EP0981622A1 - A plant disease resistance signalling gene, materials and methods relating thereto - Google Patents

Abstract

Provided are nucleic acids encoding wild-type and mutant EDS1 which may be used to modulate (either enhance or inhibit) a pathogen-resistance response in plants. Variants of the sequences are also provided as are methods of obtaining them, for instance using probes or primers. Also disclosed are wild-type, mutant and variant EDS1 proteins, which may be used (inter alia) as esterases. Vectors, host cells, plants employing heterologous EDS1 sequences, and various methods of use of these are also provided.

Description

A PLANT DISEASE RESISTANCE SIGNALLING GENE: MATERIALS AND METHODS RELATING THERETO.

The present invention relates to a plant disease resistance signalling gene and to materials and methods relating thereto. In particular the invention relates to the EDSl gene

Background Diseases of crop plants are a major cause of yield loss particularly when crop cultivars are grown as large monocultures. However, most plants are resistant to most pathogens and have evolved an array of pre-existing and inducible defences that stop pathogen invasion. Some pathogens have become specialized to overcome the plant defences and are able to rapidly colonize the plant . These pathogens cause disease and the interaction is known as "compatible" .

Resistance to diseases caused by many different microbial pathogens is determined by complementary gene pairs in the plant and the pathogen (Flor,1971) . These are referred to respectively as R (plant) genes and avr (pathogen) genes and their expression leads to a resistant plant response and an "incompatible" interaction. R gene-mediated resistance is often correlated with localized plant cell necrosis known as the "hypersensitive response" or HR (Hammond-Kosack and Jones, 1996) . Other plant defence-related responses include the accumulation of salicylic acid, an oxidative burst, cell wall reinforcements and the local and systemic induction of genes encoding pathogenesis-related (PR-) proteins (Ryals et al . , 1996). The precise role of these processes in limiting pathogen ingress is unclear.

The plant resistance response causes the pathogen to stop growing and therefore prevents it from eliciting disease symptoms, causing yield loss and spreading to other host plants. Thus, introgression of J? genes from wild relatives to crop cultivars by plant breeding techniques has been used extensively in plant disease management . A disadvantage of such a strategy is that it is normally highly specific to a pathogen race and it takes years for plant breeders to produce new resistant varieties. Also, many R genes are not durable because they can be rapidly counter-selected or "broken down" by mutations in the pathogen towards virulence (Crute and Pink, 1996) .

R genes conferring race-specific {avr gene-specific) resistance have recently been cloned from several plant species including the model crucifer, Arabidopsis thaliana . Analysis of the predicted protein sequences places these known R genes in distinct classes .

The tobacco N gene ( hitham et al . , 1994) , the flax L6 gene (Lawrence et al . , 1995), and the Arabidopsis RPS2 (Bent et al . , 1994; Mindrinos et al . , 1994) and RPM1

(Grant et al . , 1995) genes encode proteins containing a nucleotide binding site (NBS) and leucine-rich repeats (LRRs) . The proteins mediate resistance, respectively, to viral , fungal , and bacterial pathogens and are probably localized in the cytoplasm.

The tomato Cf-9 (Jones et al . , 1994) and Cf-2 (Dixon et al . , 1996) gene products specifying resistance to a fungal pathogen, also contain LRRs but are predicted to be predominantly extracytoplasmic with a C-terminal membrane anchor. The Hsl^pro1 nematode resistance gene of sugar beet is similarly membrane anchored but has a comparatively short extracellular leucine-rich region (Cai et al. , 1997) .

In contrast, the tomato Pto gene (Martin et al . , 1993; Zhou et al . , 1995), controlling resistance to a bacterial pathogen, encodes a functional serine/threonine protein kinase and is therefore quite distinct from the other two classes. However, for its function, it is now known to require an LRR-containing protein, Prf, that has sequence similarities with the NBS-LRR class of cytoplasmic R proteins (Salmeron et al . , 1996).

A fourth class is represented by the rice Xa21 gene for bacterial resistance (Song et al . , 1995). This encodes a protein that possesses predicted extracellular LRRs, a membrane spanning domain, and an intracellular serine/threonine protein kinase domain, thus possibly coupling a role in both cell surface recognition and intracellular signalling.

A limited number of motifs are shared among R proteins and this reinforces the notion that disease resistance to different pathogen types in a diverse range of plant species may operate through similar pathways. This idea was further strengthened by sequence analysis of the cloned Arabidopsis R gene RPP5 , conferring resistance to the downy mildew oomycete pathogen, Peronospora parasi tica (Parker et al . , 1997; Patent Application No: PCT/GB96/00849) . The predicted RPP5 protein shows striking similarity to the tobacco N and flax L6 products and is less similar to other Arabidopsis R proteins, RPM1 and RPS2, of the cytoplasmic NBS-LRR class (Parker et ai . , 1997) . RPP5, N and L6 share an amino-terminal region with similarity to the cytoplasmic domains of the Drosophila Toll and mammalian interleukin-1 (IL-1R) transmembrane receptors. This is designated the "TIR" domain. The predicted RPM1 and RPS2 proteins lack this similarity but possess a potential leucine zipper (LZ) consensus motif that is not present in the RPP5 , N and L6 protein sequences.

Several lines of evidence suggest that the R gene products interact directly with the pathogen Avr proteins in a specific receptor-ligand association. Three bacterial Avr proteins, AvrB (Gopalan et al . , 1996), AvrRpt2 (Leister et al . , 1996) and AvrBs3 (van der Ackervecken et al . , 1996) are recognized only within a plant cell expressing the specific corresponding R protein. This implicates a polarized translocation mechanism by which the Avr proteins may be injected into the plant cell cytoplasm from the bacterial cells that colonize the intercellular spaces. Furthermore, direct, specific interaction between the Pto protein kinase and AvrPto has been demonstrated in a yeast two hybrid assay (Scofield et al . , 1996; Tang et al . , 1996). From these results we might reasonably anticipate that other R proteins that are predicted to be cytoplasmic, such as

RPP5, function as the intracellular cognate receptors for their corresponding Avr proteins. It is envisaged that specific binding of the Avr protein by the R protein activates a resistance signalling pathway that leads to cessation of pathogen growth.

Several disease resistance signalling genes have recently been identified by mutational analyses. In tomato, the Rcrl and Rcr2 genes are required for resistance mediated by the R gene Cf-9 to Cladosporium fulvum (Hammond-Kosack et al . , 1994) . In barley, two genes, Rarl and Rar2 are necessary for resistance conferred by Mia - 12 and several other R gene specificities to the powdery mildew pathogen, Erysiphe graminis f . sp . hordei (Freialdenhoven et al . , 1994; Jorgensen, 1996). In Arabidopsis, a defective mutant allele (the πdrl mutation) of the NDR1 gene abolishes resistance specified by RPM1 , RPS2 and RPS5 to the Pseudomonas syringae avr genes, respectively, avrB, avrRpt2 , and avrPphΞ (Century et al . , 1995). Its phenotype thus suggests that the wild type NDR1 gene encodes a protein that functions in a common pathway downstream of specific R-Avr protein recognition. The ndrl mutation reduces the resistance afforded by several RPP loci to P. parasi tica isolates (Century et al . , 1995) , implicating at least a partial involvement of NDR1 in certain i?PP-specified signalling pathways. Other mutations have been identified in both positively acting genes {NPR1/NIM1 ; Cao et al . , 1994; Delaney et al . , 1995) and negatively acting genes (eg. CPR1 and Cim genes; Bowling et al . , 1994; Ryals et al . , 1996) that function in the interpretation of signals leading to systemic acquired resistance (SAR) , or in the maintenance of the SAR response. The NPR1/NIM1 gene was recently cloned and shown to encode a novel protein that contains ankyrin repeats (Cao et al . , 1997; Ryals et al . , 1997).

The EDSl gene (Enhanced Disease Susceptibility; the subject of the present application) , has been identified by mutational analysis of Arabidopsis plants. Mutagenized stocks of the landraces, assilewskija (Ws-0) and Landsberg-erecta (La-er) were inoculated with the P. parasi tica isolate Noco2 and screened for mutations that lead to a change from resistance to susceptibility. Resistance in s-0 is conferred by the RPP14 locus on chromosome 3 (Reignault et al . , 1996) and in La-er resistance is specified by RPP5 on chromosome 4 (Parker et al. , 1993; 1997) .

The first defective allele of EDSl , edsl -1 was identified in Ws-0 and a phenotypic analysis of this mutant allele has been described by Parker et al . (1996) . Briefly, edsl -1 caused a complete suppression of RPP14-mediated resistance as well as resistance conferred by RPP1 and RPP10 , that are closely linked to RPP14 but recognize different P. parasi tica isolates, edsl -1 also suppressed RPP 12 , an R locus residing on chromosome 4.

The edsl -1 mutation resulted in a partial suppression of resistance to an Albugo Candida isolate that causes disease in Brassica oleracea (cabbage) and to five P. parasi tica isolates that are pathogens of B . oleracea and are unable to cause disease on >100 different Arabidopsis landraces. Significantly, edsl -1 caused an increased ("enhanced") susceptibility to a P. parasi tica isolate and a bacterial isolate that are normally pathogenic on Ws-0, suggesting that it is an important component both of R gene-mediated resistance responses and of pathways that are involved in restricting the development in a compatible interaction.

Resistance specified by RPM1 to the bacterial avr gene, avrB, was, however, not compromised in edsl -1 plants, indicating that not all J? genes are dependent on EDSl function. Mapping of EDSl to the lower arm of chromosome 3 and further phenotypic analyses established that EDSl is not an allele of NDR1 or NPR1 , two other Arabidopsis disease resistance signalling genes described above. Plants having the edsl mutation are responsive to 2 , 6-dichloroisonicotinic acid, which is a chemical inducer of a response that leads to heightened resistance to pathogens in distal parts of the plant. This response is called a "systemic acquired resistance" or SAR and suggests that EDSl operates upstream or independently of a pathway involving NPR1 (Parker et al . , 1996) .

R gene requirements for EDSl have been examined by the isolation of two further edsl alleles from La-er, edsl -2 and edsl -3 . They both cause complete suppression of RPP5-mediated resistance to P. parasi tica isolate Noco2. In addition to RPP5 in La-er, another RPP locus, RPP21 (recognition of P. parasi tica isolate Madil, chromosome 5; Holub and Beynon, 1997) is dependent on EDSl . However, RPP8-mediated resistance (chromosome 5) to P. parasi tica isolate Emco5 (Holub and Beynon, 1997) is EDSl-independent . The edsl -2 mutation was crossed with another wild type Arabidopsis landrace, Columbia (Col-0) , and F2 plants selected that were homozygous for edsl -2 and RPP2 (recognition of P. parasi tica isolate Cala2, chr. 4; Tor et al . , 1994). This analysis established that RPP2 fully requires EDSl function. Plants were also selected from an edsl -1 Col-0 cross that were homozygous for edsl -1 and RPP4 (recognition of P. parasi tica isolate Emwal) . This analysis established that RPP4 fully requires EDSl function.

Resistance to several bacterial avr genes was tested in Ws-edsl-1 and La-edsl-2 plants. Resistances to avrB, avrRpt2 and avrPph3 , specified respectively by RPM1 , RPS2 , and RPS5 , are not suppressed by edsl . However, RPS4 recognition of avrRPS4 (Hinsch and Staskawicz, 1996) is abolished. The effect of edsl on Arabidopsis resistance to two isolates of Erysiphe (powdery mildew fungus) , E. c ichor ace arum (USC1) and E. cruciferarum (UEA1) has also been examined . Both edsl -1 (Ws-0) and edsl -2 (La-er) plants exhibited a significantly enhanced susceptibility to both isolates compared to the corresponding wild type plants .

Described here is the cloning and uses of the EDSl gene from Arabidopsis which is involved in disease resistance conferred by several different R genes. This makes it a suitable target for the manipulation of defence pathways in order to regulate (typically raise) disease resistance. The provision of the cloned gene enables the manipulations necessary to achieve such effects .

Thus broadly speaking, the present invention relates to altering a defence response in a plant and means and methods relating to this. Typically the alteration will be one of raising the defence response of a plant to provide the plant with enhanced resistance to one or more pathogens and the present invention provides for this. The present invention also provides for the (perhaps more unusual) desire to lower/cancel the defence response of a plant to render the plant more susceptible to one or more pathogens .

The invention results from the cloning of the EDSl gene and the provision of mutant alleles thereof .

According to a first aspect of the present invention there is provided a nucleic acid molecule which comprises a nucleotide sequence encoding a polypeptide with EDSl function.

From the above, it will be appreciated by those skilled in the art that "EDSl function" refers to the ability of the EDSl gene and polypeptide expression products thereof to function in the signalling pathway leading to resistance effected by the direct or indirect interaction of certain R gene products with pathogen Avr proteins. For example, an EDSl gene is able to function in the signalling pathways for resistance to the P. parasi tica isolate Noco2 conferred by the RPP14 locus on chromosome 3 in Ws-O; the RPP5 locus on chromosome 4 in La-er. It is also functional in signalling pathways conferred by other loci e.g. RPPl , RPP21 , RPP10 , RPP12 , RPP2 , RPP4 and RPS4 as described earlier. The EDSl gene is not however apparently functional in the signalling pathway for resistance specified by RPMI to the bacterial Avr gene, avrRpml . Likewise RPPS-mediated resistance to

P. arasitica isolate Emco5 is EDSl - independent . Generally speaking, phenotypic analysis of several edsl mutations has established that the wild type gene encodes a vital component of several, but not all, R gene-mediated resistance pathways. The data suggests that R genes of the ^λ TIR-NBS-LRR' type (eg RPP5) are EDSl -dependent , whereas R genes that are of the 'LZ-NBS-LRR' class (eg RPM1 ) appear to be EDSl -independent . So far the correlation between predicted R gene structures and their requirements for EDSl functions holds true. EDSl is also required to restrict pathogen growth in several compatible or partially compatible interactions in

Arabidopsis. For example, P. parasi tica isolate Cala2 is hypervirulent on La- edsl plants compared to the genetically susceptible wild type parent La-er. Similarly, Ws-edsl plants exhibit enhanced susceptibility to the Ws-compatible P. parasi tica isolate, Emwal.

Further it appears that EDSl is involved in the mediation of plant resistance to a variety of pathogen types (ie not just bacterial) eg to bacteria, fungi and possibly even insects. This is because the present applicants have shown that Ws-edsl and La- edsl plants (which carry ineffective mutant forms of the EDSl gene) display enhanced susceptibility to two fungal pathogens, Erysiphe cruciferarum and Erysiphe cichoracearum (they are unrelated to P. parasi tica and cause powdery mildew disease) . Therefore the requirements for EDSl in

Arabidopsis may extend well beyond resistance mediated by several RPP genes and by RPS4 and may include other bacterial, fungal, viral and even insect pathogens. The function of EDSl in limiting pathogen growth in these interactions may be the same as or different from its signalling function in R gene mediated responses. EDSl protein may function by processing a molecule that is synthesized or made accessible by R protein activity. Alternatively, EDSl may itself be induced or activated by the elaboration of a signal molecule or a protein conformational change. Further, EDSl activity may be induced due to an increase in synthesis of its mRNA or in stabilization of the mRNA.

The ability to function in certain resistance signalling pathways is prejudiced by certain mutations e.g. the mutation edsl -1 in the landrace Ws-0 causes complete suppression of i?PP14-mediated resistance to the P. parasi tica isolate Noco2. Thus whilst the term "EDSl function" is used to refer to sequences which dictate an EDSl phenotype in a plant (see above) , the term ⁿedsl mutant function" is used to refer to forms of EDSl sequences which suppress or cancel an EDSl phenotype in a plant . An EDSl phenotype is characterised by the resistance effects as described above. An edsl mutant phenotype is characterised by the lowering or cancelling of resistance as described above. EDSl function and edsl mutant function can be determined by assessing the level of defence responses and/or susceptibility of the plant to a pathogen as described above or other suitable alternatives known and available to those skilled in the art . Test plants may be monocotyledenous or dicotyledenous . Suitable monocots include any of barley, rice, wheat, maize or oat, particularly barley. Suitable dicots include Arabidopsis, tobacco, tomato, Brassicas, potato and grape vine .

A nucleic acid molecule according to the invention may comprise a nucleotide sequence which encodes a polypeptide comprising an amino acid sequence with the EDSl function of an amino acid sequence as shown in Figure 3 or Figure 6.. The nucleotide sequence may encode a polypeptide as shown in Figure 3 or Figure 6. Alternatively it may encode a polypeptide which is an allele, variant, fragment, derivative, mutant or homologue of a polypeptide as shown in Figure 3 or Figure 6. The allele, variant, fragment, derivative, mutant or homologue may have substantially the EDSl function of the amino acid sequence shown in Figure 3. Thus the nucleotide sequence may encode a polypeptide of Arabidopsis (eg La-er, Col-O or Ws-0) as shown in Figure 6 , or a polypeptide which is a mutant, variant, fragment, derivative, allele or homologue of an Arabidopsis polypeptide as provided. Such a mutant, variant, fragment, derivative, allele or homologue may encode a polypeptide which substantially retains the EDSl function of the polypeptide sequences disclosed.

Also encompassed by the present invention are nucleic acid molecules which comprise a nucleotide sequence which encodes a polypeptide comprising an amino acid sequence which although clearly related to a functional EDSl polypeptide (eg they are immunologically cross reactive with an EDSl polypeptide demonstrating EDSl function, or they have characteristic sequence motifs in common with an EDSl polypeptide) no longer has EDSl function. Thus the present invention provides mutants of EDSl such edsl - 2 and edsl -3 (Figure 4; Table 1 with reference to Figure 3) . Plants and plant cells carrying these mutant forms are susceptible to P. parasi tica Noco2.

Thus EDSl mutants, variants, fragments, derivatives, alleles and homologues of types which raise resistance and of types which lower resistance may both be of practical value depending on the situation. In the agronomic situation the major interest will be one of raising plant resistance to pathogens.

In particular homologues of the particular EDSl sequences provided herein (see figures 3,5, 6 and 7) are provided by the present invention as are mutants, variants, fragments and derivatives of such homologues (and comments made above in relation to such mutants etc also apply in relation to mutants etc of homologues) . Such homologues are readily obtainable by use of the disclosures made herein. Thus the present invention also extends to nucleic acid molecules which comprise a nucleic acid sequence encoding an EDSl homologue obtainable using a nucleotide sequence derived from, or as shown in Figures 3 , 5 and 7 or obtainable using the amino acid sequences shown in Figure 3 and 6. The EDSl homologue may at the nucleotide level have homology with a nucleotide sequence of Figure 3 , 5 or 7 , preferably at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% homology, or at least about 90% homology. Most preferably at least about 95% or greater homology.

In certain embodiments, an allele, variant, derivative, mutant derivative, mutant or homologue of the specific sequence may show little overall homology, say about 20%, or about 25%, or about 30%, or about 35%, or about 40% or about 45%, with the specific sequence. However, in functionally significant domains or regions the amino acid homology may be much higher. Putative functionally significant domains or regions can be identified using processes of bioinformatics, including comparison of the sequences of homologues. Functionally significant domains or regions of different polypeptides may be combined for expression from encoding nucleic acid as a fusion protein. For example, particularly advantageous or desirable properties of different homologues may be combined in a hybrid protein, such that the resultant expression product, with EDSl or edsl function, may comprise fragments of vario s parent proteins.

EDSl gene sequences from three different Arabidopsis landraces are highly conserved (Figures 5 and 6) . Conservation is also high between Arabidopsis and related cruciferous crop species as judged by hybridization signals on a genomic DNA blot using an EDSl probe (Figure 8) . It should be possible to use EDSl-derived oligonucleotide primers (as authentic or degenerate sequences) to isolate EDSl homologues from Brassica spp. and unrelated crop species such as tobacco. Once the corresponding gene is cloned it could be expressed as an antisense construct to assess its importance in resistance to agronomically important diseases such as downy mildew (P. parasi tica) and white blister {Albugo Candida) of Brassicas, blue mold of tobacco { Peronospora tabacina) , tobacco mosaic virus (TMV) , downy mildew of grape, fungal blight of potato, or blackleg disease of crucifers caused by Leptosphaeriea aculans . It should be noted that the tobacco N gene mediating resistance to TMV is of the TIR-ΝBS-LRR class and structurally very similar to RPP5. We anticipate therefore, that an EDSl homologue exists in tobacco and that it is required for N gene-mediated resistance.

The obtaining of homologues is later discussed herein, but briefly here it should be pointed out that the nucleotide sequence information provided herein, or any part thereof, may be used in a data-base search to find homologous sequences, expression products of which can be tested for EDSl (or edsl ) function. These may have ability to complement an EDSl (or edsl) phenotype in a plant or may, upon expression in a plant, confer such a phenotype . Thus the EDSl cDΝA or part of it may be used as a bait in an interaction trap assay, such as the yeast two-hybrid system, to isolate other disease resistance signalling components that are hitherto unknown. These would present further targets for pathway manipulation towards improved disease resistance.

By sequencing homologues, studying their expression patterns and examining the effect of altering their expression, genes carrying out a similar function to EDSl in Arabidopsis are obtainable. Of course, mutants, variants and alleles of these sequences are included within the scope of the present invention in the same terms as discussed above for the Arabidopsis EDSl gene.

Homology between the homologues as disclosed herein, may be exploited in the identification of further homologues, for example using oligonucleotides (e.g. a degenerate pool) designed on the basis of sequence conservation or PCR primers .

According to a further aspect, the present invention provides a method of identifying or a method of cloning an EDSl homologue, e.g. from a species other than Arabidopsis, the method employing a nucleotide sequence derived from that shown in Figure 3 , 5 or 7 or that shown in any of the other Figures herein. For instance, such a method may include providing a preparation of plant cell nucleic acid, providing a nucleic acid molecule having a nucleotide sequence substantially as shown herein (eg as in Figure 3) or complementary to a nucleotide sequence substantially as shown herein, preferably from within the coding sequence (e.g.. coding for an amino acid sequence shown in Figure 3), 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 .

Target or candidate nucleic acid may, for example, comprise genomic DNA, cDNA or RNA (or a mixture of any of these preferably as a library) obtainable from an organism known to contain or suspected of containing such nucleic acid, either monocotyledonous or dicotyledonous. Prior to any PCR that is to be performed, the complexity of a nucleic acid library may be reduced by creating a cDNA library for example using RT-PCR or by using the phenol emulsion reassociation technique (Clarke et al . (1992) NAR 20, 1289-1292) on a genomic library. Successful hybridisation may be identified and target/candidate nucleic acid isolated for further investigation and/or use. Hybridisation of nucleic acid molecule to a EDSl gene or homologue 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 EDSl are employed. However, if RACE is used only one such primer may be needed. Hybridisation may be also be determined (optionally in conjunction with an amplification technique such as PCR) by probing with nucleic acid and identifying positive hybridisation under suitably stringent conditions (in accordance with known techniques) . 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. It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain.

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 examination of restriction fragment length polymorphisms, amplification using PCR, RNAase cleavage and allele specific oligonucleotide probing.

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 by techniques such as reverse-transcriptase- PRC.

Preliminary experiments may be performed by hybridising under low stringency conditions various probes to Southern blots of DNA digested with restriction enzymes. 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. 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.

For instance, screening may initially be carried out under conditions, which comprise a temperature of about 37°C or more, 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 more and a high salt (e.g. ^XSSPE'= 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. IX 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.

PCR techniques for the amplification of nucleic acid are described in US Patent No. 4,683,195 and Saiki et al . Science 239: 487-491 (1988) . PCR includes steps of denaturation of template nucleic acid (if double- stranded) , annealing of primer to target, and polymerisation. The nucleic acid probed or used as template in the amplification reaction may be genomic DNA, cDNA or RNA. PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequences and cDNA transcribed from mRNA. References for the general use of PCR techniques include Mullis et al, Cold Spring Harbor Symp . Quant. Biol., 51:263, (1987), Ehrlich (ed) , PCR technology, Stockton Press, NY, 1989, Ehrlich et al , Science, 252:1643-1650, (1991), "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al , Academic Press, New York, (1990) .

Assessment of whether or not a PCR product corresponds to a gene able to alter a plant's resistance to a pathogen may be conducted in various ways, as discussed, and a PCR band may contain a complex mix of products . Individual products may be cloned and each screened for linkage to such known 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 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 EDSl/ edsl or other related gene. It may subsequently be analysed by transformation to assess its function on introduction into a disease sensitive variety of the plant of interest. Alternatively, the PCR band or sequences derived by analysing it may be used to assist plant breeders in monitoring the segregation of a useful resistance gene.

These techniques are of general applicability to the identification of genes able to alter a plant's resistance to a pathogen.

Preferred amino acid sequences suitable for use in the design of probes or PCR primers are sequences conserved (completely, substantially or partly) between at least two EDSl peptides or polypeptides encoded by genes involved in the signalling of a defence response in a plan . Figure 6 provides ESDI amino acid sequences conserved in Arabidopsis. Conserved nucleotide sequences may be identified from the nucleotide sequence information contained herein.

On the basis of amino acid sequence information or nucleotide sequence information, oligonucleotide probes or primers may be designed (when working from amino acid sequence information, taking into account the degeneracy of the genetic code and where appropriate, codon usage of the organism) .

A gene or fragment thereof identified as being that to which a said nucleic acid molecule hybridises, which may be an amplified PCR product, may be isolated and/or purified and may be subsequently investigated for ability to alter a plant's resistance to a pathogen. If the identified nucleic acid is a fragment of a gene, the fragment may be used (e.g. by probing and/or PCR) in subsequent cloning of the full-length gene, which may be a full-length coding sequence. Inserts may be prepared from partial cDNA clones and used to screen cDNA libraries. The full-length clones isolated may be subcloned into expression vectors and activity assayed by introduction into suitable host cells and/or sequenced. It may be necessary for one or more gene fragments to be ligated to generate a full-length coding sequence.

Molecules found to manipulate genes with ability to alter a plant's resistance to infection may be used as such, i.e. to alter a plant's resistance to a pathogen. Nucleic acid obtained and obtainable using a method as disclosed herein is provided in various aspects of the present invention.

The present application also provides oligonucleotides based on either an EDSl nucleotide sequence as provided herein or an EDSl nucleotide sequence obtainable in accordance with the disclosures and suggestions herein. The oligonucleotides may be of a length suitable for use as primers in an amplification reaction, or they may be suitable for use as hybridization fishing probes. Preferably an oligonucleotide in accordance with the invention, e.g. for use in nucleic acid amplification, has about 10 or fewer codons (e.g. 6, 7 or 8), i.e. is about 30 or fewer nucleotides in length (e.g. 18, 21 or 24) . Preferred oligonucleotide primers included those given below as EDSlf and EDSlr.

Preferred nucleic acid sequences with EDSl function are shown in Figures 3, 5 and 7. Figure 3 also shows the predicted amino acid sequence .

Nucleic acid molecules and vectors according to the present invention may be provided in a form isolated and/or purified from their natural environment, in substantially pure or homogeneous, or free or substantially free of nucleic acid and or genes of the species of interest or origin other than the relevant sequence. Nucleic acid according to the present invention may comprise cDNA, RNA, genomic DNA and maybe wholly or partially synthetic. The term "isolate" where used may encompass any of these possibilities.

Nucleic acid as herein provided or obtainable by use of the disclosures herein, may be the subject of alteration by way of one or more of addition, insertion, deletion or substitution of nucleotides with or without altering the encoded amino acid sequence (by virtue of the degeneracy of the genetic code) . Such altered forms of EDSl nucleotide sequences as herein provided or obtainable by use of the disclosures herein can be easily and routinely tested for both EDSl function and edsl function in accordance with standard techniques which basically examine plants or plant cells carrying the mutant, derivative or variant for a altered defence response to an appropriate pathogen.

The nucleic acid molecule may be in the form of a recombinant and preferably replicable vector for example a plasmid, cosmid, phage or Agrobacterium binary vector. The nucleic acid may be under the control of an appropriate promoter and regulatory elements for expression in a host cell such as a microbial, e.g. bacterial, or 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. However 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.

The nucleic acid as provided by the present invention may be placed under the control of an inducible gene promoter thus placing expression under the control of the user.

In a further aspect the present invention provides a gene construct comprising an inducible promoter operatively linked to a nucleotide sequence provided by the present invention. As discussed, this enables control of expression of the gene. The invention also provides plants transformed with said gene construct and methods comprising introduction of such a construct into a plant cell and/or induction of expression of a construct within a plant cell, e.g by application of a suitable stimulus, such as an effective exogenous inducer.

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 (which may be generated within a cell or provided exogenously) . The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus . Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic. Thus an inducible (or "switchable" ) promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero) . Upon application of the stimulus, expression is increased (or switched on) to a level which brings about the desired phenotype. One example of an inducible promoter is the ethanol inducible gene switch disclosed in Caddick et al (1998) Nature Biotechnology 16: 177-180. Many other examples will be known to those skilled in the art.

Other suitable promoters may include the, apparently constitutive, Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al , (1990a) EMBO J 9: 1677-1684); the cauliflower meri 5 promoter that is expressed in the vegetative apical meristem as well as several well localised positions in the plant body, eg inner phloem, flower primordia, branching points in root and shoot (Medford, J.I. (1992) Plant Cell 4, 1029-1039; Medford eϋ al , (1991) Plant Cell 3, 359-370) and the Arabidopsis thaliana LEAFY promoter that is expressed very early in flower development (Weigel et al , (1992) Cell 69, 843-859) .

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, 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. Specific procedures and vectors previously used with wide success upon plants are described by Bevan (Nucl. Acids Res. 12, 8711-8721 (1984)) and Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant

Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148) .

Selectable genetic markers may be used consisting of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate .

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 should 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 either will or will not occur. 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) microinj ection (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) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g. Freeman et al . Plant Cell Physiol . 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.

Thus once a 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 of the appropriate phenotype . According to a further aspect, the present invention provides a DNA isolate encoding the protein product of a gene able to alter a plant's resistance to a pathogen which has been identified by use of the presence therein of LRRs, TIRs, NBSs or LZ features as described above, or, in particular, by the technique defined above.

Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants

(Toriyama, et al . (1988) Bio / 'Technology 6 , 1072-1074; Zhang, et al . (1988) Plant Cell Rep . 7, 379-384; Zhang, et al . (1988) Theor Appl Genet 76, 835-840; Shimamoto, et al . (1989) Nature 338, 274-276; Datta, et al . (1990) Bio/Technology 8, 736-740; Christou, et al . (1991) Bio/Technology 9, 957-962; Peng, et al . (1991) International Rice Research Institute, Manila,

Philippines 563-574; Cao, et al . (1992) Plant Cell Rep . 11, 585-591; Li, et al . (1993) Plant Cell Rep . 12, 250- 255; Rathore, et al . (1993) Plant Molecular Biology 21, 871-884; Fromm, et al . (1990) Bio/Technology 8, 833-839; Gordon-Kamm, et al . (1990) Plant Cell 2, 603-618; D'Halluin, et al . (1992) Plant Cell 4, 1495-1505; Walters, et al . (1992) Plant Molecular Biology 18, 189- 200; Koziel, et al . (1993) Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology 25, 925-937; Weeks, et al . (1993) Plant Physiology 102, 1077-1084; Somers, et al . (1992) Bio/Technology 10, 1589-1594; W092/14828) . In particular, Agrojbacterium mediated transformation is now emerging also as an highly efficient alternative transformation method in monocots (Hiei et al . (1994) The Plant Journal 6 , 271-282).

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) .

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 microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233 ) . 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 reviewed in Vasil et al . , Cell Cul ture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications , Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.

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.

The invention further encompasses a host cell transformed with a vector as set forth above, especially a plant or a microbial cell. Thus, a host cell, such as a plant cell, comprising a nucleotide sequence as herein indicated is provided. Within the cell, the nucleotide sequence may be incorporated within the chromosome .

Also according to the invention there is provided a plant cell having incorporated into its genome a nucleotide sequence, particularly a heterologous nucleotide sequence, as provided by the present invention under operative control of a regulatory sequence for control of expression. The coding sequence may be operably linked to one or more regulatory sequences which may be heterologous or foreign to the gene, such as not naturally associated with the gene for its expression. The nucleotide sequence according to the invention may be placed under the control of an externally inducible gene promoter to place expression under the control of the user. A further aspect of the present invention provides a method of making such a plant cell involving introduction of nucleotide sequence or a suitable vector including the sequence of nucleotides into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome. The invention extends to plant cells containing a nucleotide sequence according to the invention as a result of introduction of the nucleotide sequence into an ancestor cell.

The term "heterologous" may be used to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, ie by human intervention. A transgenic plant cell, i.e. transgenic for the nucleotide sequence in question, may be provided. The transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. A heterologous gene may replace an endogenous equivalent gene, ie one which normally performs the same or a similar function, or the inserted sequence may be additional to the endogenous gene or other sequence. An advantage of introduction of a heterologous gene is the ability to place expression of a sequence under the control of a promoter of choice, in order to be able to influence expression according to preference.

Furthermore, mutants, variants and derivatives of the wild-type gene, e.g. with higher or lower activity than wild-type, may be used in place of the endogenous gene. Nucleotide sequences heterologous, or exogenous or foreign, to a plant cell may be non-naturally occurring in cells of that type, variety or species. Thus, a nucleotide sequence may include 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 nucleotide sequence to be placed within a cell in which it or a homologue is found naturally, but wherein the nucleotide 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. A sequence within a plant or other host cell may be identifiably heterologous, exogenous or foreign.

Plants which include a plant cell according to the invention are also provided, along with any part or propagule thereof, seed, selfed or hybrid progeny and descendants. Particularly provided are transgenic crop plants, which have been engineered to carry genes identified as stated above. Examples of 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. 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.

In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, offspring, clone or descendant.

The present invention also encompasses the polypeptide expression product of a nucleic acid molecule according to the invention as disclosed herein or obtainable in accordance with the information and suggestions herein. Also provided are methods of making such an expression product by expression from a nucleotide sequence encoding therefore under suitable conditions in suitable host cells eg E. coli . Those skilled in the art are well able to construct vectors and design protocols and systems for expression and recovery of products of recombinant gene expression.

Preferred polypeptides are as provided by Figures 3 and 6. A polypeptide according to the present invention may be an allele, variant, fragment, derivative, mutant or homologue of a polypeptide as shown in Figure 3 or Figure 6. The allele, variant, fragment, derivative, mutant or homologue may have substantially the EDSl function of the amino acid sequence shown in Figure 3.

Also encompassed by the present invention are polypeptides which although clearly related to a functional EDSl polypeptide (eg they are immunologically cross reactive with an EDSl polypeptide demonstrating EDSl function, or they have characteristic sequence motifs in common with an EDSl polypeptide) no longer has EDSl function. Thus the present invention provides variant forms of EDSl polypeptides such as edsl-2 and edsl-3 (Figure 4; Table 1 with reference to Figure 3) . Plants and plant cells carrying these mutant forms are susceptible to P. parasi tica Noco2.

"Homology" in relation to an amino acid sequence may be used to refer to identity or similarity, preferably identity. High level of amino acid identity may be limited to functionally significant domains or regions

In particular homologues of the particular EDSl polypeptide sequences provided herein (see figures 3, and 6) are provided by the present invention as are mutants, variants, fragments and derivatives of such homologues. Such homologues are readily obtainable by use of the disclosures made herein. Thus the present invention also extends to polypetides which comprise an amino acid sequence with ESDI function obtainable using sequence information as provided herein. The EDSl homologue may at the amino acid level have homology with an amino acid sequence of Figure 3 or 6 , preferably at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% homology, or at least about 90% homology. Most preferably at least about 95% or greater homology.

In certain embodiments, an allele, variant, derivative, mutant derivative, mutant or homologue of the specific sequence may show little overall homology, say about 20%, or about 25%, or about 30%, or about 35%, or about 40% or about 45%, with the specific sequence. However, in functionally significant domains or regions, the amino acid homology may be much higher. Putative functionally significant domains or regions can be identified using processes of bioinformatics, including comparison of the sequences of homologues. Functionally significant domains or regions of different polypeptides may be combined for expression from encoding nucleic acid as a fusion protein. For example, particularly advantageous or desirable properties of different homologues may be combined in a hybrid protein, such that the resultant expression product, with EDSl or edsl function, may comprise fragments of various parent proteins.

The EDSl gene encodes a novel protein with potential lipase, or other esterase, activity.

Evidence for lipase, or other esterase function, is three-fold. First, the presence of conserved amino acids at and around the three catalytic residues, Ser¹²³, Asp¹⁸⁷ and His³¹⁷, that constitute a lipase catalytic site is apparent (Hide et al . , 1992; Derewenda and Sharp, 1993; Wallace et al . , 1996) . This is shown in Figure 9. Second, the spacing of these residues in the EDSl protein is consistent with formation of the lipase catalytic triad. Third, the predicted secondary structure of EDSl derived using the Predict-Protein programme (B Rost, PHD: predicting one-dimensional protein structure by profile based neural networks. Meth. In Enzym. , 1996, 266, 525- 539) shows that the key catalytic residue, Ser¹²³ lies in a sharp loop between a .-sheet and an α-helix. This conformation is absolutely conserved with the necessary presentation of the catalytic serine in all other lipases for which two and three dimensional structures have been elucidated (Cousin et al . , 1996; Kim et al . , 1997).

Although the amino acid alignments are most suggestive of triacylglycerol lipase activity, sequence conservation across the catalytic residues is also observed with a ferulic acid esterase from Aspergillus niger (RP de Vries et al . , 1997), implicating possible esterase activity on a non-lipid substrate.

A lipid-based signalling pathway is known to operate in the activation of wound-inducible proteins such as proteinase inhibitors I and II in response to mechanical wounding and insect or herbivore attack of plants (Bergey et al . , 1996) . This gives rise to important fatty acid signalling intermediates such as jasmonic acid. However, lipase function in R gene-mediated disease resistance to microbial pathogens and in possible disease limitation caused by compatible pathogens is previously unknown. Thus, the structure of the EDSl protein and examination of its biochemical function may reveal a new aspect of disease resistance signalling in plants.

Animal and fungal lipases have a diverse range of substrates including triacylglycerides , diacylglycerides and lipoproteins (Hide et al . , 1992; Derewenda and Sharp, 1993; Wallace et al . , 1996). For example, human pancreatic lipase exhibits phospholipase activity (Dugi et al . , 1995) and several phopholipase Al enzymes possess the Ser/Asp/His consensus triad (see summary of BLAST search, Appendix 1 (Altschul, S.F., Gish, W. , Miller, W. , Myers, EW, and Lipman, D.J. (1990) J. Molecular Biology 215, 403-410) accessed at www site: http://www.ncbi.nlm.nih.gov). This raises the possibility that the EDSl substrate is a phospholipid or lipoprotein. In general, lipases are of great industrial and medical importance (Derewenda and Sharp, 1993; Cousin et al . , 1997) . They are water-soluble enzymes but act on non-soluble lipid substrates and are therefore uniquely able to function at a lipid-aqueous interface. If EDSl possesses lipase activity it may enable closer examination of plant lipid hydrolysis and reveal new substrate specificities that could be industrially important .

Purified EDSl polypeptides and mutants, variants, fragments, derivatives, alleles and homologues thereof eg 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 may be used in identifying homologues of the sequences specifically provided herein as discussed further below.

Methods of producing antibodies include immunising a mammal (eg human, mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82) . Antibodies may be polyclonal or monoclonal .

As an alternative or supplement to immunising a mammal, antibodies with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, eg using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.

Antibodies raised to a polypeptide or peptide can be used in the identification and/or isolation of homologous polypeptides, and then the encoding genes. Thus, the present invention provides a method of identifying or isolating a polypeptide with EDSl function or edsl function (in accordance with embodiments disclosed herein) , comprising screening candidate peptides or polypeptides with a polypeptide comprising the antigen- binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind an EDSl or edsl peptide, polypeptide or fragment, variant or variant thereof or preferably has binding specificity for such a peptide or polypeptide, such as having an amino acid sequence identified herein. Specific binding members such as antibodies and polypeptides comprising antigen binding domains of antibodies that bind and are preferably specific for a EDSl or edsl peptide or polypeptide or mutant, variant or derivative thereof represent further aspects of the present invention, as do their use and methods which employ them.

Candidate peptides or polypeptides for screening may for instance be the products of an expression library created using nucleic acid derived from an plant of interest, or may be the product of a purification process from a natural source .

A peptide or polypeptide found to bind the antibody may be isolated and then may be subject to amino acid sequencing. Any suitable technique may be used to sequence the peptide or polypeptide either wholly or partially (for instance a fragment of a polypeptide may be sequenced) . Amino acid sequence information may be used in obtaining nucleic acid encoding the peptide or polypeptide, for instance by designing one or more oligonucleotides (e.g. a degenerate pool of oligonucleotides) for use as probes or primers in hybridisation to candidate nucleic acid, or by searching computer sequence databases, as discussed further below.

The invention further provides a method of raising pathogen resistance in a plant which comprises expressing a heterologous nucleic acid sequence with EDSl function as discussed, within cells of the plant. Such methods may be achieved by expression from a nucleotide sequence encoding an amino acid sequence conferring an EDSl function within cells of a plant (thereby producing the encoded polypeptide) , following an earlier step of introduction of the nucleotide sequence into a cell of the plant or an ancestor thereof . Such a method may raise the plant's resistance to pathogen.

EDSl mRNA is expressed at a low level in unchallenged plants and is induced at least 2-3 fold after inoculation with an avirulent bacterial pathogen or after treatment with salicylic acid. Manipulation of expression of the EDSl transcript or EDSl protein could be used to enhance resistance to a broad spectrum of pathogens in different plants. This might be achieved by over expression using a highly active plant promoter such as the CaMV-35S promoter. Alternatively, EDSl could be attached to a pathogen-inducible promoter, allowing greater expression in challenged cells. Increased disease resistance may occur in the absence of a hypersensitive response (HR) that would have possible deleterious effects to the plant in terms of general vigour and yield.

A gene stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, cells of which descendants may express the encoded polypeptide and so may have enhanced pathogen resistance or pathogen susceptibility. Pathogen resistance may be determined by assessing compatibility of a pathogen as earlier mentioned.

The invention further provides a method which comprises expression from a nucleic acid encoding the amino acid sequence of Figure 3 or a mutant, allele or derivative of the sequence (which may have EDSl function) 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 raise the plant's resistance to one or more pathogens. The method may be used in combination with an avr 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.

In the present invention, alteration of resistance may be achieved by introduction of the nucleotide sequence in a sense orientation. Thus, the present invention provides a method of modulation of a defence response in a plant, the method comprising causing or allowing expression of nucleic acid according to the invention within cells of the plant. Generally, it will be desirable to promote the defence response, and this may be achieved by allowing EDSl gene function.

In order to down-regulate resistance signalled by EDSl , under-expression of endogenous EDSl gene may be achieved using anti-sense technology or "sense regulation".

The use of anti-sense genes or partial gene sequences to down-regulate gene expression is now well-established. Double-stranded DNA is placed under the control of a promoter in a "reverse orientation" such that transcription of the "anti-sense" strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene. The complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works. See, for example,

Rothstein et al, 1987; Smith et al , (1988) Nature 334, 724-726; Zhang et al , (1992) The Plant Cell 4, 1575-1588, English et al . , (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, 1995, and Flavell, 1994. Antisense constructs may involve 3 ' end or 5 ' end sequences of EDSl or homologues. In cases where several EDSl homologues exist in a plant species, the involvement of 5'- and 3 '-end untranslated sequences in the antisense constructs will enhance specificity of silencing. Constructs may be expressed using the natural promoter, by a constitutively expressed promotor such as the CaMV 35S promotor, by a tissue-specific or cell-type specific promoter, or by a promoter that can be activated by an external signal or agent. The CaMV 35S promoter but also the rice actinl and maize ubiquitin promoters have been shown to give high levels of reporter gene expression in rice (Fujimoto et al . , (1993)

Bio/Technology 11, 1151-1155; Zhang, et al . , (1991) Plant Cell 3, 1155-1165; Cornejo et al . , (1993) Plant Molecular Biology 23 , 567-581).

The complete sequence corresponding to the coding sequence in reverse orientation need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the level of anti- sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A suitable fragment may have about 14-23 nucleotides, e.g. about 15, 16 or 17.

Thus, the present invention also provides a method of downwardly modulating EDSl expression in a plant, the method comprising causing or allowing anti-sense transcription from nucleic acid according to the invention within cells of the plant. EDSl down- regulation may reduce a defence response. This may be appropriate in certain circumstances eg as an analytical or experimental approach.

For use in anti-sense regulation, nucleic acid comprising a nucleotide sequence complementary to a coding sequence of an EDSl gene (i.e. including homologues), or a fragment of a said coding sequence suitable for use in anti -sense regulation of expression, is provided. This may be DNA and under control of an appropriate regulatory sequence for anti-sense transcription in cells of interest .

When additional copies of the target gene are inserted in sense, that is the same, orientation as the target gene, a range of phenotypes is produced which includes individuals where over-expression occurs and some where under-expression of protein from the target gene occurs. When the inserted gene is only part of the endogenous gene the number of under-expressing individuals in the transgenic population increases. The mechanism by which sense regulation occurs, particularly down-regulation (or "silencing"), is not well-understood. However, this technique is also well-reported in scientific and patent literature and is used routinely for gene control. See, for example, van der Krol et al . , (1990) The Plant Cell 2, 291-299; Napoli et al . , (1990) The Plant Cell 2, 279- 289; Zhang et al . , (1992) The Plant Cell 4, 1575-1588. Further refinements of the gene silencing or co- suppression technology may be found in W095/34668 (Biosource) ; Angell & Baulcombe (1997) The EMBO Journal 16,12:3675-3684; and Voinnet S. Baulcombe (1997) Nature 389: pg 553.

Thus, the present invention also provides a method of downwardly modulating EDSl function in a plant, the method comprising causing or allowing expression from nucleic acid according to the invention within cells of the plant to suppress endogenous EDSl expression.

Modified versions of EDSl may be used to down-regulate endogenous EDSl function. For example mutants, variants, derivatives etc., may be employed. Reduction of EDSl wild type activity may be achieved by using ribozymes, such as replication ribozymes, e.g. of the hammerhead class (Haseloff and Gerlach, 1988, Na ture 334: 585-591; Feyter et al . Mol . , 1996, Gen . Genet . 250: 329-338) .

Another way to reduce EDSl function in a plant employs transposon mutagenesis (reviewed by Osborne et al . , (1995) Current Opinion in Cell Biology 1 , 406-413) . Inactivation of genes has been demonstrated via a

'targeted tagging' approach using either endogenous mobile elements or heterologous cloned transposons which retain their mobility in alien genomes. EDSl alleles carrying any insertion of known sequence could be identified by using PCR primers with binding specificities both in the insertion sequence and the EDSl homologue. 'Two-element systems' could be used to stabilize the transposon within inactivated alleles. In the two-element approach, a T-DNA is constructed bearing a non-autonomous transposon containing selectable or screenable marker gene inserted into an excision marker. Plants bearing these T-DNAs are crossed to plants bearing a second T-DNA expressing transposase function. Hybrids are double-selected for excision and for the marker within the transposon yielding F₂ plants with transposed elements .

Embodiments and examples relating to the present invention are now described by way of example only with reference to the below listed figures.

Figure Legends

Figure 1 A physical contig of CIC YAC clones (shaded black) and PI clones (shaded grey) in the region of the RFLP marker 118. Probes containing plant DNA derived from PI clone centromeric (black circles) or telomeric (black squares) ends are shown and their alignment with YAC clones indicated by a dotted line. PI clones 7312 and 69D23 were detected using 118 primers and the 118 probe. PI clones 105H and 5N12 extend in a centromeric direction from PI clone 7312, as shown and contain a 7 kb EcoRI fragment that detects a 0.9 kb deletion on a blot containing DNA from the edsl -2 mutant line. A dSpm element that had inserted into La-er DNA corresponding to the 7 kb EcoRI fragment of the PI clones was common to all putative transposon -induced edsl mutant plants derived from a screen for EDSl inactivation. Plant DNA flanking this dSpm element was derived by inverse-PCR and subsequently shown to correspond to part of exon 1 of EDSl .

Figure 1A

A map position of EDSl relative to RFLP markers on chromosome 3. EDSl was mapped to a position approximately 0.17cM centromeric to 118 and 0.85 cM telomeric to g4564b, based on the number of recombinant chromosomes identified between these markers and EDSl .

Figure 2 (A) Nucleotide sequence of a HindiII fragment of the La- er EDSl gene that was derived by inverse-PCR using the dSpm terminal inverted repeat primers . The sequence corresponds to part of EDSl exon 1. (B) Nucleotide sequence of the wild type La-er EDSl gene around the position of the inserted dSpm transposable element . Also shown are the corresponding sequences around the dSpm excision site that have restored EDSl function in revertant plants (a) and sequences that have failed to restore function in non-revertant plants in which dSpm excision has occurred (b) . The nucleotides marked in bold are nucleotide footprints (novel nucleotides) generated by the dSpm element. Figure 3

Nucleotide sequence of a 5742 base pair La-er Bglll fragment that contains the complete EDSl gene. The positions of four exons comprising 623 amino acids are shown with the first amino acid (a. a.), methionine (M) starting at nucleotide (nt) 1427. The three amino acids (S, 123; D 187; H 317) that form a potential lipase catalytic site are marked.

Figure 4

Structure of the EDSl gene consisting of four exons and three introns. The sizes of the exons and introns are shown in the base pairs (bp) and the nucleotide coordinates from Figure 3 for each exon indicated. Also shown are the positions of deletions in edsl mutant alleles, La- edsl -2, La- edsl -3 and La- edsl -4 . The ATG start codon and the TGA stop codon are indicated.

Figure 5 A PRETTYBOX alignment of genomic DNA sequences obtained for the wild type EDSl genes of the accession lines La-er (Edsller) , Col-0 (Edslcol) and Ws-0 (Edslws) . Nucleotide 1 of the La-er sequence corresponds to nucleotide 1 of the Bglll sequence in Figure 3. Identical nucleotides are shaded in black.

Figure 6

A PRETTYBOX alignment of the predicted amino acid sequences encoded by EDSl from the accession lines La-er (Edsller) , Col-0 (Edslws) and Ws-0 (Edslws) . Identical amino acids are shaded in black. Similarly charged amino acids are shaded in grey.

Figure 7 Nucleotide sequence of the EDSl cDNA from La-er.

Figure 8 Hybridisation signals detected with a P³²-labelled EDSl cDNA probe on a blot containing EcoRI -digested genomic DNA from Arabidopsis accession La-er (L=lμg, L2=2μg) and from other plant species (>10μg of each per lane) . Signals were obtained under moderately stringent and stringent hybridisation conditions from DNA of Brassica napus (lane 2), B . campestris (lane 3), B . oleracea (lane 4) and Sinapis alba (lane 5) . No signals were detected under the same conditions from DNA of tobacco (lane 6) , tomato (lane 7) , potato (lane 8) , pepper (lane 9) , petunia (lane 10) , antirhinnum (lane 11) , maize (lane 12) , rice (lane 13) , wheat (lane 14) , barley (lane 15) and pea (lane 16) .

Figure 9

Comparison of amino acids around the three putative lipase catalytic residues (Serine [S] 123, site 1; aspartic acid [D] , site 2; histidine [H] , site 3) of the EDSl protein and the lipase catalytic residues of the Rhizomucor miehei triacylgycerol lipase based on the protein crystal structure (Brady et al , 1990) . Identical amino acids are shown and similarly charged amino acids are indicated (+) .

Specific Description

(a) EDSl Mapping and Cloning

A total population of approximately 1200 La-edsl-2 x Col -gl F2 plants was used to construct an EDSl mapping population that consisted of 294 seedlings susceptible to P. parasi tica isolate Wandl . The Wandl isolate of P. parasi tica was chosen because it appeared to be recognized by an EDSl-dependent R locus that is non-segregating between the wild type landraces La-er and Col-gl, and therefore only segregation of EDSl would be apparent . The segregation data shows that the La-er and Col-grl R loci recognizing Wandl are segregating but still allow reliable scoring of EDSl/ edsl genotypes in most F2 plants and corresponding F3 families. The EDSl genotypes of selected recombinant F3 families were confirmed by scoring their resistance/susceptibility profiles with respect to P. parasi tica isolate Noco2 that is recognized by the EDSl-dependent RPP5 gene in La-er. Plants can be genotyped for the RPP5/ rpp5 alleles using an RPP5 gene-specific CAPS marker (Parker et al . , 1997). EDSl was mapped to a 3 cM interval between the RFLP markers, g4564b and g4014 on the lower arm of chromosome 3 (marker information obtained from the Arabidopsis RI map, web site: http://genome-www3.stanford.edu/atdb) (see Fig. 1A) . The marker, 118 (also present on the RI map) was found to be most closely linked to EDSl , with one recombinant identified in 588 F2 chromosomes. This placed EDSl < 0.2 cM centromeric to 118.

The 118 marker consists of -500 bp La-er genomic DNA that was flanking a 2.2 kb non-autonomous maize transposable element, I/dSpml8 (1-6078) that had transposed from an "in cis two-element" construct containing a stable transposase source (Aarts et al . , 1995). Plant genomic DNA flanking I/dSpml8 had been generated previously using inverse-PCR from primers annealing to the dSpm terminal inverted repeats (Aarts et al . , 1995) and was cloned into the Bluescript plasmid vector pSK+ . This is the 118 marker. Insert DNA of the I18-pSK+ clone (CPRO-DLO, ) was sequenced and the authentic plant DNA sequence derived. This allowed the construction of the following 118-specific oligonucleotide primers:

I18f 5'-AAT CAC ACC TAA AAT TTT AAA AG

I18r 5 ' - TAA GTA CTT CAA GTT TTT CTC G The 118 region of chromosome 3 is present as a physical contig in yeast artificial chromosomes (YACs) from the Arabidopsis CIC library (Creusot et al . , 1995). Using PCR, the 118 primers were used to amplify a 350 bp sequence from La-er and Col-0 genomic DNA. The 118 primers were used to test the presence of 118 DNA in the candidate YAC clones, 3B10, 11D12, 3D2 and 7A9. Only 3B10 and 11D12 gave positive signals which were confirmed by DNA hybridisation to the corresponding yeast clones with the ³²P-labelled 118 PCR-amplification product (118 probe) . Using PCR, the 118 primers were also used to identify two PI clones, 7312 and 69D23, from 96 pools of an Arabidopsis (Lui et al 1995) PI library containing Col-0 genomic DNA (Lui et al . , 1995) as shown in Figure 1. End fragments derived from 7312 and 69D23 were made by Thermal Asymmetric Interlaced (TAIL-) -PCR (Lui and Whittier, 1995) . The 69D23 end products were developed into RFLP markers and mapped using selected recombinants in the EDSl region. This enabled orientation of 69D23 relative to EDSl (Figure 1) . 7312 extended in a centromeric direction from the EDSl-proximal end of 69D23. The 7312 centromeric end TAIL-PCR product was used to identify by hybridization two other PI clones, 105H5 and 5N12 that extended the PI contig in the EDSl direction and possibly encompassing the EDSl gene (Figure 1) . End probes derived from the PI clones were further cross-referenced to yeast strains carrying the YAC clones 11D12, 3D2 and 7A9 to confirm their relative positions in the EDSl region (Figure 1) .

Since the 118 marker was derived from a non-autonomous dSpm element, this offered an opportunity to isolate the EDSl gene by transposon tagging (G. Coupland (1992) Transposon tagging in Arabidopsis. In: Koncz, C, Chua, N.H., and Schell, J. (eds) Methods in Arabidopsis research. World Scientific, Singapore, pp 290-309). A dSpm insertion in EDSl can be identified by inactivation of the gene with a consequential change from Noco2 -resistance (conferred by RPP5) to susceptibility. 50 La-er seed that were homozygous or hemizygous for dSpm/118 were germinated under sterile conditions on an agarose-based germination medium containing 20 μg/ml hygromycin (Aarts et al . , 1995). All seedlings were hygromycin resistant, suggesting that they were homozygous for the stable transposase source . Genomic DNA was prepared from 35 of these plants, digested with HindiII and the digested DNA probed on a DNA gel blot with the 118 probe. This showed that a dSpm element was present at its original 118 site (a diagnostic 5 kb hybridizing band) in all plants but, as expected, a new hybridizing band of - 3kb was evident in all plants indicating that the dSpm element was actively transposing from 118 in these lines. Seed from each of the 35 plants (SI generation) was harvested (self progeny) (SI generation) and 70 000 third generation (S3) seedlings screened for susceptibility to Noco2. Five Noco2 -susceptible plants were identified. Allelism tests established that these were mutated at EDSl . FI seeds (approximately 1500) were made between each of the candidate dSpm-insertional edsl lines (or selfed Noco2-susceptible progeny derived from them) and the stable fast neutron-derived mutant line, edsl -3 . The FI progeny of these crosses was then tested for reversion to resistance due to excision of the dSpm element by inoculating the FI seedlings with P. parasi tica isolate Noco2. Revertant seedlings were observed at a low frequency (0.5%) indicating that the mutation observed at edsl was unstable and most likely caused by a transposable element.

The five original Noco2-susceptible plants that had a probable dSpm-insertion within EDSl contained a common novel dSpm-hybridising band of 2.4 kb on DNA gel blot analysis of Hindlll-digested DNA. This band was absent from sibling plants that were not mutated at EDSl . Plant DNA flanking this element was derived by inverse-PCR from an agarose gel-enriched fraction of Hindlll digested DNA using the dSpm terminal inverted repeat primers. The IPCR product (EDSl-I) obtained is 198 nt long and its sequence is shown in Figure 2A.

EDSl-I was used as a probe on the PI clones that formed a contig from 118 (Figure 1) and was found to hybridize to a 7 kb EcoRI fragment and a 5.7 kb Bglll fragment that overlap and lie internally within the inserts of PI clones 105H5 and 5N12. Thus, physical mapping of the IPCR fragment centromeric to 118 was consistent with the genetic location of EDSl . Both the PI EcoRI and EDSl-I fragments were used to probe DNA gel blots of

EcoRI-digested genomic DNA from La-er, Col-0, and from the two stable fast neutron-derived edsl mutant lines, edsl -2 and edsl -3 . Both mutant lines had a deletion detected by the 7 kb EcoRI DNA fragment as follows :

edsl -2 = -0.9 kb edsl -3 = -0.5 kb

Altogether, these data showed that the candidate EDSl-I fragment is almost certainly derived from EDSl and that the EDSl gene from Col-0 lies, at least in part, on the 7 kb EcoRI genomic DNA fragment shared by PI clones 105H5 and 5N12.

The 7 kb EcoRI fragment from 105H5 was subcloned into pGEM3Zf (+) (available from Promega UK) and double stranded DNA sequence obtained using an automated ABI 377 sequencing system. Sequence was obtained around the dSpm insertion site and outwards in both directions. Contiguous sequence was constructed using the Xbap alignment programme (Staden package) on a UNIX workstation. Two possible ORFs with coding probability were identified and one of these is shown as an EDSl-HindiII fragment in Figure 2A. The two ORFs could be joined by splicing out a putative small intron, predicted by the NetPlantGene Programme. Two cosmid clones, A19 and M4 , from a La-er genomic DNA library constructed in the binary cosmid vector 04541 were identified by PCR using the following EDSl-I forward and reverse primers:

EDSIf : 5' AAG CTT ACC TAA CCG AGC GCT

EDSlr: 5' AAG CTT CGT TAA CAG TAG CTA C

The cosmid clones were confirmed to contain EDSl sequence by hybridisation to the EDSl-I probe. A 5.7 kb Bglll fragment (see Figure 1) from clone M4 that was anticipated to contain the complete La-er EDSl gene was subcloned into pGEM3Zf (+) and double stranded DNA sequence obtained as described above . Figure 3 shows the complete nucleotide sequence of the La-er Bglll fragment and the corresponding amino acids that comprise 4 exons and 3 introns of the EDSl gene. A DNA sequence with 82% identity to EDSl at the nucleotide level (EDSl-horn.1) lies adjacent to the 3' end of EDSl and is partly contained within the Bglll fragment. The homology to EDSl starts -150 bp upstream from the EDSl ATG codon and extends to the end of the 5.5 kb Bglll fragment (see Figure 1) . This lies halfway through the exon 4 of EDSl-hom.l. Two pieces of evidence suggest that EDSl-hom.l is not functional. First, EDSl-hom.1-specific primers did not detect any cDNA candidate clones when used in a PCR-screen of a La-er cDNA library. Second, the EDSl homology in EDSl-hom.l has a 2 bp deletion in the first third of exon 2, causing a frameshift and leading to a stop codon 25 bp 3' to the deletion.

Final proof that the sequence in Figure 3 corresponds to the La-er EDSl gene was obtained by sequencing across the known dSpm excision sites in FI revertant plants obtained from the dSpm- edsl mutant x edsl -3 cross (described above) . The presence of a deletion in edsl -3 made this analysis easier since the edsl -3 allele could be easily distinguished from wild type and dSpm- insertion alleles on DNA gel blots. It could also be eliminated from PCR amplification by the choice of EDSl-specific primers corresponding to sequence within the edsl -3 deletion. Plants that had reverted to Noco2 -resistance were shown by DNA gel blot analysis to have lost the dSpm element from EDSl . Sequence analysis of three independent plants established that DNA footprints left by transposon excision maintained the EDSl ORF, as shown in Figure 2B . In contrast, eight independently derived FI plants that had lost the dSpm element but retained full susceptibility to Noco2 were shown to have sequence frameshifts caused by imperfect dSpm excision (Figure 2B) .

(b) Characterization of EDSl

The structure of the EDSl gene showing the position of the dSpm insertion that inactivated the gene and the deletion in edsl -2 is given in Figure 4. The edsl -3 deletion has not been precisely mapped but eliminates - 500 bp from the 5' untranslated region and from part of exon 1.

EDSl genomic DNA sequence obtained for the parental landraces, La-er, Col-0, and Ws-0 is compared in a

PRETTYBOX alignment in Figure 5 PRETTYBOX and FASTA are part of the GCG package of sequence analysis tools (Wisconsin computer group, Madison, USA) . This reveals a very high level of sequence conservation. A PRETTYBOX alignment of the corresponding predicted amino acid sequences is shown in Figure 6 . La-er EDSl protein is 98% identical to both the Col-0 and Ws-0 wild type EDSl alleles. The nucleotide coordinates for defective edsl alleles analyzed so far are given in Table 1. The La-er EDSl cDNA nucleotide sequence is shown in Figure 7. The cDNA appears to be full length, encoding the complete EDSl protein and hybridizing in a Northern gel blot analysis to a single 2.2 kb RNA from La-er polyadenylated RNA.

Table 1. Sequence changes in edsl alleles

^a Mutagens were ethane methyl sulphonate (EMS) or fast neutrons (FN) ^b Numbering of nucleotides is according to the Bglll nucleotide sequence in Figure 3. The La-er cDNA was used to probe a DNA blot of EcoRI -digested DNA from Arabidopsis and different dicotyledenous and monocotyledonous species. As shown in Figure 8, hybridization signals were observed under high and medium stringency conditions in the cruciferous plant species Brassica napus, B . campestris , B . oleracea and Sinapis alba . This indicates the presence of highly related genes in these species. Signals were not detected in the other species under the same conditions suggesting that any similar sequences in these plants are probably more diverged than in the related species .

(c) Structural analysis of EDSl

EDSl encodes a novel 71.6 kD protein that is predicted to be cytoplasmic. No predicted signal peptide or transmembrane regions were found using several programmes. Also no homologous sequences were identified using the EDSl amino acid sequence in searches of Expressed Sequence Tag "EST" databases comprising ESTs from invertebrates, mammals and plants. A single Col-0 EST (T45498) was found in the Arabidopsis EST database (dbEST) that corresponds to the Col-0 EDSl cDNA.

References

Aarts et al (1995). Mol. Gen. Genet. 247, 555-564.

Bent et al (1994). Science 265, 1856-1860. Bergey et al (1996). Proc. Natl. Acad. Sci. USA 93,

12053-12058.

Bowling et al (1994) . The Plant Cell 6, 1845-1857.

Brady et al (1990) Nature 343, 767-770.

Cai et al (1997) Science 275, 832-834. Cao et al (1994) The Plant Cell 6, 1583-1592.

Century et al (1995) Proc. Natl. Acad. Sci. USA 92, 6597-

6601.

Cordes and Barsch (1994) Cell 79, 1025-1034.

Cousin et al (1997) Nucleic Acids Res. 25, 143-146. Creusot et al (1995) Plant Journal, 8, 763-770.

Crute and Pink (1996) Plant Cell, 8, 1747-1755.

Delaney et al (1995) Proc. Natl. Acad. Sci. USA 92,

6602-6606.

Derewenda and Sharp (1993) Trends in Biochem. Sci. 18, 20-25.

Dixon et al (1996) Cell 84, 451-459.

Dugi et al (1995) J. of Biological Chemistry 270, 25396-

25401.

Flor (1971) Ann. Rev. Phytopathol . 9, 275-296. Freialdenhoven et al (1994) The Plant Cell 6, 983-994.

Gopalan et al (1996) Plant Cell 8, 1095-1105.

Grant et al (1995) Science 269, 843-846.

Hammond-Kosack and Jones (1996) Plant Cell, 8, 1773-1791.

Hide et al (1992) Journal of Lipid Research 33, 167-178. Hinsch and Staskawizc (1996) Mol. Plant-Microbe Interact

9, 55-61.

Holub and Beynon (1997) Advances in Botanical Research

24, 228-273.

Jones et al (1994) Science 266, 789-793. Jorgensen (1996) Genome 39, 492-498.

Kim et al (1997) Structure 5, 173-185.

Lawrence et al (1995) The Plant Cell 7, 1195-1206. Leister et al (1996) Proc. Natl. Acad. Sci. USA 93,

15497-15502.

Liu and Whittier (1995) Genomics 25, 674-681.

Martin et al (1993) Science 262, 1432-1436. Mindrinos et al (1994) Cell 78, 1089-1099.

Parker et al (1996) Plant Cell 8, 2033-2046.

Parker et al (1993) The Plant Journal 4, 821-831.

Parker et al (1997) Plant Cell 9, 879-894.

Reignault et al (1996) Mol. Plant -Microbe Interact 9, 464-473.

Reimmann et al (1995) Physiol. Mol. Plant Pathol. 46, 71-

81.

Ryals et al (1996) Plant Cell, 8, 1809-1819.

Ryals et al (1997) Plant Cell 9, 425-439. Salmeron et al (1996) Cell 86, 123-133.

Scofield et al (1996) Science 274, 2063-2065.

Song et al (1995) Science 270, 1804-1806.

Tang et al (1996) Science 274, 2060-2063.

Tor et al (1994) Mol. Plant-Microbe Interact. 7, 214-222 Van der Ackerveken et al (1996) Cell 87, 1307-1316. de Vries et al (1997) Applied and Environmental

Microbiology 63: 4638-4644.

Wallace et al (1996) Protein Science 5, 1001-1013.

Whitham et al (1994) Cell 78, 1011-1115. Zhou et al (1995) Cell 83, 925-935.

APPENDIX 1

1. BLASTP: protein sequences searched with the complete EDSl peptide. Several triacylglycerol acyhyldrolases

(lipases) have sequence similarity with EDSl over the lipase catalytic sites (see below) .

2. PROSITE database: searched with the complete EDSl peptide identified a lipase motif.

3. SWISS-PROT: a search using the ScanProsite programme with a degenerate sequence motif around the conserved serine of the lipase catalytic site:

[LIV] -X- [LIVFY] - [LIVST] -G- [HYWV] -S-X-G- [GSTAC]

identified among other lipase/esterases, several phospholipase Al enzymes and two rice sequences (040708 and 043360) (Reimmann et al . , 1995) encoding lipase-like proteins Pir7a and Pir7b of unknown function.

4. FASTA: amino acid sequence alignment (allowing gaps) searched with the complete EDSl peptide identified sequence similarity with a petal -abundant lipase-like protein Pn47p from Ipomoea nil (Japanese morning glory) .

5. A manual inspection of predicted genes encoded by BAC clones that are being sequenced as part of the Arabidopsis Genome Initiative, identified a lipase isolog (T06B20.10) that has strong homology to the Ipomoea nil amino acid sequence.

6. Manual inspection of the EDSl peptide revealed three possible bipartite nuclear localization sequences (NLSs) .

7. A central domain of EDSl (amino acids 301 to 453 in Figure 3) aligns with part of the C-terminal domain of a leucine zipper (LZ) mouse transcription factor gene, kr (Cordes and Barsch, 1994), as shown in Appendix 1. EDSl, however, contains no putative LZ motif, and the alignment extends beyond the basic DNA-binding domain of kr to the domain containing the candidate EDSl histidine residue of a potential lipase catalytic site.

Claims

Claims

1. An isolated nucleic acid molecule encoding a polypeptide with EDSl function.

2. A nucleic acid as claimed in claim 1 wherein the EDSl polypeptide comprises any one of the amino acid sequences shown in Fig 3 , or shown in Fig 6 and designated Edsller, Edslcol or Edslws.

3. A nucleic acid as claimed in claim 1 or claim 2 comprising any one of the nucleotide sequences shown in Fig 5 and designated Edller, Edslcol or Edslws, or shown in Fig 7 and designated EDSl cDNA, or shown in Fig 3 and designated complete Bglll fragment, or being degeneratively equivalent thereto.

4. A nucleic acid encoding an EDSl variant polypeptide, which nucleic acid is an allele, fragment, derivative, mutant or homologue of the nucleic acid of claim 2 or claim 3, and wherein the EDSl variant polypeptide is immunologically cross reactive with the polypeptide encoded by the nucleic acid of claim 2 or claim 3.

5. A nucleic acid as claimed in claim 4 encoding an EDSl variant polypeptide which is an EDSl homologue obtainable using a nucleic acid as claimed in claim 2 or claim 3, said homologue having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95% homology with the nucleic acid of claim 2 or claim 3.

6. A nucleic acid as claimed in claim 4 or claim 5 wherein the EDSl variant polypeptide has EDSl function.

7. A nucleic acid as claimed in claim 5 comprising any one of the nucleotide sequences shown in Table 1 with reference to Fig 3 and designated: Ws edsl - 1 , Ler edsl -2 , Ler edsl -3 , Ler edsl - 4 , Ws edsl -5, Ws edsl - 6, Ws edsl - 7, Ws edsl - 8 .

8. A method for identifying or isolating a nucleic acid as claimed in any one of claims 1 to 7, the method comprising the steps of:

(i) providing a preparation of plant cell nucleic acid, (ii) providing a nucleic acid molecule which is a probe or primer having a nucleotide sequence comprising all or part of a nucleotide sequence as claimed in claim 2 or claim 3, or complementary to that sequence, (iii) contacting nucleic acid in said preparation with said probe or primer under conditions for hybridisation, (iv) identifying any nucleic acid hybridised to said probe or primer.

9. A method as claimed in claim 8 wherein the nucleic acid is identified by amplification.

10. A method as claimed in claim 8 or claim 9 for cloning the EDSl homologue of claim 5.

11. A method as claimed in claim 8 or claim 9 for monitoring the segregation of a resistance gene.

12. An oligonucleotide for use as a nucleic acid probe or primer for use in the method claimed in any one of claims 8 to 11; said oligonucleotide comprising

(i) a nucleotide sequence encoding an amino acid sequence which is conserved, or substantially conserved, between at least two EDSl polypeptides encoded by the nucleic acid molecules of claim 2 or claim 3, or

(ii) a nucleotide sequence which is complementary to said conserved sequence .

13. An oligonucleotide as claimed in claim 12 comprising at least about 18, 21, 24 or 30 nucleotides.

14. An oligonucleotide as claimed in claim 13 selected from: EDSlf (5 'AAG CTT ACC TAA CCG AGC GCT) or EDSlr (5 'AAG CTT CGT TAA CAG TAG CTA C) .

15. A recombinant vector comprising the nucleic acid of any one of claims 1 to 7.

16. A vector as claimed in claim 15 wherein the nucleic acid is under the control of a promoter.

17. A vector as claimed in claim 16 further comprising one or more of the following: a terminator sequence; a polyadenylation sequence; an enhancer sequence; a marker gene .

18. A vector as claimed in claim 16 or claim 17 wherein the promoter is an inducible gene promoter.

19. A host cell comprising a nucleic acid as claimed in any one of claims 1 to 7.

20. A host cell transformed with a vector as claimed in any one of claims 15 to 18.

21. A host cell having incorporated into its genome a heterologous nucleic acid as claimed in any one of claims 1 to 7.

22. A host cell as claimed in any one of claims 19 to 21 which is a plant cell.

23. A method of making the plant cell of claim 22, the method comprising the steps of:

(i) introducing a vector as claimed in any one of claims 15 to 18 into the plant cell, and,

(ii) causing or allowing recombination between the vector and the plant cell genome to introduce a nucleic acid as claimed in any one of claims 1 to 7 into the genome.

24. A plant including a plant cell as claimed in claim 22.

25. A plant which is a clone; selfed or hybrid progeny, or other off-spring or descendant of the plant of claim 24.

26. A plant as claimed in claim 25 including the nucleic acid of any one of claims 1 to 7.

27. A plant as claimed in any one of claims 24 to 26 which is a crop plant .

28. A cutting, part, or seed, or other propagule of a plant as claimed in any one of claims 24 to 27.

29. A polypeptide expression product of a nucleic acid molecule of any one of claims 1 to 7.

30. A polypeptide as claimed in claim 29 which is an EDSl polypeptide comprising any one of the amino acid sequences shown in Fig 3 or shown in Fig 6 and designated Edsller, Edslcol or Edslws.

31. A polypeptide as claimed in claim 29 which is an EDSl variant polypeptide which is a variant, fragment, derivative, mutant or homologue of the EDSl polypeptide of claim 30 and which is immunologically cross reactive with that polypeptide.

32. An EDSl variant polypeptide as claimed in claim 31 which has EDSl function.

33. A method of making the polypeptide of any one of claims 29 to 32 by causing or allowing expression from a nucleic acid encoding the polypeptide in a suitable host cell.

34. The use of a polypeptide as claimed in any one of claims 29 to 32 as an esterase

35. The use as claimed in claim 34 wherein the esterase is a lipase.

36. The use as claimed in claim 35 of the polypeptide in a lipid-based signalling pathway.

37. Use of a polypeptide as claimed in any one of claims 29 to 32 to raise an antibody.

38. An antibody having specific binding affinity for the polypeptide claimed in any one of claims 29 to 32.

39. A polypeptide comprising the antigen-binding site of an antibody as claimed in claim 38.

40. A method of identifying or isolating a polypeptide as claimed in any one of claims 29 to 32, said method comprising the step of screening candidate polypeptides with a polypeptide or an antibody as claimed in claim 38 or claim 39.

41. A method of modulating the defence response in a plant, the method comprising causing or allowing expression of nucleic acid as claimed in any one of claims 1 to 7 within the cells of the plant, following an earlier step of introducing the nucleic acid into a cell of the plant or an ancestor thereof.

42. A method as claimed in claim 41 wherein the nucleic acid is expressed under the control of a pathogen- inducible promoter.

43. A method as claimed in claim 41 or claim 42 for raising pathogen resistance to a plant which comprises expressing a nucleic acid as claimed in any one of claims 1 to 7 within the cells of a plant.

44. A method as claimed in any one of claims 41 to 43 wherein the pathogen resistance is mediated by an R gene of the TIR-NBS-LRR type.

45. A nucleic acid which is the complement of a nucleic acid as claimed in any one of claims 1 to 7.

46. A method of downwardly modulating EDSl expression in a plant, the method comprising any of the following: (i) causing or allowing transcription from a nucleic acid as claimed in claim 45 within the cells of a plant, (ii) causing or allowing transcription from a nucleic acid as claimed in any one of claims 1 to 7 within the cells of a plant, such as to co-suppress endogenous EDSl expression,

(iii) use of a nucleic acid encoding a ribozyme specific for a nucleic acid as claimed in any one of claims 1 to 7.