WO1995031564A2 - Procede d'introduction d'une resistance aux agents pathogenes chez les vegetaux - Google Patents

Procede d'introduction d'une resistance aux agents pathogenes chez les vegetaux Download PDF

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Publication number
WO1995031564A2
WO1995031564A2 PCT/GB1995/001075 GB9501075W WO9531564A2 WO 1995031564 A2 WO1995031564 A2 WO 1995031564A2 GB 9501075 W GB9501075 W GB 9501075W WO 9531564 A2 WO9531564 A2 WO 9531564A2
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Prior art keywords
gene
plant
nucleotide sequence
derivative
sequences
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PCT/GB1995/001075
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English (en)
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WO1995031564A3 (fr
Inventor
Jonathan Dallas George Jones
Kim Elizabeth Hammond-Kosack
David Allen Jones
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John Innes Centre Innovations Limited
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Priority claimed from GB9409394A external-priority patent/GB9409394D0/en
Priority claimed from PCT/GB1994/002812 external-priority patent/WO1995018230A1/fr
Priority claimed from GBGB9506658.5A external-priority patent/GB9506658D0/en
Priority claimed from GBGB9507232.8A external-priority patent/GB9507232D0/en
Application filed by John Innes Centre Innovations Limited filed Critical John Innes Centre Innovations Limited
Priority to JP7529439A priority Critical patent/JPH10500010A/ja
Priority to AU24154/95A priority patent/AU703644B2/en
Priority to EP95918096A priority patent/EP0759086A1/fr
Publication of WO1995031564A2 publication Critical patent/WO1995031564A2/fr
Publication of WO1995031564A3 publication Critical patent/WO1995031564A3/fr

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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8281—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 bacterial 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • the present invention relates to a method of introducing pathogen resistance in plants, particularly broad spectrum pathogen resistance, and plants which may be obtained by said method and which show
  • Crop plants are constantly challenged by potentially pathogenic microorganisms. Plants are constantly challenged by potentially pathogenic microorganisms. Crop plants are
  • Pathogens must specialize to circumvent the defence mechanisms of the host, especially those biotrophic pathogens that derive their nutrition from an intimate association with living plant cells. If the pathogen can cause disease, the interaction is said to be compatible, but if the plant is resistant, the interaction is said to be incompatible.
  • HR hypersensitive response
  • SAR systemic acquired resistance
  • SAR has also been correlated with increased levels of salicylic acid in plants which have been challenged by pathogens (Malamy et al . , 1990; Metraux et al . , 1990) which has been confirmed by studies that show that a supply of exogenous salicylic acid to unchallenged plants can result in SAR (Ward et al . , 1991; Hennig et al . , 1993).
  • Transgenic plants designed so that salicylic acid accumulation is prevented by expression of a salicylate hydroxylase gene show reduced SAR compared to non-transgenic plants. where salicylic acid accumulation is not prevented (Gaffney et al . , 1993).
  • SAR can also be induced by many
  • Ciba-Geigy such as 2,6-dichloroisonicotinic acid (INA) (Uknes et al . , 1992).
  • INA 2,6-dichloroisonicotinic acid
  • SAR is an attractive method by which broad spectrum disease control can be achieved.
  • two major drawbacks hinder its commercial exploitation: SAR is not a heritable trait and so the phenomenon has to be successfully induced into every plant in the crop stand; to be effective throughout the crop's life, the SAR phenotype has to be re-boosted at regular
  • R genes encode products that enable plants to detect the presence of pathogens, provided said pathogens carry the corresponding AVR gene (Gabriel and Rolfe, 1990). This recognition is then transduced into the activation of a defence response.
  • the mlo allele of the Mlo gene of barley is the one example of a recessive disease resistance gene currently widely used in plant breeding. Lines that are homozygous for the recessive allele of this gene activate the defence response (comprising formation of cell wall appositions) even in the absence of the pathogen (Wolter et al , 1993).
  • the mlo mutation causes a defence mimic phenotype, also known as a necrotic or disease lesion mimic phenotype, and appears to deregulate the defence response, so that it is activated precociously, or is regulated on more of a "hair trigger".
  • hydrophilic proteins with no homology to other classes of protein, while others carry repeating units whose number can be modified to change the range of plants on which they exhibit avirulence (Keen, 1992; Long and Staskawicz, 1993). Additional bacterial genes (hrp genes) are required for bacterial Avr genes to induce HR, and also for pathogenicity (Keen, 1992; Long and Staskawicz, 1993). It is not clear why pathogens make products that enable the plant to detect them. It is widely believed that certain easily discarded Avr genes contribute to but are not required for pathogenicity, whereas other Avr genes are less dispensable (Keen, 1992; Long and Staskawicz, 1993).
  • tobacco mosaic virus coat protein is the avirulence determinant for the N' gene product.
  • potato virus X coat protein appears to be the avirulence determinant for the Rx and Nx genes (Kavanagh et al . , 1992; Santa-Cruz et al . , 1993; Kohm et al . , 1993; Goulden et al . , 1993).
  • PCT/GB94/02812 describes a method for generally identifying and cloning plant resistance genes.
  • Targets include (amongst others) rust resistance genes in maize, Antirrhinum and flax (by transposon tagging); downy mildew resistance genes in lettuce and Arabidopsis (by map based cloning and T-DNA tagging); Cladosporium fulvum (Cf) resistance genes in tomato (by tagging, map based cloning and affinity labelling with avirulence gene products);
  • Tomato (Lycopersicon esculentum) is susceptible to disease caused by the leaf mould fungal pathogen Cladosporium fulvum.
  • the Avr9 gene of C. fulvum which confers avirulence on C. fulvum races that attempt to attack tomato varieties that carry the Cf-9 gene, encodes a secreted cysteine-rich peptide with a final processed size of 28 amino acids.
  • the R genes (Cf-genes) that act against C. fulvum have been identified and bred into cultivated varieties, often from related species of tomato
  • C. fulvum contains Avr genes that confer recognition by plants which contain the Cf-genes , leading to activation of host defence mechanisms to attack the disease (incompatibility).
  • the Avr4 and Avr9 genes encode small peptides that are secreted by the pathogen into the intercellular spaces of infected leaves, from which they can be extracted. This has enabled the purification and sequencing of these peptides and the isolation of the genes that encode them (De Wit, 1992; Joosten et al . , 1994).
  • C. fulvum race 4 can overcome Cf-4;
  • C. fulvum race 5 can overcome Cf-5 and
  • C. fulvum race 2.4.5.9 can overcome Cf-2 , Cf-4 , Cf-5 and Cf-9.
  • WO 91/15585 describes a hypothetical method whereby if a Cf-9 gene and/or an Avr9 gene were
  • polynucleotide sequences could be used either as the resistance gene or as an actual promoter which would be suitably affected by a broad range of pathogens.
  • a further problem with this proposed method is that necrosis induced by the Cf-9 and AvrS gene combination could lead to further induction of Avr9 and/or Cf-9 leading to spreading of the necrosis and severe
  • promoters such as promoters for plant defence genes and other genes involved in the defence response such as PR genes (pathogenesis related genes), are induced in both a compatible and an incompatible interaction. Therefore, even if a promoter exists which is effectively induced by a broad range of pathogens, the method would not be viable unless the promoter is only induced by the appearance of a
  • the present invention has resulted from
  • 35S:SP:Avr9 and Cf-9*Ds were somatic excision of Ds from the Cf-9*Ds gene, somatically restoring Cf-9 function and giving rise to localised activation in cells of plant defence responses due to recognition of the constitutively expressed Avr-9 peptide. These cells died and gave rise to small necrotic sectors, the plants phenotypically showing variegation for a defence-related necrosis, similar to somatic flecks of necrosis that are associated with the induction of SAR in plants challenged with necrotising pathogens. Further work showed that plants that variegate for somatic sectors of plant defence response in this way have increased resistance to a range of pathogens.
  • a first aspect of the present invention relates to a method of providing pathogen resistance, in particular broad spectrum pathogen resistance, in plants by induction of variegation in which genes are expressed or suppressed resulting in the activation of necrosis.
  • a method according to the present invention comprises: (i) inactivating a nucleotide sequence which contributes to plant cell necrosis or inactivating one or more nucleotide sequences forming part of a
  • nucleotide sequences which contribute to plant cell necrosis are preferably defence-related plant cell necrosis.
  • a second aspect of the present invention relates to a method of providing pathogen resistance in plants by induction of variegation in which genes are
  • a plant defence response which comprises: (i) inactivating a nucleotide sequence which contributes to the plant defence response or inactivating one or more nucleotide sequences forming part of a combination of nucleotide sequences which contribute to the plant defence response; (ii) introducing said nucleotide sequence or sequences into the genome of a plant; and (iii) restoring said inactivated nucleotide sequence or sequences to a functional form to result in pathogen resistance.
  • the variegation will generally be for somatic sectors.
  • Pathogen resistance will generally be
  • the nucleotide sequence or sequences comprise one or more genes.
  • the plant defence response and/or plant cell necrosis occurs on expression of the gene or genes.
  • the defence response and/or. plant cell necrosis can be conditional or unconditional on the expression of one or more interacting genes.
  • a substance or a combination of substances may result in increased pathogen resistance. Examples are discussed further below.
  • the nucleotide sequence or sequences may comprise a gene encoding either a substance which leads to necrosis, e.g. through activation of the plant defence response, or a substance which leads to a plant defence response with no sign of necrosis.
  • sequence or sequences may comprise a plant pathogen resistance gene (R), an avirulence gene (Avr) or other elicitor or ligand gene (L) of an R gene, or both and R gene and an L gene.
  • R plant pathogen resistance gene
  • Avr avirulence gene
  • L ligand gene
  • response and/or plant cell necrosis is preferably effected by insertion of a transposable genetic element into the nucleotide sequence or one or more of the nucleotide sequences forming a combination of
  • the transposable genetic element is preferably a transposon or a nucleotide sequence flanked by specific nucleotide sequences so that transposon excision gives rise to activation of the plant defence response and/or necrosis.
  • insertion of a genetic lesion into the nucleotide sequence disrupts the gene to prevent expression of a product able to function in contributing to the plant defence response and/or plant cell necrosis.
  • the gene may be expressed to produce a functional product, i.e. gene function is restored.
  • the lesion may be inserted into the part of the gene coding for the expression product, or may be in a regulatory sequence such as a promoter required for expression of the product.
  • re-activation within the plant is preferably carried out by restoraration of the inactivated nucleotide sequence or sequences resulting in activation of a plant defence response and/or necrosis.
  • Such restoration may be caused or allowed by culturing of the plant.
  • the plant genome should contain at least one nucleotide sequence coding for a corresponding transposon activation system (for example, comprising a transposase).
  • the inactive form could be flanked by recombinase recognition sequences that are acted on by a site specific recombination system (comprising a specific recombinase) so that recombination activates the inactive form of the gene.
  • a site specific recombination system comprising a specific recombinase
  • inactivated nucleotide sequence or sequences are introduced into the plant genome somatic excision of the transposon or recombination of the nucleotide sequence occurs in some cells leading to activation of the plant defence response and/or necrosis in specific clones of cells.
  • the number of cells in which restoration of function occurs may vary. As discussed further below, certain measures are available for optimising the system, e.g. by controlling the frequency of
  • the present invention further provides transgenic plants having increased pathogen resistance obtainable by the method of the present invention, and any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed.
  • the invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on.
  • Derivatives of plants are also provided by the present invention.
  • a derivative is any functional unit derived therefrom howsowever achieved (e.g. functional allele of gene made by mutagenesis, recombinant DNA, synthesis, or plant which could not have been produced without the use or manufacture of the plant from which it is derived.)
  • Transgenic plants in accordance with the present invention may demonstrate increased pathogen resistance since the induced plant defence response and/or
  • necrosis of plant cells may cause other cells, such as adjacent cells, to acquire pathogen resistance.
  • the activation of, for example, a plant resistance gene in a plant cell is inherited by the progeny and
  • the expression of one or more plant pathogen resistance gene may either lead to initiation of the defence response only resulting in variegation for small somatic sectors in which the plant defence response is activated or of plant cell necrosis which is not related to the plant defence response resulting in variegation for small somatic sectors in which plant cell necrosis is activated.
  • the plant may acquire resistance to a broad range of pathogens and not only to the pathogen associated with the gene or genes contributing to necrosis, for example, C. fulvum in the case of the Cf-9/Avr gene combination.
  • a transgenic tomato plant according to the present invention may demonstrate resistance against a broad range of
  • pathogens such as one or more bacterial plant pathogens (for example, Xanthomonas campestris, Pseudomonas syringae) , fungal plant pathogens (for example,
  • Phytophthora infestans Fusarium oxysporum, Botrytis cinerea, Verticillium dahliae, Al tenaria solani ,
  • Rhizoctonia solani and viral pathogens (for example, TMV, PVX, PVY, TSWV) .
  • other transgenic plants such as transgenic tobacco, Arabidopsis and potato plants may display resistance to a large number of major diseases of important crop species such as, Peronospora, Phytophthora, Puccinia, Erysiphe and Botrytis.
  • a plant or any part thereof, which is phenotypically variegated, with clones of cells expressing a first phenotype and other cells expressing a second phenotype which is increased pathogen resistance compared with wild-type.
  • the first phenotype is preferably necrosis and/or a plant defence response phenotype.
  • plants variegated by somatic sector for such a phenotype may have enhanced pathogen resistance as a result of a second phenotype in cells, which may be adjacent to the cells with the first phenotype which are necrotic and/or in which a plant defence response is .activated.
  • the phenotypic variegation is likely to result from expression in cells with the first phenotype of a gene or gene, or nucleic acid comprising a gene or genes, which
  • the present invention provides a host cell, such as a plant or microbial cell, or a plant comprising at least one such cell, containing (i) nucleic acid encoding one or more nucleotide sequences which cause or contribute to the plant defence response and/or cell necrosis, at least one of the nucleotide sequences being reversibly inactivated, for example by insertion of a transposable element such as a transposon, and (ii) nucleic acid encoding a molecule able to reverse the inactivation, such as, in the case of a transposon, a transposase.
  • a host cell such as a plant or microbial cell, or a plant comprising at least one such cell, containing (i) nucleic acid encoding one or more nucleotide sequences which cause or contribute to the plant defence response and/or cell necrosis, at least one of the nucleotide sequences being reversibly inactivated, for example by insertion of
  • the cell may comprise a plant resistance gene or other gene involved in the plant defence response or able to kill a cell when expressed therein (either alone or incombination with one or more sequences, for example in the case of an R gene the corresponding elicitor), the gene being inactivated by insertion therein of a transposon, and the cell further
  • the genome of the cell comprises the gene Cf-9, or a mutant, derivative, variant or allele thereof which retains Cf-9 function, inactivated by insertion therein of a transposon, the genome also comprising the Avr-9 gene, or a mutant, derivative, variant or allele thereof which retains Avr-9 function, and a gene encoding a transposase able to excise the transposon from the Cf-9 gene or
  • resistance genes may be employed, as may genes which do not require the presence of an elicitor molecule to cause cell
  • the cell may comprise the nucleic acid encoding the various genes by virtue of introduction into the cell or an ancestor thereof of the nucleic acid, e.g. by transformation, using any suitable technique available to those skilled in the art.
  • plants which comprise such cells, and seed therefore may be produced by crossing suitable parents to create a hybrid whose genome contains the required nucleic acid, in accordance with any available plant breeding technique. For example, a parent strain comprising within its genome a plant resistance gene containing a transposon or other inactivating lesion may be crossed with a second strain comprising within its genome a gene encoding the elicitor molecule for the plant resistance gene and a suitable transposase for excision of the transposon.
  • At least a proportion of the hybrid progeny of the parents, i.e. seed or plants grown therefrom, will comprise the required nucleic acid for activation in the plant of, in this example, the plant resistance gene and, following interaction with the elicitor, the plant defence response and/or plant cell necrosis.
  • Plants according to this aspect of the present invention will be variegated genetically. Clones of cells will have one or more nucleotide sequences which cause or contribute to the plant defence response and/or cell necrosis reactivated by removal of the inactivating lesion such as a transposon, so that a first phenotype such as necrosis is shown, while in other cells the sequence or sequences will remain inactivated so these cells will not show the first phenotype.
  • the nucleic acid may be incorporated within the chromosome.
  • a gene stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, so such decendants should show the desired phenotypic variegation and so may have enhanced pathogen
  • the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed.
  • the invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on.
  • nucleic acid e.g. a vector
  • nucleic acid comprising (i) nucleic acid encoding one or more nucleotide sequences which cause or contribute to the plant defence response and/or cell necrosis, at least one of the nucleotide sequences being reversibly inactivated, for example by insertion of a transposable element such as a transposon, and/or (ii) nucleic acid encoding a molecule able to reverse the inactivation, such as, in the case of a transposon, a transposase into a plant cell.
  • nucleic acid (i) may be any nucleic acid encoding one or more nucleotide sequences which cause or contribute to the plant defence response and/or cell necrosis, at least one of the nucleotide sequences being reversibly inactivated, for example by insertion of a transposable element such as a transposon, and/or (ii) nucleic acid encoding a
  • nucleic acid ii
  • introduction may be followed by recombination between the nucleic acid and the plant cell genome to introduce the sequence of nucleotides into the genome.
  • Descendants of cells into which nucleic acid has been introduced are included within the scope of the present invention.
  • the level of the plant defence response and/or plant cell necrosis in the small somatic sectors should be sufficient to result in the induction of acquired resistance or the induction of other defence
  • nucleotide sequence or sequences which contribute to the plant defence response and/or plant cell necrosis may be under control of any suitable promoter, such as a constitutive promoter or, in the case of R genes, their own endogenous promoter, or a cell type specific promoter. Furthermore, the restoration of the nucleotide sequence or sequences which contribute to the plant defence response and/or plant cell necrosis, for example the avirulence and plant resistance genes, may be under control of any suitable promoter, such as a constitutive promoter or, in the case of R genes, their own endogenous promoter, or a cell type specific promoter. Furthermore, the restoration of the nucleotide sequence or sequences which contribute to the plant defence response and/or plant cell necrosis, for example the avirulence and plant resistance genes, may be under control of any suitable promoter, such as a constitutive promoter or, in the case of R genes, their own endogenous promoter, or a cell type specific promoter. Furthermore, the restoration of the nucleotide sequence
  • nucleotide sequence or sequences for example by the somatic excision of a transposon, gives rise to
  • the present invention may be used for many applications and is suitable for deployment in Fl hybrid seed production system.
  • one of the parents should be homozygous, for example, for the transposase or recombinase gene.
  • this parent in a system where two components are required for inducing the necrosis such as in the Avr9/Cf- 9 gene combination for example, this parent should also be homozygous for the constitutively expressed genes.
  • the other parent should be homozygous for the gene that encodes the non-autonomous inactivation system, such as the transposon or recombinase-recognition sequences.
  • the present invention also provides in further aspects various compositions of matter comprising combinations of nucleotide sequences encoding various substances employed herein.
  • Such combinations of nucleotide sequences which may be introduced into cells in accordance with the present invention follow:
  • A activator of transposition of genetic insert.
  • R may encode a substance whose presence in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, with I being a genetic insert able to inactivate R and A encoding a substance able to reactivate R inactivated by I :
  • R and L may encode substances whose presence together in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, I being a genetic insert able to inactivate R and/or L and A encoding a substance able to
  • Also provided by the present invention is a method of producing a plant, or a part, propagule, derivative or descendant thereof, containing nucleic acid comprising a nucleotide sequence or nucleotide sequences encoding R, I and A, wherein R encodes a substance whose presence in a plant results in a plant defence response, necrosis and/or increased pathogen resistance, I is a genetic insert able to inactivate R and A encodes a substance able to reactivate R
  • I inactivated by I, comprising crossing plant lines whose genomes comprise any of R, I, A and combinations thereof, to produce the plant or an ancestor thereof.
  • a further aspect provides a method of producing a plant, or a part, propagule, derivative or descendant thereof, containing nucleic acid comprising a
  • I is a genetic insert able to inactivate R and/or L and A encodes a substance able to reactivate R and/or L inactivated by I, comprising crossing plant lines whose genomes comprise any of R, L, I, A and combinations thereof, to produce the plant or an ancestor thereof.
  • Said plant lines may contain nucleic acid
  • receptor is a product encoded by a gene capable of interacting with another product, the ligand.
  • nucleotide sequences in which at least one of the sequences is inactivated are numerous and may include an engineered allele of a ubiquitin conjugating enzyme (Becker et al . , 1993), the CaMV gene VI protein
  • Genes coding for substances leading to rapid cell death such as BARNASE (Mariani et al . , 1990) or diphtheria toxin (Thorsness et al ., 1993) may be usable to induce the changes that lead to GAR even though cell death in these latter examples is not caused by BARNASE (Mariani et al . , 1990) or diphtheria toxin (Thorsness et al ., 1993) may be usable to induce the changes that lead to GAR even though cell death in these latter examples is not caused by
  • a preferred example of the present invention is the use of the Cf-9/Avr9 gene system.
  • This can involve the matching of a transposon inactivated allele of the Cf-9 gene to constitutive expression of the Avr9 gene.
  • This system can be replaced by similar combinations of related genes for example the Avr4 and Cf-4 gene, sequence provided herein (cloning of Cf-4 is described in a co-pending GB application filed simultaneously with the present application); the Avr2 and the Cf-2 gene, sequence provided herein (cloning of Cf-2 is described in GB 9506658.5, priority from which is claimed herein); the Avr5 and the Cf-5 gene, or by cloning resistance genes and corresponding avirulence genes from other systems, such as RPP5, sequence provided herein (cloning of RPP5 is described in GB 9507232.8, priority from which is claimed herein). It certain cases it may be possible to provoke a suitable response in plant cells expressing an R gene in the absence of corresponding Avr
  • Avr or other elicitor gene may not be required. Instead a fragment may be employed, representing a part of the elicitor molecule which interacts to provoke a plant defence response and/or plant cell necrosis.
  • the nucleotide sequence comprises the inactivated R gene, the inactivated Avr gene or both, or comprises both the R and Avr gene wherein one of the genes is inactivated.
  • the plant defence response and/or plant cell necrosis may be dependent on the expression of both genes and so one example would be that the R gene could be constitutively expressed and the Avr gene could exhibit somatic variegation for expression due to somatic excision and restoration of Avr9 gene
  • Nucleotide sequences employed in the present invention may encode a wild-type sequence (e.g. gene) selected from those available, or a mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence.
  • An alteration to or difference in a nucleotide sequence may or may not be reflected in a change in encoded amino acid sequence, depending on the degeneracy of the genetic code.
  • Preferred mutants, derivatives and alleles are those which retain a functional characteristic of the protein encoded by the wild-type gene, in the present context the ability to contribute to a plant defence response and/or plant cell necrosis.
  • changes to the nucleic acid which make no .difference to the encoded amino acid sequence are included.
  • homologues of the various genes whose use is disclosed herein from other species or races may be employed, as may mutants, variants and derivatives of such homologues.
  • a method according to the present invention may employ any of a variety of transposon systems known to the skilled person, including the maize
  • Ac/Ds system Activator/Dissociation
  • En/Spm Enhancer/Suppressor imitator
  • Antirrhinum Tam1 and Tam3 systems (Coen et al . , 1989).
  • any modified recombination systems which are engineered to yield the appropriate results may be employed, such as, the bacterial Cre-Loxp (Odell et al , 1990) or the "FLP/FRT” system (Lloyd and Davis, 1994).
  • transposon, recombination or other system used to inactivate the nucleotide sequence or sequences which encode substances leading to the plant defence response and/or plant cell necrosis is not essential to or a limitation of the present
  • a transposon or recombination system might be so active that an unacceptable level of necrosis is seen. If encountered, this may be overcome by engineering alleles of the transposon or recombinase recognition sequence in which the frequency at which activated nucleotide sequences arise is reduced, such as with Ac(C1a) (Keller et al . , 1993). Alternatively, chemical or site-directed mutagenesis may be used to recover alleles of the necrosis-inducing genes which are less active and therefore result in less severe levels of plant cell necrosis (Hammond-Kosack et al . , 1994).
  • transposition or recombination may be inefficient resulting in too few activated nucleotide sequences leading to an insufficient level of plant cell necrosis. This may be overcome by constructing suitable promoter fusions to the
  • a form of the Cf-9 gene may be constructed so that it activates the defence response even in the absence of its ligand.
  • the original disease resistance gene may be mutated so that it binds to a defined chemical such as an agrichemical and this chemical activates Cf-9 to initiate the defence response and/or necrosis.
  • agrichemical binds to a defined chemical
  • Cf-9 activates Cf-9 to initiate the defence response and/or necrosis.
  • genotypic variegation for excision activating the gene may occur, without initiation of the somatic necrotic reaction due to the defence response.
  • the defence response would be initiated when the agrichemical is applied and
  • the inactivated nucleotide sequence or
  • the nucleic acid may be in the form of a
  • recombinant vector for example a plasmid or
  • the nucleic acid may be under the control of an appropriate promoter and regulatory elements for expression in a plant cell.
  • genomic DNA this may contain its own promoter and regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter and regulatory elements for expression in the host cell.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory
  • sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer
  • the nucleic acid to be inserted may be assembled within a construct which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material may or may not occur according to different embodiments of the invention.
  • the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
  • the target cell type should be such that cells can be regenerated into whole plants.
  • Plants transformed with a DNA segment containing pre-sequence may be produced by standard techniques which are already known for the genetic manipulation of plants.
  • DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718, NAR 12(22) 8711 - 87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966), electroporation (EP 290395, WO 8706614) or other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611).
  • a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718, NAR 12(22) 8711 -
  • Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Although Agrobacterium has been reported to be able to transform foreign DNA into some monocotyledonous species (WO 92/14828), microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or
  • Agrobacterium coated microparticles EP-A-4862344 or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).
  • Selectable genetic markers may be used consisting of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron,
  • methotrexate methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate (Herrera-Estrella et al , 1983; van den Elzen et al , 1985).
  • the present invention is particularly beneficial for use in crop and amenity plants.
  • suitable plants include tobacco, potato, pepper, cucurbits, carrot, vegetable brassicas, lettuce, strawberry, oil seed brassicas, sugar beet, wheat, barley, maize, rice, soybeans, peas, sunflower, carnation, chrysanthemum, other ornamental plants, turf grass, poplar, eucalyptus and pine.
  • Figure 1 schematically depicts the Cf-9 gene, showing tagged alleles.
  • X marks a probable promoter.
  • Figure 2 illustrates genetic acquired resistance to C. fulvum induced following necrotic sector
  • Figure 3 illustrates genetic acquired resistance to Phytophthora infestans (late blight of tomato and potato).
  • panel A the appearance of leaves from the mutant 50 experiment 7 days after inoculation is shown.
  • panel B the rate of leaf abscission (in days after inoculation) in the various genotypes inoculated is given.
  • Figure 4 illustrates genetic acquired resistance to Phytophthora infestans (late blight of tomato and potato).
  • GAR+ and GAR- plants from Cf-9*Ds, mutant lines M31 and M50 and Cf0 plants were spray inoculated with 100 sporangiospores/mL.
  • panel A the appearance of leaves from the mutant 50 (GAR+ - right-hand) experiment 7 days after inoculation is shown, compared with GAR- (left-hand).
  • panel B the rate of
  • sporulating lesion formation on the various plant genotypes inoculated is given, with the mean number of sporulating lesions/leaflet given at 5, 7, 10, 13 and 16 days after inoculation.
  • Figure 5 shows genetic acquired resistance to Oidium lycopersici (powdery mildew disease).
  • GAR+ and GAR- plants from Cf-9*Ds, mutant lines M31 and M50 and Cf0 plants were painted with equivalent numbers of spores.
  • panel A the appearance of leaves 14 days after inoculation is shown, GAR- on the left, GAR+ on the right.
  • B the rate of chlorotic lesion (upper panel) and sporulating lesion (lower panel) formation on the various plant genotypes is given for Mutant 31: mean number of lesions given at 7, 10, 14, 21, 24 and 30 days after inoculation.
  • C shows equivalent results for Mutant 50.
  • Figure 6 shows the appearance of tomato fruits on GAR + ( sAc, Cf-9*Ds - right-hand) and GAR- ( sAc, Cf-9*Ds, Avr-9 - left-hand) plants from mutant line M23 at 2, 3, 4, 5, 6 and 7 weeks after flower pollination. Dark green sectors formed on the GAR + but not GAR- fruits by 5 weeks. These dark green sectors were not visible on the red fruit.
  • Figure 7 shows levels of defence-related gene expression in GAR+ and GAR- plants from Cf-9*Ds mutant lines M23, M31 and M50 just prior to the pathogen inoculation experiments.
  • Northern analysis shows in panel A the levels of a basic ⁇ -1,3 glucanase gene transcript and in panel B the levels of an anionic peroxidase gene transcript.
  • Figure 8 illustrates functional expression of the Cf-9 gene under the control of its own promoter in tobacco and potato.
  • panel A a tobacco leaf that has been injected with intercellular fluid (IF) either containing the Avr9 peptide or lacking the Avr9 peptide.
  • IF intercellular fluid
  • Avr9+ IF was obtained from transgenic tobacco or a compatible C. fulvum - tomato interaction
  • Figure 9 shows development of the necrotic lethal phenotype in seedlings from the tobacco cross cv.
  • Petite Havana 6201A 35S;SP;Avr9)homozygote x cos 34.1 (genomic Cf-9) heterozygote.
  • dsp seed planting
  • Figure 10 shows development of the necrotic lethal phenotype in seedlings from the Arabidopsis cross 6201B4 (35S:SP:Avr9) heterozygote x cos 138
  • FIG 11 shows a single T-DNA construct systems to apply GAR to potato plants.
  • the T-DNA contains a Cf-9 gene sequence under the control of its own
  • Figure 12 shows a photograph of three leaves, two of which are diseased with C. fulvum and one which is expressing GAR and is resistant to the same inoculum of C. fulvum.
  • Figure 13 illustrates how GAR + plants may be made by crossing stable lines (1) comprising a Cf-9 gene, inactivated by insertion of a Ds transposon, and an Avr-9 gene and (2) an Ac transposase gene, as described in Example 1.
  • Figure 14 illustrates basic simplified haploid crossing schemes to produce plants with increased disease resistance.
  • T 1 /P 1 line comprising in its genome at
  • T 1,2 /P 1,2 line comprising in its genome at
  • T 3 /P 3 line comprising in its genome at
  • T 3,4 /P 3,4 line comprising in its genome at
  • T 1,2,3 /P 1,2,3 line comprising in its genome at least one of each of three of the four genes R,L,I or A
  • T 4 /P 4 line comprising in its genome at
  • SEQ ID NO. 1 shows the genomic DNA sequence of the Cf-9 gene. Features: Nucleic acid sequence - Translation start at nucleotide 898; translation stop at nucleotide 3487; polyadenylation signal (AATAAA) at nucleotide 3703-3708; polyadenylation site at
  • nucleotide 3823 a 115 bp intron in the 3' non-coding sequence from nucleotide 3507/9 to nucleotide 3622/4.
  • Predicted Protein Sequence - primary translation product 863 amino acids; signal peptide sequence amino acids 1-23; mature peptide amino acids 24-863.
  • SEQ ID NO. 2 shows Cf-9 protein amino acid sequence.
  • SEQ ID NO. 3 shows the sequence of one of the Cf-9 cDNA clones. Translation initiates at the ATG at position +58.Cf-9 genomic sequence
  • SEQ ID NO. 4 shows the amino acid sequence and DNA sequence of the preferred form of the chimaeric Avr9 gene used as described herein.
  • SEQ ID NO. 5 shows the genomic DNA sequence of the Cf-2.1 gene.
  • SEQ ID NO. 6 shows Cf-2 protein amino acid sequence, designated Cf-2.1.
  • SEQ ID NO. 7 shows the amino acid sequence encoded by the Cf-2.2 gene. Amino acids which differ between the two Cf-2 genes are underlined.
  • SEQ ID NO. 8 shows the sequence of an almost full length cDNA clone which corresponds to the Cf2-2 gene.
  • SEQ ID NO. 9 shows the genomic DNA sequence of the RPP5 gene. Anticipated introns are shown in non-capitalised letters. Features: Nucleic acid sequence - Translation start at nucleotide 966; translation stop at nucleotide 5512.
  • SEQ ID NO. 10 shows predicted RPP5 protein amino acid sequence.
  • SEQ ID NO. 11 shows genomic DNA sequence of Cf-4. Features of this sequence include: translation start site at nucleotide 201, translation stop beginning at nucleotide 2619, consensus polyadenylation sequence beginning at nucleotide 2835, splice donor sequence in 3' untranslated sequence at 2641, splice acceptor sequence ending at nucleotide 2755, proposed site of polyadenylation at nucleotide 2955.
  • SEQ ID NO. 12 shows the predicted Cf-4 amino acid sequence.
  • the predicted protein sequence is composed of a primary translation product of 806 amino acids, signal peptide sequence amino acids 1-23, mature peptide amino acids 24-806.
  • SEQ ID NO. 13 shows double-stranded nucleic acid and deduced amino acid sequence of a ClaI/SalI DNA fragment encoding the PRla signal peptide sequence fused to a sequence proposed to encode the mature processed form of C. fulvum AVR4.
  • SLJ10512 (Scofield et al 1992) which contains (a) a beta-glucuronidase (GUS) gene (Jefferson et al 1987) to monitor T-DNA segregation and (b) stable Ac (sAc) that expresses transposase and can trans-activate a Ds, but which will not transpose (Scofield et al 1992).
  • GUS beta-glucuronidase
  • sAc stable Ac
  • the line FT33 did not carry a Cf-9 gene. We had to obtain recombinants that placed Cf-9 in cis with the T-DNA in FT33 in order to carry out linked targeted tagging. Two strategies were pursued simultaneously:
  • Kanamycin resistant progeny were tested for the presence of Cf- 9 ; 5 C. fulvum resistant individuals were obtained among 180 .
  • FT33 T-DNA a transposable Ds element is cloned into a hygromycin resistance gene, preventing its function. The somatic transactivation of this Ds element, which only occurs in the presence of transposase gene expression, results in activation of the hygromycin resistance.
  • RFLP marker was available, designated CP46, that enabled us to distinguish between homozygotes and heterozygotes for the Cf-9 gene
  • a likely frequency for obtaining any desired mutation in a gene tagging experiment is less than 1 in 1000, and often less than 1 in 10,000 (Döring, 1989). To avoid screening many thousands of plants for
  • the sequence of the 28 amino acids of the mature Avr9 protein is known (van Kan et al 1991). It is a secreted protein and can be extracted from
  • oligonucleotides to assemble a gene that carried a 30 amino acid plant signal peptide, from the Prla gene (Cornelissen et al 1987) preceding the first amino acid of the mature Avr9 protein (see SEQ ID NO. 4).
  • the preferred Avr9 gene sequence depicted in SEQ ID NO. 4 shows a chimaeric gene engineered from the Pr-la signal peptide sequence (Cornelissen et al , 1987) and the Avr9 gene sequence (van Kan et al , 1991). This reading frame was fused to the 355 promoter of
  • cauliflower mosaic virus (Odell et al 1984), and the 3' terminator sequences of the octopine synthase gene (DeGreve et al 1983), and introduced into binary plasmid vectors for plant transformation, using
  • section (iv) Individuals that were homozygous for the Avr 9 gene (section (iv)) were used as male parents to pollinate individuals that were homozygous for Cf- 9 , and carried both sAc and the Ds in the FT33 T-DNA
  • DNA was obtained from survivors and subjected to Southern blot analysis using a Ds probe. It was observed that several independent mutations were correlated with insertions of the Ds into a BglII fragment of a consistent size. This suggested that several independent mutations were a consequence of insertion of the Ds into the same DNA fragment.
  • DNA adjacent to the Ds in transposed Ds-carrying mutant #18 was amplified using inverse PCR (Triglia et al 1988). This DNA was used as a probe to other mutants, and proved that in independent mutations, the Ds had inserted into the same 6.7 kb BglII fragment.
  • the Ds in FT33 contains a bacterial replicon and a chloramphenicol resistance gene as. a bacterial selectable marker (Rommens et al 1992).
  • plant DNA carrying this transposed Ds can be digested with a restriction enzyme that does not cut within the Ds (such as BglII), the digestion products can be recircularized, and then used to transform E. coli .
  • Chloramphenicol resistant clones can be obtained that carry the Ds and adjacent plant DNA. This procedure was used to obtain a clone that carried 1.8 kb of plant DNA on the 3' side of the Ds, and 4.9 kb of plant DNA on the 5' side of the Ds .
  • a series of primers (F1, 2, 3, 4, 5, 6, 7, 12, 13, 10, 26, 27 and 25, indicated in Figure 1) was used to characterise a large number of independent mutations by PCR analysis in combination with primers based on the sequence of Ds. Therefore, these primers were used in polymerase chain reactions with primers based on the maize Ac/Ds transposon sequence, to characterise the locations of other mutations of Cf-9 that were caused by transposon insertion.
  • Mutants E, #55, #74 and #100 gave incomplete survival and showed a necrotic phenotype, and based on the available sequence information, they are 5' to the actual reading frame and might permit enough Cf9 protein expression to activate an incomplete defence response.
  • oligonucleotide primers were designed that could be used in polymerase chain reactions in combination with primers based on the sequence of the Ds element, to characterize both the location and the orientation of other transposon insertions in the gene. These are shown on Figure 1. Based on the results of such experiments, the map positions of 17 other Ds
  • necrotic sectors were visible on cotyledons, leaves, stems, petioles, sepals, and green fruits throughout plant development. Also, the necrotic sectors formed in both the lower and upper epidermis, in all mesophyll layers and in the cells surrounding the vascular tissue. The size of the necrotic sector and the frequency of their formation was determined by both the position of the Ds element in the Cf-9 sequence and the orientation of the Ds .
  • Sensitivity to the pathogen was measured by counting the number of sporulating pustules that were visible on each genotype 14 days and 21 days after inoculation. Samples were also taken for microscopic analysis. The results of the assay after 14 days are shown in Figure 2, and typical infections on each genotype after 21 days are shown in Figure 12.
  • Figure 2 shows a histogram in which the
  • sensitivity of different individual tomato plants is expressed on the y axis as the number of sporulating pustules per leaf.
  • the Ds carried a GUS gene.
  • M20, M23, M30 and M31 show C. fulvum growth on plants resulting from crosses between Cf-9*Ds and sAc, and derive from Cf-9*Ds #20, Cf-9*Ds #23, Cf-9*Ds #30 and Cf-9*Ds #31, respectively.
  • Cf0 carries no R genes and M20, M23, M30 and M31 GUS- plants have lost by segregation both Cf-9*Ds and sAc and are thus
  • FIG. 2 shows that in these experiments, Cf0 plants (lacking the Cf-9 gene) exhibited about 38 pustules per leaf and non-variegating individuals derived from Cf-9*Ds #20, Cf-9*Ds #23 or Cf-9*Ds #31 also showed about 38 pustules per leaf.
  • the non-variegated individuals that carried Cf- 9*Ds #30 showed about 17 pustules per leaf indicating some residual action of the tagged Cf-9 allele.
  • variegated individuals that carried Cf-9*Ds #20, Cf-9*Ds #23, Cf-9*Ds #30 or Cf-9*Ds #31 showed 1-3 pustules per leaf.
  • In total seventy variegated individuals were assessed. These results demonstrate a very significant level of disease control by this method.
  • Figure 12 shows three leaves.
  • Leaf 1 and Leaf 2 are infected with C. fulvum which confers the white fluffy appearance.
  • Leaf 1 is Cf0 and
  • Leaf 2 is a disease sensitive sib from Cf-9*Ds #23.
  • Leaf 3 showing minimal sporulation is a necrotic individual (small sectors of necrosis are discernible) that carried Cf-9*Ds #23, sAc and 35S:Avr9.
  • Leaf 3 is therefore expressing GAR.
  • each leaf was inoculated by brushing with an artist paintbrush the spores from a single 14 day old sporulating pustule over an entire upper surface.
  • the inoculated plants were then kept under diffuse light conditions at 20°C during the 16 h photoperiod and at 18°C during the dark period. The RH was maintained at 70%.
  • C. largenarium are hemibiotroph that initially forms simple haustoria but later on kills host cells in both the epidermal and mesophyll layers.
  • Homozygous Cf-9*Ds, 35S: SPAvr9 lines have been established for the tomato lines M31 and M50.
  • the F 1 backcross progeny derived from crosses to a homozygous sAc source may be assessed for their resistance to various pathogens, including:
  • Potato virus X Pseudomonas syringae pv. tomato, Necrotrophic fungi - Botrytis spp, Colletotrichum spp, Nematodes - Meloidogyne incognata, Aphids - Green Peach Aphid, and fruit, pod, root or tuber attacking
  • GAR Acquired Resistance
  • transgenic tobacco expression Cf-9 is crossed to transgenic tobacco plants engineered to express Avr9 peptide constitutively, the F1 seedlings die within 2 days of seed germination ( Figure 9).
  • the system is based around a single T-DNA
  • Figure 11 containing, a Cf-9 gene sequence under the control of its own promoter which has been inactivated by an autonomous Ac element that is only capable of a low level of excision (the Ac (Cla) element (Keller et al. 1993), and the 355:SP:Avr9 transgene).
  • the Ac element is inserted at various positions in the Cf-9 sequence and in both orientations in order to determine the best configuration to produce a high frequency of small somatic sectors where Cf-9 function has been restored.
  • Placing the Cf-9 sequence or other R gene sequence under the control of a cell-type specific promoter may enhance the GAR phenotype.
  • Potential target cellular sites include the epidermis and the vascular parenchyma cells.
  • the Cf-4 gene has been tested in transgenic plants in a number of ways: firstly by inoculation with a race of C. fulvum containing the corresponding avirulence gene Avr4 to test if that race gives an incompatible response on the transgenic plant; secondly by injecting leaves of a transformed plant with
  • fulvum race 2,5 using primers to the published sequence and fused a sequence encoding the proposed mature polypeptide to a DNA sequence encoding the N-terminal signal peptide of the tobacco PR1a protein. This would facilitate targeting of AVR4 to the intercellular space in transgenic plants where it is expressed.
  • This chimeric gene (SPAvr4) was inserted into a cDNA copy of potato virus X, as a ClaI/SalI DNA fragment (SEQ ID NO. 13) as described previously (Hammond-Kosack et
  • transcripts of the recombinant virus were generated by in vi tro transcription. All nucleic acid manipulations were performed using standard techniques well known to those skilled in the art.
  • Cf0 plants developed visible symptoms of virus infection at 7-10 d.p.i.
  • PVX SPAvr4 .
  • Transgenic plants were propagated by cuttings so that Cf-4 activity could be detected by inoculation with PVX:SPAvr4 on 12 tomato transformants.
  • Transgenic tomato plants containing Cf-4 exhibited leaf necrosis on inoculated leaves 3-4 d.p.i. This necrosis
  • VaI GIy Cys lie Pro Lys GIy Lys Gin Phe Asp Ser Phe GIy Asn Thr
  • GIu His lie lie Thr Thr Lys Met Lys Lys His Lys Lys Arg Tyr
  • GATTGTGTAA AACTTGTATT CCTTATGCTA TATACCTTTC TCTGTCAACT TGCTTTATCC 120
  • ATGTTTACCA TTAATCCTAA TGCTTCTGAT TATTGTTACG ACATAAGAAC ATACGTAGAC 240
  • CAATCCCTTC ATTTATCAGT CAATCCCCAG CTCACGGTTA GGTTTCCCAC AACCAAATGG 840
  • GGTCATATTC CAAGCATTAT TGGAGATCTT GTTGGACTTC GTACGTTGAA CTTGTCTCAC 2160

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Abstract

Des végétaux panachés présentent une résistance accrue aux agents pathogènes: des cellules du végétal expriment un phénotype qui peut comprendre la nécrose et/ou une réponse de défense du végétal, et d'autres cellules n'exprimant pas ce phénotype possèdent une résistance accrue aux agents pathogènes. Dans certains modes de réalisation de l'invention, on utilise divers gènes, notamment les gènes de résistance au pathogène Cladosporium fulvum, qui sont inactivés, par exemple, par suite de l'insertion d'un élément génétique transposable, puis réactivés dans les cellules végétales afin de provoquer la nécrose et/ou la réponse de défense du végétal, conférant à ce dernier une résistance accrue aux agents pathogènes. L'invention concerne des cellules, des végétaux ainsi que d'autres compositions de matière qui comprennent les diverses combinaisons de gènes impliquées dans ce système.
PCT/GB1995/001075 1994-05-11 1995-05-11 Procede d'introduction d'une resistance aux agents pathogenes chez les vegetaux WO1995031564A2 (fr)

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AU703644B2 (en) 1999-03-25
EP0759086A1 (fr) 1997-02-26

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