EP1078086A2 - Gene de resistance tire d'une plante - Google Patents

Gene de resistance tire d'une plante

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Publication number
EP1078086A2
EP1078086A2 EP99917014A EP99917014A EP1078086A2 EP 1078086 A2 EP1078086 A2 EP 1078086A2 EP 99917014 A EP99917014 A EP 99917014A EP 99917014 A EP99917014 A EP 99917014A EP 1078086 A2 EP1078086 A2 EP 1078086A2
Authority
EP
European Patent Office
Prior art keywords
plant
nucleic acid
resistance
polypeptide
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99917014A
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German (de)
English (en)
Inventor
Abdelhafid Bendahmane
David Charles Baulcombe
Konstantin Valerievich Univ. of Bristol KANYUKA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Plant Bioscience Ltd
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Plant Bioscience Ltd
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Publication date
Application filed by Plant Bioscience Ltd filed Critical Plant Bioscience Ltd
Publication of EP1078086A2 publication Critical patent/EP1078086A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi

Definitions

  • the present invention relates to the Rx resistance gene from potato. It further relates to methods and materials employing the gene, and processes for identifying or producing other related genes. It also relates generally to methods for identifying novel genes or markers within clusters of repetitive DNA sequences.
  • Rx is a resistance gene from potato conferring extreme resistance against potato virus X (PVX) .
  • PVX potato virus X
  • Rx genes in potato are widely used in breeding programmes to confer resistance against PVX because :
  • Rx is highly durable with only one natural isolate able to overcome the resistance.
  • the resistance is extreme resistance according to the classification of Cooper (Cooper and Jones, 1983) .
  • ER Extreme resistance
  • Rx-mediated resistance is active in protoplasts wherein it suppresses viral replication or promotes degradation of the viral RNA.
  • HR resistance is not expressed in protoplasts but is thought to be a tissue-related phenomenon requiring cell-to-cell contact (Adams et al , 1986) .
  • the Rx-mediated resistance against PVX is thought to conform to an elicitor-receptor model. According to the model there are two phases in the Rx resistance mechanism: a recognition phase that is believed to be highly specific for potato virus X and a response phase that prevents accumulation of a broad spectrum of plant viruses, including those taxonomically unrelated to PVX (K ⁇ hm et al . , 1993) . Published work has also demonstrated that the PVX coat protein (CP) is the elicitor of Rx-mediated resistance (Bendahmane et al . , 1995; Goulden et al . , 1993).
  • CP the elicitor of Rx-mediated resistance
  • Rx loci there are two Rx loci, Rxl and Rx2 (on chromosomes XII and V respectively) . These loci function independently of each other and have the same specificity for the PVX CP. It has been suggested that Rx may have been introgressed into potato cultivars from a wild Solanum accession (see also Arntzen et al (1994)) .
  • Rx lies within an introgressed chromosomal segment would also be expected to hinder map based cloning efforts because recombination is believed to be suppressed in such regions (see Liharska et al (1996)).
  • BAC bacterial artificial chromosome
  • BAC77 spanned Rx. There was a chromosomal recombination in one plant of the mapping population that separated the BAC77 right end from Rx. On the other side of Rx it was found that the BAC77 left end was outside the region of DNA carrying Rx that had been introgressed into potato cv Amaryl from Solanu tuberosu ssp. andigena CPC1673. The fact that BAC77 did indeed span Rx was shown using novel, transient expression assays, and by transgenic expression in Nicotiana tabacum, Nicotiana benthamiana, Lycopersicon esculentum and potato.
  • the Rx gene has been sequenced and the expression product shown to share some motifs with other, known, resistance proteins, as well as having some distinctive sequence regions. The inventors have demonstrated that it can be used to introduce PVX resistance into plants, including L. esculentum, N. tajbacum, N. Jbei-fcainiana and potato.
  • portions of the Rx sequence can be used to identify Rx homologues, some of which may be closely-linked to the Rx locus.
  • Rx gene (or modifications or homologues thereof) can be used to engineer resistance traits, preferably broad spectrum ER, into plants using a variety of innovative formats.
  • Rx can be activated by certain non-PVX viruses, particularly those comprising coat proteins sharing sequence homology with that of PVX.
  • non-PVX viruses include (but are not limited to) Narcissus mosaic virus (NMV) ; Nandina virus X (NVX) ; Viola mosaic virus (VMV) ; Cymbidium mosaic virus (CyMV) ; Poplar mosaic virus (PopMV) and White clover mosaic virus (WClMV) .
  • NMV Narcissus mosaic virus
  • NVX Nandina virus X
  • VMV Viola mosaic virus
  • Cymbidium mosaic virus Cymbidium mosaic virus
  • PopMV Poplar mosaic virus
  • WClMV White clover mosaic virus
  • nucleic acid molecule encoding a polypeptide which is capable of conferring extreme resistance against a pathogen, such as a plant virus, in a plant into which said polypeptide is expressed.
  • nucleic acid molecules according to the present invention may be provided in recombinant 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.
  • the nucleic acid molecules (and their encoded polypeptide products) may also be (i) isolated and/or purified from their natural environment (although not necessarily in pure form per se) , or (ii) in substantially pure or homogeneous form.
  • Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and may be wholly or partially synthetic ('constructs'). Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed.
  • a particular aspect of the invention is nucleic acid having the sequence all or part of the sequence shown in Annex 1 (Seq ID No 1) including (where appropriate) both coding and/or non-coding regions.
  • Seq ID No 1 there is apparently a large open reading frame (ORF) .
  • ORF open reading frame
  • Subsequent comparison of the genomic DNA sequence with the sequence of cDNAs revealed that the gene contains three exons and two introns in the 3 ' end (234 bp and 111 bp) .
  • the introns are marked as shown in Annex 1.
  • the final exon contains only two nucleotides of coding sequence plus the TAG stop codon and the 3 ' non-coding region of mRNA.
  • Rx polypeptide sequence is shown in Fig 1 - designated Seq ID No 2) .
  • Rx appears to contain 937 residues and have a molecular weight of 107.5 kD.
  • nucleic acids of this aspect of the invention include those encoding the Rx protein product and cDNA, believed to be base 2249-5404 excluding the introns marked as shown (4945-5178 and 5290-5400 inclusive) .
  • the Rx-coding (cDNA) nucleic acid sequence is designated Seq ID No 3. This is shown as Annex II.
  • Rx is similar to that of the NBS-LRR (Jones and Jones, 1997) class of R proteins, in which the resistance is associated with an HR.
  • the highest degree of similarity is between Rx and a subclass of NBS-LRR resistance proteins represented by Rps2 , Rpml and Prf (Jones and Jones, 1997) .
  • These Arabidopsis and tomato proteins contain a putative four to six heptad amphipathic leucine zipper (LZ) motif at the N-terminus (Jones and
  • the putative NBS domain (domain 11; Figure 1) of Rx comprises three motifs: kinase 1A or ' P-loop' (residues 168-180) , kinase 2 (residues 237-247) , and kinase 3a (residues 265-273) .
  • the putative NBS is followed by a domain with unknown function that includes GLPL, CFLY and the MHD motifs. These motifs are characteristic of all NBS- LRR R gene products thus far identified (Hammond-Kosack and Jones, 1997; van der Biezen and Jones, 1998) .
  • the putative LRR domain of Rx (residues 473-868) comprises 14-16 imperfect copies of the LRR motif. This motif shows a good match to the cytoplasmic LRR consensus sequence motif (Jones and Jones, 1997) and most closely resembles the LRR domain of the tomato Prf protein (Salmeron et al . , 1996) .
  • the C-terminal part of Rx contains three unique motifs with unknown functions: an amide-rich region (residues 869-893), a short basic region (residues 894-902) and an 'acidic tail 1 region (residues 903-937) .
  • the acidic tail is encoded entirely within the short second exon of Rx. This domain is rich in aspartic and glutamic acid residues and contains two copies of 10 amino acid residues in direct repeat. These features are not present in any previously described products of R genes.
  • Rx homologues such as allelic variants, paralogues (from the same species, but at a different location e.g. pseudoalleles at linked loci) , or orthologues (related genes from different species) . Examples of these are shown below.
  • the variant encodes a product which is homologous (similar) to Rx, which may be isolated or produced on the basis of that sequence, and is capable of conferring pathogen resistance against one or more pathogens .
  • Resistance gene activity can be tested by conventional methods known in the art, as appropriate to the nature of the resistance being investigated. Example methods can be found in the following publications: bacterial (Grant et al , 1995); fungal (Dixon et al , 1996; Jones, 1994; Thomas et al, 1997) ; nematode and viral (Whitham et al , 1994) .
  • activity is tested by complementation of trait in a plant. This can be achieved by coupling the putative active variant to a promoter and terminator for expression in plants and transforming it into a 'susceptible' plant that lacks a given resistance trait. The activity of the Rx variant is then confirmed by challenge with the appropriate pathogen.
  • a transient expression assay can be used to test for activation of the Rx variant analogous to the assay described in the Examples below or that used by Mindrinos et al (1994) .
  • the putative active Rx variant is co- expressed from a plasmid with a pathogen-derived gene which is an elicitor of the resistance specified by the putative Rx homologue (in the case of Rx this can be PVX-CP) and a reporter gene (e.g. GUS or CP) . If the variant is activated by the continuous expression of the pathogen derived gene, then an HR would result and the reporter gene activity would be abolished. If no activity was initiated, then the reporter gene would be detectable.
  • Similarity or homology between the variant and Rx may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J. Mol . Biol . 215: 403-10, which is in standard use in the art, or, and this may be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . Comparisons herein have used DNASTAR software using the CLUSTAL method with PAM250 residue weight table (gap penalty 10, gap length 10) .
  • Homology may be at the nucleotide sequence and/or the expressed amino acid sequence level.
  • the nucleic acid and/or amino acid sequence shares homology with the coding sequence or the sequence encoded by the nucleotide sequence of Annex I or other sequences set out herein, preferably at least about 50%, or 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.
  • Homology may be over the full-length of the relevant sequence shown herein, or may more preferably be over a contiguous sequence of about or greater than about e.g. 20, 100, 200, 300, 500, 600 or more amino acids or codons, compared with the relevant amino acid sequence or nucleotide sequence as the case may be .
  • Naturally occurring Rx variants may be isolated, in the light of the present disclosure, without burden from any suitable source (e.g. genomic or cDNA) .
  • the putative resistance genes can be obtained using materials (e.g. primers or probes) based on regions peculiar to Rx, for instance designated Rx gene signature' in Fig 1. It should be noted that previously identified sequence motifs that characterise resistance genes (see e.g. Kanazin et al . , 1996; Leister et al . , 1996; Leister et al . , 1998) are not useful for identification of these Rx variants because they do not discriminate between the Rx variants and homologues of other resistance gene homologues in the genome.
  • a further aspect of the present invention provides a method of identifying and/or cloning homologous Rx variants from a plant, which method employs all or part of a nucleotide sequences as described above.
  • nucleotide sequence information provided herein may be used in a data-base (e.g. of ESTs, or STSs, or other genomic sequence information) search to find homologous sequences, expression products of which can be tested for pathogen resistance activity e.g. using methods based on the transient assays of the present invention, or conventional phenotype assays in transgenic plants.
  • a data-base e.g. of ESTs, or STSs, or other genomic sequence information
  • probes based on the sequence may be used e.g. in southern blotting.
  • DNA may be extracted from cells taken from plants displaying the appropriate resistance trait and digested with different restriction enzymes. Restriction fragments may then be separated (e.g. by electrophoresis on an agarose gel) before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined.
  • Preliminary experiments may be performed by hybridising under low stringency conditions.
  • 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.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes - 1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
  • T ra 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • the T ra is 57°C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.
  • suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0.1X SSC, 0.1% SDS.
  • suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0.1X SSC, 0.1% SDS.
  • Binding of a probe to target nucleic acid may be measured using any of a variety of techniques at the disposal of those skilled in the art.
  • probes may be radioactively, fluorescently or enzymatically labelled.
  • Other methods not employing labelling of probe include amplification using PCR (including, where appropriate, RACE PCR), RN'ase protection and allele specific oligonucleotide probing.
  • the identification of successful hybridisation is followed by isolation of the nucleic acid which has hybridised, which may involve one or more steps of PCR or amplification by cloning in a vector that replicates in a suitable host.
  • clones e.g. lambda, cosmid, plasmid, BACs, biBACS
  • fragments identified in the search can be extended or supplemented.
  • the original DNA source e.g. a clone library, mRNA preparation etc.
  • the original DNA source e.g. a clone library, mRNA preparation etc.
  • sequences, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence.
  • the genes are then tested for functionality as described above.
  • one scheme for isolating Rx homologues is as follows :
  • Resistance gene coding activity is then tested as described above. A variation on this procedure would employ bulked segregants in an intermediate stage, as described previously (Michelmore et al . , 1991).
  • Rx-genes may be linked in clusters.
  • Clustering of R-genes in potato has already been reported (Leister et al . 1996; De Jong et al . 1997) .
  • One of the large R-gene clusters is on the short arm of potato chromosome 5.
  • This cluster comprises of at least four R loci : Rl conferring resistance to Phytophthora infestans, Nb conferring HR type resistance to potato virus X (PVX) and Gpa and Grpl conferring resistance to the potato cyst nematode.
  • Rx-homologues a number of the genes and D ⁇ A sequences linked to Rx represent Rx-homologues . It is likely that one or more of these homologues are R genes against viruses, fungi, bacteria or nematodes . For instance, Gpa2 (which bestows a specific resistance response to a small set of populations of the potato cyst nematode Globodera pallida) is believed to be tightly linked to the Rx locus on chromosome 12.
  • the S. acaule Rx-homologue is likely to be on chromosome V linked to Rx2 and, using the agrobacterium infiltration assay, has been shown to function as a resistance gene against PVX. This result shows that the sequence of Rx can be used to isolate other resistance genes without reference to the conserved motifs that have been used previously to define resistance gene homologues (Leister et al . , 1996).
  • Rx variants providing different R traits
  • D ⁇ A used for the initial amplification step is taken from members of the population in which the required R trait co-segregates with Rx itself (or a previously identified Rx variant) .
  • Rx is similar to the sequence of the otherwise unrelated Rps2 that confers resistance in Arabidopsis against a bacterial pathogen.
  • sequence of Rx could be modified e.g. by site-directed or random mutation, to produce Rx mutants or other derivatives which can confer resistance against (i.e. is switched on by) pathogens that are quite different from PVX. This can be achieved as described below, with Rx mutants being tested with the transient expression assay methods described above .
  • the nucleic acid molecule which is the mutant or other derivative is generated either directly or indirectly (e.g. via one or amplification or replication steps) from an original nucleic acid corresponding to all or part of the sequence shown in Seq ID No 1 or other sequences disclosed herein.
  • a further aspect of the present invention is a method of producing a nucleic acid encoding an Rx derivative comprising the step of modifying a nucleic acid encoding Rx.
  • the derivative may include changes to the nucleic acid which make no difference to the encoded amino acid sequence (i.e. 'degeneratively equivalent') .
  • 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 substitution of one or more amino acids in the encoded polypeptide.
  • a variant nucleic acid may encode an amino acid sequence including additional amino acids at the C-terminus and/or N- terminus .
  • parts or fragments (however produced) corresponding to portions of the sequences provided, and which encode polypeptides having biological activity - for instance pathogen resistance or the ability to raise Rx-binding antibodies .
  • changes may be desirable for a number of reasons, including introducing or removing the following features: restriction endonuclease sequences; codon usage; other sites which are required for post translation modification; cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide for glycosylation, lipoylation etc.
  • Leader or other targeting sequences may be added to the expressed protein to determine its location following expression. All of these may assist in efficiently cloning and expressing an active polypeptide in recombinant form (as described below) .
  • Other desirable mutation may be random or site directed mutagenesis in order to alter the activity (e.g. specificity) or stability of the encoded polypeptide.
  • homology at the amino acid level is determined in terms of amino acid similarity or identity. Similarity allows for conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine .
  • substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another
  • one polar residue for another such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine .
  • altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which
  • homologues having non-conservative substitutions are also included.
  • substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide ' s three dimensional structure.
  • regions which are critical in determining the peptides conformation or activity such changes may alter the properties of the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity, in particular broader specificity. Mutants having these properties can then be selected as described above .
  • Rx sequences may include mixing or incorporating sequences from related resistance genes into the Rx sequence.
  • restriction enzyme fragments of Rx could be ligated together with fragments of an Rx homologue or even of an unrelated gene to generate recombinant versions of Rx.
  • An alternative strategy for modifying Rx would employ PCR as described above (Ho et al . , 1989) or DNA shuffling (Crameri et al. , 1998) .
  • the methods of the invention may include hybridisation of one or more (e.g. two) probes or primers based on the Rx sequence either to screen for Rx homologues or to produce Rx derivatives.
  • probes or primers form a further part of the present invention.
  • An oligonucleotide for use in probing or PCR may be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24) .
  • Generally specific primers are upwards of 14 nucleotides in length.
  • primers of 16-24 nucleotides in length may be preferred.
  • probing can be done with entire restriction fragments of the gene disclosed herein which may be 100' s or even 1000 's of nucleotides in length.
  • the nucleic acid described above is in the form of a recombinant and preferably replicable vector.
  • Vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrojbacteriu_ ⁇ binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication) .
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eucaryotic (e.g. higher plant, mammalian, yeast or fungal cells) .
  • a vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome .
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell.
  • a host cell such as a microbial, e.g. bacterial, or plant cell.
  • the vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA) .
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • this aspect of the invention provides a gene construct, preferably a replicable vector, comprising a promoter operatively linked to a nucleotide sequence provided by the present invention, such as the Rx gene, or a variant (e.g mutant, derivative or allele) thereof.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • a gene construct preferably a replicable vector, comprising an inducible promoter operatively linked to a nucleotide sequence provided by the present invention.
  • inducible as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on” or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic.
  • an inducible (or “switchable” ) promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero) .
  • expression is increased (or switched on) to a level which brings about the desired phenotype .
  • Plant vectors Particularly of interest in the present context are plant vectors. Specific procedures and vectors previously used with wide success upon plants are described by Bevan (Nucl . Acids Res. 12, 8711-8721 (1984)) and Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148) .
  • Rx Since the natural in vivo activation of Rx is believed to be post-transcriptional , it may be preferred that a constitutive promoter is used.
  • Suitable promoters which operate in plants include the Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al, 1990a and 1990b); the cauliflower meri 5 promoter that is expressed in the vegetative apical meristem as well as several well localised positions in the plant body, e.g. inner phloem, flower primordia, branching points in root and shoot (Medford, 1992; Medford et al , 1991) and the Arabidopsis thaliana LEAFY promoter that is expressed very early in flower development (Weigel et al , 1992) .
  • Other promoters include the rice actin promoter.
  • an inducible promoter may be used.
  • the GST-II -27 gene promoter which has been shown to be induced by certain chemical compounds which can be applied to growing plants.
  • the promoter is functional in both monocotyledons and dicotyledons. It can therefore be used to control gene expression in a variety of genetically modified plants, including field crops such as canola, sunflower, tobacco, sugarbeet, cotton; cereals such as wheat, barley, rice, maize, sorghum; fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, and melons; and vegetables such as carrot, lettuce, cabbage and onion.
  • field crops such as canola, sunflower, tobacco, sugarbeet, cotton
  • cereals such as wheat, barley, rice, maize, sorghum
  • fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, and melons
  • vegetables such as carrot, lettuce, cabbage and onion.
  • the GST-II-27 promoter is also suitable for use in a variety of tissues, including roots, leaves, stems and reproductive tissues.
  • Other promoters include the patatin promoter (tubers) , ubiquitin promoter (wheat embryos) .
  • the promoter may include one or more sequence motifs or elements conferring developmental and/or tissue-specific regulatory control of expression.
  • vectors of the present invention may include the Rx gene or a variant thereof, in addition to various sequences required to give them replicative, integrative and/or expression functionality.
  • Such vectors can be used, for instance, to make plants into which they are introduced resistant to PVX or other viruses.
  • PVX CP post-transcriptionally silenced elicitor
  • pathogens which suppress gene silencing which may, for instance be PVY or related viruses - see Pruss et al . , 1997) the PVX CP would be unsilenced in the region infiltrated by PVY, and the HR would be limited to that region.
  • the present invention also provides methods comprising introduction of the Rx constructs discussed above (such as vectors) into a host cell and/or induction of expression of a construct within a plant cell, by application of a suitable stimulus, an effective exogenous inducer.
  • the vectors described above may be introduced into hosts by any appropriate method e.g. conjugation, mobilisation, transformation, transfection, transduction or electroporation, as described in further detail below.
  • a host cell containing nucleic acid or a vector according to the present invention, especially a plant or a microbial cell.
  • 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
  • Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (Toriyama, et al . (1988) Bio/Technology 6, 1072-1074; Zhang, et al . (1988) Plant Cell Rep . 1 , 379-384; Zhang, et al . (1988) Theor Appl Genet 76, 835-840; Shimamoto, et al . (1989) Nature 338, 274- 276; Datta, et al . (1990) Bio/Technology 8, 736-740; Christou, et al .
  • Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective.
  • 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 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, gentamycin, spectinomycin, imidazolinones and glyphosate .
  • a further aspect of the present invention provides a method of transforming a plant cell involving introduction of a vector comprising a nucleic acid of the present invention (e.g. Rx or Rx variant) into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome .
  • a nucleic acid of the present invention e.g. Rx or Rx variant
  • the invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention, especially a plant or a microbial cell.
  • the transgenic plant cell i.e. transgenic for the nucleic acid in question
  • the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome.
  • heterologous is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, i.e. by human intervention.
  • a heterologous gene may be additional to a corresponding endogenous gene.
  • Nucleic acid heterologous, or exogenous or foreign, to a plant cell may be non-naturally occurring in cells of that type, variety or species.
  • the heterologous nucleic acid may comprise a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant .
  • a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al . , Cell Culture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.
  • Plants which include a plant cell according to the invention are also provided, along with any part or propagule thereof, seed, selfed or hybrid progeny and descendants.
  • a plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders' Rights. It is noted that a plant need not be considered a "plant variety” simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.
  • the present invention embraces all of the following: a clone of such a plant, seed, selfed or hybrid progeny and descendants (e.g. FI and F2 descendants) and any part of any of these, such as cuttings, seed.
  • the invention also provides a plant propagule from such a plant, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on.
  • the sequences disclosed herein may be used to facilitate selection of plants into which it is desired to introduce the resistance trait using conventional plant breeding methods. Progeny from crosses which carry the gene may be readily identified by screening on the basis of the Rx sequence, particularly the Rx signature sequence.
  • the methods disclosed herein for identifying proximal markers to the Rx locus may be generally applicable to other genes found in clusters (e.g. plant derived resistance genes) . Such methods are characterised in that they employ a step using low stringency PCR with non-degenerate primers which avoid conserved sequence motifs.
  • the general approach may be summarised as follows :
  • the present invention also encompasses the expression product of any of the Rx or variant nucleic acid sequences disclosed above, and methods of making the expression product by expression from encoding nucleic acid therefor under suitable conditions, which may be in suitable host cells.
  • Antibodies may be raised to a purified Rx/variant polypeptide or peptide by any method known in the art (for an overview see e.g. "Immunology - 5th Edition” by Roitt, Brostoff, Male: Pub 1998 - Mosby Press, London).
  • Such antibodies, or fragments or derivatives thereof can be used to bind Rx, or in the identification and/or isolation of proteins homologous to Rx (i.e. which share epitopes therewith) , which in turn can provide the basis of an alternative method to those described above to isolate their encoding genes .
  • the invention further provides a method of influencing or affecting a resistance trait (preferably ER trait) in a plant, whereby the method includes the step of causing or allowing expression of a heterologous nucleic acid sequence as discussed above (e.g. Rx or Rx variant, in each case plus an optional elicitor) within cells of the plant.
  • a resistance trait preferably ER trait
  • Rx activity it may be desirable to down-regulate Rx activity. This may be achieved, for instance used antisense technology (which is reviewed in Bourque, (1995) , Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496) .
  • An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same,_orientation as the target gene, to achieve reduction in expression of the target gene by co- suppression. See, for example, van der Krol et al . , (1990) The Plant Cell 2, 291-299; Napoli et al . , (1990) The Plant Cell 2 , 279-289; Zhang et al . , (1992) The Plant Cell 4, 1575-1588, and US-A-5 , 231 , 020.
  • the invention provides a method which includes expressing Seq ID No 3 or a variant thereof within the 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
  • an Rx-mediated resistance ER or HR
  • an appropriate viral elicitor or other initiator or inducer elicitor or other initiator or inducer.
  • the elicitor or other trigger may be encoded directly by the invading virus (such as the coat proteins of PVX or certain other Potex- and Carlaviruses) .
  • the invading virus such as the coat proteins of PVX or certain other Potex- and Carlaviruses
  • it may be expressed by a separate construct or transgene which is itself triggered or upregulated by the viral infection.
  • modification of the Rx (variant) sequence may allow triggering by a non-natural elicitor, if this is preferred.
  • the expression of the elicitor is decoupled from that of the Rx e.g. by use of a strong, constitutive promoter .
  • Annex I shows the sequence derived from BAC77 (designated Seq ID No 1) and containing the Rx gene. Putative initiation codon (ATG) of Rx gene is given in bold and underlined. Two introns in the 3 ' -end of the Rx gene are underlined.
  • Annex II shows a partial nucleotide sequence of the Rx cDNA including the Rx-coding nucleic acid sequence (designated Seq ID No 3) .
  • Fig 1 shows the putative Rx polypeptide sequence (designated Seq ID No 2) . Regions corresponding to characterised domains are given in bold and underlined. conserveed aliphatic residues (L, V, I, F, M) in the LRR region are underlined. Two duplicated acidic fragments in the C-terminus of Rx are indicated by arrows.
  • Fig 2 shows a sequence alignment between Rx and products of four R genes encoding proteins containing putative leucine zipper/nucleotide binding site/leucine-rich (LZ-NBS-LRR) repeat motifs.
  • Rx - Rx protein of the present invention PRF fragment - C-terminal fragment (residues 841 - the end) of the Lycopersicon esculentum Prf protein (SPTREMBL accession Q96485) ; RPS2 - Arabidopsis thaliana Rps2 protein (PIR accession A54809) ; RPM1 - A. thaliana Rpml protein (PIR accession A57072) ; I2C-1 - L. esculentum resistance complex I2C-1 protein (SPTREMBL accession 024015; Ori et al (1997) Plant Cell, 9: 521-532.).
  • Fig 3 High resolution map of the Rx locus based on the Experiments disclosed herein (not drawn to scale) .
  • the top panel shows a simplified genetic map of potato chromosome XII (denoted by a horizontal line) in which the area left of the arrow is reversed in the potato and tomato genetic maps (Tanksley et al . , 1992).
  • Vertical lines indicate positions of previously mapped RFLP markers
  • the middle panel shows the genetic map of the GP34—218L interval (denoted by a horizontal line) .
  • Positions of the RFLP marker GP34 and the AFLP markers IPM3, IPM4-a and IPM5 are indicated by vertical lines.
  • the positions of BAC end- derived markers are indicated by arrows.
  • the positions of markers enclosed in parentheses (e.g. 45L-b) have not been determined precisely.
  • the numbers in square brackets indicate the numbers of SI Cara individuals containing recombination events in each marker interval .
  • the predicted position of Rx is indicated by v « ' symbol.
  • the bottom panel shows positions of BAC clones in GP34—218L interval.
  • Each open rectangle represents one BAC insert DNA.
  • the estimated insert size in kb except for the BAC29
  • ⁇ R' denoting the BAC ends that had been mapped relative to Rx.
  • Fig 4 Alignments of putative peptide sequences derived from Rx-linked markers.
  • Fig 5 Introgressed DNA in the vicinity of Rx.
  • the diagrams show chromosomal DNA in the IPM3—IPM5 interval from potato cv Cara and clone SH83 and from individuals in our SI Cara mapping population (identified in left hand column) .
  • These plants were either Rx (R) or rx (S) genotypes and the chromosomal region introgressed from S . tuberosum spp. andigena is shown as a close rectangle.
  • the S. tuberosum spp. tuJberosum DNA is shown as a thin line.
  • DNA markers used to map the introgressed DNA are identified over the vertical lines, and the region containing Rx is delineated by the horizontal arrow.
  • Fig 6 shows regions of the BAC77 clone which were used in transient assay experiments (A) and also a construct (PVX- TK) used in the experiments (B) .
  • Fig 7 shows a sequence alignment between Rx (from S . andigena) , Rx2 (from S . acaule) and other Rx homologues.
  • Rxl indicates sequence from Rx from S. andigena.
  • Acl5 and Ac64 indicate two sequences from two independent colonies carrying Rx2 from S . acaule .
  • lllhl and 221h2 indicate two Rx homologous sequences from BAC111 and BAC221 respectively.
  • K39.hom indicate an Rx homologous sequence from the natural potato hybrid S.
  • X juzepczukii carrying Rx juz (A) is a DNA sequence alignment
  • (B) is a protein sequence alignment.
  • a BAC library of 160000 clones from plant SC-781 which is a progeny of selfed cv Cara carrying Rx in the duplex condition (Rx, Rx, rx, rx) , was prepared. Based on the average insert size of 100 kb in the BAC clones (data not shown) , a haploid genome size of 900000 kb in potato (Arumuganathan and Early, 1991) and taking into account the presence of Rx in the duplex condition we estimated that there was more than a 99% probability that this library carried the DNA of the Rx (or any other) locus.
  • N Ln (1-P)/Ln (1-l/G) , where I is the size of the average cloned fragment in base pairs, and G is the size of the genome, in base pairs (Clark and Carbon, 1976) .
  • I the size of the average cloned fragment in base pairs
  • G the size of the genome, in base pairs (Clark and Carbon, 1976) .
  • BAC117, BAC191, Rx The 191L marker was separated from Rx by only a single chromosomal recombination event (in plant #1146) in a mapping population of 1720 plants. In the same population, 117L and IPM3 markers were separated from Rx by two and three recombination events respectively whereas the GP34 marker, present in BAC167, was separated from Rx by thirteen recombinations ( Figure 3) . The BAC library did not contain additional BACs extending further towards Rx from the 191L marker.
  • the IPM4 marker was previously mapped at 0.06 cM from Rx on the side away from IPM3 (Bendahmane et al . , 1997). Screening of the BAC library with IPM4 identified two clones: BAC73 and BAClll, with inserts of -70 kb each. The Tagl digestion of the IPM4 CAPS marker in these clones suggested that BAClll was linked in cis to the Rx locus whereas BAC73 carries DNA insert from the rx chromosome (data not shown) .
  • markers are allele-specific (i.e. marker primers are able to amplify only an allele linked in cis to Rx) . These markers do not require restriction enzyme digestion.
  • BACs containing IPM5 would allow us to orientate the 111L and 73L markers relative to Rx ( Figure 3) .
  • These analyses identified BAC218, carrying an allele of IPM5 identified by Pstl digestion as being linked in cis to Rx (data not shown) .
  • the end sequences of BAC218 insert DNA were converted into the CAPS markers, 218L and 218R, and mapped genetically to the recombination events between GP34 and IPM5. Marker 218L was positioned at 8 recombination events (0.48 cM) from Rx, between IPM5 and CT129.
  • the 218R marker was positioned between IPM4 and IPM5 at five recombination events (0.30 cM) from Rx.
  • CAPS analysis revealed that each of these markers in the BAC pool #29 is represented by the allele linked in cis to Rx (data not shown) .
  • BAC pool #29 contains a single BAC clone, BAC29, with DNA insert linked in cis to Rx. Therefore, BAC29 provided a link between BAC218 and the IPM4 BAC contig and allowed us to orientate the markers from the IPM4 contig in the following order: Rx, 111L, IPM4, 73L ( Figure 3).
  • BAC221 which carries an insert DNA of 50 kb and is linked in cis to Rx (data not shown) .
  • the left end sequence of BAC221 is located inside of BAClll whereas the right end sequence of BAC221 extends further towards Rx ( Figure 3) .
  • the marker 221R co-segregated with IPM4 ( Figure 3) in our mapping population and was separated from Rx by the recombination event in plant #761.
  • BAC45 which has an insert DNA of 50 kb and is linked in cis to Rx (data not shown) .
  • the right end sequence of BAC45 is located inside of BAC221, whereas the left end sequence of BAC45, 45L, extends further towards Rx ( Figure 3) .
  • BAC45 does not contain Rx as the CAPS marker 45L is genetically separated from Rx by the recombination event in plant #761. Additional PCR screening of the BAC library with the 45L marker failed to identify any new BAC clones therefore leaving a gap between the IPM3 and IPM4 BAC contigs ( Figure 3) .
  • the low stringency PCR products obtained with 45L primers and digested with Ddel produced two new fragments from the R pool DNA template in addition to those produced from the S pool and from BAC45. These fragments were also obtained after Ddel digestion of the BAClll-derived PCR product (data not shown) .
  • the previously identified 45L marker is referred to as 45L-a and the newly identified locus in BAClll as 45L- b ( Figure 3) .
  • the Cos98 primers were based on a sequence in the BAC221 insert DNA (A.B., K.K. and D.C.B., unpublished data).
  • IPM4-a IPM4 locus
  • IPM4 restriction fragments that had not previously been detected (data not shown) .
  • One of these fragments was nonpolymorphic in the R and S pools. This fragment must have originated from BAC221 as the Tagl restriction fragment of similar size was also detectable after digestion of IPM4-b allele derived from this BAC (data not shown) .
  • second new DNA fragment was polymorphic between R and S pools and was not detected after digestion of either IMP4-a or IPM4-b alleles derived from BAClll and
  • the potato BAC library was screened by PCR with IPM4 primers using conditions for detection of the IPM4-C allele.
  • One new BAC clone with DNA insert of -50 kb, BAC77, carrying the IPM4-C locus was identified (data not shown) .
  • the end fragments of BAC77 DNA insert were cloned, sequenced and converted into the CAPS markers, 77L and 77R.
  • the marker 77L co-segregated with both IPM4-C and Rx whereas 77R was separated from Rx by one recombination event in the recombinant individual #761 (based on analysis of 1720 segregants; see Figure 3 and Figure 5) .
  • the BAC77 DNA was combined in a single plasmid, with a PVX-GUS cDNA, under the control of a CaMV 35S promoter.
  • an avirulent (PVX-TK) or a virulent (PVX- KR) strain of PVX under the control of 35S promoter was cloned into Srfl unique site of BAC77 to obtain BAC77 (PVX- TK) and BAC77 (PVX-KR) respectively.
  • To monitor virus replication the GUS gene was expressed from duplicated PVX coat protein promoter - expression of GUS requires efficient accumulation of PVX-GUS in infected cells.
  • a mutation within the PVX ORF encoding the 25-kD movement protein ensured that GUS expression would only report virus replication in cells also expressing the candidate Rx.
  • BAC77 regions used in the assays, and an Example construct (PVX-TK) are shown in Fig 6(A) and (B) respectively.
  • the BAC77/PVX-GUS plasmid was introduced into tobacco cells and susceptible potato cells by biolistic transformation using coated gold particles. Results showed that there were approximately ten fold more GUS+ cells if the PVX-GUS was based on an Rx-breaking strain of PVX-KR than with a common, Rx-avirulent, strain of PVX-TK. This is consistent with activation of Rx resistance by expression of the avirulent
  • PVX-TK virus leading to suppression of the PVX-GUS activity.
  • the result confirmed the gene-for-gene relationship between BAC77 and virulent inducer (PVX-KR) .
  • Example 3 - Rx is active in transient expression studies in plants which are not PVX hosts
  • Rx/PVX-GUS constructs were assembled in which Rx was under control of the 35S promoter. These constructs were bombarded into leaves of Arabidopsis plants. As with the experiments described above there were approximately ten fold more GUS+ cells if the PVX-GUS was based on an Rx-breaking strain of PVX than with a common strain of PVX. This result shows that the cloned Rx was active in Arabidopsis which is not a normal host of PVX.
  • Example 4 Transiently expressed Rx can suppress PVX
  • N. tabacum, N. benthamiana, L . esculentum and potato cells using Agrobacterium-mediated transformation were transferred into a binary Ti plasmid vector in Agrobacterium tu efaciens .
  • the Agrobacterium cells were then infiltrated into leaves of the test plants.
  • the Ti plasmid constructs had different regions of BAC77 and in some instances, as in the biolistic experiments, included PVX-GUS cDNA from common strain or Rx-breaking strain PVX.
  • PVX-GUS in these constructs was from a resistance breaking strain of PVX there was abundant GUS expression in all samples tested, indicating that the infiltration method was an efficient procedure for inoculation of PVX.
  • the plant transformation was carried out using SLJ7292 binary vector (Jones et al , 1992) .
  • the larger 18 kb FR9 Rx fragment was used in order to ensure that the sequence encoded all of the required regulatory sequence.
  • Infectivity assays were performed using mechanical inoculation of PVX strains. Seven of eight independent transgenic lines carrying the FR9 insert were resistant to Rx-avirulent PVX-TK and susceptible to Rx-virulent PVX-KR. This resistance was manifested as the absence of PVX-TK symptoms and complete lack of PVX-TK accumulation in inoculated and systemic leaves. This resistance reaction is phenotypically similar to that observed in the wild type potato cultivar CARA.
  • Example 6 The Rx transgene also confers resistance in heterologous plants species
  • Plants of 18 transgenic lines (six each of L. esculentum, N. taJbacum and N. jben .ha_ ⁇ uana) were rub inoculated with Rx- virulent and Rx-avirulent strains of PVX.
  • PVX-TK indicating that PVX could cross the graft union.
  • Rx cDNA under the control of the CaMV 35S constitutive promoter in N. taJbacum.
  • control we prepared a second construct in which Rx cD ⁇ A was expressed from its weak wild type promoter.
  • Rx constructs were introduced into binary pBinl9 plasmid vector (Bevan, 1984) and transformed into A . tumefaciens .
  • the A . tumefaciens cultures were then infiltrated into leaves of wild type N. taJbacum via agroinfiltration (see general methods below) .
  • the constructs also had regions of BAC77 but, instead of the PVX-GUS insert there was a 35S-PVX coat protein (CP) construct. This uncoupled the CP expression from PVX replication.
  • CP 35S-PVX coat protein
  • TMV-KR does not encode the elicitor of Rx-mediated resistance.
  • Transcripts of the TMVTK and TMV-KR cDNAs were initially inoculated onto N. benthamiana and sap extracts were produced from the inoculated leaves at 8 days after inoculation. Tobacco plants carrying the N gene either alone or in combination with Rx were inoculated with these extracts containing high concentration of virus, without dilution. After 3 to 4 days the TMV-KR and TMV-TK induced HR on N. taJbacum expressing the N gene. There was also an HR when TMV-KR was inoculated onto plants of the N, Rx- genotype. However, when these plants were inoculated with TMV-TK there was no HR.
  • Repeated DNA loci that do not get PCR-amplified from a genomic DNA template may get amplified from cloned DNA because the primer-complementary sequences are more abundant in the cloned DNA than in genomic DNA and because there is no potential for competition for the primers between similar loci.
  • Example 7 overexpression of the Rx gene led to cell death in susceptible species.
  • the modulation of the level of Rx expression may be used as a strategy to engineer disease resistance into plants.
  • overexpression of the Rx gene in susceptible species may be advantageous because:
  • Example 8 Our interpretation of the results in Example 8 is based on a "hair trigger" hypothesis.
  • PVX infected cells and in the experiments described in the Examples above, the production of PVX CP from the viral genome would have activated Rx-mediated resistance at an early stage in the infection cycle because Rx is so sensitive to the presence of the coat protein elicitor. Virus accumulation would have been arrested before CP accumulation had reached a high level.
  • Other work has confirmed that the induction of the resistance mechanism by CP does not require de novo transcription, and that the Rx-mediated resistance includes a mechanism that prevents growth or differentiation in cells.
  • Rx mediated resistance would have no effect on elicitor production in cells infiltrated with the 35S CP construct. There would be continuous, decoupled, activation of the Rx mediated resistance rather as if the "hair trigger" of a repeating gun was held in place with a rubber band. This continuous activation of the resistance mechanism would expose secondary resistance responses (eg HR induced necrosis) that would not normally be evident in resistant plants after inoculation owing to the effectiveness of the primary ER.
  • secondary resistance responses eg HR induced necrosis
  • Rx shares sequence similarity to many disease resistance genes, it is likely that variations of this model are relevant to other disease resistance interactions in plants .
  • Rx loci There are two Rx loci mapped in potato conferring strain- specific resistance to PVX (Cockerham, 1970) . These loci are on chromosome V (Rx2) and chromosome XII (Rxl) (Ritter et al, 1992; Bendahmane et al , 1997) but are functionally identical : both confer extreme resistance which is only overcome by the same natural or mutant isolates of PVX (Querci et al . , 1995) .
  • Rxl originates from S andigena and is the same as Rx in potato cultivar CARA.
  • Rx2 originates from S acaule. In this section we describe the isolation of Rx2 using primers based on Rx sequence (cloned from potato cultivar CARA) following the strategy described earlier. The basic scheme was as follows:
  • the Rx2 paralogue contains acidic and amide motifs in the C- terminal domains which are also present in the Rx gene and other paralogues on potato chromosome V as well as on chromosome XII.
  • the Rx2 paralogue is functionally identical to, but independent of, the Rx gene described in the Examples above.
  • Fig 7 also shows sequences lllhl and 221h2; these are two Rx homologous sequences from BAClll and BAC221 respectively.
  • Example 10 Modification of Rx to give broad specificity This can be achieved as follows.
  • a plasmid is assembled based on the Rx information disclosed herein, a pathogen-derived gene (selected in accordance with the desired specificity) and a reporter gene (GUS or GFP) .
  • the pathogen-derived gene may be the PVX CP . If the CP is from an avirulent isolate of PVX the Rx hair trigger will be activated leading to an HR. As a result of the HR there would be no GUS or GFP activity (the cells would be dead) . In contrast if the CP is from a virulent strain there would be no HR and there would be a high level of GUS or GFP. However, a mutant Rx would recognize the virulent CP and there would be an HR and loss of GFP or GUS. To identify a mutant Rx that can recognize the virulent CP, Rx is mutagenised using PCR. The mutant forms of Rx will be inserted into the transient expression plasmid and expressed in plant cells and those capable of recognising the virulent CP will be diagnosed by the absence of GUS or GFP.
  • mutant forms of Rx are tested in an analogous manner for the ability to recognize proteins or molecules that are completely unrelated to the PVX CP.
  • Rx-mediated resistance could be activated by any agent, for example a potyvirus, that is able to suppress post transcriptional gene silencing (Pruss et al . , 1997).
  • a potyvirus that is able to suppress post transcriptional gene silencing
  • Narcissus mosaic virus (NMV) , Nandina virus X (NVX) , Viola mosaic virus (VMV) , Cymbidium mosaic virus (CyMV) , Poplar mosaic virus (PopMV) and White clover mosaic virus (WCIMV) were able to elicit Rx-mediated resistance. In these tests there were extreme resistance against NMV, NVX and WCIMV and HR type resistance against VMV. Against CyMV and PopMV there was a weak elicitation of Rx-mediated resistance. These weak elicitation was not able to block the systemic mouvment of CyMV and PoMV. Their was no resistance against Foxtaim mosaic virus (FoMV) .
  • FI seeds from the cross between potato cv Cara (Rx, rx, rx, rx) and cv Huinkel (rx, rx, rx, rx) were obtained from the Plant Breeding Institute, Cambridge.
  • the progeny of this cross (370 individuals) were used initial experiments (Bendahmane et al . , 1997) to identify the chromosomal position of Rx, and to assign RFLP markers linked to Rx.
  • the selfed (SI) progeny of cv Cara (1350 plants) were used as a mapping population to position Rx with respect to RFLP, AFLP and BAC-derived PCR markers.
  • High molecular weight DNA was prepared in agarose plugs from potato protoplasts essentially as described in Bendahmane et al . (1997) .
  • the agarose plugs were dialysed three times for 30 min against TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) , once at room temperature and twice at 4°C.
  • the plugs were then equilibrated in HindiII buffer (10 mM Tris-HCl, 10 mM MgCl 2 , 50 mM NaCl , 1 mM DTT, pH 7.9) twice on ice for 1 h.
  • the size selected potato DNA (25-50 ng) was ligated to 25-50 ng of Hindlll -digested and dephosphorylated pBeloBACll vector kindly provided by Dr H.Shizuya (University of Southern California, Los Angeles; Shizuya et al . , 1992) using 400 to 800 units of T4 DNA LIGASE (New England BioLabs, USA) at 16°C for 24 hours in a total volume of 50 ⁇ l .
  • the ligation products were dialysed against 1 X TE using 0.025 ⁇ M MF-MILLIPORE MEMBRANE FILTER (Millipore, UK) at 4°C for 2 h and 30 min at room temperature using the "drop dialysis" method of Maruzyk and Sergeant (1980) .
  • Transformation of E. coli DH10B cells was carried out by electroporation using a BRL CELL-PORATOR SYSTEM (Life Technologies Ltd, UK) . To 20 ⁇ l of electro-competent cells, 0.5-3 ⁇ l of ligation mixture was added. After electroporation, E. coli cells were mixed with 1 ml SOC medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl , 2.5 mM KC1, 10 mM MgCl 2 , 10 mM MgS0 4 , 20 mM glucose) and incubated at 37°C for 1 h with gentle shaking (80 rpm) .
  • SOC medium 0.5% yeast extract, 2% tryptone, 10 mM NaCl , 2.5 mM KC1, 10 mM MgCl 2 , 10 mM MgS0 4 , 20 mM glucose
  • the cells were spread on Luria broth (LB) agar plates containing chloramphenicol (12.5 ⁇ g/ml), 5-bromo-4-chloro-3-indolyl- ⁇ - D-galactoside (Xgal) (40 ⁇ g/ml) and isopropyl-1-thio- ⁇ -D- galactoside (IPTG) (0.12 mg/ml) . Plates were incubated at 37°C for 24 hours.
  • LB Luria broth
  • Xgal 5-bromo-4-chloro-3-indolyl- ⁇ - D-galactoside
  • IPTG isopropyl-1-thio- ⁇ -D- galactoside
  • LB freezing buffer 36 mM K 2 HP0 4 , 13.2 mM KH 2 P0 4 , 1.7 mM citrate, 0.4 mM MgS0 4 , 6.8 mM (NH 4
  • the potato BAC library was screened with IPM3, IPM4 and IPM5 CAPS markers corresponding to the AFLP markers PM3 , PM4 and PM5 flanking the Rx locus (Bendahmane et al . , 1997).
  • the IPM4F primer turned out to correspond to a portion of the C terminal part of the protein in a non-conserved part of the leucine rich repeat (bases 2522-2545 on Figure 3) .
  • the IMP4R primer corresponded to a region immediately downstream of the stop codon (base 2875 on Figure 3) , extending into the intron immediately 5' of that codon.
  • the protocol was as follows .
  • the plasmid DNA was isolated using the standard alkaline lysis protocol (Hilor et al . , 1997) from pooled bacteria of each plate to produce 240 plate pools. Aliquots of these plate pools were combined to prepare 26 'superpools' each of which contains DNA from 9 plate pools, and one superpool containing DNA from 6 plate pools. To identify individual BAC clones carrying the CAPS markers the superpools and then the corresponding plate pools were screened. Once an individual plate had been identified the clones corresponding to each of the 24 columns of the positive plate were grown for 3-4 h at 37°C in LB medium and PCR was carried out on 3 ⁇ l of bacteria. After identification of a positive column a colony PCR on each of the corresponding 16 colonies of this column was carried out leading to identification of a single positive BAC clone.
  • the second part of the library was stored as one hundred pools of approximately 1000 clones.
  • the plasmid DNA was isolated from each pool of clones using standard alkaline lysis protocol and PCR was carried out to identify the positive pool.
  • the bacteria corresponding to the positive pool were diluted, plated on LB agar plates and then colony hybridisation was carried out as described in Sambrook et al . (1989) using 32 P-labelled DNA probes corresponding to CAPS markers .
  • PCR with the corresponding CAPS primers was used to distinguish hybridising colonies carrying the markers that had been previously mapped from the homologues located elsewhere in the genome.
  • BACs containing potato DNA were isolated from 5 ml overnight cultures (LB medium supplemented with 12.5 mg/ml chloramphenicol) using the standard alkaline lysis miniprep protocol (Engebrecht et al . , 1997) and resuspended in 50 ⁇ l TE. Plasmid DNA (10 ⁇ l) was digested with Notl for 3 h at 37°C to free the genomic D ⁇ A from the pBeloBACll vector.
  • the digested D ⁇ A was separated by CHEF electrophoresis in a 1% agarose gel in 0.5 X TBE at 4°C using a BIO-RAD CHEF DR II system (Bio-Rad Laboratories, USA) at 150 volts with a constant pulse time of 14 sec for 16 h.
  • IPCR Inverse polymerase chain reaction
  • BAC DNA was isolated and digested separately with Nlalll , Hpall, ⁇ fsel , HinPlI, PvuII, Haelll (for isolation of a left end sequence) or with Rsal , Sacl , EcoRI , Haelll, Maell, Msel , PvuII, HinPlI (for isolation of a right end sequence) for 4 h at 37°C and recircularised by self ligation for 2 h at 20°C.
  • Ligations were carried out using 5-50 ng of digested D ⁇ A and 5-10 units of T4 D ⁇ A LIGASE (Boehringer Mannheim, Germany) in a total volume of 100 ⁇ l .
  • PCR amplification of the recircularised D ⁇ A was carried out using 3 ⁇ l of self-ligated D ⁇ A as the template.
  • AB1 (5'- CCTAAATAGCTTGGCGTAATCATG-3' ) and AB2 (5'- TGACACTATAGAATACTCAAGCTT-3' ) primers were used for PCR amplification of the left end sequence of insert D ⁇ A
  • AB3 (5 ' -CGACCTGCAGGCATGCAAGCTT-3 '
  • AB4 (5 ' - -
  • ACTCTAGAGGATCCCCGGGTAC-3' primers were used for PCR amplification of the right end sequence of insert D ⁇ A.
  • PCR conditions were as follows: 94°C for 15 sec, 60°C for 15 sec, 72 °C for 90 sec - for 35 cycles.
  • PCR products were digested simultaneously with Hindlll and the restriction enzyme used in the preparation of IPCR D ⁇ A template.
  • the released insert ends were gel purified and cloned into pGEM- 3Z(f+) (Promega, USA).
  • Sequences of the clones containing -1-2 kb inserts were determined using a 377 or 373 D ⁇ A SEQUENCING SYSTEM (Applied Biosystems, UK) , and these sequences were used to design PCR primers for amplification of the BAC end region in potato genomic DNA.
  • the sequences of primers and PCR conditions used in the amplification of each BAC end sequence are summarised in Table 1.
  • the BAC end sequences were named according to the name of the BAC, followed by the letter L for the left end or by the letter R for the right end sequence.
  • the left and the right end sequences of BAC218 are 218L and 218R, respectively.
  • PVX-TK and PVX-KR constructs are cD ⁇ As of potato virus X (PVX) isolate CP4.
  • PVX-TK has codons ACA (specifying T) and MA (specifying K) at positions 121 and 127 of the coat protein (CP) gene and is the same as wild-type CP4.
  • the construct PVX-KR has MA (K) and AGG (R) at the coat protein codons 121 and 127, respectively. These constructs were described previously (Goulden et al . , 1993).
  • the tobacco mosaic virus (TMV) constructs were derived from the TMV vector (TMV/odontoglossum ringspot virus; construct TB2) described previously (Donson et al . , 1991).
  • the TMV-TK and TMV-KR were made by cloning PVX CP from PVX-TK and PVX-KR, respectively, in the Xhol site of TB2. Schematic structure of these constructs is described in Fig 6.
  • the TMV-GFP was made by cloning the green fluorescent protein (GFP) open reading frame in the Xhol site of TB2.
  • PVX-TK/BAC77 and PVX-KR/BAC77 were made by digestion of the unique Srfl site in BAC77 and ligation with the cD ⁇ As of the Rx-avirulent or the fix-virulent strain of PVX expressed under the control of the cauliflower mosaic vinus (CaMV) 35S promoter. PVX in these constructs was defective in cell-to- cell movement and modified to express a 13-glucuronidase (GUS) reporter gene. The cell-to-cell movement defect was due to a sequence deletion between nucleotides 4588 and 4945 of the 25-kD protein open reading frame (Angel1 et al . , 1 996) .
  • PVX-TK/BAC77 and PVX- KR/BAC77 All the clones derived from PVX-TK/BAC77 and PVX- KR/BAC77 were designated PVX-TK/BAC* and PVX-KR/BAC* where the asterisks is used depending on the insert derivatives of BAC77 (see Figure 6) .
  • Bacterial artificial chromosome (BAC) plasmid DNAs were isolated using an alkaline lysis method (Leonardo and Sedivy, 1990) and purified on cesium chloride gradients (Sambrook et al . , 1989). One hundred micrograms of plasmid DNA was precipitated onto 10 mg of 0.95-lim diameter gold particles as described previously (McCabe et al . , 1988). Leaf discs from 6-week-old plants, placed onto Murashige and Skoog medium (Imperial, UK) containing 3% (w/v) sucrose were bombarded with the gold particles coated with plasmid DNAs as described previously (McCabe et al . , 1988). The leaf discs were incubated at room temperature in the dark for 48 hr and then stained for GUS activity as described previously (Angell and Baulcombe, 1997) .
  • BAC Bacterial artificial chromosome
  • the CP genes from the avirulent or the virulent strain of PVX were inserted between the 35S promoter and the transcriptional terminator of CaMV and transferred into the binary vector pBinl9 (Bevan, 1984) to create pBIN35S-TK and pBIN35S-KR, respectively.
  • These constructs were transformed into Agrobacterium strain C58C1 carrying the virulence helper plasmid pCH32 (Hamilton et al . , 1996).
  • pCH32 expresses VirG and VirE and was used to enhance T-DNA transfer.
  • Agrobacterium cells were inoculated into 5 mL L broth medium supplemented with 50 ⁇ g/mL kanamycin and 5 ⁇ g/mL tetracycline and grown at 28°C overnight.
  • L broth medium (50 mL) supplemented with 50 ⁇ g/mL kanamycin, 5 ⁇ g/mL tetracycline, 10 mM MES pH 5.6, and 20 ⁇ M acetosyringone was then inoculated with the 5-mL overnight cultures and grown at 28°C for 1 day.
  • Infectious viral RNA were electroporated into potato protoplasts and RNA gel blot analysis was used to measure the accumulation of viral RNA as described previously (Kohm et al., 1993) .
  • the probe used to detect PVX RNA was antisense RNA probe transcribed from the T7 promoter of pHB- RP plasmid linearized with Apal and was described previously (Kohm et al. , 1993) .
  • Graft Inoculation Graft inoculations involved the grafting of healthy scions to infected stocks.
  • seedlings of N. Jbenthamiana were sap inoculated with PVX strains.
  • the apex of a healthy plant to be tested was grafted onto the infected stock.
  • a diagonal cut was made in the stem of the third internode to insert the infected scion.
  • the 2 to 6 cm scions were given a V shape at the end that would be inserted into the cut on the stock plant to ensure good contact between vascular systems.
  • the graft was secured with Parafilm M (Sigma) and the whole plant was covered with a thin transparent plastic bag to avoid dehydration of the scion.
  • the bag was removed 7 days after grafting.
  • the presence of virus in the stock and the scion was determined 4 weeks after grafting by using R ⁇ A gel blot analysis. All graft inoculation experiments were repeated at least twice.
  • the Fspl-Pvull fragment of BAC9 (18,285 bp) was cloned into pSLJ7292 binary vector (Jones et al . , 1992) digested with Ecll3611 to create pSLJ9. This clone was introduced into Agrobacterium strain LBA4404. Transformation of N. Jbenthamiana and N. tabacum were carried out by Agrobacteriummediated leaf disc transformation (Horsch et al., 1985) . Transformation of potato cultivar Maris Bard was carried out as described previously (Gilbert et al . , 1998). The transformation of N.
  • benthamiana with the N disease resistance gene was carried out using the Agrobacterium strain AGLl which carries the binary vector pTG34, and was kindly provided by B. Baker (University of California, Berkeley, CA; Whitham et al . , 1994). 13 DNA sequencing and analysis
  • a shotgun cloning strategy was used for sequencing BAC9.
  • Three aliquots of 15 ⁇ g of CsCI purified DNA were sonicated for 30, 60 and 120 sec respectively by using Soniprep 150 (MSE, UK) .
  • DNA fragments in the range of 1-kb and 4-kb were gel purified, blunt ended by T4 DNA polymerase and cloned into pGEM3Zf+ (Promega) digested with Small Clones carrying potato insert DNA were selected by colony hybridization using insert DNA from BAC9 as probe.
  • the sequencing reactions were performed using a dye terminator cycle sequencing reaction kit (Perkin-Elmer) and M13 universal forward and reverse primers.
  • sequence reactions were resolved on ABI377 automated sequencer (Applied Biosystems ABI, La Jolla, CA) . Sequence contigs were assembled using UNIX versions of the Staden programs package (R. Staden, Medical Research Councel, Cambridge, UK). Homology searches were done using the BLAST software. The genomic DNA sequence has EMBL accession number AJ011801.
  • the 5 ' and 3 ' ends of the RxcDNA were determined by rapid amplification of cDNA ends (RACE) using the MARATHON cDNA amplification kit (Clontech Laboratories, Palo Alto, CA) . To obtain specific RACE products, two consecutive rounds of amplification (35 cycles each) were necessary. Conditions for the first round of polymerase chain reaction (PCR) were as recommended by the manufacturer. PCR samples of the first amplification round were diluted 1:10 with distilled water, and a 1-11L aliquot was used as a template for the second round of PCR (15 sec at 94°C, 15 sec at 65°C, and 1 min at 72 °C) . Two sets of oligonucleotides were used in combination with the adapter primers (API and AP2) of the kit: K3 (5'- ACTCGAGGTCCTTATACTATCATGG-3 ' ) and K4 (5'-
  • pBl binary vector is a modified pBIN19 binary vector that carries a transcription cassette having an Rx promoter and Rx terminator.
  • the Rx promoter was PCR-amplified using the primers RxP4 (TCG GGG TAC CTC TAT TGA AGA ATT GAG ATC CAA G) and RxP2 (CTC AGT ATC TAG ATG AAC AAA TTG CC) and the PCR product was digested with Xbal .
  • the Rx terminator was PCR- amplified with primers RxTl (CAG CTG TAA GCT CGT TGA TAT AGA GG) and RxT2 (GGT GTT CTA GAG ACT AGC CAG AGC TCT GAA AT) and the PCR product was digested with Xbal and Kpnl .
  • BAC77 DNA (Bendahmane et al 1999) was used as template for both PCR reactions.
  • the digested PCR products were ligated to a modified pBIN19 plasmid vector digested with Kpnl and Ecll36 to create pBl .
  • the modified pBIN19 plasmid is identical to the one published previously except the unique Xbal site was deleted.
  • tumefaciens strain C58C1 carrying the virulence helper plasmid pCH32 (Bendahmane et al 1999) . Independent colonies were agroinfiltrated into tobacco leaves that express transgenically PVX coat protein (Spillane et al 1997) and screened for elicitation of hypersensitive response. We screened 200 colonies and 6 led to HR. Two positive clones pBAC15 and pBAC65 were sequenced and were found to carry the same insert DNA. There was only one amino acid polymorphic between pBAC15 and pBAC65, which may well have been introduced by the PCR.
  • the Smal-Pvu II fragment of pBAC15 was cloned into pSLJ7292 binary vector (Jones et al . , 1992) digested with Eel 136II to create pSLJAC15. This clone was introduced into Agrobacterium tumefaciens strain LBA4404. Transformation of N. benthamiana and N. taJbacum were carried out by A . tumefaciens-mediated leaf disc transformation (Horsch et al . , 1985) . The transgenic plants were tested for resistance to virulent and avirulent strain of PVX by mechanical inoculation. Four independent transgenic lines of ⁇ . tabacum and two independent transgenic lines of ⁇ .
  • benthamiana carrying the Smal-Pvu II fragment of pBAC15 regenerated were resistant to Rx-avirulent PVX-TK and susceptible to virulent PVX-KR. This resistance was manifested as the absence of PVX-TK accumulation in the inoculated and systemic leaves.
  • pBIN35-Rx binary vector was carried out using pBIN61 binary vector (see 15 above) .
  • Rx cDNA was PCR amplified with the primers Rxl (GGC AAT TTG TTC ATC TAG ATA CTG AGA GA) and Rxac4 (TAT TTC AGA GCT CTG GCT AGT CCT CAG AAC ACC) .
  • the PCR product was digested with Xbal and ligated with pBIN61 digested with Xbal and Smal to create pBIN35-Rx.
  • pBl-Rx binary vector The Construction of pBl-Rx binary vector was carried out using pBl binary vector.
  • pBl binary vector is a modified pBIN19 binary vector that carry a tanscriprion cassette constituted of Rx promoter and Rx terminator.
  • RxP4 TCG GGG TAC CTC TAT TGA AGA ATT GAG ATC CAA G
  • RxP2 CTC AGT ATC TAG ATG AAC AAA TTG CC
  • the Rx terminator was PCR-amplified with primers RxTl (CAG CTG TAA GCT CGT TGA TAT AGA GG) and RxT2 (GGT GTT CTA GAG ACT AGC CAG AGC TCT GAA AT) and the PCR product was digested with Xbal and Kpnl.
  • BAC77 DNA (Bendahmane et al 1999) was used as template for both PCR.
  • the digested PCR products were ligated to a modified pBIN19 plasmid vector digested with
  • the modified pBIN19 plasmid is identical to the one published previuosely except the unique Xbal site was deleted.
  • Rx cDNA was PCR amplified with the primers Rxl (GGC AAT TTG TTC ATC TAG ATA CTG AGA GA) and Rxac4 (TAT TTC AGA GCT CTG GCT AGT CCT CAG AAC ACC) .
  • the PCR product was digested with Xbal and Sad and ligated with pBl digested with Xbal and Sad to create pBl-Rx.
  • pBIN35-Rx and pBl-Rx were transformed into A .
  • pCH32 expresses VirG and VirE and was used to enhance T-DNA transfer.
  • Agrobacterium cells were inoculated into 5 ml L-broth medium supplemented with 50 ⁇ g/ml kanamycin and 5 ⁇ g/ml tetracycline and grown at 28°C overnight.
  • the Cells were then precipitated and resuspended to a final concentration of 0.5 OD 600 in a solution containing 10 mM MgCl 2 , 10 mM MES pH 5.6 and 150 ⁇ M acetosyringone.
  • the cultures were incubated at room temperature for 2 hr before agroinfiltration into N. taJbacum leaves.
  • All the viruses used in this experiment were maintaned in wild type N. Jbenthamiana.
  • To carry out the resistance test for each virus, one leaf of an infected N. benthamiana plant was ground in a pestle and mortar in presence of 1 ml of 10 mM phosphate buffer and sand. The solution was centrifuged for 2 min at 2000 g and the supernatant was used as inoculum to infect Rx transgenic N. Jbenthamiana. As a control we also inoculated wild type N. Jbenthamiana. The infected plants were scored for resistance 3 weaks post-inoculation.
  • the plant were considered resistant if there were no symptoms and no virus accumulation in the inoculated and the systemic leaves of infected Rx transgenic plants and symptoms and systemic movement of the virus in the control plant (infected wild type N. Jbenthamiana) .
  • Annex I - Rx DNA sequence - nt 1-5820 - rows of 80
  • CATGAGATTCACTATGTGCA TCACCCACATCGTCTAAGTATTTTATGGCAATCAAGCCCTACAACTAGCTTCTTCTTTAT
  • GATCGAAAGAAGCTGTTCCC TCATCAATAATAGACATTCCTCTATCGATATCAAGCCTATGCTATCTGCAAACTTTTAAA
  • AAAGAAGTTGCTTCTTGCCG CAATAATGT CGAGTAGTTAAGGTGTTCTGAGGACTAGCCAGTTCTCTGA AATAAATGTCAAATCAGAAGCCAAATGTGTGAGTGTTTGTTTTGTTCGTTTTCATTTTTT CTGCATAAGGTGGCAGGATG
  • Pruss et al (1997) Plant Cell 9, 859-868. Querci et al (1995) Phytopathology 85, 1003-1010.

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Abstract

La présente invention concerne des acides nucléiques codant des polypeptides qui sont capables de conférer une extrême résistance (ER) contre des pathogènes de plantes et qui sont déclenchés par ces pathogènes de plantes. Ces pathogènes sont notamment des virus tels que les PVX et les isolats apparentés à ces PVX. En l'occurrence, des acides nucléiques préférés codent le polynucléotide Rx issu de Solanum tuberosum, ou une variété de ses homologues, naturels ou résultant d'obtentions, et notamment les 111h1, 221h2, Ac15, Ac64 et K39.hom. L'invention concerne également des procédés particuliers permettant d'activer la résistance par utilisation de combinaisons de gènes de résistance et de d'éliciteurs, ce qui, dans certains cas, conduit à une réaction d'hypersensibilité. Sous d'autres aspects, l'invention se rapporte non seulement à des amorces spécifiques, des vecteurs, des cellules hôtes, des polypeptides, des anticorps et des plantes transgéniques, mais également à des procédés de production et d'utilisation correspondants, plus particulièrement lorsqu'on cherche à agir sur les caractéristiques de résistance d'une plante.
EP99917014A 1998-04-16 1999-04-16 Gene de resistance tire d'une plante Withdrawn EP1078086A2 (fr)

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AU8751898A (en) * 1998-07-31 2000-02-21 Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro - Dlo) Engineering nematode resistance in solanacae
GB9924483D0 (en) * 1999-10-15 1999-12-15 Plant Bioscience Ltd Modified resistance genes
CA2480564C (fr) * 2002-03-27 2009-06-02 University Of Tsukuba Nouveau gene resistant au nematode cecidogene et leur application
CN101415828B (zh) * 2006-02-28 2012-12-26 关键基因公司 抗病植物
RU2393227C2 (ru) * 2006-02-28 2010-06-27 Киджин Н.В. Болезнеустойчивые растения
WO2015153839A1 (fr) * 2014-04-03 2015-10-08 The Penn State Research Foundation Procédé d'identification de gènes liés à une résistance aux pathogènes améliorée chez des plantes

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