WO2000008189A2 - Plant resistance gene - Google Patents

Abstract

Disclosed are isolated nucleic acid molecules comprising an RPP1 nucleotide sequence which encodes a Peronospora parasitica resistance gene of the RPP1 complex locus, such as the RPP1 homologues A, B and C from Arabidopsis thaliana Wassilewskija various of which have specificities corresponding to RPP1, 10 and 14. Also disclosed are variants of these sequences, which may be natural homologues or derivatives. The invention further provides probes and primers, vectors, host cells, and transgenic plants, plus corresponding methods of producing and employing the same.

Description

Plant resistance gene

The present invention relates to methods and materials, particularly nucleic acids, for manipulating the resistance of plants to downy mildew { Peronospora parasi tica) . It further relates to plants which have been modified using such methods and materials.

PRIOR ART

R-Avr interactions

Genotype specific disease resistance in plants depends on the expression of complementary avirulence (Avr) genes in the pathogen and resistance (R) genes in the host

(Staskawicz et al., 1995; Bent, 1997). The final outcome of a matched R-Avr interaction is incompatibility i.e. containment of the pathogen at the site of penetration, and is commonly associated with a hypersensitive response (HR) of the penetrated host cells.

Characterization of a number of pathogen Avr genes suggests that some are derived from pathogenicity (or virulence) determinants that have become vulnerable to detection by a "surveillance system" in plants specified by" evolving R genes and coupled in some way to rapid defence activation (Baker et al . , 1997) . A powerful selection pressure is therefore exerted on the pathogen to mutate from avirulence to virulence, as has been demonstrated in several fungal (Joosten et al . , 1994; Rohe et al., 1995) and bacterial (Kearney et al . , 1988; Bogdanove et al . , 1998) plant pathogens. It is crucial that the host plant is able to respond by generating novel recognition capabilities.

The majority of cloned R genes encode proteins that possess variable numbers of leucine-rich repeats (LRRs) (Jones and Jones, 1997) . Several recent reports suggest that the LRR motif permits the recurrent generation of novel protein/protein recognition specificities. This can occur through hypervariability in predicted solvent exposed amino acids in a putative parallel beta sheet recognition surface and/or through variation in LRR copy number (Thomas et al., 1997, Parniske et al . , 1997). Structural comparisons between the tomato Cf-9 and Cf-4 R proteins revealed significant sequence divergence only within the N-terminal portion of their LRRs implicating this domain in specific Avr recognition (Thomas et al . , 1997) . Molecular analysis of allelic variants at the flax L locus also suggest that the LRR domain is partly responsible for recognition specificity (Ellis et al . , 1997) .

Additionally, the majority of R genes reside at complex loci and the structure of these may influence the rate of R gene diversification (Pryor and Ellis, 1993) . The maize Rpl locus (Sudapak et al., 1993), the tomato Cf-4/9 locus (Thomas et al . , 1997; Parniske et al . , 1997), the flax M locus (Anderson et al . , 1997), and the lettuce Dm3 locus

(Anderson et al., 1996) all consist of genetically linked resistance specificities. Furthermore, Rpl gene instability and the creation of novel resistance specificities was shown to be associated with the exchange of flanking markers (Sudapak et al . , 1993; Richter et al . , 1995), suggesting that unequal crossing- over contributes to sequence variation at R gene loci. Molecular analysis of genes comprising the Cf-4/9 locus in tomato revealed a number of distinct R gene specificities (Parniske et al . , 1997). Here, evidence was presented for diversifying selection on amino acids within the LRRs that are predicted to be solvent exposed and therefore may mediate ligand binding.

The characterisation and cloning of R genes, particularly those having novel specificities or recognitions, allows the pathogen resistance traits arising from those genes to be manipulated. This is particularly important when dealing with commercially significant pests.

RPP specificities

Extensive genetic variation exists in the interaction between the model plant, Arabidopsis, and the biotrophic oomycete, Peronospora parasi tica (downy mildew) , indicative of a highly co-evolved pathosystem (reviewed by Holub and Beynon, 1997) . More than twenty downy mildew resistance specificities have been mapped to RPP (Recognition of P. parasi tica) loci on six of the ten chromosome arms of Arabidopsis using differential responses of four standard accessions to a diverse collection of P. parasi tica isolates.

Although clustering of RPP loci has been observed in linkage groups of up to 15 cM, often coinciding with additional LRR-containing genes (Botella et al, 1997; Aarts et al, 1998a) , an example of a complex locus such as those described in crop species (genetic scale <lcM) conferring multiple recognition specificities, hitherto had not been clearly demonstrated in Arabidopsis.

One characterised RPP gene is RPP5 (Parker et al . , 1997) . The nature of repeated LRR sequence blocks in the

Arabidopsis RPP5 protein suggests that these have arisen through intragenic duplications . Thus it appears that LRR domains are amenable to expansion and contraction by insertion or deletion of complete LRR units, potentially giving rise to novel recognition surfaces.

A further three downy mildew specificities (.RPPl, RPP10 and RPP14) have previously mapped to the same locus on the bottom arm of chromosome 3 :

RPPl was characterized first as a specificity in accession Niederzenz (Nd-1) conferring resistance to the P. parasi tica isolate Emoy2 (Holub et al . , 1994; Tor et al . , 1994) . s-0 resistance to Emoy2 was inseparable by genetic recombination from the RPPl locus identified in

Nd-1 (Holub et al . , 1994; Holub and Beynon, 1997), suggesting that an RPPl allele exists in s-0.

RPP10 was designated as a second specificity in Ws-0 conferring resistance to the Nd-compatible isolate Cala2. Although RPP10 has not been separated by genetic recombination from RPPl -Emoy2 in Ws-0, a mutation affecting resistance to Cala2 was identified which confirmed that Ws-0 carried at least two downy mildew resistance genes at the RPP1/10 locus (Holub, 1997) .

RPP14 was designated as a third specificity to explain resistance in Ws-0 to a Nd-compatible isolate, Noco2 (Reignault et al., 1996). This isolate remains incompatible in the Vts-rppl O mutant, suggesting that either a third Noco2-specific gene exists in Ws-0 at the RPPl/ 10/ 14 locus, or that the RPPl -Nd and RPPl -Ns alleles defined by Emoy2 have different specificities as defined by Noco2.

Although these specificities were defined in the prior art, the gene or genes giving rise to the specificities had not been accurately mapped or cloned.

DISCLOSURE OF THE INVENTION

The present inventors have investigated the RPPl/ 10/ 14 locus in the Arabidopsis accession Wassilewskija (Ws) in order to determine its specificities with respect to the three P. parasitica isolates Emoy2, Cala2 and Noco2, plus also a fourth isolate Maks9 which had previously been mapped to the same locus (Holub, 1997) .

They demonstrated through high-resolution mapping that RPPl/ 10/ 14 was a complex locus (hereinafter the RPPl locus or region) carrying at least three functional genes (designated RPPl -WsA, RPPl -WsB and RPPl -WεC) that differ in their ability to detect the four P. parasi tica isolates . The genes encode functional products of the NB (Nucleotide Binding) -LRR R protein class. They possess a TIR (Toll, χnterleukin-1, Resistance) domain that is characteristic of certain other NB-LRR type R proteins and are highly sequence related. However, their distinct, but partially overlapping, resistance profiles indicate that they recognize different pathogen avirulence (Avr) genes or alleles. One of the R proteins contains a N- terminal putative signal anchor whereas the other two proteins lack the signal anchor but encode a novel and unique hydrophilic N-terminus. Together, the three RPPl genes account for the spectrum of resistance previously assigned to the RPPl region, and thus comprise a complex

R locus that contains a functionally variable family of

R-genes . The distinct resistance capabilities of these genes is best explained by the hypothesis that each recognizes a different pathogen avirulence determinants .

Further work done by the inventors suggests that positive selection for diversification of the predicted ligand binding domain has affected the evolution of the RPPl gene family.

Thus in a first aspect of the present invention there is disclosed a nucleic acid molecule encoding a Peronospora parasitica resistance gene of the RPPl complex locus.

Nucleic acid molecules 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 other nucleic acids of the species of origin. Where used herein, the term "isolated" encompasses all of these possibilities.

The nucleic acid molecules may be wholly or partially synthetic . In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e.g. using an automated synthesiser.

By " Peronospora parasi tica resistance gene" is meant a gene encoding a polypeptide capable of recognising and activating a defense response in a plant in response to challenge with a P parasi tica isolate or an elicitor or Avr gene product thereof. Preferably the plant is one of the cultivated crucifer relatives (Brassicas) . In particular the resistance gene, or combinations of the resistance genes discussed herein, may (either individually or in combination) be capable of mediating a response against one or more isolates of downy mildew, such as the following P parasi tica isolates: Emoy2, Cala2, Noco2 , Maks9.

The activity of the encoded polypeptide may be tested, for instance, by challenging a plant in which the corresponding gene has been introduced e.g. by analogy with the methods of Holub et al (1994) or Parker et al (1997) .

Plants to which the invention may be most advantageously applied include any which are susceptible to downy mildew e.g. lettuces (lactuca sativa) , grape vines, maize, and late flowering potato. Also preferred may be brassicas such as B . Napus, B Oleracea (green cabbage, white cabbage, broccoli, brussels sprouts, and curly kale etc.)

Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs . 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.

Thus in one embodiment of this aspect of the invention, there is disclosed a nucleic acid comprising any one or more of the following nucleotide sequences in Figures 3A- 3K:

Seq ID No 1 labelled RPPl-WsA genomic

Seq ID No 2 labelled RPPl-WsA cDNA

Seq ID No 4 labelled RPPl-WsB genomic

Seq ID No 5 labelled RPPl-WsB cDNA

Seq ID No 7 labelled RPPl-WsC genomic

Seq ID No 8 labelled RPPl-WsC cDNA

Equally a nucleic acid of the present invention may encode one or more of the following amino acid sequences in Figures 3A-3K. Seq ID No 3 : labelled RPPl-WsA peptide Seq ID No 6: labelled RPPl-WsB peptide Seq ID No 9: labelled RPPl-WsC peptide

In a further aspect of the present invention there are disclosed nucleic acids which are variants of the sequences of the first aspect.

A variant nucleic acid molecule shares homology with, or is identical to, all or part of the the coding sequence discussed above. Generally, variants encode, or be used to isolate or amplify nucleic acids which encode, polypeptides which are capable of mediating a response against a pathogen, particularly P parasi tica .

Variants of the present invention can be artificial nucleic acids (i.e. containing sequences which have not originated naturally) which can be prepared by the skilled person in the light of the present disclosure. Alternatively they may be novel, naturally occurring, nucleic acids, isolatable using the sequences of the present invention.

Thus a variant may be a distinctive part or fragment (however produced) corresponding to a portion of the sequence provided. The fragments may encode particular functional parts of the polypeptide, e.g. TIR, NB-ARC, or LRR regions, or the hydrophobic or hydrophilic termini.

Equally the fragments may have utility in probing for, or amplifying, the sequence provided or closely related ones. Suitable lengths of fragment, and conditions, for such processes are discussed in more detail below.

Also included are nucleic acids which have been extended at the 3 ' or 5 ' terminus .

Sequence variants which occur naturally may include alleles (which will include polymorphisms or mutations at one or more bases) .

Artificial variants (derivatives) may be prepared by those skilled in the art, for instance by site directed or random mutagenesis, or by direct synthesis. Preferably the variant nucleic acid is generated either directly or indirectly (e.g. via one or amplification or replication steps) from an original nucleic acid having all or part of the sequences of the first aspect. Preferably it encodes a Peronospora parasi tica resistance gene.

The term 'variant' nucleic acid as used herein encompasses all of these possibilities. When used in the context of polypeptides or proteins it indicates the encoded expression product of the variant nucleic acid.

Some of the aspects of the present invention relating to variants will now be discussed in more detail.

Similarity (or homology, or identity - the terms are interchangeable herein) 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) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman.

Homology may be at the nucleotide sequence and/or encoded amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shares at least about 50%, or 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology with any one of Seq ID Nos 1 to 9 as appropriate.

As shown in Figure 4, the closest sequence of the prior art (RPP5) has a much lower level of amino acid sequence identity than this (37% with respect to Seq ID No 3) . Additionally, no hybridization was detected between a YAC encompassing the Col-0 RPP14 region identified by the present inventors and RPP5 under low stringency conditions . Thus a variant polypeptide in accordance with the present invention may include within the sequence shown in Seq ID No 3, 6 or 9, a single amino acid or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes. In addition to one or more changes within the amino acid sequence shown, a variant polypeptide may include additional amino acids at the C-terminus and/or N- ter inus . As discussed below, it may be desirable to insert additional LRR repeats into the sequence.

Regarding nucleic acid variants, changes to the nucleic acid which make no difference to the encoded polypeptide (i.e. ' degeneratively equivalent ' ) are naturally included within the scope of the present invention.

Thus in a further aspect of the invention there is disclosed a method of producing a derivative nucleic acid comprising the step of modifying the coding sequence of a nucleic acid comprising any one sequences Seq ID No 1, 2, 4, 5, 7, or 8.

Changes to a sequence, to produce a 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.

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 (e.g. binding sites) . Leader or other targeting sequences (e.g. hydrophobic anchoring regions) may be added or removed from 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.

Changes may be by way of 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. As is well known to those skilled in the art, 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 is critical in determining the peptides conformation.

Also included are variants having non-conservative substitutions . As is well known to those skilled in the art, 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.

In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity. As is discussed in more detail hereinafter, the work of the present inventors has strongly indicated that manipulation of the LRR regions of the polypeptides encoded by the nucleic acids of the present invention may allow the production of novel resistance specificities e.g. with respect to existing or novel downy mildew isolates. Other motifs and domains identified by the inventors include the TIR domain; the NB-ARC domain; residues which appear to correspond to the β-strand/β-turn structural motif in the porcine ribonuclease inhibitor (PRI) ; a conserved kinase-la (P- loop) domain (GPPGIGKTT) , a kinase-2 domain (FLVLDE) , a kinase-3a domain (FGSGSR) , and a conserved hydrophobic motif (ELPLGL) .

Other methods for generating novel specificities may include mixing or incorporating sequences from related resistance genes into the RPP sequences disclosed herein. For example restriction enzyme fragments of RPPl could be ligated together with fragments of an RPP5 or even of an unrelated gene to generate recombinant derivatives. An alternative strategy for modifying RPP sequences would employ PCR as described below (Ho et al . , 1989, Gene 77, 51-59) or DNA shuffling (Crameri et al., 1998, Nature 391 ) .

In a further aspect of the present invention there is provided a method of identifying and/or cloning a nucleic acid variant from a plant which method employs any of Seq ID Nos 1, 2, 4, 5, 7, or 8 a derivative thereof (e.g. fragment, or complementary sequence) .

In each case, if need be, clones or fragments identified in the search can be extended. For instance if it is suspected that they are incomplete, the original DNA source (e.g. a clone library, mRNA preparation etc.) can be revisited to isolate missing portions e.g. using sequences, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence.

In one embodiment, nucleotide sequence information provided herein may be used in a data-base (e.g. of expressed sequence tags, or sequence tagged sites) search to find homologous sequences, such as those which may become available in due course, and expression products of which can be tested for activity as described below.

In a further embodiment, a variant in accordance with the present invention is also obtainable by means of a method which includes:

(a) providing a preparation of nucleic acid, e.g. from plant cells,

(b) providing a nucleic acid molecule having a nucleotide sequence shown in or complementary to any of Seq ID Nos 1, 2, 4, 5, 7, or 8,

(c) contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue in said preparation, and identifying said gene or homologue if present by its hybridisation with said nucleic acid molecule.

Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated 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. DNA for probing may be prepared from RNA preparations from cells.

Test nucleic acid may be provided from a cell as genomic DNA, cDNA or RNA, or a mixture of any of these, preferably as a library in a suitable vector. If genomic DNA is used the probe may be used to identify untranscribed regions of the gene (e.g. promoters etc.), such as is described hereinafter. Probing may optionally be done by means of so-called 'nucleic acid chips' (see Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review) .

Preliminary experiments may be performed by hybridising under low stringency conditions. For probing, 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.

For instance, screening may initially be carried out under conditions, which comprise a temperature of about 37°C or less, a formamide concentration of less than about 50%, and a moderate to low salt (e.g. Standard

Saline Citrate ('SSC') = 0.15 M sodium chloride; 0.15 M sodium citrate; pH 7) concentration. Alternatively, a temperature of about 50°C or less and a high salt (e.g. 'SSPE'= 0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4) . Preferably the screening is carried out at about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5 X SSC, or a temperature of about 50°C and a salt concentration of about 2 X SSPE. These conditions will allow the identification of sequences which have a substantial degree of homology (similarity, identity) with the probe sequence, without requiring the perfect homology for the identification of a stable hybrid.

Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na₂HP0₄, pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0.1X SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65°C in 0.25M Na₂HP0₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0.1X SSC, 0.1% SDS.

It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e.g. cDNA libraries representative of expressed sequences, may be searched. Those skilled in the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on.

Binding of a probe to target nucleic acid (e.g. DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled. Other methods not employing labelling of probe include amplification using PCR (see below) or RN'ase cleavage. 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 of a vector in a suitable host.

In a further embodiment, hybridisation of nucleic acid molecule to a variant may be determined or identified indirectly, e.g. using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR) . PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of glycosyltransferases are employed. Using RACE PCR, only one such primer may be needed (see "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, Academic Press, New York, (1990)).

Thus a method involving use of PCR in obtaining nucleic acid according to the present invention may be carried out as described above, but using a pair of nucleic acid molecule primers useful in (i.e. suitable for) PCR, at least one of which has a nucleotide sequence shown in or complementary to any of Seq ID Nos 1, 2, 4, 5, 7, or 8.

The methods described above may also be used to determine the presence of one of the nucleotide sequences of the present invention within the genetic context of an individual plant, optionally a transgenic plant such as a Brassica plant which may be produced as described in more detail below. This may be useful in plant breeding programmes e.g. to directly select plants containing alleles which are responsible for desirable traits in that plant species, either in parent plants or in progeny (e.g hybrids, FI, F2 etc.) . Thus use of the markers defined in the Examples below, or markers which can be designed by those skilled in the art on the basis the nucleotide sequence information disclosed herein, forms one part of the present invention.

An oligonucleotide for use in probing or amplification reactions 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. For optimum specificity and cost effectiveness, primers of 16-24 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, 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.

As used hereinafter, unless the context demands otherwise, the term ".RPPl" is intended to cover any of the nucleic acids of the invention described above, including functional variants.

In one aspect of the present invention, the RPPl 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 Agrobacterium 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) .

Specifically included are 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 .

Preferably 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. 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

By "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.

Thus 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 RPPl-WsA, B or C or a variant thereof .

Generally speaking, 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 sequences, 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 .

Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis (see above discussion in respect of variants) , sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular

Biology, Second Edition, Ausubel et al . eds . , John Wiley & Sons, 1992. The disclosures of Sambrook et al . and Ausubel et al . are incorporated herein by reference .

In one embodiment of this aspect of the present invention, there is provided a gene construct, preferably a replicable vector, comprising an inducible promoter operatively linked to a nucleotide sequence provided by the present invention, such as an Rppl Ws sequence.

The term "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 .

Particular of interest in the present context are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148) .

Suitable promoters which operate in plants include the Cauliflower Mosaic Virus 35S (CaMV 35S) . Other examples are disclosed at pg 120 of Lindsey & Jones (1989) "Plant Biotechnology in Agriculture" Pub. OU Press, Milton

Keynes, UK. The promoter may be selected to include one or more sequence motifs or elements conferring developmental and/or tissue-specific regulatory control of expression. Inducible plant promoters include the ethanol induced promoter of Caddick et al (1998) Nature Biotechnology 16: 177-180.

It may be desirable to use a strong constitutive promoter. RPP gene dosage has previously been shown to influence the interaction phenotype, for instance it was possible to discriminate phenotypically between plants homozygous or heterozygous at the RPP14 (Reignault et al., 1996) and RPP5 (Parker et al . , 1993) loci.

If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to antibiotics or herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate) .

The present invention also provides methods comprising introduction of such a construct into a host cell, particularly a plant cell.

In a further aspect of the invention, there is disclosed a host cell containing a heterologous construct according to the present invention, especially a plant or a microbial cell .

The term "heterologous" is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question (an .RPPl gene) 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 replace an endogenous equivalent gene, i.e. one which normally performs the same or a similar function, or the inserted sequence may be additional to the endogenous gene or other sequence .

Nucleic acid heterologous to a plant cell may be non- naturally occurring in cells of that type, variety or species. Thus 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 further possibility is for a nucleic acid sequence to be placed within a cell in which it or a homolog is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.

The host cell (e.g. plant cell) is preferably transformed by the construct, which is to say that the construct becomes established within the cell, altering one or more of the cell's characteristics and hence phenotype e.g. with respect to downy mildew resistance.

Nucleic acid can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718 , NAR 12(22) 8711 - 87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al . (1987) Plant Tissue and Cell

Cul ture, Academic Press), electroporation (EP 290395, WO 8706614 Gelvin Debeyser) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g. Freeman et al . Plant Cell Phyεiol . 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U. S.A. 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech . Adv. 9: 1-11.

Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species . Recently, there has also been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (see e.g. Hiei et al . (1994) The Plant

Journal 6, 271-282)). Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium alone 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 microprojectile 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, nor is the choice of technique for plant regeneration.

Thus a further aspect of the present invention provides a method of transforming a plant cell involving introduction of a construct as described above into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce a nucleic acid according to the present invention into the genome .

The invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention (e.g comprising several RPPl sequences) especially a plant or a microbial cell . In 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. There may be more than one heterologous nucleotide sequence per haploid genome.

Generally speaking, following transformation, 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 reviewd 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 Weiεsbach and Weissbach, Methods for Plant Molecular Biology,

Academic Press, 1989. The generation of fertile transgenic plants has been achieved in the cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5 , 158-162.; Vasil, et al . (1992) Bio/Technology 10, 667-674; Vain et al . , 1995,

Biotechnology Advances 13 (4) : 653-671; Vasil, 1996, Nature Biotechnology 14 page 702) .

Plants which include a plant cell according to the invention are also provided.

In addition to the regenerated plant, 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 descendents) and any part of any of these.

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 invention also provides a plant propagule from such plants, 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 influencing or affecting the degree of resistance of a plant to a pathogen, particularly downy mildew, more particularly to one of the isolates discussed above, the method including the step of causing or allowing expression of a heterologous nucleic acid sequence as discussed above within the cells of the plant.

The step may be preceded by the earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof . The methods may also include the manipulation of other genes e.g. which may be involved in transduction of the resistance signal, or in generating a resistance response. For instance, certain RPP genes in Arabidopsis are dependent on a second gene, EDS1 , for resistance function (Aarts et al . , 1998b).

The foregoing discussion has been generally concerned with uses of the nucleic acids of the present invention for production of functional RPPl and variant polypeptides in a plant, thereby increasing its pathogen resistance. However the information disclosed herein may also be used to reduce the activity or levels of such polypeptides in cells in which it is desired to do so. For instance the sequence information disclosed herein may be used for the down-regulation of expression of genes e.g. using anti-sense technology (see e.g. Bourque, (1995), Plant Science 105, 125-149); sense regulation

[co-suppression] (see e.g. Zhang et al . , (1992) The Plant Cell 4, 1575-1588) . Further options for down regulation of gene expression include the use of ribozymes, e.g. hammerhead ribozymes, which can catalyse the site- specific cleavage of RNA, such as mRNA (see e.g. Jaeger (1997) "The new world of ribozymes" Curr Opin Struct Biol 7:324-335.

Nucleic acids and associated methodologies for carrying out down-regulation (e.g. complementary sequences) form one part of the present invention.

The present invention also encompasses the expression product of any of the functional nucleic acid sequences disclosed above, plus also methods of making the expression product by expression from encoding nucleic acid therefore under suitable conditions, which may be in suitable host cells.

Following expression, the recombinant product may, if required, be isolated from the expression system. Generally however the polypeptides of the present invention will be used in vivo (in particular in planta) . Purified RPPl or variant protein produced recombinantly by expression from encoding nucleic acid therefor, may be used to raise antibodies employing techniques which are standard in the art. Antibodies and polypeptides comprising antigen-binding fragments of antibodies form a further part of the present invention, and may be used in identifying homologues from other plant species.

The invention will now be further described with reference to the following non-limiting Figures and

Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

FIGURES

Figure 1: Physical delineation of RPP14

(A) Alignment of yeast artificial chromosomes (YAC) clones relative to molecular markers flanking RPP14. Individual YAC clones were positioned by hybridization with the RFLP flanking markers ve021 and pAT3-89.1. End probes from the YACs (solid for the right ends and open for the left ends) were obtained as described in Methods and used to orientate the YAC clones .

(B) Two YAC end probes, 4D9LE and 8D7LE were used to isolate overlapping PI clones. Two of the PI clones, Pl- 37C7 and P1-53P2, that are marked with asterisks, were found to overlap. Random sequence of these clones identified a fragment, 37C7-5 (shaded box) with homology to RPP5.

(C) DNA gel blot analysis of five Arabidopsis accessions reveals a small, polymorphic gene family. The blot was hybridized at high stringency and probed with the 37C7-5 fragment. DNA from the accessions Landsberg-erecta (L) ,

Columbia (C) , Wassilewskija (W) , Niederzenz (N) and Oystese (0) were digested with EcoRI and Hindlll as indicated.

Figure 2: Physical organization of RPPl /10/14 candidate genes in Ws-0 (A) Three different genes were identified containing

37C7-5-homologous sequence and assembled in two contigs. Only informative clones are shown. Minimal constructs for homolog A (psub-Hom.A) , homolog B (psub-Hom.B) and homolog C (psub-HomC) were derived from ws-cos 65.1, ws- 69.12, and ws-phage 1.1 (see Methods) EcoRI restriction sites are indicated by open circles and Hindlll sites by filled circles. The 1.1 kb HindiII probe of the 5' end from Hom-A that was used in the DNA gel blot analysis shown in (B) is indicated with an asterisk.

(B) DNA from the Ws-0 (W) and Col-0 (C) Arabidopsis accessions was digested with Hindlll or EcoRI. The filter was probed with a 1.1 kb Hindlll fragment from the 5' end of Horn. A. The identity of fragments derived from the

Hom-A, Hom-B and Hom-C genes are indicated on the left hand side of the blot. Hybridizing bands derived from Hom-D are also shown.

Figure 3 : Sequences of .RPPl and RPPl homologs

Seq ID No 1 labelled RPPl-WsA genomic

Seq ID No 2 labelled RPPl-WsA cDNA

Seq ID No 3 labelled RPPl-WsA peptide S Seeaq I IDD N Noo 4 4: labelled RPPl-WsB genomic

Seq ID No 5 labelled RPPl-WsB cDNA

Seq ID No 6 labelled RPPl-WsB peptide

Seq ID No 7 labelled RPPl-WsC genomic

Seq ID No 8 labelled RPPl-WsC cDNA S Seeqq I IDD N Noo 9 9: labelled RPPl-WsC peptide

Figure 4 : Structure of the .RPPl gene family members and their predicted proteins

(A) Sequence features of genes RPPl -WsA, RPPl -WsB and

RPPl -WsC. El to E8 represent exons 1 to 8. The predicted

ATG and the Stop codons are shown. Exons showing high homology are indicated with shading according to the key shown .

(B) Schematic comparison of the RPPl protein family with related JR gene products . The percentage amino acid sequence identity between RPPl-WsA and RPPl-WsB, RPPl- WsC, RPP5, N or L6 is shown. Intron positions are marked by arrowheads . Similar protein domains are indicated with identical shading according to the key shown. TIR domain has similarity with the cytoplasmic domains of Toll and IL-1R (see also Figure 5) . NB-ARC domain contains motifs that constitute a nucleotide binding site and domains with homology to APAF-1 and CED-4, regulators of cell death. LRR corresponds to the C-terminal part of the proteins that contain the LRRs. N-termini correesponds to the extension of the proteins observed in RPPl-WsB and RPPl-WsC. SD correspond to the signal domains observed in RPPl-WsA and L6.

Figure 5 : Alignment and hydrophobicit plot of the TIR domains of RPPl-WsA, RPPl-WsB, RPPl-WsC. RPP5 , N and L6

(A) Prettybox representation of a Pileup analysis of the TIR region of RPPl-WsA (exon 1) , RPPl-WsB (exons 1 and

2), RPPl-WsC (exons 1 and 2), RPP5 (exon 1), N (exon 1) and L6 (exon 1) . The N-terminal extensions of the RPPl family and L6 proteins relative to RPP5 and N are shaded and indicated by an arrow. The asterisk indicates the position of the introns in RPPl-WsB and RPPl-WsC.

(B) Hydrophobicity analysis of the sequences shown in

(A) . The N-terminal extensions shown above are shaded and indicated by an arrow. Analysis was performed as detailed in Methods.

Figure 6 : Structural domains of the RPPl protein family and analysis of their LRRs

(A) The RPPl family of proteins can be divided into several domains. The TIR domain (exonl in RPPl-WsA, RPP5 , N and L6 ,- exon 2 and exon 3 in RPPl-WsB and RPPl-WsC) is similar to the cytoplasmic domains of Drosophila Toll and mammalian Interleukin-1-receptors . The NB-ARC domain (exon2 in RPPl-WsA, RPP5, N and L6 ; exon 4 in RPPl-WsB and RPPl-WsC) has similarity to the nematode CED-4 and mammalian APAF-1 proteins, both activators of apoptotic proteases (Van der Biezen and Jones, 1998) . The LRR domain (exons 3, 4, 5 and 6 in RPP5 ; exons 3 and 4 in N and L6; exon 4 in RPPl-WsA, exon6 in RPPl-WsB) is envisaged to mediate specific protein-protein interaction. Residues corresponding to the β-strand/β- turn structural motif in the porcine ribonuclease inhibitor (PRI) are delimited by vertical lines in the RPPl-WsA sequence.

The conserved kinase-la (P-loop) domain (GPPGIGKTT) , the kinase-2 domain (FLVLDE) , the kinase-3a domain (FGSGSR) , and the hydrophobic motif conserved in this class of proteins (ELPLGL) are underlined. An 11 amino acid deletion in the Ws-rpplO mutant is highlighted by a box within the NB-ARC domain. A deletion of one LRR in RPP1- WsC LRR is highlighted by a box. The arrow in the TIR domain indicates the start of homology between RPPl-WsA and proteins, RPPl-WsB and RPPl-WsC.

(B) Amino acid differences within the LRR domains between the RPPl family of proteins. Amino acids changes are more prevalent in the predicted β-strand/β-turn structural motif that is predicted to interact with the cognate ligand (Jones and Jones, 1997) .

(C) Synonymous and non-synonymous nucleotide substitutions in different coding regions of the RPPl family of genes coding sequences . The values shown are calculated as described previously (Parniske et al . , 1997) .

EXAMPLES

Example 1: High resolution Mapping of RPP14

The RPP14 specificity in Ws-0 was chosen as the first target for positional cloning using the corresponding P. parasi tica isolate Noco2 to characterize recombinants from a cross between Ws-0 and the Noco2-compatible accession Col-0. RPP14 was previously mapped to a 3.2 cM interval between the RFLP markers ve021 (centromeric) and pAT3-89.1 (telomeric) on chromosome 3 (Reignault et al . , 1996) . The flanking markers were used to identify yeast artificial chromosome (YAC) clones from the available Arabidopsis CIC YAC library containing DNA inserts derived from Col-0 (Creusot et al . , 1995; Botella et al . , 1997) . These YAC clones were orientated relative to each other and the RFLP markers, revealing a maximum physical interval of 800 kb between the two flanking RFLP markers, as shown in Figure 1A. The position of the RPP14 locus in Col-0 was further delimited by positioning two YAC- derived end-probes on recombinant plants (see Methods below) and using the end-probes to identify overlapping PI clones (Liu et al., 1995; Figure IB). EcoRI restriction fragments from the clones P1-37C7 and P1-53P2 were cloned and sequenced. One of the subclones (37C7-5, Figure IB) derived from P1-37C7 contained an open reading frame with similarity to the leucine-rich-repeat (LRR) region of RPP5 (Parker et al., 1997), although no hybridization was detected in the YAC encompassing the Col-0 RPP14 region when RPP5 was used in low stringency hybridization (data not shown) . Based on this homology, we considered it likely that the 37C7-5 sequence was part of an rppl4 allele in Col-0.

Gel blot analysis of DNA from several Arabidopsis accessions using the Col-0 37C7-5 fragment as a probe, showed the presence of a highly polymorphic multigene family (Figure 1C) . All polymorphic family members cosegregated as two separate groups corresponding with phenotypic classes of the two parental accessions, Ws-0 or Col-0. In addition, all Col-0 hybridizing bands were contained on YAC clones CIC6B1 and CIC8D7 indicating that they spanned a maximum physical distance of approximately 400 kb, the distance encompassed by the YAC clones. Three or four gene members were found in all accessions analyzed with the exception of (Nd-1) which appears to contain a more complex gene family (Figure 1C) . Most importantly, molecular evidence for a gene family in Ws-0 suggested that it should be possible to determine which genes may explain the RPPl , RPP10 and RPP14 specificities .

Example 2: Isolation of RPPl /10/14 gene candidates from Ws-0 Clones from a binary vector cosmid library (Arabidopsis Biological Resource Center, Ohio) containing Ws-0 genomic were identified using the 37C7-5 Col-0 DNA probe and assembled into two contigs, as shown in Figure 2A. Two members of the gene family were identified in this library including homologue A (Hom-A) contained in cosmid ws-cos 65.1, and Hom-B contained in cosmid ws-cos 69.12. A third family member, Hom-C (contained in phage 1.1), was identified by screening a Ws-0 genomic DNA phage library (Dietrich et al . , 1997). A 1.1 kb Hindlll DNA fragment from ws-cos 65.1 was identified that distinguished between the different gene copies (Figure 2A) . Gel blot analysis of DNA from Arabidopsis accessions Ws-0 and Col-0 using this probe revealed four hybridizing bands (Figure 2B) . Therefore, we concluded that there is a fourth member of the gene family, designated Hom-D .

However, no cosmid or phage clones were identified that contained this homologue. The EcoRI and Hindlll fragment patterns deduced from the sequence of the Ws-0 candidate genes correspond to the hybridizing fragments assigned to Ho -A, Hom-B, and Hom-C in Figure 2B.

Example 3: Determining function of Hom-A. Hom-B and Hom-C

A plant line from the Col-0 x Ws-0 mapping cross was selected that was homozygous for the disease sensitive alleles from Ws-0 at RPP2 and RPP4 and from Col-0 at RPPl/ 10/ 14 , using PCR-based markers that defined these regions on each chromosome. We confirmed that progeny from this line were uniformly susceptible to isolates Noco2 , Emoy2 and Cala2 as well as to a fourth Ws- incompatible isolate Maks9 (data not shown) . The susceptible line is denoted CW84 and was used as a recipient for Agrobacterium-mediated transformation with each of the candidate genes .

Stable transformants (T_α plants) of CW84 were obtained with binary cosmid clone ws-cos 65.1 (Hom-A; Figure 2A) , and Ws-0 genomic DNA fragments containing only Ho -A,

Hom-B or Hom-C that had been cloned into the binary vector pSLJ755I5 (see Methods) . Selfed T₂ progeny and homozygous T₃ progeny of several independent transformants for each construct were inoculated with each of the Ws- incompatible P. parasi tica isolates (Table 1:).

Table 1. Interaction phenotypes of different wild type, transgenic and mutant Arabidopsis accessions following inoculations with five isolates of Peronospora parasitica.

Arabi- Construct³ No. of No. of Resistant lines to the dopsis lines following P. parasi tica line analyzed isolates

Noco2 Emoy2 Cala2 Maks9 Emco5 1 1 1 1 0

WS- 0

Col- 0 1 0 1 1 NT 0

CW84 1 0 0 0 0 0

WS -0 1 1 1 0 NT NT rppl O

CW84 ws-65 . 1 6 6 6 6 6 0

Hom-A 12 11 11 11 11 0

Hom-B¹ 12 12 12 0 12 0

Hom-C 12 12 0 0 0^C 0

CW84 - 2 NT

Hom.A

X eds 1

F2^{d a}The constructs used for transformation are shown on

Figure 2A. The complete ws-cosmid was used in the transformation. "The interaction phenotypes in these lines were characterized for a low level of P. parasitica . sporulation in most of the lines. Only 4 lines were tested for the interaction phenotype. ^dA Ler-eds 1 mutant was used in the cross (Parker et al . , 1996) . NT: not tested .

Hom-A conferred resistance to all four isolates. Hom-B conferred partial resistance (see below) to the isolates Noco2, Emoy2 and Maks9, but was fully susceptible to

Cala2 ; and Hom-C specified resistance only to Noco2. All transgenic lines were also tested against Emco5 (Table 1) , an isolate that is compatible in both Ws-0 and Col-0, thus confirming that the transgenes were specific only to the Ws-0-incompatible isolates. Resistance in Ws-0 to isolates Noco2, Emoy2 and Cala2 is known to require another component encoded by the EDS1 gene (Parker et al., 1996; Aarts et al., 1998b). Analysis of F₂ progeny from a cross between a CW84 containing multiple copies of Hom-A and a Ws-0 edsl mutant line, edsl -1 , showed that

EDS1 was also necessary for the function of this transgene (Table 1) .

The response phenotypes of wild-type Ws-0, CW84 and CW84 transgenic lines containing Ho -A, Hom-B or Hom-C after inoculation with isolate Noco2 were assessed in nine- day-old seedlings, 6 days after spray-inoculation with P. parasitica isolate Noco2. Phenotypes were monitored macroscopically with a hand lens and microscopically by observing lactophenol-trypan blue stained leaves (data not shown) . As in leaves of wild-type Ws-0, Hom-A conferred effective resistance to Noco2 in transgenic CW84. Asexual sporulation was never observed in leaves following inoculations of either transgenic Hom-A or wild-type resistant plants. A similar phenotype with no sporulation was observed in transgenic Hom-A plants inoculated with isolates Emoy2 , Maks9 and Cala2 (not shown) . Resistance specified by Hom-C to Noco2 appeared to be as strong in transgenic CW84 as in Hom-A . In contrast, Hom-B conferred only partial resistance to Noco2, Emoy2 and Maks9, allowing sparse asexual sporulation of all three isolates. Sporulation was nevertheless markedly less compared with heavy sporulation observed on non-transformed CW84. The different resistance phenotypes were confirmed by microscopic examination of inoculated leaves that had been stained with lactophenol-trypan blue (Reignault et al., 1996). In leaves of Ws-0 and transgenic Hom-A plants, hyphal growth was restricted to a few host cells. Hyphal growth extended further in transgenic Ho -B leaves, but this was associated with necrosis of penetrated host cells and was not as extensive as pathogen development in non-transformed CW84. Col-0 was transformed with Hom-A or Hom-B, and these transgenic plants produced the response phenotypes to Noco2 as observed in CW84, indicating that the different phenotypic expressions of Hom-A and Hom-B were not a consequence of the recombinant background of CW84.

The pathology data demonstrated that three members of the gene family are functional downy mildew resistance genes and that each exhibits a unique specificity for recognizing different combinations of P. parasitica isolates. Hereafter, we refer to each of the functional homologues as RPPl -WsA, RPPl -WsB and RPPl -NsC for Hom-A,

Hom-B and Hom-C, respectively. This nomenclature in no way limits the scope of the invention, but will be useful in the future for designating functional homologues from other accessions, such as may be isolated using these sequences. The locus and family of genes, regardless of accession, is designated hereafter as the "RPPl complex locus" and "RPPl gene family".

Example 4 : DNA structure of three functional genes within the RPPl complex locus A 2.8 kb EcoRI fragment from RPPl -WsA was used as a probe to isolate cDNAs from a Ws-0 silique cDNA library and several partial cDNAs were characterized (see Methods) to confirm the location of introns within the three functional genes. Their structure and organization is shown in Figure 4A. The positions of intron-exon splice junctions are conserved in all three genes except at the 5' termini. Here, two additional introns were identified in the RPPl -WsB and RPPl -WsC alleles, one located in the 5' untranslated region and the second 181 bp (RPPl -WsB) and 169 bp (RPPl -WsC) downstream from their predicted start codons .

Example 5 : The predicted protein products of the RPPl gene family

These three genes encode similarly sized proteins of 1189, 1221, and 1217 amino acids, respectively, that possess a high overall identity (Figure 4B) . Comparison of their predicted amino acid sequences with Genbank database sequences showed that they are most similar to the products encoded by RPP5 (Parker et al., 1997), N

(Whitham et al., 1994) and LS (Lawrence et al . , 1995) that are all functional R proteins containing a nucleotide binding site (ΝB) and carboxy-terminal leucine-rich-repeats (LRRs) . This affiliation includes amino-terminal similarity to the cytoplasmic domains of the Drosophila Toll and mammalian Interleukin-1 receptors, the so-called "TIR" domain (Figure 4B, see also Figure 5) . Like RPP5 , Ν and L6 , the kinase-la (P loop; GPPGIGKTT) , kinase2 (FLVLD) and kinase3a (FGPGSR) consensus motifs that constitute a predicted ΝBS (Traut, 1994) are contained entirely within exon 2 of RPPl-WsA or exon 3 of RPPl-WsB and RPPl-WsC (Figure 4) . The spacing between these three motifs is consistent with that found in known ATP and GTP binding proteins (Traut, 1994) . The C-terminal portion of the RPPl family of proteins can be divided into two regions (Figure 4B; Figure 6) . The first is composed of imperfect LRRs conforming to the canonical LRR consensus for cytoplasmic LRR proteins (Jones and Jones, 1997) . Like other TIR-NB-LRR products a cysteine residue is frequently found in the β-turn region after the LXXLXLXX motif (Jones and Jones, 1997) . Ten LRRs varying in length from 21 to 24 amino acids were identified in RPPl-WsA and RPPl-WsB, and nine LRRs were identified in RPPl-WsC (Figure 6) . The second part in the carboxy-terminus does not contain LRRs and the proportion of leucine residues is only 12% compared to 22% in the LRR region.

The most striking differences in the predicted proteins were found at their N-termini (Figure 4B; Figure 5A) . A hydrophobic domain, predicted to be a membrane anchor (Von Heijne, 1986) was located in the RPPl-WsA protein that extends its N-terminus by 40 amino acids relative to RPP5 and N. An N-terminal signal peptide has also been predicted for the L6 protein (Lawrence et al., 1995) although no sequence homology was found in this domain between RPPl-WsA and L6 (Figure 5A) . An additional exon in RPPl-WsB and RPPl-WsC (Figure 4B, Figure 5A ) extended the N-terminal ends of the encoded proteins into a strong hydrophilic region beyond the TIR domain (Figure 5B) . A BLAST search using the first exon of RPPl-WsB and RPPl- WsC did not reveal significant homology in the database and its role is unclear.

Example 6 : Identification of mutant alleles at the RPPl complex locus

Ws-0 seedlings derived from ethane-methyl sulphonate- (EMS-) mutagenized M₂ seeds and the Feldmann Ws-0 T-DNA tagged lines (see Methods) were inoculated with Noco2, Emoy2 or Cala 2 and screened for mutations from disease resistance to susceptibility. No mutations to Noco2 and Emoy2 were found that mapped to the .RPPl complex locus .

This can be explained by the presence of more than one gene conferring resistance to those isolates (Table 1) . However, a mutant line that had lost resistance to Cala 2 but retained the wild-type resistance response to Noco2 and Emoy2 was identified (Table 1) . Analysis of F₂ progeny derived from backcrossing the mutant with Ws-0 showed that the recessive mutant phenotype did not co-segregate with the presence of the T-DNA. The mutation was shown to be linked to the morphological marker gl -1 , which is tightly linked to the RPP1/10/14 locus. Based on complementation analysis of the wild-type RPPl homologues (Table 1) we anticipated that this mutant should contain a defective allele of RPPI O . Sequence analysis of RPPIO in the mutant revealed the presence of an in frame deletion of 33 nucleotides encoding 11 amino acids in the NBS domain (Figure 6A) that precede a hydrophobic amino acid motif highly conserved in NBS/LRR resistance proteins (Parker et al., 1997; Hammond-Kosack and Jones 1997) . We have therefore designated this Ws-0 mutant as rppl-IVsA.

Example 7 : Variation among .RPPl gene family members in non-synonymous nucleotide substitutions encoding the solvent-exposed residues of the LRRs

The LRR domain is proposed to confer recognition specificity of ligand binding due to its capacity to evolve new configurations by deletion, insertions and nucleotide substitutions (Jones and Jones, 1997; Parniske et al., 1997) . For example, a deletion of a complete LRR was found in RPPl-WsC compared with RPPl-WsA and RPPl-WsB (Figure 6) . Further comparison of amino acid differences in the LRRs among RPPl-WsA, RPPl-WsB and RPPl-WsC from Ws-0 showed that most changes are found in the residues predicted to be solvent exposed (37-42%) while only 5-8% take place in the rest of the LRR domain (Figure 6B) . Interestingly, in the LRR domain, it is only in solvent exposed positions that amino acid substitutions in all three proteins are found.

Comparison of synonymous (Ks) and non-synonymous (Ka) substitution rates per synonymous/non-synonymous site can be used to determine the type of selection acting on a gene family. When the Ka/Ks ratio is 1 or similar, a ratio that has been observed in pseudogenes, no selection pressure is operating (Hughes, 1995) . A Ka/Ks ratio greater than 1 suggests that diversifying selection has influenced the evolution of the gene family (Hughes and Nei, 1988; Parniske et al . , 1997). The rates of synonymous and non-synonymous nucleotide substitutions among members of the RPPl gene family were calculated for the XX(L)X(L)XX sequence of the LRRs, the non-consensus amino acids of which are predicted to be solvent exposed (Kobe and Deisenhofer, 1993, 1994; Parniske et al . , 1997; Thomas et al., 1997) . The rate of non-synonymous substitutions (Ka) in this region was more than seven times higher than that observed in other regions of the protein. However, the rate of synonymous substitutions was found to be similar (Figure 6 C) . The value for the Ka/Ks ratio in the XX(L)X(L)XX region is greater than 1, suggesting that positive selection for diversification in the predicted ligand binding domain has affected the evolution of the .RPPl gene family.

Example 8: Introduction of RPPl genes into Brassica

Transformation of B . Napus Plants

Transformation of B . Napus cotyledons may be achieved with GV3101 according to the method of Maloney et al (1989) Plant Cell Reporter 8, 238-242. Transformed cotyledon cells are selected by plating the cotyledons on agar containing phosphophenylthricane (PPT) . Transformed plants are grown to maturity and seeds harvested. Seeds are germinated and screened for resistance to P.

Parasitica isolate R3 , in a seedling assay according to the method of Luca J.A et al 1988 Plant Pathology 37, 538-545. Resistant plants are selected.

Alternatively, B napus may be transformed using the methodology of Schroder et al (1994) Physiologia Plantarum 92: 37-46.

Transformation of B . Oleracea Plants

Transformation of B . Oleracea cotyledons (such as broccoli and cabbage) may be achieved with GV 3101 following the methods outlined above. Seeds are germinated and screened for resistance to P. Parasitica isolates and isolates of the closely related pathogen Albugo Candida (causative agent of the disease White

Blister) in a seedling assay according to the method of Leckie D., et al., 1994, Acta Horticulturae, 407, 95-102. Resistant plants are selected.

Alternatively, and more preferably, transformation of B . oleracea is achieved using an Agrobacterium rhizogenes- mediated co-transformation system. An optimised procedure for co-transformation of brassica explants is described by Puddephat et al (1997) "Agrobacterium rhizogenes-mediated co-transformation of Brassica oleracea: selection of transgenic roots by GUS assay". ISHS Symposium on Brassicas, 10th Crucifer Genetics Workshop, 23-27 September 1997 Renne-France . Book of Abstracts pl44. This technique permits the production of transgenic lines within a common genetic background that are free from ancillary DNA sequences, including marker genes such as antibiotic resistance genes. Selection of transformation events is achieved by screening explants 4-5 weeks after Agrobacterium inoculation for expression of a reporter e.g. β-glucuronidase or green fluorescent protein. The technology permits the pyramiding of transgenes in the same genetic background while minimising the build up of homologous DNA sequences (ie presence of multiple copies of the same gene sequence) which could lead to instabilities in gene expression (Puddephat et al, 1996, "Transformation of Brassica oleracea L. :a critical review."

Molecular Breeding, 2, 185-210) . Other techniques for transforming B . oleracea include that used by Bhalla and Smith (1998) Molecular Breeding 4: 531-541.

Transformation of Lettuce (Lactuca sativa)

Transformation of Lactuca sativa cotyledons is achieved with GB 3101 according to the method of Enomoto S., et al., 1990, Plant Cell Reporter, 9, 6-9 and Dede Y and Buchanan-Wollaston V, 1997, Turkish Journal of Agriculture and Forestry 21, 543-550.

Transformed cotyledon cells are selected by plating the cotyledons on agar containing PPT. Transformed plants are grown to maturity and seeds harvested. Seeds are germinated and screened for resistance to Bremia lactucae

(the causative agent of downy mildew in lettuce) in a seedling assay according to the method of Crute I.R and Lebeda A 1981 Theoretical and Applied Genetics 60, 185- 189. Bremia lactucae resistant plants are selected.

Mul tiple transgenes

Plants transformed as outlined above may be transformed individually with RPPl, A, B and C in individual constructs . To combine these individual genes in a single plant, crosses are made and individuals containing any two gene copies or all three genes are identified by amplifying parts of the resistance genes using PCR from primers designed to distinguish the individual genes.

DISCUSSION OF EXAMPLES 1 TO 7

At least three Arabidopsis RPP specificities to P. parasi tica isolates Emoy2 (RPPl ) , Cala2 (RPPIO) and Noco2

(RPP14) have been shown to be closely linked on the long arm of chromosome 3 in accession Ws-0 (Holub et . al 1994; Reignault et al, 1996 ) . In the present study we have cloned and assigned a recognition function to three out of four genes that belong to a physically tightly linked R gene family. The resistance phenotypes conferred by these three genes can account for the three RPP specificities identified in this region. The isolation and characterization of the RPPl gene family now permits detailed analysis of the molecular mechanisms underpinning parasite recognition and the selective forces directing the evolution of novel recognition specificities at a complex R gene locus in Arabidopsis. The fourth gene may be analysed by methods analogous to those disclosed herein.

Three RPPl gene family members have distinct but overlapping functions

The recognition specificities conferred by the three RPP genes cloned and individually analyzed in this study were distinguished using four Ws-0- incompatible P. parasi tica isolates (Table 1) . All three genes (RPPl -WsA, RPPl -WsB and RPPl -WsC) conferred resistance to Noco2 , two genes conferred resistance to Emoy2 and Maks9 (RPPl -WsA and JRPPI-JVSJB) , and only one gene (RPPl -WsA) specified resistance to Cala2. These results suggest that the RPPl- WsA protein recognizes an AVR determinant that is common to all four isolates. Thus, RPPl-WsB is likely to be interacting with a different AVR determinant that is present in Noco2, Emoy2 and Maks9 but absent from Cala2. Similarly, RPPl-WsC must be recognizing an AVR determinant that is unique to Noco2. Thus, including RPP5, R genes are now in hand that recognize four different Avr gene products of Noco2.

Differences between RPPl -WsA . RPPl -WsB and RPPl -NsC resistance phenotypes

Interestingly, RPPl -WsA and RPPl -NsC conferred a strong resistance whereas RPPl -WsB specified only partial resistance. Although the different recognition specificities conferred by RPPl -WsA, RPPl -WsB and RPP1 - WsC indicate genetic variation Avr genes in the parasite isolates used in this study, the phenotype of the R gene- mediated response is also likely to be influenced by a number of other factors. R and AVR protein expression levels, the timing of AVR gene expression during pathogen development, the zygotic condition of alleles at the Avr loci (P. parasi tica is a diploid organism) , and the efficiency of delivery of the parasite's AVR or compatibility signals to the plant, could all affect the phenotype .

Predicted functional domains of the RPPl proteins

The three RPPl gene family members identified and characterized here add to the set of predicted NBS-LRR proteins that have been assigned recognition function in plant disease resistance (Bent, 1996) . These proteins possess the structural attributes that would fulfil two anticipated functions: recognition specificity with potential for adaptive variability within the LRRs (see also below) , and conserved motifs that may direct transduction of common signals in the defence response .

Highest RPPl amino acid sequence conservation was observed with RPP5, N and L6 over the TIR domain (Figures 5B and 6) . Therefore, RPPl-WsA, RPPl-WsB and RPPl-WsC can be considered to belong to the TIR-NBS-LRR subclass of NB-LRR proteins that possesses N-terminal similarity to Toll and Interleukin-1 (Hammond-Kosack and Jones, 1997 ) . Members of this subclass that have been characterized in Arabidopsis are dependent on a second gene, EDS1 , for resistance function (Aarts et al . , 1998b). An unexpected observation was the presence of additional introns at the 5 ' end of the .RPPl and RPP14 genes that give rise to hydrophilic N-terminal extensions beyond the TIR domain and the N-termini of the predicted RPP5, N and L6 proteins (Figures 4 and 5) . The role of these extensions is not clear and no proteins with homology to these regions have been detected in the databases . Unlike RPPl and RPP14, and like L6, RPP10 possesses an N-terminal extension of 40 amino acids relative to both RPP5 and N. This region is a potential signal anchor, suggesting that the protein may become membrane-associated. Further studies are required to establish whether the differences in the respective N-termini are reflected in different cellular locations. Whatever their role, they appear not to correlate with the strength of the resistance phenotypes observed here or with a differential dependence on EDS1 , since resistance to Noco2 was fully abolished in a Ws-edsl background (Parker et al . , 1996) .

It is predicted that the NB domain binds ATP or GTP as part of a conserved and essential feature of pathogen recognition specified by the NB-LRR R protein class . Like other NB-LRR R proteins, RPPl proteins carry extended homology to APAF-1 and CED-4, regulators of cell death in animal cells (Van der Biezen and Jones, 1998) . It is postulated that this region, designated the "NB-ARC" domain by Van der Biezen and Jones (1998) , may serve an adaptor function that transduces information from an LRR- modulated recognition event to a common signaling element such as the TIR domain.

Evolutionary forces operating at the .RPPl locus

Molecular analysis of three out of four genes comprising the .RPPl complex locus points to at least two tandem duplications followed by sequence divergence giving rise to homologues A, B, C and D. Duplicated functional genes may provide several selective advantages . Their arrangement could allow multiple specificities to be assembled and retained in a single haplotype, thus preserving the potential for variation and the evolution of novel specificities through mis-pairing, intergenic recombination, and gene duplication (Parniske et al . , 1997; Thomas et al . , 1997) . Such mechanisms would be expected to generate rapidly novel gene variants. The higher number of homologous RPPl sequences present in the

Nd-1 accession (Figure 2C) probably indicates a locus- dependent and intraspecific copy number expansion. Genetic and restriction fragment length polymorphism (RFLP) analysis in Nd-1 demonstrated that all polymorphic bands mapped to the same location as the members of the Ws-0 .RPPl locus (data not shown) . The presence of functional genes at the RPPl complex locus can be assumed to be a result of an active selection pressure from the pathogen. RPPl -WsA, RPPl -WsB and RPPl -WsC all recognize

Noco2 and this redundancy would allow diversification of one of the genes by relaxing the selection pressure on the remaining genes in the complex. If, as we surmise, the RPP genes in the cluster recognize different AVR determinants, their presence would provide a clear selective advantage against pathogen variants that have lost individual Avr genes through mutation. There is evidence that sequence exchange has taken place between different RPPl genes of Ws-0 (data not shown) . Shuffling of sequences is considered a major force in generating gene diversity. Indeed, DNA shuffling has been proven to be a powerful process for directed evolution by recombination to combine useful variation from homologous genes (Crameri et al., 1998).

It is envisaged that the LRR domain confers specificity in pathogen recognition due to its capacity to evolve new configurations (Jones and Jones, 1997) . Analysis of several alleles from the L locus and chimeric genes in flax supports this view (Ellis et al . , 1997) . Two key mechanisms are proposed to create sequence diversity within the LRRs. In the first, deletion or insertion of complete LRRs may occur by intragenic crossing over. This was revealed in a mutant allele of RPP5 that has an in- frame intragenic duplication of four LRRs without loss of function (Parker et al . , 1997). In the second, an enhanced rate of non-synonymous nucleotide substitutions in the solvent-exposed residues of the LRRs appears to operate, as shown for members of the Cf4/9 locus (Jones and Jones, 1997; Parniske et al . , 1997) . Evidence for both mechanisms were observed in the three genes of the .RPPl complex. A deletion of a complete LRR was found in

RPPl-WsC (Figure 6) . Deletion of a different LRR was also observed in a Col-0 rppl allele.

We observed a high degree of sequence variation in the interstitial amino acid residues that are predicted to be solvent exposed in the LRR β-strand/β-turn region, based on the structure of the porcine ribonuclease inhibitor (Kobe and Deisenhofer, 1995) . Within the genes of the RPPl complex locus, the high degree of polymorphism observed in those residues supports a role for these sequences in recognition specificity, as was shown previously for genes of the tomato Cf4/9 complex. Here we present evidence that sequence diversity is positively selected for (as indicated by a high Ka/Ks ratio) in a region predicted to be involved in specificity. In contrast, the remainder of the protein exhibits a Ka/Ks ratio of around 1. We conclude that evolution of the LRRs in different structural classes of R gene product from different plant species has been brought about by very similar mechanisms and selective forces .

GENERAL METHODS IN EXAMPLES 1 TO 7

Cultivation of Arabidopsis plants and pathogenicity tests

The origins of Columbia (Col-0) and Wassilewskija (Ws-0) were as reported by Reignault et al. (1996) . Col-0 x Ws-0 F₂ seed were used for the mapping of RPP14 . The Vls-rppl O mutant line was selected from the K. Feldmann T-DNA lines (Feldmann, 1992) . The conditions for plant cultivation, maintenance of P. parasi tica and pathogenicity tests were as described previously (Holub et al . , 1994; Parker et al. , 1997) .

Analysis of recombinants

Analysis of 90 F2 seedlings (or corresponding F3 families) generated from a cross between Col-σl and Ws-0 and 48 F2 seedlings generated from a cross between Col-grl and Pr-0 showed linkage of RPP14 to the marker Gl - 1 on chromosome 3. The Col-g x Ws-0 and Col-gl x Pr-0 populations were considered together in our efforts to clone RPP14 because an allelism test between Ws-0 and Pr-

0 showed a complete cosegregation between the two resistance loci (Reignault et al . , 1996).

Plant genomic DNA preparations

Large-scale plant genomic DNA preparations and rapid, small-scale DNA samples were made as described previously (Parker et al . , 1993; Reignault). The method of Klimyuk et al., (1993) was used to prepare denatured DNA suitable for polymerase chain reaction (PCR) amplification.

DNA manipulations

A binary Ws-0 library (Arabidopsis Biological Resource Center, Ohio) and phage Ws-0 genomic libraries (UNC, Chapel Hill) were screened using the 37C7-5 probe. Three binary cosmid clones were obtained that contained Ho -A and two cosmid clones were obtained that contained Hom-B .

A genomic phage (1.1) was obtained containing Hom-C.

Cosmid subclones were inserted into the binary cosmid vector pSLJ755I5 and cultured in the presence of 50 mg/L tetracycline. PI clones were cultured in the presence of 50 mg/L kanamycin. DNA of these clones were obtained using the alkaline lysis method (Liu et al . , 1995) . Fingerprinting of cosmids and PI clones was performed by digestion of the DNA with several restriction enzymes, end-labeling with P³³ and separating the fragments on a sequencing gel. Several partial cDNAs were identified in a Ws-0 cDNA library derived from Arabidopsis siliques (Castle and Meinke, 1994, Arabidopsis Biological Resource Center, Ohio) using a 2.8 kb EcoRI probe from Hom-A (see Figure 2A) . Yeast artificial chromosome DNA manipulations

YACs from the CIC library (Creusot et al . , 1995) were identified as described previously (Botella et al., 1997) . Left-end or right-end YAC DNA probes were generated by inverse-PCR using YAC vector nested primers, as described previously (Schmidt and Dean, 1995) or thermal-asymmetric-interlaced (TAIL) -PCR (Liu and Whittier, 1995) . The end probes derived from the YAC clones were agarose gel-purified and then used to probe plant genomic DNA blots . YACs were orientated relative to each other probing YAC ends with filters containing YAC clones DNA.

Sequence Analysis

A shotgun cloning approach was employed to determine the nucleotide sequence of the three genes. Escherichia coli

(DH5α) transformants harbouring recombinant clones containing Arabidopsis DNA subcloned in pUC18 were identified by hybridization to insert DNA isolated from the cosmids ws-cos 65.1, ws-cos 69.12 and phage 1.1 (Figure 2A) . M13 universal forward and reverse primers were employed to determine end sequences, using the Dye Deoxy terminator cycle sequencing method (Applied

Biosystems [ABI] , La Jolla, CA) and an ABI model 377 sequencing system. Sequence contigs were assembled using UNIX versions of the Staden programs package (Roger Staden, MRC, Cambridge, UK) . Computer-aided sequence similarity searches were made with Blast (Altchul et al . , 1990) programs and the National Center for Biotechnology Information (Bethesda, MD) nucleotide and peptide sequences databases. Secondary structure predictions and determination of signal peptides were made with the predict programs

(http: //www. cmpharm.ucsf .edu/^~nomi/nnpredict .html) and (http: //cookie. imcb.osaka-u.ac.jp/nakai/psort .html) Construction of subclones containing Hom-A. Hom-B and Hom-C

All subclones containing members of the RPPl gene family were constructed in the binary vector pSLJ755I5

(http://www.uea.ac.uk/nrp/jic/s3d_plas.htm). For psub- Hom-A, a 7 kb DNA fragment originating from a partial Tspel digestion of the 65.1 ws-cosmid clone, was subcloned into pUC118. A Kpnl fragment obtained from Hom- A from a Kpnl site located 2756 bp upstream of the predicted ATG translation start codon and the Kpnl site of the pUCllδ polylinker was subcloned into the Kpnl site of the pOK12 vector (Vieira and Messing, 1991) . The Spel fragment liberated from the vector was subcloned into the compatible Xbal site of pSLJ755I5. For psub-Hom-B, an 11 kb Pst fragment derived from ws-cos 69.12 was subcloned into the PstI site of the binary vector. For psub-Hom-C, a Sail-Spel fragment and a Spel-EcoRI fragment derived from phage 1.1 were subcloned into the Xhol-EcoRI site of the binary vector using a 3-way ligation. Arabidopsis transformation

The whole plant infiltration method of Bechtold et al . (1993) was used for the transformation experiments. Transformed (T_τ) seedlings were selected on Arabidopsis medium (Bechtold et al . , 1993) containing 50 mg/L of hygromycin for selection of ws-cos 65.1 transformants. For all the constructs made using the pSLJ755I5 vector, transformed (T_:) seedling were selected by spraying L- phosphinotricin at 100 mg/L on young seedlings.

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Claims

1 An isolated nucleic acid molecule comprising an RPPl nucleotide sequence which encodes a Peronospora parasi tica resistance gene of the RPPl complex locus.

2 A nucleic acid as claimed in claim 1 comprising an RPPl nucleotide sequence which encodes one of the following amino acid sequences in Figures 3A-3K.

Seq ID No 3 labelled RPPl-WsA peptide Seq ID No 6 labelled RPPl-WsB peptide Seq ID No 9 labelled RPPl-WsC peptide

3 A nucleic acid as claimed in claim 1 or claim 2 comprising an .RPPl nucleotide sequence identical to or degeneratively equivalent to any one of the following nucleotide sequences in Figures 3A-3K:

Seq ID No 1 labelled .RPPl-WsA genomic Seq ID No 2 labelled .RPPl-WsA cDNA Seq ID No 4 labelled RPPl-VlsB genomic Seq ID No 5 labelled RPPl-WsB cDNA Seq ID No 7 labelled RPPl-WsC genomic Seq ID No 8 labelled RPPl-WsC cDNA

4 An isolated nucleic acid which comprises a variant .RPPl nucleotide sequence which variant shares at least about 50%, 60%, 70%, 80% or 90% homology with the RPPl nucleotide sequence of claim 2 or claim 3.

5 A nucleic acid as claimed in claim 4 wherein the variant RPPl nucleotide sequence is an active P. parasi tica resistance gene which encodes a polypeptide capable of recognising and activating a defence response in a plant in response to challenge with a P. parasi tica isolate or an elicitor or Avr gene product thereof.

6 A nucleic acid as claimed in claim 4 or claim 5 wherein the variant RPPl nucleotide sequence is a naturally occurring homolog of the RPPl nucleotide sequence of claim 2 or claim 3.

7 A nucleic acid as claimed in claim 6 wherein the variant .RPPl nucleotide sequence is obtainable from

Arabi dopsi s thaliana .

8 A nucleic acid as claimed in claim 7 wherein the variant .RPPl nucleotide sequence is an allele of the RPPl nucleotide sequence of claim 2 or claim 3.

9 A nucleic acid as claimed in claim 4 or claim 5 wherein the variant RPPl nucleotide sequence is an artificial derivative by way of one or more of addition, insertion, deletion or substitution of one or more nucleotides of the RPPl nucleotide sequence of claim 2 or claim 3.

10 A nucleic acid as claimed in claim 9 which encodes a modified RPPl polypeptide by way of addition, insertion, deletion or substitution of one or more amino acids.

11 A nucleic acid as claimed in claim 10 wherein an LRR region of the polypeptide has been modified such as to alter the specificity of the polypeptide with respect to a P. parasi tica isolate or an elicitor or Avr gene product thereof .

12 A nucleic acid as claimed in claim 5 or claim 11 wherein the P. parasi tica isolate is selected from: Emoy2 , Cala2 , Noco2.

13 An isolated nucleic acid which comprises a complementary RPPl nucleotide sequence which is the complement of the RPPl nucleotide sequence of any one of the preceding claims .

14 A method of producing the nucleic acid of any one of claims 9 to 11 comprising the step of modifying the nucleic acid of claim 2 or claim 3.

15 An isolated nucleic acid which is an RPPl probe or primer consisting of a sequence of at least about 15, 18, 21, 24 or 30 contiguous nucleotides of the .RPPl nucleotide sequence of claim 2 or claim 3 or the complement thereof .

16 A method of identifying, cloning or amplifying an .RPPl nucleic acid of any one of claims 1 to 13, which method employs a nucleic acid of claim 15.

17 A method as claimed in claim 16 comprising the steps of:

(a) providing a preparation of nucleic acid from plant cells,

(b) providing probe or primer as claimed in claim 15,

(c) contacting nucleic acid in said preparation with said probe or primer under conditions for hybridisation, and

(d) identifying the .RPPl nucleic acid if present by its hybridisation with said probe or primer.

18 A method as claimed in claim 17 wherein the RPPl nucleic acid is amplified by PCR using a pair of primers, at least one of which is a primer as claimed in claim 15.

19 A nucleic acid as claimed in any one of claims 1 to 13 which is a vector.

20 A nucleic acid as claimed in claim 19 wherein the vector comprises a promoter operably linked to the RPPl nucleotide of any one of claims 1 to 13.

21 A nucleic acid as claimed in claim 19 or claim 20 which is a plant vector.

22 A method comprising the step of introducing the nucleic acid of any one of claims 1 to 13 into a host cell.

23 A host cell containing a heterologous nucleic acid of any one of claims 1 to 13.

24 A method of transforming a host cell, which method comprises introducing the vector of any one of claims 19 to 21 into the host cell, and causing or allowing recombination between the vector and the host cell genome .

25 A host cell transformed with a vector of any one of claims 19 to 21.

26 A host cell as claimed in claim 23 or claim 25 which is a plant cell.

27 A method for producing a transgenic plant, which method comprises the steps of:

(a) performing a method as claimed in claim 24,

(b) regenerating a plant from the transformed plant cell.

28 A transgenic plant which is obtainable by the method of claim 27, or which is a clone, or selfed or hybrid progeny or other descendant of said transgenic plant, which in each case includes the plant cell of claim 26. 29 A part or propagule of a plant of claim 28 which includes the plant cell of claim 26.

30 A method of influencing or affecting the degree of resistance of a plant to a fungus, the method including the step of causing or allowing expression of a heterologous nucleic acid as claimed in any one of claims 1 to 13 within the cells of the plant.

31 A method as claimed in claim 30 wherein the fungus is P. parasi tica

32 A polypeptide which is encoded by the .RPPl nucleotide sequence of any one of claims 1 to 12.