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WO1996030518A1 - Genes de resistance aux agents pathogenes vegetaux et leurs utilisations - Google Patents

Genes de resistance aux agents pathogenes vegetaux et leurs utilisations Download PDF

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Publication number
WO1996030518A1
WO1996030518A1 PCT/GB1996/000785 GB9600785W WO9630518A1 WO 1996030518 A1 WO1996030518 A1 WO 1996030518A1 GB 9600785 W GB9600785 W GB 9600785W WO 9630518 A1 WO9630518 A1 WO 9630518A1
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WIPO (PCT)
Prior art keywords
nucleic acid
plant
sequence
gene
encoding
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PCT/GB1996/000785
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English (en)
Inventor
Mark Stewart Dixon
David Allen Jones
Jonathan Dallas George Jones
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John Innes Centre Innovations Limited
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Publication date
Application filed by John Innes Centre Innovations Limited filed Critical John Innes Centre Innovations Limited
Priority to EP96908261A priority Critical patent/EP0817850A1/fr
Priority to AU51576/96A priority patent/AU709028B2/en
Priority to JP8529099A priority patent/JPH11503310A/ja
Priority to NZ304401A priority patent/NZ304401A/xx
Publication of WO1996030518A1 publication Critical patent/WO1996030518A1/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
    • C12N15/8282—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 pathogen resistance in plants and more particularly the identification and use of pathogen resistance genes. It is based on cloning of the tomato Cf-2 gene.
  • Plants are constantly challenged by potentially pathogenic microorganisms. Crop plants are particularly vulnerable, because they are usually grown as genetically uniform monocultures; when disease strikes, losses can be severe. However, most plants are resistant to most plant pathogens. To defend themselves, plants have evolved an array of both preexisting and inducible defences. 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.
  • Race specific resistance is strongly correlated with the hypersensitive response (HR) , an induced response by which (it is hypothesized) the plant deprives the pathogen of living host cells by localized cell death at sites of attempted pathogen ingress.
  • HR-associated disease resistance is often (though not exclusively) specified by dominant genes (i? genes) . Flor showed that when pathogens mutate to overcome such R genes, these mutations are recessive. Flor concluded that for R genes to function, there must also be corresponding genes in the pathogen, denoted avirulence genes (Avr genes) . To become virulent, pathogens must thus stop making a product that activates R gene-dependent defence mechanisms (Flor, 1971) .
  • 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) .
  • He toxin Helininthosporiu-n carJbonum races that express a toxin (He toxin) infect maize lines that lack the Hml resistance gene. Mutations to loss of He toxin expression are recessive, and correlated with loss of virulence, in contrast to gene-for-gene interactions in which mutations to virulence are recessive. A major accomplishment was reported in 1992, with the isolation by tagging of the Hml gene (Johal and Briggs, 1992) . Plausible arguments have been made for how gene-for-gene interactions could evolve from toxi -dependent virulence.
  • Pathogen avirulence genes are still poorly understood.
  • Several bacterial Avr genes encode 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,
  • 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) ; virus resistance genes in tomato and tobacco (by map based cloning and tagging) ; nematode resistance genes in tomato (by map based cloning) ; and genes for resistance to bacterial pathogens in Arabidopsis and tomato (by map based cloning) .
  • W093/11241 reports the sequence of a gene encoding a polygalacturonase inhibitor protein (PGIP) that has some homology with Cf-9 and, as we have now discovered, Cf-2 (the subject of the present invention) .
  • Cf-9 , Cf-2 and others ⁇ Cf -4 , 5 etc. are termed by- those skilled in the art "pathogen resistance genes” or “disease resistance genes” .
  • PGIP-encoding genes are not pathogen resistance genes.
  • a pathogen resistance gene (R) enables a plant to detect the presence of a pathogen expressing a corresponding avirulence gene (Avr) . When the pathogen is detected, a defence response such as the hypersensitive response (HR) is activated.
  • HR hypersensitive response
  • the PGIP gene of W093/11241 is a gene of the kind that is induced in the plant defence response resulting from detection of a pathogen by an R gene.
  • a pathogen resistance gene may be envisaged as encoding a receptor to a pathogen-derived and Avr dependent molecule.
  • PGIP is involved in the defence the plant mounts to the pathogen once detected and is not a pathogen resistance gene.
  • Expression of a pathogen resistance gene in a plant causes activation of a defence response in the plant. This may be upon contact of the plant with a pathogen or a corresponding elicitor molecule, though the possibility of causing activation by over-expression of the resistance gene in the absence of elicitor has been reported.
  • the defence response may be activated locally, e.g.
  • Activation of a defence response in a plant expressing a pathogen resistance gene may be caused upon contact of the plant with an appropriate, corresponding elicitor molecule, e.g. as produced by a Cladosporium fulvum avr gene as discussed.
  • the elicitor may be contained in an extract of a pathogen such as Cladosporium fulvum, or may be wholly or partially purified and may be wholly or partially synthetic.
  • An elicitor molecule may be said to "correspond" if it is a suitable ligand for the R gene product to elicit activation of a defence response.
  • Cf-x /"Avrx” terminology is standard in the art.
  • the Cf resistance genes and corresponding fungal avirulence genes (Avr) were originally defined genetically as interacting pairs of genes whose measurable activities fall into mutually exclusive interacting pairs.
  • Avr9 elicits a necrotic response on Cf-9 containing tomatoes but no response on Cf-2 containing tomatoes, the moeity recognised by Cf-2 being different from that recognised by Cf-9.
  • Expression of Cf-2 function in a plant may be determined by investigating compatibility of various C. fulvum races.
  • a race of C. fulvui ⁇ that carries functional copies of all known Avr genes (race 0) will grow (compatible) only on a tomato which lacks all the Cf genes. It will not grow (incompatible) on a plant carrying any functional Cf gene. If the C. fulvum race lacks a functional Avr2 gene (race 2) it will be able to grow not only on a plant lacking any Cf genes but also a plant carrying the Cf -2 gene. A race also lacking a functional Avr4 gene (race 2,4) will also be able to grow on a plant carrying the Cf-4 gene. A race only lacking a functional Avr4 gene (race 4) will not be able to grow on a plant carrying Cf-2. Similarly, a C.
  • fulvuin race 5 (lacking a functional Avr5 gene) will not be able to grow on a plant carrying a Cf-2 gene. Neither a race 4 nor a race 2,4 will be able to grow on a plant carrying any of the other Cf genes.
  • Various races are commonly available in the art, e.g. from the Research Institute for Plant Protection (IPO-DLO) , PO Box 9060, 6700 GW Wageningen, The Netherlands.
  • a race 4 is available under accession number IPO10379 and a race 2,4 available under Accession number IPO50379.
  • the tomato Cf-2 genes were isolated by map-based cloning.
  • the locus that confers resistance is mapped at high resolution relative to restriction fragment length polymorphism (RFLP) markers that are linked to the resistance gene.
  • RFLP restriction fragment length polymorphism
  • Two independent overlapping clones conferred disease resistance and the region of overlap contains a reading-frame which shows remarkable structural resemblance to the Cf- 9 gene. Since this sequence is the primary constituent of the DNA that overlaps the two clones that complement, we are confident that this sequence must correspond to the Cf-2 gene.
  • a second almost identical region on one of the cosmids was also able to confer disease resistance, indicating that there are two functional Cf-2 genes) .
  • the present invention provides a nucleic acid isolate encoding a pathogen resistance gene, the gene being characterized in that it comprises nucleic acid encoding the amino acid sequence shown in SEQ ID NO 2 or SEQ ID NO. 3 or a fragment thereof.
  • the nucleic acid isolate may comprise DNA, and may comprise the sequence shown in SEQ ID NO 1 or a sufficient part to encode the desired polypeptide (eg. from the initiating methionine codon to the first in frame downstream stop codon) .
  • the DNA comprises a sequence of nucleotides which are the nucleotides 1677 to 5012 of SEQ ID NO 1, or a mutant, derivative or allele thereof.
  • a further aspect of the invention provides a nucleic acid isolate encoding a pathogen resistance gene, or a fragment thereof, obtainable by screening a nucleic acid library with a probe comprising nucleotides 1677 to 5012 of SEQ ID NO 1, or a fragment, derivative, mutant or allele thereof, and isolating DNA which encodes a polypeptide able to confer pathogen resistance to a plant, such as resistance to Cladosporium fulvum (eg. expressing Avr2) .
  • the plant may be tomato. Suitable techniques are well known in the art .
  • Nucleic acid according to the present invention may encode the amino acid sequence shown in SEQ ID NO 2 or a mutant, derivative or allele of the sequence provided e.g. SEQ ID NO 3.
  • Preferred mutants, derivatives and alleles are those which retain a functional characteristic of the protein encoded by the wild-type gene, especially the ability to confer pathogen resistance and most especially the ability to confer resistance against a pathogen expressing the Avr2 elicitor molecule.
  • Changes to a sequence, to produce a mutant or derivative may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or subsitution of one or more amino acids.
  • nucleic acid encoding the amino acid sequences shown in Figure 3 (SEQ ID NO.'s 2 and 3) include encoding sequences shown in Figures 2 and 4, respectively.
  • DNA may comprise a nucleotide sequence shown in Figure 4 (SEQ ID NO. 5) .
  • nucleic acid comprising a sequence of nucleotides complementary to a nucleotide sequence hybridisable with any encoding sequence provided herein. Another way of looking at this would be for nucleic acid according to this aspect to be hybridisable with a nucleotide sequence complementary to any encoding sequence provided herein.
  • DNA is generally double-stranded and blotting techniques such as Southern hybridisation are often performed following separation of the strands without a distinction being drawn between which of the strands is hybridising.
  • the hybridisable nucleic acid or its complement encode a polypeptide able to confer pathogen resistance on a host, i.e., includes a pathogen resistance gene.
  • Preferred conditions for hybridisation are familiar to those skilled in the art, but are generally stringent enough for there to be positive hybridisation between the sequences of interest to the exlucsion of other sequences.
  • the polypeptides encoded by the Cf -2 and Cf-9 genes share a high degree of homology, the genes themselves are not sufficiently homologous to identify each other in genomic Southern blotting using a stringency of 2 x SSC at 60°C. In a BLASTN search, the highest level of identity between the DNA sequences of Cf-2 and Cf-9 is 69% over a 428 base region.
  • Nucleic acid according to the present invention may be distinguished from Cf - 9 by one or more of the following: not being sufficiently homologous with Cf- 9 tor the nucleic acid of the invention and Cf-9 to identify each other in Southern blotting using a stringency of 2 x SSC at 60°C; having greater than 70%, preferably greater than about 75%, greater than about 80%, greater than about 90% or greater than about 95% homology with the encoding • sequence shown in Figure 2 as nucleotides 1677-5012; eliciting s_» defence response, in a plant expressing the nucleic acid, upon contact of the plant with Avr2 elicitor molecule, e.g.
  • a Cladosporium fulvum race expressing Avr2 eliciting a defence response, in a plant expressing the nucleic acid, upon contact of the plant with the C. fulvum race 4 deposited at and available from the Research Institute for Plant Protection (IPO-DLO) , PO Box 9060, 6700 GW Wageningen, The Netherlands, under accession number IPO10379, or an extract thereof, but not eliciting a defence response in the plant upon its contact with the C.
  • IP-DLO Research Institute for Plant Protection
  • fulvum race 2,4 deposited at and available from the same institute under Accession number IPO50379, or an extract thereof; not eliciting a defence response, in a plant expressing the nucleic acid, upon contact of the plant with Avr9 elicitor molecule, e.g. as provided by a Cladosporium fulvum race or other organism expressing
  • the nucleic acid isolate which may contain DNA encoding the amino acid sequence of SEQ ID NO 2 or SEQ ID NO 3 as genomic DNA or cDNA, may be in the form of a recombinant vector, for example a phage or cosmid vector.
  • the DNA may be under the control of an appropriate promoter and regulatory elements for expression in a host cell, for example a plant cell. In the case of 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 seuqences, enhancer sequences, marker genes and other sequences as appropriate.
  • appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation seuqences, enhancer sequences, marker genes and other sequences as appropriate.
  • Nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of nucleic acid or genes of the species of interest or origin other than the sequence encoding a polypeptide with the required function.
  • Nucleic acid according to the present invention may comprise cDNA, RNA, genomic DNA and may be wholly or partially synthetic. The term “isolate” encompasses all these possibilities.
  • 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 target cell type must be such that cells can be regenerated into whole plants.
  • Plants transformed with the DNA segment containing the 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 ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg. bombardment with Agrobacterium coated microparticles (EP-A-486234) or mircoprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233) .
  • a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention.
  • a Cf-2 gene and modified versions thereof (alleles, mutants and derivatives thereof) , and other nucleic acid provided herein may be used to confer resistance in plants, in particular tomatoes, to a pathogen such-as C. fulvum. This may include cloned DNA from Lycopersicon pimpinellifolium which has the same chromosomal location as the Cf-2 gene or any subcloned fragment thereof.
  • a vector as described above may be used for the production of a transgenic plant.
  • a plant may possess pathogen resistance conferred by the Cf-2 gene.
  • the invention thus further encompasses a host cell transformed with such a vector, especially a plant or a microbial cell.
  • a host cell such as a plant cell, comprising nucleic acid according to the present invention is provided.
  • the nucleic acid may be incorporated within the chromosome.
  • a vector comprising nucleic acid according to the present invention need not include a promoter, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.
  • a plant cell comprising, e.g. having incorporated into its genome a sequence of nucleotides as provided by the present invention, under operative control of a promoter for control of expression of the encoded polypeptide.
  • a further aspect of the present invention provides a method of making such a plant cell involving introduction of a vector comprising the sequence of nucleotides into a plant cell. Such introduction may be followed by recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome. The polypeptide encoded by the introduced nucleic acid may then be expressed.
  • a plant which comprises a plant cell according to the invention is also provided, along with any part or clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of these, such as cuttings, seed.
  • the invetion 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.
  • the invention further provides a method of comprising expression from nucleic acid encoding the amino acid sequence SEQ ID NO 2 or SEQ ID NO 3, or a mutant, allele or derivative of either sequence, within cells of a plant (thereby producing the encoded polypeptide) , following an earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof.
  • a method may confer pathogen resistance on the plant.
  • This may be used in combination with the Avr2 gene according to any of the methods described in W091/15585 (Mogen) or, more preferably, PCT/GB95/01075 (published as WO 95/31564) , or any other gene involved in conferring pathogen resistance.
  • the Cf-2 and Cf- 9 genes function in a similar manner in that they both confer a resistance to tomato that prevents the growth of tomato leaf mould C. fulvum. They, however, by recognition of different Avr products and have subtle differences in the speed with which they stop growth of the pathogen and stimulate a resistance response (Hammond-Kosack and Jones 1994; Ashfield et al 1994) . These differences may be exploited to optimise applications disclosed herein.
  • a gene stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, cells of which decendants may express the encoded polypeptide and so may have enhanced pathogen resistance.
  • Pathogen resistance may be determined by assessing compatibility of a pathogen (eg. Cladosporium fulvum) or using recombinant expression of a pathogen avirulence gene, such as Avr- 2 or delivery of the Avr-2 gene product.
  • Cf-2 DNA sequence encoding leucine-rich repeat (LRR) regions and homology searching has revealed strong homologies to other genes containing LRRs.
  • LRR leucine-rich repeat
  • the present invention provides a method of identifying a plant pathogen resistance gene comprising use of an oligonucleotide which comprises a sequence or sequences that are conserved between pathogen resistance genes such as Cf-9 and Cf-2 to search for new resistance genes.
  • nucleic acid comprising a pathogen resistance .
  • gene encoding a polypeptide able to confer pathogen resistance
  • hybridisation of an oligonucleotide (details of which are discussed herein) or a nucleic acid molecular comprising such an oligonucleotide to target/candidate nucleic acid.
  • Target or candidate nucleic acid may, for example, comprise a genomic or cDNA library obtainable from an organism known to encode a pathogen resistance gene. Successful hybridisation may be identified and target/candidate nucleic acid isolated for further investigation and/or use.
  • Hybridisation may involve probing nucleic acid and identifying positive hybridisation under suitably stringent conditions (in accordance with known techniques) and/or use of oligonucleotides as primers in a method of nucleic acid amplification, such as PCR.
  • stringent conditions in accordance with known techniques
  • oligonucleotides as primers in a method of nucleic acid amplification, such as PCR.
  • preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further. It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain.
  • oligonucleotides designed to amplify DNA sequences may be used in PCR reactions or other methods involving amplification of nucleic acid, using routine procedures. See for instance "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al . 1990, Academic Press, New York.
  • Preferred amino acid sequences suitable for use in the design of probes or PCR primers are sequences conserved (completely, substantially or partly) between polypeptides able to confer pathogen resistance such as those encoded by Cf-2 and Cf-9.
  • oligonucleotide probes or primers may be designed, taking into account the degeneracy of the genetic code, and, where appropriate, codon usage of the organism from the candidate nucleic acid is derived.
  • Preferred nucleotide sequences may include those comprising or having a sequence encoding amino acids (i) SGEIPOO; (ii) YE/OGNDG; (iii) FEGHIPS; or (iv) SGEIPOOLASLTSLE, or a sequence complementary to these encoding sequences. Suitable fragments of these may be employed.
  • Preferred oligonucleotide sequences include:- (i) TCX-GGX-GAA/G-AAT.C.A-CCX-CAA/G-CA; (ii) TAT/C-G/CAA/G-GGX-AAT/C-GAT/C-GGX-CTX-CG; and (iii) CG-XAG-XCC-A/GTC-A/GTT-XCC-T/CTC/G-A/GTA. (All sequences given 5' to 3' ,- see Figure 6) . Sequences (ii) and (iii) are complementary: (iii) is useful as a back (reverse) primer in PCR.
  • oligonucleotide in accordance with the invention e.g. for use in nucleic acid amplification, has about 10 or fewer codons (e.g. 6, 7 or 8) , i.e. is about 30 or fewer nucleotides in length (e.g. 18, 21 or 24) .
  • a PCR band may contain a complex mix of products. Individual products may be cloned and each sreened for linkage to known disease resistance genes that are segregating in progeny that showed a polymorphism for this probe. Alternatively, the PCR product may be treated in a way that enables one to display the polymorphism on a denaturing polyacrylamide
  • DNA sequencing gel with specific bands that are linked to the resistance gene being preselected prior to cloning Once a candidate PCR band has been cloned and shown to be linked to a known resistance gene, it may be used to isolate clones which may be inspected for other features and homologies to Cf- 9 , Cf-2 or other related gene. It may subsequently be analysed by transformation to assess its function on introduction into a disease sensitive variety of the plant of interest. Alternatively, the PCR band or sequences derived by analysing it may be used to assist plant breeders in monitoring the segregation of a useful resistance gene. These techniques are of general applicability to the identification of pathogen resistance genes in plants.
  • genes that can be identified in this way include Phytophthora resistance in potatoes, mildew resistance and rust resistance in cereals such as barley and maize, rust resistance in Antirrhinum and flax, downy mildew resistance in lettuce and Arabidopsis, virus resistance in potato, tomato and tobacco, nematode resistance in tomato, resistance to bacterial pathogens in Arabidopsis and tomato and Xanthomonas resistance in peppers.
  • the present invention provides a DNA isolate encoding the protein product of a plant pathogen resistance gene which has been identified by use of the presence therein of LRRs or, in particular, by the technique defined above.
  • the invention provides transgenic plants, in particular crop plants, which have been engineered to carry pathogen resistance genes which have been identified by the presence of LRRs or by nucleic acid hybridisation as disclosed.
  • suitable plants include tobacco, cucurbits, carrot, vegetable brassica, lettuce, strawberry, oilseed brassica, sugar beet, wheat, barley, maize, rice, soyabeans, peas, sorghum, sunflower, tomato, potato, pepper, chrysanthemum, carnation, poplar, eucalyptus and pine.
  • Figure 1 shows a physical map of the tomato Cf-2 locus generated from overlapping cosmids (38, 82, 89, 90, 92, 94, 96 and 141) isolated from the Cf -2/Cf-9 cosmid library. Also included are the modified cosmids (1126- ⁇ and 112B 2 ) which contain sequences derived from cosmid 94 (also known as 2.2) . The extent of each cosmid and location of the Cf-2 genes are shown schematically. Also indicated is the predicted direction of transcription (arrow) . The boxed regions represent expanded views of areas encoding Cf -2 genes. The open boxes show regions not sequenced, the hatched boxes show the sequenced regions.
  • Figure 2 shows the genomic DNA sequence of the Cf -2 . 1 gene (SEQ ID NO 1) .
  • Predicted Protein Sequence primary translation product 1112 amino acids; signal peptide sequence amino acids 1-26; mature peptide amino acids 27-1112.
  • Figure 3A shows Cf-2 protein amino acid sequence, designated Cf-2, 1 (SEQ ID NO 2) .
  • Figure 3B shows the amino acid sequence encoded by the Cf-2.2 gene (SEQ ID NO 2) .
  • Figure 4 shows shows the sequence of an almost full length cDNA clone (SEQ ID NO. 4) which corresponds to the Cf2 -2 gene.
  • Figure 5 shows a comparison of the carboxy-terminal regions of the Cf-2 and Cf-9 genes (SEQ ID No's 5 and 6, respectively) .
  • the protein sequences are aligned according to predicted protein domains. Identical amino acid residues are indicated by bold type.
  • Figure 6 shows an alignment of part of the Cf-2 and Cf-9 proteins (SEQ ID NO's 7 and 8, respectively). Two identical regions are shown in bold type and are also shown as PEP SEQ 1 (SEQ ID NO. 9) and PEP SEQ 2 (SEQ ID NO. 10) respectively.
  • OLIGO 1 SEQ ID NO. 11
  • OLIGO 2 show the sequence of degenerate oliggonucleotides which encode these regions of protein similarity.
  • Figure 7 shows the primary amino acid saequence of Cf-2 (SEQ ID NO. 2) divided into domains of predicted differing functions.
  • the Cf-2 gene was cloned using a map-based cloni'ng strategy similar in principle to that used for the isolation of the tomato Pto gene, described briefly earlier.
  • RFLP restriction fragment length polymorphism
  • MG112A and CT119 is only 40 kb.
  • MG112B a weak homologue of MG112A
  • SC3-8 a weak homologue of MG112A
  • tomato crosses were set up to look for recombination between the Cf-2 and Cf-5 resistance genes.
  • a plant that was heterozygous for both Cf-2 and Cf-5 was crossed to a C. fulvum-sensitive tomato line.
  • Approximately 12,000 resulting FI progeny were screened for resistance to C. fulvum, and a single sensitive plant was identified.
  • DNA from this plant was analysed with the molecular markers which map closely to the Cf-2 gene and this plant was found to carry a chromosome that was recombinant between MG112A and CT119. This analysis strongly indicated that the RFLP marker MG112B identified DNA which mapped very closely linked to the Cf-2 gene or was the Cf-2 gene itself (Dixon et al 1996) .
  • DNA sequences were isolated from a plant that carried the Cf-2 gene and transformed into Cf- 0 tomato plants.
  • a genomic DNA library was constructed from a stock that carried both the Cf-9 gene on chromosome 1, and the Cf-2 gene on chromosome 6, so that the library could be used for isolating both genes.
  • the library was constructed in a binary cosmid cloning vector pCLD04541, obtained from Dr C. Dean, John Innes Centre, Colney Lane, Norwich (see also Bent et al 1994) .
  • This vector is essentially similar to pOCAl ⁇ (Olszewski et al 1988) . It contains a bacteriophage lambda cos site to render the vector packageable by lambda packaging extracts and is thus a cosmid (Hohn and Collins, 1980) . It is also a binary vector (van den Elzen et al 1985) , so any cosmid clones that are isolated can be introduced directly into plants to test for the function of the cloned gene.
  • High molecular weight DNA was isolated from leaves of 6 week old greenhouse-grown plants by techniques well known to those skilled in that art (Thomas et al 1994) and partially digested with Mbol restriction enzyme.
  • the partial digestion products were size fractionated using a sucrose gradient and DNA in the size range 20-25 kilobases (kb) was ligated to BamHI digested pCLD04541 DNA, using techniques well known to those skilled in the art.
  • the cosmids were introduced into a tetracycline sensitive version (obtained from Stratagene) of the Stratagene Escherichia coli strain SURETM . Recombinants were selected using the tetracycline resistance gene on PCLD04541.
  • the library was randomly distributed into 144 pools containing about 1500 clones per pool, cells were grown from each pool and from 10 ml of cells, 9 ml were used for bulk plasmid DNA extractions, and 1 ml was used after addition of 0.2 ml of glycerol, to prepare a frozen stock. Plasmid DNA from the pools was isolated by alkaline lysis (Birnboim and Doly, 1979) , and DNA samples were analyzed by hybridisation in "slot blots" with the molecular marker MG112B. Pools 38, 82, 89, 90, 92, 94, 96 and 141 proved positive by this assay. "94” is also known as "2.2" .
  • the function of a putative cloned Cf-2 gene was assessed in transformed tomato by testing transformants for resistance to Avr2-carrying C. fulvum.
  • the DNA sequence of the 6.5 kb region representing the central core of the cosmid 82/94 overlap has been determined. Two small regions of 2.3 and 1.1 kb corresponding to the extremities of the cosmid overlap have not been sequenced (Fig. 1) .
  • the central core sequence carries a single major open reading frame which upon conceptual translation has revealed an interesting motif (the leucine rich repeat, or LRR) that may be diagnostic of other resistance genes, as previously noted for the Cf-9 gene (WO 95/18320) .
  • LRR leucine rich repeat
  • the open reading frame initiates with the translation start codon (ATG) at position 1677 and finishes with the translation termination codon TAG at position 5012 with an intervening 3336 bp sequence that encodes a 1112 amino acid protein. This is the Cf-2. 1 gene.
  • the sequence of the region labelled MG112B 2 carried on cosmid 112B 2 has also been determined.
  • This sequence also carries a single open reading frame which differs by only 3 nucleotides from the Cf -2. 1 gene sequence.
  • this also encodes a 1112 amino acid protein which differs by only 3 amino acids from the Cf-2. 1 protein.
  • These amino acid differences are all clustered in the carboxy-terminal region of the protein and are indicated as underlined residues in Figure 3B (SEQ ID NO. 3) . We therefore designate this to be Cf - 2 . 2.
  • NCBI Biological Information
  • LRRs The Cf-2 gene identifies Cf-9 with a blast score of 483.
  • Other homologies include the Arabidopsis genes TMK1 (Chang et al 1992), TMKLl (Valon et al 1993), RLK5 (Walker, 1993) , as well as expressed sequences with incomplete sequence and unknown function (e.g. Arabidopsis thaliana transcribed sequence [ATTS] 1447) .
  • the presence of LRRs has been observed in other genes, many of which probably function as receptors (see Chang et al [1992] for further references) .
  • TMK1 and RLK5 genes have structures which suggest they encode transmembrane serine/threonine kinases and carry extensive LRR regions. As yet no known function has been assigned to them.
  • Disease resistance genes are known to encode gene products which recognize pathogen products and subsequently initiate a signal transduction chain leading to a defence response. It is known that another characterized disease resistance gene (Pto) is a protein kinase (Martin et al 1993) .
  • Pto protein kinase
  • Cf-2 there is no apparent protein kinase domain based on genomic DNA and cDNA sequence analysis. The predicted Cf-2 amino acid sequence can be divided into 7 domains (Fig. 7) .
  • Domain A is a 26 amino acid probable signal peptide.
  • Domain B is a 37 amino acid region with some homology to polygalacturonase inhibitor proteins.
  • Domain C is a 930 amino acid comprising 33 perfect copies and 5 imperfect copies of a 24 amino acid leucine rich repeat (LRR) .
  • Domain D is a 30 amino acid domain with some homology to polygalacturonase inhibitor proteins.
  • Domain E is a 28 amino acid domain rich in negatively charged residues.
  • Domain F is a 24 amino acid hydrophobic domain encoding a putative transmembrane domain.
  • Domain G is a 37 amino acid domain rich in positively charged residues.
  • the Cf-2 and Cf-9 proteins are predicted to have the same general features in that they can both be sub ⁇ divided into the above 7 domains. They are, however, very different in length, 1112 verses 863 amino acids respectively. The majority of this size difference resides in the number of LRRs in domains C. Although the LLRs are characterised by specific conserved amino acids (mainly leucine) , they are generally spaced apart such that no block of conserved amino acids exists. Additionally, leucine can be encoded by 6 different codons and as a result it would be difficult to exploit the similarities of the conserved amino acids in the LLR domain at the level of DNA hybridisation to identify new related genes. Indeed, at the level of genomic Southern hybridisation the Cf-2 and Cf-9 genes did not identify each other under the conditions we used.
  • conserved amino acids mainly leucine
  • oligonucleotide primers like those indicated in Figure 6 might be produced. These primers correspond to the different DNA sequences which potentially encode amino acids conserved between the Cf-2 and Cf -9 polypeptides. These synthetic oligonucleotide primers may be used in a PCR to identify related sequences from any species which contains them.

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Abstract

On a cloné le gène Cf-2 de la tomate et on a établi sa séquence, ainsi que la séquence codée d'acides aminés. L'ADN codant le polypeptide, ainsi que ses allèles, ses mutants et ses dérivés, peuvent être introduits dans des cellules végétales et le polypeptide codé peut être exprimé, ce qui confère une résistance aux agents pathogènes végétaux comprenant lesdites cellules, ainsi qu'à leurs descendants. La séquence de Cf-2 comprend des répétitions riches en leucine et la présence de ces répétitions permet d'identifier d'autres gènes de résistance aux agents pathogènes végétaux. Des homologies de Cf-9 révèlent des motifs utiles pour identifier d'autres gènes de résistance aux agents pathogènes végétaux.
PCT/GB1996/000785 1995-03-31 1996-04-01 Genes de resistance aux agents pathogenes vegetaux et leurs utilisations WO1996030518A1 (fr)

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JP8529099A JPH11503310A (ja) 1995-03-31 1996-04-01 植物病原体耐性遺伝子及びその使用
NZ304401A NZ304401A (en) 1995-03-31 1996-04-01 Plant pathogen resistance genes and uses thereof based upon cloning of the tomato cf-2 gene

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WO1997043429A1 (fr) * 1996-05-09 1997-11-20 Plant Bioscience Limited Genes resistant aux pathogenes pour des plantes et utilisation de ces genes
WO2000012736A2 (fr) * 1998-08-31 2000-03-09 Monsanto Co. Nouvelle technique d'identification de genes non hotes de resistance aux maladies dans les plantes

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WO2007051483A1 (fr) * 2005-11-01 2007-05-10 Universiteit Utrecht Holding B.V. Plantes résistantes aux maladies

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997043429A1 (fr) * 1996-05-09 1997-11-20 Plant Bioscience Limited Genes resistant aux pathogenes pour des plantes et utilisation de ces genes
US6225532B1 (en) 1996-05-09 2001-05-01 Plant Bioscience Limited Tomato CF-5 gene encoding a disease resistance polypeptide
WO2000012736A2 (fr) * 1998-08-31 2000-03-09 Monsanto Co. Nouvelle technique d'identification de genes non hotes de resistance aux maladies dans les plantes
WO2000012736A3 (fr) * 1998-08-31 2000-10-05 Monsanto Co Nouvelle technique d'identification de genes non hotes de resistance aux maladies dans les plantes
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