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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)

Definitions

• 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.
• the invention relates to the EDSl gene
• 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) .
• HR hypersensitive response
• 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.
• 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 .
• genes encode proteins containing a nucleotide binding site (NBS) and leucine-rich repeats (LRRs) .
• NBS nucleotide binding site
• LRRs leucine-rich repeats
• 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) .
• 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.
• 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.
• 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.
• 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.
• a defective mutant allele (the ⁇ drl mutation) of the NDR1 gene abolishes resistance specified by RPM1 , RPS2 and RPS5 to the Pseudomonas syringae avr genes, respectively, avrB, avrRpt2 , and avrPph ⁇ (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 .
• NDR1/NIM1 nuclear factor-binding protein 1
• SAR systemic acquired resistance
• 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) .
• edsl -1 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.
• edsl -1 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.
• 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.
• 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.
• RPP21 another RPP locus, RPP21 (recognition of P. parasi tica isolate Madil, chromosome 5; Holub and Beynon, 1997) is dependent on EDSl .
• RPP8-mediated resistance (chromosome 5) to P. parasi tica isolate Emco5 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 .
• the present invention relates to altering a defence response in a plant and means and methods relating to this.
• 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 .
• nucleic acid molecule which comprises a nucleotide sequence encoding a polypeptide with EDSl function.
• 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.
• 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 .
• P. arasitica isolate Emco5 is EDSl - independent .
• 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 ⁇ 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
• P. parasi tica isolate Cala2 is hypervirulent on La- edsl plants compared to the genetically susceptible wild type parent La-er.
• Ws-edsl plants exhibit enhanced susceptibility to the Ws-compatible P. parasi tica isolate, Emwal.
• 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.
• pathogen types ie not just bacterial
• Ws-edsl and La- edsl plants which carry ineffective mutant forms of the EDSl gene
• Ws-edsl and La- edsl plants 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
• EDSl may function by processing a molecule that is synthesized or made accessible by R protein activity.
• 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.
• EDSl function is used to refer to sequences which dictate an EDSl phenotype in a plant (see above)
• n edsl 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.
• 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.
• 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.
• a mutant, variant, fragment, derivative, allele or homologue may encode a polypeptide which substantially retains the EDSl function of the polypeptide sequences disclosed.
• 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.
• 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.
• 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.
• the major interest will be one of raising plant resistance to pathogens.
• homologues of the particular EDSl sequences provided herein 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.
• 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.
• 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.
• 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.
• 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.
• TMV tobacco mosaic virus
• 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 .
• EDSl cD ⁇ A 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.
• homologues 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 .
• oligonucleotides e.g. a degenerate pool
• 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.
• 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..).
• 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.
• genomic DNA e.g., genomic DNA
• cDNA or RNA or a mixture of any of these preferably as a library
• 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) .
• 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 may be measured using any of a variety of techniques at the disposal of those skilled in the art.
• probes may be radioactively, fluorescently or enzymatically labelled.
• Other methods not employing labelling of probe include 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.
• 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.
• SSC Standard Saline Citrate
• 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 .
• Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42 °C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS.
• suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0.1X SSC, 0.1% SDS.
• 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.
• 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.
• 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. conserveed nucleotide sequences may be identified from the nucleotide sequence information contained herein.
• 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 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• an inducible (or “switchable” ) promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero) .
• expression is increased (or switched on) to a level which brings about the desired phenotype.
• 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.
• 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.
• CaMV 35S Cauliflower Mosaic Virus 35S
• Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate .
• appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate .
• 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 .
• 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 .
• 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 .
• 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,
• 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
• Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective.
• a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233 ) .
• 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 invention further encompasses a host cell transformed with a vector as set forth above, especially a plant or a microbial cell.
• a host cell such as a plant cell, comprising a nucleotide sequence as herein indicated is provided.
• the nucleotide sequence may be incorporated within the chromosome .
• 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.
• 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.
• nucleotide sequences heterologous, or exogenous or foreign, to a plant cell may be non-naturally occurring in cells of that type, variety or species.
• 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.
• 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.
• transgenic crop plants which have been engineered to carry genes identified as stated above.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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
• homologues of the particular EDSl polypeptide sequences provided herein 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• lipase function in R gene-mediated disease resistance to microbial pathogens and in possible disease limitation caused by compatible pathogens is previously unknown.
• 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).
• 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.
• EDSl substrate is a phospholipid or lipoprotein.
• 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 .
• 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 .
• 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.
• 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.
• an antibody for example whole antibody or a fragment thereof
• 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.
• 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.
• 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.
• HR hypersensitive response
• 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.
• 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.
• alteration of resistance may be achieved by introduction of the nucleotide sequence in a sense orientation.
• 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.
• it will be desirable to promote the defence response and this may be achieved by allowing EDSl gene function.
• under-expression of endogenous EDSl gene may be achieved using anti-sense technology or "sense regulation".
• 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,
• 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.
• a constitutively expressed promotor such as the CaMV 35S promotor
• 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)
• 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.
• 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.
• 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.
• This may be DNA and under control of an appropriate regulatory sequence for anti-sense transcription in cells of interest .
• 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.
• 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) .
• '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.
• 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 F 2 plants with transposed elements .
• FIG. 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 .
• 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 footprints (novel nucleotides) generated by the dSpm element.
• 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 7 Nucleotide sequence of the EDSl cDNA from La-er.
• 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.
• 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.
• 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:
• 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.
• 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) .
• 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
• 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:
• 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.
• EDSl-hom.l 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.
• 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.
• 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.
• 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 .
• 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.
• EST Expressed Sequence Tag
• lipases have sequence similarity with EDSl over the lipase catalytic sites (see below) .
• PROSITE database searched with the complete EDSl peptide identified a lipase motif.
• SWISS-PROT a search using the ScanProsite programme with a degenerate sequence motif around the conserved serine of the lipase catalytic site:
• 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) .
• 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 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.

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