytique d'une telle kinase, et un promoteur qui commande l'expression de la séquence codant l'ADN dans la cellule végétale. L'invention concerne enfin des végétaux issus de la régénération de telles cellules transformées et de semences de tels végétaux transformés.
PCT/US2000/021624 1999-08-06 2000-08-07 Augmentation de la resistance de vegetaux a des pathogenes Ceased WO2001011036A1 (fr)

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WO2002038780A3 (fr) * 2000-11-08 2003-02-13 Agronomique Inst Nat Rech Utilisation d'un acide nucleique pour conferer a une plante une resistance a l'agression par un pathogene

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