AU709028B2 - Plant pathogen resistance genes and uses thereof - Google Patents

Description

WO 96/30518 PCT/GB96/00785 PLANT PATHOGEN RESISTANCE GENES AND USES THEREOF 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, plantshave 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 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.

It has long been known that HR-associated disease resistance is often (though not exclusively) specified by dominant genes (R genes). Flor showed that when pathogens mutate to overcome such R genes, these WO 96/30518 PCr/GB96/00785 2 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). A broadly accepted working hypothesis, often termed the elicitor/receptor model, is that R genes encode products that enable plants to detect the presence of pathogens, provided said pathogens carry the corresponding Avr gene (Gabriel and Rolfe, 1990).

This recognition is then transduced into the activation of a defence response.

Some interactions exhibit different genetic properties. Helminthosporium carbonum races that express a toxin (Hc toxin) infect maize lines that lack the Hml resistance gene. Mutations to loss of Hc 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 toxin-dependent virulence.

For example, plant genes whose products were the target of the toxin might mutate to confer even greater sensitivity to the toxin, leading to HR, and the conversion of a sensitivity gene to a resistance gene.

However, this does not seem to be the mode of action of WO 96/30518 PCT/GB96/00785 3 Hml, whose gene product inactivates He toxin.

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, 1993). It is not clear why pathogens make products that enable the plant to detect them. It is widely believed that certain easily discarded Avr genes contribute to but are not required for pathogenicity, whereas other Avr genes are less dispensable (Keen, 1992; Long and Staskawicz, 1993). The characterization of one fungal avirulence gene has also been reported; the Avr9 gene of Cladosporium fulvum, which confers avirulence on C.

fulvum races that attempt to attack tomato varieties that carry the Cf-9 gene, encodes a secreted cysteine-rich peptide with a final processed size of 28 amino acids but its role in compatible interactions is not clear (De Wit, 1992).

The technology for gene isolation based primarily on genetic criteria has improved dramatically in recent years, and many workers are currently attempting to clone a variety of R genes. Targets include (amongst others) rust resistance genes in maize, Antirrhinum and flax (by WO 96/30518 PCT/GB96/00785 4 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).

The map based cloning of the tomato Pto gene that confers "gene-for-gene" resistance to the bacterial speck pathogen Pseudomonas syringae pv tomato (Pst) has been reported (Martin et al, 1993). A YAC (yeast artificial chromosome) clone was identified that carried restriction fragment length polymorphism (RFLP) markers'that'were very tightly linked to the gene. This YAC was used to isolate homologous cDNA clones. Two of these cDNAs were fused to a strong promoter, and after transformation of a disease sensitive tomato variety, one of these gene fusions was shown to confer resistance to Pst strains that carry the corresponding avirulence gene, AvrPto.

These two cDNAs show homology to each other. Indeed, the Pto cDNA probe reveals a small gene family of at least six members, 5 of which can be found on the YAC from which Pto was isolated, and which thus comprise exactly the kind of local multigene family inferred from genetic analysis of other R gene loci.

WO 96/30518 PCT/GB96/00785 The Pto gene cDNA sequence is puzzling for proponents of the simple elicitor/receptor model. It reveals unambiguous homology to serine/threonine kinases, consistent with a role in signal transduction Intriguingly, there is strong homology to the kinases associated with self incompatibility in Brassicas, which carry out an analogous role, in that they are required to prevent the growth of genotypically defined incompatible pollen tubes. However, in contrast to the Brassica SRK kinase (Stein et al 1991), the Pto gene appears to code for little more than the kinase catalytic domain and a potential N-terminal myristoylation site that could promote association with membranes. It would be surprising if such a gene product could act alone to accomplish the specific recognition required to initiate the defence response only when the AvrPto gene is detected in invading microrganisms. The race-specific elicitor molecule made by Pst strains that carry AvrPto is still unknown and needs to be characterized before possible recognition of this molecule by the Pto gene product can be investigated.

Since the isolation of the.Pto gene a number of other resistance genes have been isolated. The isolation of the tobacco mosaic resistance gene N from tobacco was reported by Whitham et al (1994). The isolation of the Arabidopsis thaliana gene for resistance to Pseudomonas syringae RPS2 was reported by Bent et al (1994) and by Mindrinos et al (1994). These genes probably encode WO 96/30518 PCT/GB96/00785 6 cytoplasmic proteins that carry a P-loop and a leucinerich repeat. The ligands with which they interact are uncharacterised and it is not known what other plant proteins they interact with to accomplish the defence response. Our own laboratory has reported the isolation of the tomato Cf-9 which confers resistance against the fungus Cladosporium fulvum. This is a subject of a previous patent application (PCT/GB94/02812 published as WO 95/18230) and has been reported in Jones et al (1994).

Cf-9 and Avr9 sequences, and sequences of the encoded polypeptides are given in W095/18320 and Jones et al (1994).

We have now cloned Cf-2 genes.

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 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. By such means a plant may deprive the pathogen of living cells by localised cell death at sites of attempted pathogen ingress. On the other hand, the PGIP gene of W093/11241 WO 96/30518 PCT/GB96/00785 7 (for example) is a gene of the kind that is induced in the plant defence response resulting from detection of a pathogen by an R gene.

Thus, a pathogen resistance gene may be envisaged as encoding a receptor to a pathogen-derived and Avr dependent molecule. In this way it may be likened to the RADAR of a plant for detection of a pathogen, whereas 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. at a site of contact of the plant with pathogen or elicitor molecule, or systemically. 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.

WO 96/30518 PCT/GB96/00785 8 The "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. fulvum 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. fulvum 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 WO 96/30518 PCT/GB96/00785 9 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.

We have now isolated two almost identical tomato genes, Cf-2.1 and Cf-2.2, which confer resistance against the fungus Cladosporium fulvum and we have sequenced the DNA and deduced the amino acid sequence from these genes.

(Both genes are almost identical and any statement made herein about one should be considered as applying to both, unless context demands otherwise.) The DNA sequence of the tomato Cf-2.1 genomic gene is shown in Figure 2 (SEQ ID NO. 1) and the deduced amino acid sequences (for both genes) are shown in Figure 3A and B (SEQ ID NO's 2 and 3).

As described in more detail below, the tomato Cf-2 genes were isolated by map-based cloning. In this technique 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. We identified a marker that appeared to be absolutely linked to the resistance gene and used probes corresponding to this marker to isolate binary vector cosmid clones from a stock that carried the Cf-2 gene locus. Two independent overlapping clones conferred disease resistance and the region of overlap contains a reading-frame which shows remarkable structural WO 96/30518 PCT/GB96/00785 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).

According to one aspect, 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). In one embodiment 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 WO 96/30518 PCT/GB96/00785 11 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. Of course, changes to the nucleic acid which make no difference to the encoded amino acid sequence are included.

Preferred embodiments of 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. To encode amino acid SEQ ID NO. 3 (Figure 3b), DNA may comprise a nucleotide sequence shown in Figure 4 (SEQ ID NO. Also provided by an aspect of the present invention is nucleic acid comprising a sequence of nucleotides complementary to a nucleotide sequence hybridisable with WO 96/30518 PCT/GB96/00785 12 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.

Of course, 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. Preferably the hybridisable nucleic acid or its complement encode a polypeptide able to confer pathogen resistance on a host, 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.

Although 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 0 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, for instance mutants, derivatives and alleles of the specific sequences disclosed herein, may be distinguished from Cf- 9 by one or more of the following: not being sufficiently homologous with Cf-9 for the WO 96/30518 PCT/GB96/00785 13 nucleic acid of the invention and Cf-9 to identify each other in Southern blotting using a stringency of 2 x SSC at 60 0

C;

having greater than 70%, preferably greater than about 75%, greater than about 80%, greater than about or greater than about 95% homology with the encoding* sequence shown in Figure 2 as nucleotides 1677-5012; eliciting a-defence response, in a plant expressing the nucleic acid, upon contact of the plant with Avr2 elicitor molecule, e.g. as provided by 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 IP010379, or an extract thereof, but not eliciting a defence response in the plant upon its contact with the C. fulvum race 2,4 deposited at and available from the same institute under Accession number IP050379, 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 Avr9 (de Wit, 1992), the amino acid and encoding nucleic acid sequences of chimaeric forms of which are given for example in W095/18230 as SEQ ID NO 3 and in W095/31564 as WO 96/30518 PCT/GB96/00785 14 SEQ ID NO 4.

comprising 38 leucine rich repeats (LRR's).

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.

Those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. 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. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press.

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 WO 96/30518 PCT/GB96/00785 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.

When introducing a chosen gene construct into a cell, certain considerations must be taken into account, well known to those skilled in the art. 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.

Finally, as far as plants are concerned 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 WO 96/30518 PCT/GB96/00785 16 175966), electroporation (EP 290395, WO 8706614) or other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611). 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).

The particular choice of 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.

WO 96/30518 PCTGB96/00785 17 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. For this purpose a vector as described above may be used for the production of a transgenic plant. Such 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. Thus, a host cell, such as a plant cell, comprising nucleic acid according to the present invention is provided. Within the cell, 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.

Also according to the invention there is provided 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 WO 96/30518 PCT/GB96/00785 18 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. Such 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 WO 96/30518 PCT/GB96/00785 19 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.

Sequencing of the Cf-2 gene has shown that like the Cf-9 gene it includes DNA sequence encoding leucine-rich repeat (LRR) regions and homology searching has revealed strong homologies to other genes containing LRRs. The Cf-2 and Cf-9 genes contain all the same general features and as such form a new class of disease resistance genes separate from other disease resistance genes characterised to date. As discussed in WO 95/18230, and validated herein, the presence of LRRs may be characteristic of many pathogen resistance genes and the presence of LRRs may be used in identifying further pathogen resistance genes.

Furthermore, there are some striking homologies between Cf-9 and Cf-2. These homologies may also be used to identify further resistance genes of this class, for WO 96/30518 PCT/GB96/00785 example using oligonucleotides a degenerate pool) designed on the basis of sequences conserved (preferably at the amino acid level) between the Cf-9 and the Cf-2 genes.

According to a further aspect, 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. Thus, a method of obtaining nucleic acid comprising a pathogen resistance .gene (encoding a polypeptide able to confer pathogen resistance) is provided, comprising 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. For probing, preferred conditions are those which are WO 96/30518 PCT/GB96/00785 21 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.

As an alternative to probing, though still employing nucleic acid hybridisation, 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.

On the basis of amino acid sequence information, 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 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:- WO 96/30518 PCT/GB96/00785 22

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 see Figure 6).

Sequences (ii) and (iii) are complementary: (iii) is useful as a back (reverse) primer in PCR.

Preferably in oligonucleotide in accordance with the invention, e.g. for use in nucleic acid amplification, has about 10 or fewer codons 6, 7 or i.e. is about 30 or fewer nucleotides in length 18, 21 or 24).

Assessment of whether or not a PCR product corresponds to a resistance genes may be conducted in various ways. 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 WO 96/30518 PCT/GB96/00785 23 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.

Examples of the type of 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.

Once a pathogen resistance gene has been identified, it may be reintroduced into plant cells using techniques well known to those skilled in the art to produce transgenic plants. According to a further aspect, 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.

According to a yet further aspect, 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. Examples of WO 96/30518 PCT/GB96/00785 24 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.

Modifications to these and further aspects and embodiments of the present invention will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

As already indicated, the present invention is based on the cloning and sequencing of the tomato Cf-2 genes and this experimental work is described in more detail below with reference to the following figures.

Figure 1 shows a physical map of the tomato Cf-2 locus generated from overlapping cosmids (38, 82, 89, 92, 94, 96 and 141) isolated from the Cf-2/Cf-9 cosmid library. Also included are the modified cosmids (112B 1 and 112B2) which contain sequences derived from cosmid 94 (also known as 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 Features: Nucleic acid WO 96/30518 PCT/GB96/00785 sequence Translation start at nucleotide 1677; translation stop at nucleotide 5012; a consensus polyadenylation signal (AATAAA) exists in the characterised sequence downstream of the translation stop starting at nucleotide 3586. 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 Figure 3B shows the amino acid sequence encoded by the Cf-2.2 gene (SEQ ID NO. Amino acids which differ between the two Cf-2 genes are underlined.

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. respectively. OLIGO 1 (SEQ ID NO. 11) and OLIGO 2 (SEQ ID NO. 12) show the sequence of degenerate oliggonucleotides which encode these regions of protein WO 96/30518 PCT/GB96/00785 26 similarity.

Figure 7 shows the primary amino acid saequence of Cf-2 (SEQ ID NO. 2) divided into domains of predicted differing functions.

Cloning of the tomato Cf-2 gene The Cf-2 gene was cloned using a map-based cloning strategy similar in principle to that used for the isolation of the tomato Pto gene, described briefly earlier.

Assignment of Cf- gene-map locations We have mapped several Cf genes, including Cf-2, to their chromosomal locations (Dickinson et al 1993; Jones et al 1993; Balint-Kurti et al 1994). We showed that Cf-4 and Cf-9 map to approximately the same location on the'short arm of chromosome 1, and Cf-2 and Cf-5 map to approximately the same location on chromosome 6.

(ii) High resolution mapping of the physical location of the Cf-2 gene We have ordered a number of restriction fragment length polymorphism (RFLP) markers by examining the DNA isolated from recombinant tomato plants. In this way, we have assembled a detailed linkage map of the location of the Cf-2 gene on tomato chromosome 6 (Dixon et al 1995]) We determined that the Cf-2 gene maps between the RFLP WO 96/30518 PCT1GB96/00785 27 markers MG112A and CT119. These RFLP markers were made available from the laboratory of S. Tanksley (Cornell).

Using available YACs, also made available by the Tanksley laboratory, we have also shown that in tomato (L.

esculentum) the physical distance between the markers MG112A and CT119 is only 40 kb. We isolated two further RFLP markers, MG112B (a weak homologue of MG112A) and SC3-8 that were shown to map to this region and as such represented candidate Cf-2 genes.

To determine more precisely the position of the Cf-2 gene, 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 Fl 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).

(iii) Isolation of binary cosmid vector clones that carry a genomic Cf-2 gene To determine whether DNA identified by the molecular marker MG112B carried the Cf-2 gene, DNA sequences were WO 96/30518 PCT/GB96/00785 28 isolated from a plant that carried the Cf-2 gene and transformed into Cf-O tomato plants.

A genomic DNA library was constructed from a stock that carried both the Cf-9 gene on chromosome i, 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 pOCA18 (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 MboI 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. After in vitro packaging using Stratagene packaging extracts, the cosmids were introduced into a tetracycline sensitive version (obtained from Stratagene) of the Stratagene Escherichia coli strain SURE Recombinants WO 96/30518 PCT/GB96/00785 29 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 For each pool, approximately 10,000 colonies were plated out and inspected for MG112B homology by colony hybridisation with a radioactive MG112B probe, and from each pool, single clones were isolated that carried such homology. These techniques are all well known to those skilled in the art.

These clones have been further characterized by Southern blot hybridisation using a MG112B probe, and by restriction enzyme mapping. Our current assessment of the extent of contiguous DNA around MG112B, as defined by these overlapping cosmids is shown in Figure 1. These cosmids revealed two regions with very similar restriction maps that hybridised to MG112B (now labelled MG112B 1 and B 2 respectively). Four of these cosmids (82, 89, 94, 141) were subsequently used in plant WO 96/30518 PCT/GB96/00785 transformation experiments, selecting for plant cells transformed to kanamycin resistance, using techniques well known to those skilled in the art. Transgenic tomato and tobacco plants were produced (Fillatti et al 1987; Horsch et al 1985) with at least one of each of cosmids 82, 89, 94 and 141.

(iv) Assessment of cosmid function in transgenic tomato The function of a putative cloned Cf-2 gene was assessed in transformed tomato by testing transformants for resistance to Avr2-carrying C. fulvum.

Most transgenic plants containing cosmid 94 (11 of 16) or cosmid 82 (all of 4) were resistant to C. fulvum carrying Avr2. All transgenic plants containing cosmid 89 or 141 were sensitive to C. fulvum. These data indicate that the genomic DNA which carries the Cf-2 gene is that piece which corresponds to the overlap between cosmids 82 and 94. Thus the Cf-2 gene lies in one of the regions identified by marker MG112B 1 The region on cosmid 94 identified by MG112B 2 has many similarities with the cosmid 82/94 overlap. This region was subcloned to generate cosmid 112B 2 (Figure 1) which was also transformed into a sensitive tomato line.

Of the transgenic tomato plants carrying cosmid 112B 2 16 of 18 were resistant to C. fulvum carrying Avr2. The overlap between cosmids 82 and 112B 2 is very small and unlikely to carry the Cf-2 gene. Additionally, the region identified as MG112B

I

was subcloned to generate WO 96/30518 PCT/GB96/00785 31 cosmid 112B 1 (Figure 1) which was transformed into a sensitive tomato line. This cosmid 112B 1 only contains the sequences characterised in figure 2 (SEQ ID NO. 1).

Of the transgenic tomato carrying cosmid 112B 1 ,all (5 out of 5) were resistant to C. fulvum carrying Avr-2.

Therefore, these data indicate the presence of 2 functional Cf-2 genes (Cf-2.1 and Cf-2.2 respectively) characterised by the molecular probe MG112B (Dixon et al 1996). The results of all transformation experiments are summarized in Table 1.

Progeny from all resistant nonpolyploid transformed plants were screened with matched races of C. fulvum either carrying or lacking Avr-2 (race 5,9 compared with race Races of C. fulvum are named after the resistance genes they can overcome. All progeny were susceptible to C. fulvum lacking Avr-2 (race 2,5,9) whereas approximately 75% of progeny from each transformant were resistant to C. fulvum carrying Avr-2 (race These data confirm the race-specific nature of the resistance genes cloned as Cf-2.

DNA sequence analysis of the regions characterised by MG112B.

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).

WO 96/30518 PCT/GB96/00785 32 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). 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. Upon conceptional translation 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. We therefore designate this to be Cf- 2.2.

An almost full length cDNA clone (SEQ ID NO. 4) has been isolated corresponding to the Cf-2 gene (Fig. 4).

this confirms the predicted amino acid sequence of the Cf-2 genes as it is colinear with the genomic sequence through the entire open reading frame. The cDNA clone lacks the most 5' sequences including any untranslated leader sequence and the codon encoding the initiator WO 96/30518 PCT/GB96/00785 33 methionine. The first full codon of this cDNA encodes for Valine which is amino acid number 4. A single intron of 182 bases exists in the 3' untranslated sequences (Dixon et al 1996).

Genbank accession numbers for the sequences reported within are U42444 for Cf-2.1 and U42445 for Cf-2.2.

(vi) Identification of a leucine-rich repeat region in Cf-2.

A genomic DNA sequence of the Cf-2.1 gene is shown in Figure 2 (SEQ ID NO. The deduced amino acid sequence of the Cf-2 protein is shown in Figure 3A (SEQ ID NO. 2).

Homology searching of the resulting sequence against sequences in the databases at the US National Centre of Biological Information (NCBI) reveals strong homologies to other genes that contain leucine rich repeat regions (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), TMKL1 (Valon et al 1993), (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).

The TMK1 and RLK5 genes have structures which suggest they encode transmembrane serine/threonine WO 96/30518 PCT/GB96/00785 34 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). However, in 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.

Domains E, F and G together comprise a likely membrane anchor.

The Cf-2 and Cf-9 proteins are predicted to have the WO 96/30518 PCT/GB96/00785 same general features in that they can both be subdivided 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.

(vii) Comparison of the amino acid sequence of Cf-2 and Cf-9 Comparison of the amino acid sequence of Cf-2 and Cf-9 shows a remarkable degree of homology at the end of Domain C and in Domain D. These are shown in bold in Figure 6. Regions of Cf-2 such as the sequence F E G H I P S (SEQ ID NO. 13) starting at position 915 and the sequence S G E I P Q Q L A S L T S L E (SEQ ID NO. 14) starting at position 965 are absolutely identical to Cf- 9. Other regions of identity or strong conservation also exist. Since Cf-2 and Cf-9 are on different chromosomes, it seems likely that they did not diverge recently. This conservation then suggests functional conservation due to selection of the maintenance of an association between the amino acids in Cf-9 and Cf-2 and some other protein required for Cf-9 or Cf-2 to provide disease resistance.

Accordingly, these homologies may be used in the isolation of further disease resistance genes. Using techniques well known to those skilled in the art these sequences and fragments thereof may be used to design oligonucleotide probes/primers for the purpose of isolating further disease resistance genes that carry these amino acid sequence motifs.

For example, based upon the identities between the Cf-2 and Cf-9 genes (Figures 5 and synthetic degenerate 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 9 may be:used in a PCR to identify related sequences from any species which contains them.

Throughout this specification and the claims which follow, unless the context requires otherwise, the work "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or S, steps.

;7 WO 96/30518 WO 96/05 18PCT/GB96/00785 37 TABLE 1 Cosrnid Cosmid Cosmid Cosinid Cosmid Cosmid Line 94 82 89 141 112B 1 112B 2 A S R S S R

R

B S R S S R

R

C R R S S R

R

D R R S S R

R

E R S S R

R

F R S S

R

G R S

R

H R S

R

IR S

R

JR

R

K R

R

L R

R

M S

R

N R

S

0 S

R

P S

R

Q

S

R

R

The reponse of transgenic tomato plants (primary transformants) carrying different cosmids. Tomato transformants were tested for resistance or susceptibility to a race of C. fulvum carrying Avr-2 (Race 4 GUS).

WO 96/30518 WO 9630518PCrIGB96OO785 38

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Claims (4)

1. 2. Nucleic acid according to claim 1 wherein said activation is upon contact of the plant with a pathogen or corresponding elicitor molecule.
2. 3. Nucleic acid according to claim 1 wherein the sequence of nucleotides comprises an encoding sequence shown in Figure 2 or Figure 4.
3. 4. Nucleic acid according to claim 1 wherein the sequence of nucleotides comprises an allele, derivative or mutant, by way of addition, insertion, deletion or substitution of one or more nucleotides, of an encoding sequence shown in Figure 2 or Figure 4. Nucleic acid encoding a pathogen resistance gene whose expression in a plant can cause activation of a defence response in the plant, comprising a sequence of nucleotides encoding a polypeptide, the polypeptide comprising an amino acid sequence which comprises an allele, derivative or mutant, by way of addition, WO 96/30518 PCT/GB96/00785 41 insertion, deletion or substitution of one or more amino acids, of the amino acid sequence shown in Figure 3A or Figure 3B; with the proviso that the nucleic acid has one or more of the following features: it is not sufficiently homologous with Cf-9 for it and Cf-9 to identify each other in Southern blotting using a stringency of 2 x SSC at 60 0 C; it has at least 70% homology with the encoding sequence shown in Figure 2 at nucleotides
4. 1677-5012; it can cause said activation of a defence response upon contact with an Avr2 molecule; it can cause said activation of a defence response upon contact of the plant with the Cladosporium fulvum race 4 deposited under the Accession number IP010379, or an extract thereof, but does not cause said activation of a defence response upon contact of the plant with the Cladosporium fulvum race 2,4 deposited under Accession number IP050379, or an extract thereof; it does not activate said defence response upon contact with an Avr9 molecule; the encoded polypeptide comprises 38 leucine rich repeats (LRR's). 6. Nucleic acid according to claim 5 wherein said activation is upon contact of the plant with a pathogen or corresponding elicitor molecule. 42 7.A nUc-leiC aci~d -solate co-mprising a pathogen r esistance gene whose expression i,1 a lncncas activation of a defence response in he plant, comprising a sequence of nucl1ectides ccnplementa-vy to a nucleotide sequence hybridisable with an encodin~g sequence shown in Figure 1 or Figure 4 on a Scuth1ern- blcz using a str~ingency of 2 x SSC ac W0C. 8. Nucleic acid according t~o clairm 7 wherein said activaticn is -upon contact of the plant with a pathogen or. corresponding a74Citor M'olecule. 9. Nucleic ac-Id which is a vector -cmprising nucilic acid according to any one of claims I to S. 1s Nucleic acid according to claim 9 further- comprising regulatory sequences for expression of said pclypeptide. 11. Use of nucleic acid according to any one off the precedingB claiMS in production of a tranageni4c plan. A host cell comprising exogenous nucleic acid according to any one of claims I to IS. A host cell accor-ding to claim 12 which is microbial. 14. A host cell according to claim 13 which is a plant Cell. Q:\OPER\MRO\1932842.168 22/6/99 -43- A plant or any part thereof comprising a cell according to claim 14. 16. A method which comprises introduction of nucleic acid according to any one of claims 1 to 10 into a host cell. 17. A method according to claim 16 wherein the host cell is a plant or microbial cell. 18. A method according of conferring pathogen resistance on a plant, comprising expression from nucleic acid according to any one of claims 1 to 10, within cells of the plant, following an earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof. 19. A method according to claim 18 wherein the nucleic acid encodes an amino acid sequence shown in Figure 3. An oligonucleotide which comprises at least about 30 nucleotides in length derived from the nucleic acid isolate according to any one of claims 1 to 10, wherein said oligonucleotide encodes an amino acid sequence conserved between pathogen resistance genes or is complementary thereto. 21. The oligonucleotide according to claim 20 wherein the pathogen resistance genes are Cf-2 (amino acid sequence of Figure 3A or 3B) and Cf-9 (W095/18230) of tomato. 22. An oligonucleotide comprising a nucleotide sequence encoding any one of the amino acid sequences: SGEIPQQ; (ii) YE/QGNDG; (iii) FEGHIPS; (iv) SGEIPQQLASTSLE; or a nucleotide sequence complementary to a said encoding sequence. Q:\OPER\MRO\ 1932842.168 22/6/99 -44- 23. An oligonucleotide comprising a sequence selected from: TCX-GGX-GAA/G-ATT/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. 24. An oligonucleotide which comprises a sequence which is a variant or derivative, by way of addition, insertion, deletion or substitution of one or more nucleotides, of the sequence of an oligonucleotide according to any one of claims 20 to 23. A method of obtaining nucleic acid comprising a plant pathogen resistance gene whose expression in a plant can cause activation of a defence response in the plant, comprising hybridisation of an oligonucleotide according to any one of claims 20 to 24, or a nucleic acid molecule comprising a said oligonucleotide, said nucleic acid molecule not being the Cf-9 gene of tomato (W095/18230), to target nucleic acid. 26. A method according to claim 25 involving use of nucleic acid amplification. 27. A method according to claim 25 or claim 26 wherein the hybridisation is followed by identification of successful hybridisation and isolation of target nucleic acid. 28. A method wherein following the obtaining of nucleic acid using the method of any of claims 25 to 27 a nucleic acid molecule comprising all or part of the sequence of the obtained nucleic acid is used in the production of a transgenic plant. 29. A method according to claim 28 wherein said nucleic acid molecule us used to confer pathogen resistance on said plant. Use of an oligonucleotide according to any of claims 20 to 23 for obtaining nucleic acid comprising a plant pathogen resistance gene whose expression in a plant can cause activation of a defence response in the plant. Q:\OPER\MRO\1932842.168 22/6/99 31. The nucleic acid isolate according to any one of claims 1 to 10 substantially as hereinbefore described with reference to the Figures and/or Examples. 32. The host cell according to any one of the claims 12 to 14 substantially as hereinbefor e described with reference to the Figures and/or Examples. 33. The plant of claim 15 substantially as hereinbefore described with reference to the Figures and/or Examples. 34. The method according to any one of claims 16 to 19 or any one of claims 25 to 29 substantially as hereinbefore described with reference to the Figures and/or Examples. 35. The oligonucleotide according to any one of claims 20 to 24 substantially as hereinbefore described with reference to the Figures and/or Examples. 36. The use according to any one of claims 11 to 30 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this TWENTY SECOND day of JUNE, 1999. Plant Bioscience Limited by DAVIES COLLISON CAVE Patent Attorneys for the Applicants .u 1