WO2000008160A2 - A plant disease resistance signalling gene: materials and methods relating thereto - Google Patents

Abstract

Plant disease resistance signalling gene Rar1 from barley, rice, Arabidopsis and homologues from other species. Nucleic acid and encoded polypeptides are useful in modulating the signalling pathway in plants leading to a plant pathogen defence response and/or cell death, or pathogen resistance effected by interaction of R gene products with pathogen Avr proteins.

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 Rarl gene of barley and homologues thereof from other species.

Plant resistance to pathogens is known to be associated with the induction of a battery of defence-related responses including the production of antimicrobial compounds, the activation of pathogenesis-related (PR) genes, cross-linking of the plant cell wall, and the production of reactive oxygen species (Dixon and Lamb 1990; Hammond-Kosack and Jones 1996) . In most cases resistance is associated with the activation of a host cell death response at the site of attempted infection, termed the hypersensitive response (HR) . It has proven difficult, however, to show the significance of a particular response in arresting growth of the intruder using biochemical and physiological techniques. In contrast, genetic studies have defined loci, the R genes, that contribute key roles in the response to microbial pathogens carrying corresponding avirulence determinants (Flor 1971) . Therefore knowledge of these resistance gene products and their signalling pathways promises deeper insights into plant defence mechanisms. In recent years a large number of R genes have been isolated (Martin et al . 1993; Bent et al . 1994; Jones et al . 1994; Mindrinos et al . 1994; Grant et al . 1995; Lawrence et al . 1995; Song et al . 1995; Anderson et al . 1997; Cai et al . 1997; Ori et al . 1997; Yoshi ura et al . 1998) . Most R genes isolated from plants fall into two classes, encoding either a variable stretch of leucine rich repeats and a putative nucleotide binding site or encode a leucine rich repeat domain but no nucleotide binding site. A third and fourth class, each having only one representative so far, encode proteins with leucine rich repeats and a serine-threonine protein kinase domain, or a protein kinase only. The precise action of J? genes and the downstream pathways they affect, however, are largely unknown.

The complexity of plant defence responses suggested that other components beside the J? gene are involved in resistance reactions. First genetic evidence for additional components in race-specific resistance was obtained in the barley- powdery interaction by the identification of Rarl and Rar2, two components required for the action of the R gene Mla-12 (previously designated Nar-1 and Nar-2 ; Torp and Jørgensen, 1986; Jørgensen 1988; Freialdenhoven et al . 1994). Later studies in other plant pathogen interactions revealed similar observations (Hammond-Kosack et al . 1994; Salmeron et al . 1994; Century et al . 1995; Parker et al . 1996) and two of these components have recently been isolated in the simpler dicot model species Arabidopsis thaliana (unpublished PCT/GB98/01406; Century et al . 1997). Both of these Arabidopsis genes, EDS1 and NDR1 , are required for the function of multiple R genes to different pathogens, i.e. bacterial and fungal pathogens. NDR1 encodes a putative integral membrane protein with unknown biochemical function whereas EDS1 is predicted to encode a putative novel plant lipase .

To date, evidence indicates that mutants in barley Rarl suppress most tested powdery mildew race-specific resistance specificities encoded at the Mia locus on chromosome 1H (Mla- 6, Mla-9, Mla -12, Mla -13 , Mla -14 , Mla-22, and Mla -23) as well as resistance specificities to powdery mildew at other loci (Mlat, Mlh, Mlk, Mlra, and Mlg) (Jørgensen 1996; Peterhansel et al . 1997) . However, in some cases, Mla-1 , Mla - 7 and mlo, no suppression of a resistance gene function was observed (Jørgensen, 1996; Peterhansel et al . 1997). Two chemically- induced allelic mutants in Rarl , designated rarl -1 (mutant M82) and rarl -2 (mutant M100) , have been characterized

(Freialdenhoven et al . 1994). Each of these two recessive mutant alleles enables the powdery mildew pathogen to complete its life cycle in the presence of a the above mentioned powdery mildew R genes. The epidermal single cell HR, a characteristic early event of the Mla-12 specified resistance response, is abolished in the presence of rarl -1 or rarl -2 . However, at later stages, susceptible infection phenotypes in the presence of rarl -1 or rarl -2 can be easily discriminated. In the presence of the former defective allele less aerial fungal mycelium and less sporulation is observed, and infection sites are frequently surrounded by necrotic host tissue. In contrast infection phenotypes in the presence of rarl -2 are hardly distinguishable from a fully susceptible barley genotype. The inventors have interpreted these findings such that the rarl -1 allele retains residual gene activity and have recently demonstrated that rarl -2 does too.

The genetic data described above provide strong evidence that Rarl represents a point of convergence in the signalling of powdery mildew resistance triggered by a multitude of powdery mildew R genes.

There is in the literature a lack of any reliable biochemical test procedures enabling a conventional purification of the Rarl protein.

The present inventors have succeeded in cloning the Rarl gene from barley using positional cloning, this despite the fact that map-based isolation of genes from the highly complex barley genome (5.3 x 10⁹ bp/haploid genome; (Bennett and Smith 1991) ) poses a major experimental challenge primarily due to unfavourable ratio of genetic and physical distances and due to a high percentage of repetitive noncoding DNA sequences. To date, there has been only one report of a successful map- based isolation of a barley gene (Bϋschges et al . 1997) .

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

In various aspects the invention relates to nucleic acid encoding a polypeptide with Rarl function. "Rarl function" refers to the ability of the Jarl gene and polypeptide expression products thereof to function in the signalling pathway leading to a plant pathogen defence response and/or cell death and preferably pathogen resistance effected by the direct or indirect interaction of R gene products with pathogen Avr proteins. The term "Rarl function" may be used to refer to sequences which dictate an Rarl phenotype in a plant, the term "Rarl mutant function" or " rarl function" may be used to refer to forms of Rarl sequences which suppress or cancel an Rarl phenotype in a plant. An rarl mutant phenotype is characterised by the lowering or cancelling of pathogen resistance and/or plant pathogen defence response. Rarl function and rarl 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 polynucleotide according to the invention may encode a polypeptide including the amino acid sequence shown in Figure 1. The coding sequence may be that shown included in Figure 1 or it may be a mutant, variant, derivative or allele of the sequence shown. The sequence may differ from that shown by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to a nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code. Thus, nucleic acid according to the present invention may include a sequence different from the sequence shown in Figure 1 yet encode a polypeptide with the same amino acid sequence (i.e. the coding sequence may be "degeneratively equivalent").

A polynucleotide according to the invention may include one or more sequences identified as an exon in Figure 4.

Also encompassed by the present invention are nucleic acid molecules which include a nucleotide sequence which encodes a polypeptide including an amino acid sequence which although clearly related to a functional Rarl polypeptide (e.g. is immunologically cross reactive with an Rarl polypeptide demonstrating Rarl function, or has characteristic sequence motifs in common with an Rarl polypeptide) no longer has Rarl function. Thus the present invention provides mutants of Rarl which do not promote a plant pathogen defence response or cell death, and/or pathogen resistance. Plants and plant cells carrying these mutant forms are susceptible to pathogen infection.

Thus Rarl 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 Rarl sequences provided herein (see e.g. Figure 3) 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 include a nucleic acid sequence encoding an Rarl homologue obtainable using a nucleotide sequence derived from, or as shown in Figure 1 or Figure 4, or obtainable using amino acid sequence shown in Figure 1 or Figure 4. The Rarl homologue may at the nucleotide level have homology with a nucleotide sequence of Figure 1, or may encode a polypeptide which has homology with the polypeptide of which the amino acid sequence is shown in Figure 1, 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. (Determination of homology at the amino acid level is discussed further below. )

In certain embodiments, a polypeptide 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 amino acid sequence of Figure 1. 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 Rarl or Rarl function, may include fragments of various parent proteins.

Fragments according to further aspects of the present invention are shown in Figure 5A and Figure 5C, with encoding nucleotide sequences shown in Figure 5B and Figure 5D respectively .

Further domains and fragments of the present invention are shown in Figure 6, with encoding nucleotide sequences shown in Figure 7. The domains and fragments may be employed in various aspects and embodiments of the present invention disclosed herein

Each nucleotide sequence of Figure 7 represents a further aspect of the present invention, and polynucleotides comprising a sequence as shown may be employed in various aspects and embodiments disclosed herein.

Rarl-derived oligonucleotide primers (as authentic or degenerate sequences) may be used to isolate Rarl homologues from many different plants, including monocots and dictors, such as barley, wheat, maize, oats, rice, tomatoes, melons, cucurbi taceae, Brassicaceae, capsicums, lettuces, grape vines, ornamentals. Once a corresponding gene is cloned it may be expressed as an antisense construct to assess its importance in resistance to agronomically important diseases such as Puccinia hordei (leaf rust) , Rhynchosproium secalis (scald) , Pyrenophera teres (net blotch) , Heterodera avenae (barley cereal cyst namatode) , Drechslera teres, Powdery mildew and yellow dwarf virus (e.g. barley yellow dwarf virus) .

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 Rarl (or rarl) function. These may have ability to complement an Rarl (or rarl) phenotype in a plant or may, upon expression in a plant, confer such a phenotype. Thus the .Rarl cDNA 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 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 Rarl 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 Barley Rarl gene, although it should be noted that homologue sequences pre-existing on databases, such as any identified herein including in Figure 3, may be excluded from one or more aspects or embodiments of the present invention while included in one or more other aspects .

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 .

Primers useful in aspects of the present invention include "AtRarl 5'" and "AtRarl 3'", the sequences of which are given below.

According to a further aspect, the present invention provides a method of identifying or a method of cloning an Rarl homologue, e.g. from a species other than Barley, the method employing a nucleotide sequence derived from that shown in Figure 1 or Figure 4. 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 or complementary to a nucleotide sequence substantially as shown herein, preferably from within the coding sequence (e.g. a sequence coding for the amino acid sequence shown in Figure 1) 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, include 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 Rarl 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 Rarl 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-PCR.

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

Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na₂HP0₄, pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0. 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. IX SSC, 0.1% SDS.

In general, hybridizations may be performed according to the method of Sambrook et al . (below) using a hybridization solution comprising: 5X SSC (wherein SSC = 0.15 M sodium chloride; 0.15 M sodium citrate; pH 7), 5X Denhardt's reagent, 0.5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes - 1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is (Sambrook et al. (1989)): T_m = 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex.

As an illustration of the above formula, using [Na+] = [0.368] and 50-% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57°C. The T_m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C. Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.

It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. Other 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. IX SSC, 0.1% SDS. An alternative, which may be particularly appropriate with plant nucleic acid preparations, is a solution of 5x SSPE (final 0.9 M NaCI , 0.05M sodium phosphate, 0.005M EDTA pH 7.7), 5X Denhardt's solution, 0.5% SDS, at 65°C overnight, (for high stringency, highly similar sequences) or 50°C (for low stringency, less similar sequences). Washes in 0.2x SSC/0.1% SDS at 65°C for high stringency, alternatively at 50-60°C in lx SSC/0.1% SDS for low stringency.

The present invention extends to nucleic acid selectively hybridisable under high stringency with nucleic acid identified herein, e.g. the coding sequence of Figure 1, the sequence of Figure 4 or the sequence of Figure 5C or Figure 5D.

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 Rarl/Rarl 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 Rarl peptides or polypeptides encoded by genes involved in the signalling of a defence response in a plant . Conserved sequences may be identified using information contained herein, for instance in Figure 3.

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 Rarl nucleotide sequence as provided herein or an Rarl 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) . A probe or primer may be about 20-30 nucleotides in length. 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 include cDNA, RNA, genomic DNA and may be 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 Rarl nucleotide sequences as herein provided or obtainable by use of the disclosures herein can be easily and routinely tested for both Rarl function and Rarl 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 binary vector, e.g. suitable for use with Agrobacterium. 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. Thus, the nucleotide sequence of Figure 1 (for example) may be placed under the control of a promoter other than that of the Barley Rarl gene. Similarly, a Rarl homologue sequence from another species may be operably linked to a promoter other than that with which it is naturally associated. However a vector including 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 including 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 including 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 Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al ,

(1990) 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, e.g. inner phloem, flower primordia, branching points in root and shoot (Medford, J.I. (1992) Plant Cell 4, 1029-1039; Medford et 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) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al . (1987) Plant Tissue and Cell Cul ture, Academic Press) , electroporation (EP 290395, WO 8706614) 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 .

Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Production of stable, fertile transgenic plants in almost all economically relevant monocot plants is also now routine: (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, Agrobacterium mediated transformation is now a 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) . Wan and Lemaux (1994) Plant Physiol . 104: 37-48 describe techniques for generation of large numbers of independently transformed fertile barley plants.

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, e.g. 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 XXX, 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, including 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, melons, capsicums, grape vines, 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 e.g. 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 preferred polypeptide includes the amino acid sequence shown in Figure 1. A polypeptide according to the present invention may be an allele, variant, fragment, derivative, mutant or homologue of a polypeptide as shown in Figure 1. The allele, variant, fragment, derivative, mutant or homologue may have substantially the Rarl function of the amino acid sequence shown in Figure 1 or may be a rarl mutant .

Also encompassed by the present invention are polypeptides which although clearly related to a functional Rarl polypeptide (e.g. they are immunologically cross reactive with an Rarl polypeptide demonstrating Rarl function, or they have characteristic sequence motifs in common with an Rarl polypeptide) no longer have Rarl function. Thus the present invention provides variant forms of Rarl polypeptides, such as those resulting from the rarl -1 and rarl -2 mutations identified herein. Plants and plant cells carrying these mutant forms are susceptible to pathogen ingress.

"Homology" in relation to an amino acid sequence may be used to refer to identity or similarity, preferably identity. As noted already above, high level of amino acid identity may be limited to functionally significant domains or regions, e.g. any of the domains identified herein (e.g. see Figure 6) .

In particular homologues of the particular Rarl 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. Thus the present invention also extends to polypetides which include an amino acid sequence with Rarl function obtainable using sequence information as provided herein. The Rarl homologue may at the amino acid level have homology with the amino acid sequence of Figure 1, 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 85 %, or at least about 88% 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 Rarl or Rarl function, may include fragments of various parent proteins. Individual domains and fragments of Rarl polypeptide are shown in Figure 6 and these, also derivatives, variants and homologues as noted, are useful in various aspects and embodiments of the invention, for instance in the activation of cell death and/or downstream resistance responses.

Similarity of amino acid sequences may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J^". Mol . Biol . 215: 403-10, which is in standard use in the art. In particular, TBLASTN 2.0 may be used with Matrix BL0SUM62 and GAP penalties: existence: 11, extension: 1. Another standard program that may be used is BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Adv. Appl . Math . (1981) 2: 482-489) . Other algorithms include GAP, which uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. As with any algorithm, generally the default parameters are used, which for GAP are a gap creation penalty = 12 and gap extension penalty = 4. Alternatively, a gap creation penalty of 3 and gap extension penalty of 0.1 may be used. The algorithm FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448) is a further alternative.

Use of either of the terms "homology" and "homologous" herein does not imply any necessary evolutionary relationship between compared sequences, in keeping for example with standard use of terms such as "homologous recombination" which merely requires that two nucleotide sequences are sufficiently similar to recombine under the appropriate conditions. Further discussion of polypeptides according to the present invention, which may be encoded by nucleic acid according to the present invention, is found below.

Purified Rarl polypeptides and mutants, variants, fragments, derivatives, alleles and homologues thereof e.g. 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 including 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 (e.g. 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, e.g. 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 Rarl function or Rarl function (in accordance with embodiments disclosed herein) , including screening candidate peptides or polypeptides with a polypeptide including the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind an Rarl or Rarl 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 including antigen binding domains of antibodies that bind and are preferably specific for a Rarl or Rarl 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 promoting cell death and/or a plant pathogen defence response in a plant which includes expressing a heterologous nucleic acid sequence with Rarl function as discussed, within cells of the plant .

The invention further provides a method of raising pathogen resistance in a plant which includes expressing a heterologous nucleic acid sequence with Rarl 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 Rarl 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.

Manipulation of expression of the Rarl transcript or Rarl protein may be used to enhance resistance to a broad spectrum of pathogens in different plants. This may be achieved by over expression using a highly active plant promoter such as the CaMV-35S promoter. Alternatively, Rarl may be attached to a pathogen-inducible promoter (see discussion below) , allowing greater expression in challenged cells. Increased disease resistance may occur in the absence of a hypersensitive response (HR) that may 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 includes expression from a nucleic acid encoding the amino acid sequence of Figure 1 or a mutant, allele or derivative of the sequence (which may have Rarl 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 including 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 Rarl gene function.

In order to down-regulate resistance signalled by Rarl , under-expression of endogenous Rarl 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 Rarl or homologues. In cases where several Rarl 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 Rarl expression in a plant, the method including causing or allowing anti-sense transcription from nucleic acid according to the invention within cells of the plant. Rarl down-regulation may reduce a defence response. This may be appropriate in certain circumstances e.g. as an analytical or experimental approach.

For use in anti-sense regulation, nucleic acid including a nucleotide sequence complementary to a coding sequence of an Rarl 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 Vomnet & Baulcombe (1997) Nature 389: pg 553.

Suitable fragments may be about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 nucleotides in length.

Thus, the present invention also provides a method of downwardly modulating Rarl function in a plant, the method including causing or allowing expression from nucleic acid according to the invention within cells of the plant to suppress endogenous Rarl expression. Modified versions of Rarl may be used to down-regulate endogenous Rarl function. For example mutants, variants, derivatives etc., may be employed.

Reduction of Rarl wild type activity may be achieved by using ribozymes, such as replication ribozymes, e.g. of the hammerhead class (Haseloff and Gerlach, 1988, Nature 334 : 585-591; Feyter et al . Mol . , 1996, Gen . Genet . 250: 329- 338) .

Another way to reduce Rarl function in a plant employs transposon mutagenesis (reviewed by Osborne et al . , (1995) Current Opinion in Cell Biology 7, 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. .Rarl alleles carrying any insertion of known sequence could be identified by using PCR primers with binding specificities both in the insertion sequence and the Rarl 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 following figures.

Brief Description of the Figures

Figure 1 shows the nucleotide and deduced amino acid sequences of the barley Rarl cDNA. The nucleotide and the deduced amino acid sequence are based on the combined data of RT-PCR and RACE obtained from experiments using RNA of cultivar Ingrid Rarl . The stop codon is marked by an asterisk and the detected termini of RACE products are indicated by arrows above the sequence.

Figure 2 illustrates the Rarl gene structure. The structure of the barley Rarl gene is given schematically. Exons are highlighted by black boxes. Positions of introns and exons were identified by comparison of RT-PCR products with genomic sequences. Positions of mutational events are indicated for mutants rarl -1 and rarl -2.

Figure 3 shows an alignment of deduced peptide sequences in genes from various species indicating relatedness to barley Rarl. Regions of homology are highlighted in black (identity) , dark grey (highly conservative exchange) or light grey (conservative exchange) . Sequence data were analyzed with the Genetics Computer Group, Wisconsin Program, version 8 (GCG; Devereux, 1984) . Display of aligned deduced amino acid sequences were carried out by using the "prettybox" option in the extended GCG software. Numbers on the left indicate GenBank accession mumbers of each peptide sequence.

Figure 4 shows 10,000 nucleotides of the Rarl genomic gene sequence, including coding exons and introns . The rarl -1 and rarl -2 mutations (both G->A) are marked. Underlined sequences represent Rarl exon sequences and nucleotides in bold represent 5' and 3' consensus splice sequences.

Figure 5A shows the amino acid sequence of a N-terminal fragment of the Rarl polypeptide of Figure 1.

Figure 5B shows a nucleotide sequence encoding the Rarl polypeptide fragment of Figure 5A.

Figure 5C shows the amino acid sequence of a C-terminal fragment of the Rarl polypeptide of Figure 1.

Figure 5D shows a nucleotide sequence encoding the Rarl polypeptide fragment of Figure 5C. Figure 6 shows the amino acid sequence of fragments and domains I, II and III of the Barley Rarl protein, representing particular aspects of the present invention.

Figure 7 shows nucleotide sequences encoding the fragments and domains I, II and III of Figure 6, polynucleotides with these sequences, and polynucleotides comprising these sequences, representing further aspects of the present invention.

Figure 8A shows AtRarl cDNA sequence, including coding sequence .

Figure 8B shows the AtRarl protein sequence (also shown in Figure 3 as ab010074) .

Figure 8C shows the encoding nucleotide sequence for an AtRarl protein N-terminal fragment.

Figure 8D shows the ArRarl protein N-terminal fragment encoded by the nucleotide sequence of Figure 8D.

Figure 8E shows the encoding nucleotide sequence for an AtRarl protein internal fragment . Figure 8F shows the ArRarl protein internal fragment encoded by the nucleotide sequence of Figure 8E.

Figure 8G shows the encoding nucleotide sequence for an AtRarl protein C-terminal fragment.

Figure 8H shows the ArRarl protein C-terminal fragment encoded by the nucleotide sequence of Figure 8G.

Figure 9 shows alignment of various "CHORD" sequences ("Cysteine and Histidine Rich Domain") and a consensus sequence .

All documents cited herein are incorporated by reference.

EXAMPLE 1

Cloning and Characterisation of Rarl From Barley and

Homologues From Other Species

High resolution genetic mapping of Rarl

A previous low resolution interval mapping procedure located Rarl on barley chromosome 2, flanked by RFLP loci CMWG694 and MWG503 within a 5 cM interval (Freialdenhoven et al . 1994) . Because 1 cM in barley corresponds to approximately 3 Mb we decided as a first step towards the isolation of Rarl to establish a local high resolution genetic map. We aimed at a resolution of approximately 0.01 cM which corresponds to an average physical distance of 30 kb.

Due to the low frequency of DNA polymorphism especially in spring types (Russel et al . 1997), we reasoned that the availability of alternative crosses would be advantageous because it increased the likelihood to find a polymorphism with a given probe. Therefore both mutants were crossed with three Mla -12 backcross (BC) lines (Mla -12 BC Ingrid, Mla -12 BC Pallas, Mla -12 BC Siri; (Freialdenhoven et al . 1994; Kølster et al . 1986; Kølster and Stølen 1987) representing different genetic backgrounds of barley spring types.

The stages of the high resolution mapping were, in outline:

Using the initial RFLP map which was based on 50 plants (Freialdenhoven et al . 1994), the CAPS markers MWG876, MWG892 and MWG2123 were integrated into the genetic map. A phenotypic screen was used to analyse 1040 plants for recombination events between Ant2 and Rarl . The observed recombinants were used to reveal that MWG892 maps distal in relation to Rarl . A subsequent CAPS-based recombinant screen of 1063 additional plants was performed in the marker interval MWG892 - cMWG694 . Analysis of the observed recombinants positioned MWG876 proximal in relation to Rarl . Finally another 2207 plants were investigated in the marker interval MWG892 - MWG876. Two AFLPs R25/48 and A25/43 were identified and tested on DNA of pool individuals. R25/48 was identified in resistant bulked segregants. PCR products were analysed on a 4.5% denaturing polyacrylamide gel. Presence of the linked AFLP signal was found in one of the susceptible pool individuals, indicating a recombination event between the AFLP locus R25/48 and Rarl , which was not displayed in the susceptible pool (Bs) . The two recombinants identified with R25/48 and A25/43 revealed cosegregation of Rarl and MWG876, thus both AFLP loci must be located distal from MWG876. Genetic distances (cM) were calculated on the basis of two-point estimates.

In more detail :

RFLP markers MWG503 and CMWG694 which define an approximately 5 cM interval containing Rarl (Freialdenhoven et al . 1994) were sequenced, oligonucleotides for amplification of the corresponding loci were derived and polymorphisms between the susceptible (rarl-1 , rarl-2 ) and resistant parents (Mla-12 BC Ingrid, Mla-12 BC Pallas, Mla-12 BC Siri) were determined. This involved display on ethidium bromide stained 2.5% agarose gel of restriction enzyme digested amplification products using M82, MlOO, Mla -12 BC Ingrid, Mla-12 BC Pallas and Mla -12 BC Siri as template DNA. Amplification and t

54 digestion were carried out as described below in relation to Table 2. The displayed CAPS markers corresponded to RFLP loci MWG87, MWG503, CMWG694, MWG876, and MWG2123. PCR- primers for locus MWG892 enabled allele-specific discrimination of PCR products without subsequent restriction digestion. Minor bands were due to incomplete Bell digests of the PCR amplicons.

To increase marker density adjacent to Rarl , we selected three further RFLP markers which map close to the above mentioned RFLP loci from a general RFLP map (MWG876, MWG892 and MWG2123 [Graner, 1991] . Each of these RFLPs was converted to a cleavable amplifiable polymorphic sequence (CAPS) and was mapped relative to Rarl based on a population of 50 segregants . In this population MWG876 and MWG892 showed cosegregation with Rarl whereas MWG2123 was positioned distal to CMWG694.

Recombinant screen

Cultivar Sultan-5 (Mla-12, Rarl) from which both Rarl mutants (rarl-1, rarl-2 ) are derived, contains an anthocyanin pigmentation deficiency (ant2) whereas the three resistant Mla-12 BC lines used for mapping (Mla-12 BC Ingrid, Mla-12 BC Pallas, Mla-12 BC Siri) carry the Ant2 wild type allele. The Ant2 locus was previously shown to map at a distance of approximately 0.5 cM proximal to Rarl (Freialdenhoven et al . 1994) . To identify rare recombinants in the small interval between Rarl and Ant2 we selected 1,040 susceptible F₂ individuals (rarl/ rarl) and screened for presence of the anthocyanin wild type allele Ant2. A total of 14 recombinants were found and these were tested for alleles in MWG87, MWG876, and MWG892. Analysis of the 14 recombinants showed cosegregation of Rarl , MWG87 and MWG876. Marker MWG892 was positioned between Ant2 and Rarl , separated from the former by two recombination events. The different crosses between the two allelic Rarl mutants and the three Mla-12 BC lines did not reveal significantly different recombination frequencies. Therefore we restricted our search for further recombinants to one cross only (MlOO x Mla-12 BC Ingrid) . This enabled us also to use the identified recombinants ajacent to Rarl for a targeted AFLP- based marker screen described below.

To increase the genetic resolution in the vicinity of the target locus another 1,063 F₂ plants were screened for recombination events flanking Rarl by utilising CAPS makers MWG892 and CMWG694. Subsequent investigation of MWG87 and MWG876 alleles revealed complete linkage for MWG87 and Rarl but one recombination event between MWG876 and the target gene, positioning this RFLP distal to Rarl . We attempted to separate MWG87 genetically from Rarl by testing a further 2,207 F₂ plants for recombination events in the marker interval MWG876 - MWG892 by CAPS analysis. However, investigation of the observed recombinant plants still revealed cosegregation of Rarl and MWG87. The tight genetic linkage of MWG87, MWG876 and Rarl may indicate small physical distances between these loci but could also be a result of a low recombination frequency in this genomic segment. To investigate these possibilities and to enrich the interval with additional DNA markers, we initiated an AFLP-based marker search. If the tight genetic linkage of Rarl , MWG87 and MWG876 is caused by a suppression of recombination, then the large physical interval would be expected to reveal a large number of linked AFLP markers in the target region.

Targeted AFLP marker search

We employed the AFLP technology (Vos et al . 1995) in conjunction with a bulked segregant analysis (Giovannoni,

1991; Michelmore et al . 1991) by using selected F₂ progenies of the cross rarl-2 x Mla-12 BC Ingrid. To minimise detection of AFLP markers which are not tightly linked to Rarl we used DNA marker-selected recombinants for the construction of DNA pools which we identified in the CAPS- based recombinant screen described above. Both DNA pools comprised each 10 F₂ plants. At the time we initiated the AFLP marker screen we had analysed only 500 F₂ individuals in the marker interval MWG892-cMWG694 and the recombinant between MWG876 and Rarl had not yet been identified. In consequence, MWG876 could not be used to select suitable recombinants. The susceptible pool (rarl-2 / rarl-2 ) contained three individuals with a recombination between cMWG694 and Rarl , four individuals with a recombination between MWG892 and Rarl and three susceptible individuals without a recombination event in the investigated marker interval. The selection of recombinants for the resistant pool was based on DNA markers only. By using plants which show the allelic pattern of the resistant parent for CMWG694 and MWG892 we could ensure homozygosity in the corresponding genetic interval. Therefore linked AFLP markers are expected in trans and cis . To narrow down the target interval of the resistant pool we employed plants carrying a recombination event between MWG503 and MWG892 (two plants) or CMWG694 and MWG2123 (two plants) . In addition to the recombinant individuals we used six plants without a detectable recombination event in the investigated marker interval.

The genome-wide frequency of AFLP-polymorphisms between MlOO and Mla-12 BC Ingrid was found to be 7%. Each AFLP primer combination displayed, on average, 100 DNA fragments.

Therefore, using seven Pstl + 2- and 56 Msel + 3 -primers in 392 combinations, approximately 40,000 loci were inspected. Only two primer combinations identified AFLP markers linked to Rarl in the DNA pools. Analysis of these on individuals of each pool revealed that they are separated from the target gene by one (R25/45) and two (A25/46) recombination events and map distal relative to Rarl .

The small amount of identified AFLP markers linked to Rarl is certainly influenced by the way we assembled the DNA pools. It may also indicate that the small genetic interval in which we searched for DNA markers is physically not excessively large. To obtain more precise estimates on the relationship of genetic and physical distances, we performed PFGE Southern analysis in combination with rare cutting restriction enzymes and RFLP probes linked to Rarl .

Long range physical mapping

Fragment sizes after restriction with seven different rare cutting restriction endonucleases were determined using the cosegregating probe MWG87 and flanking probes MWG892 and MWG876. The analysis revealed a single co-migrating lul restriction fragments hybridising to MWG87 and MWG876 (Table 1) . This may indicate a maximal physical distance of 550 kb between MWG876 and MWG87. Fragments of common size were also detected using the probe/restriction enzyme combinations MWG876/NotI , Sail and Smal (90 kb) and MWG87/Sf^"iI and Smal (100 kb) . These fragments of common size using one probe and different endonucleases are possibly caused by a clustering of restriction sites which has been reported before in vertebrates (Bickmore et al . 1992; Larsen et al. 1992) .

Physical delimi tation of Rarl on barley yeast artificial chromosomes (YACs)

Screening of a barley YAC library

Significant in the high resolution genetic mapping of Rarl was the identification of a 0.7 cM interval bordered by loci MWG892 and MWG876 encompassing the target locus. Locus MWG87 was found to cosegregate with the target on the basis of more than 8,000 meiosis. This provided a rationale to initiate a screening of a barley YAC library to isolate large insert genomic clones containing MWG87. PCR-based screening with the cleaved amplified polymorphic sequence (CAPS) MWG87 resulted in the identification of five yeast clones, each generating the expected PCR product. To confirm that the obtained amplicons represented the MWG87 locus, we sequenced each PCR product and found identical sequences in all YAC- derived fragments. Two of the five YACs also contained the CAPS marker MWG876 which maps 0.015 centimorgan (cM) distal to Rarl .

YAC insert analysis by PFGE and inverse PCR (IPCR)

We used the probe MWG87 in PFGE Southern analysis to determine the insert sizes of the isolated YACs; 680 kb (Y18) , 340 kb (Y30) , 1,100 kb (Y31) , 720 kb (Y73) and 300 kb (Y113) respectively. Two independent Southern analyses showed a significantly reduced signal intensity for YAC Y113 indicating disturbed inheritance of this yeast artificial chromosome. To establish a local contig with the identified YACs, we isolated their left (L) and right (R) termini by IPCR and determined their nucleotide sequence. Sequence analysis of those YAC ends revealed that the YAC terminus Y31L has about 95% identity to the barley BARE-1 retrotransposon, a highly repetitive sequence which comprises about 6.7% of the barley genome (Suoniemi et al . 1996) . Further sequence stretches with high relatedness to the BARE- 1 retrotransposon were found in Y18R, Y18L, and Y73L. In addition, the YAC end Y31R revealed about 64% sequence identity to the maize retrotransposon Opie-2 (SanMiguel et al . 1996) indicating a novel element which has so far not been described in barley.

Overlap analysis of YAC inserts End probes from each yeast clone were tested against all YACs to determine their relationship. Based on the results of the nucleotide blast, YAC-end specific oligonucleotides were designed in sequence stretches representing supposedly non- repetitive DNA. This was not possible for YAC end Y31L which comprises multicopy DNA only. Subsequently, PCR analysis was employed to determine the presence or absence of these sequences in the respective yeast clones (Table 2) . Two anonymous YACs (Yl, Y2) were included to uncover primer pairs which still recognised repetitive DNA. Amplification products corresponding to YAC terminus Y31L in yeast clones Yl and Y2 corroborated the multicopy character of this YAC end. YAC end Y113L gave rise to amplification products in YAC Y73 and Y113. However the length of the PCR product in Y73 was different from the expected size which was detected in Y113. Therefore we concluded that the locus corresponding to Y113L was not present in YAC Y73. All other YAC-end specific primers detected clear absence/presence polymorphisms on the different YAC clones and did not amplify fragments in Yl or Y2 , indicating their suitability for YAC contig analysis.

If all YAC inserts are colinear with the source DNA, each end probe should detect the yeast clone it was derived from plus any YAC covering this area. For two YAC termini, marking the ends of the contig, only the yeast clone they were derived from should be detected. These end probes define now the termini of the YAC contig. Since we determined four YAC ends which are not amplified in any YAC, but the one they are derived from, at least two of the four YAC ends must be derived from chimeric YACs. However based on this information, it remained uncertain which two of the four YACs are chimeric. Genetic mapping of the YAC ends could resolve this lack of clarity but is only possible if the end probes are single or low copy markers.

Copy number of YAC ends

With the exception of Y31L all YAC end specific markers proved suitable for contig establishment based on YAC DNA. Next, we determined whether the YAC-end derived oligonucleotides amplified single loci from barley genomic DNA, a prerequisite for genetic mapping. We performed PCR analysis with the respective YAC-end specific primer pairs using genomic DNA of cultivar Ingrid, the source DNA of the YAC library. Primers corresponding to Y113L revealed multiple amplicons of different lengths matching to nonspecific PCR products also observed in YAC Y73. In contrast, oligonucleotides corresponding to YAC termini Y18L, Y18R, Y30L, Y30R, Y31R, Y73R and Y113R (Table 3) resulted in the amplification of uniformly sized fragments. However, cloning and sequencing of the PCR products (three independent clones for each YAC end) revealed that the oligonucleotides corresponding to Y30L and Y73L generated at least three different amplicons which show about 5% sequence divergence. This indicated that primers corresponding to Y30L and Y73L recognise multiple loci with high degree of sequence similarity. The sequence analysis was used for the selection of endonucleases recognising nucleotide stretches which are polymorphic between the three characterised subclones and the YAC-end derived sequence. Restriction digest of the PCR products with these diagnostic endonucleases resulted in a more complex banding pattern than predicted for an amplicon derived from a single locus of known sequence. Therefore endonuclease based analysis of the PCR products from genomic DNA confirmed heterogeneity of the amplification products corresponding to Y30L and Y73L and may be in general a useful tool to determine if a certain marker detects a single copy locus . Sequence analysis of the subclones corresponding to YAC termini Y18L, Y18R, Y30R, Y31R and Y113R indicated that these amplicons are homogenous.

Assignment of YAC ends to barley chromosomes

To determine the chromosomal location of the isolated YAC ends we used the wheat/barley diteleosomic addition lines, each containing a known chromosome arm of the barley cultivar Betzes (Shepherd and Islam 1981; Islam 1983) . The diteleosomic wheat/barley addition lines facilitate a rapid assignment of a given barley sequence to its corresponding chromosome arm if barley specific signals can be discriminated from wheat specific signals. We used PCR primers derived from the barley cultivar . Ingrid to assign Y18R and Y18L to barley chromosome 2HL, Y31R to chromosome 5HS and Y73R to chromosome 6HS . This indicated chimerism of YAC Y31 and Y73. YAC end Y113R could not be mapped since the primers derived from cultivar Ingrid did not amplify a fragment from cultivar Betzes, the barley DNA donor for the addition lines. Similarly the YAC terminus Y30R could not be assigned because it generated fragments of identical size in wheat and barley.

High-resolution genetic mapping of YAC ends

The wheat/barley diteleosomic addition lines facilitate identification of chimeric YACs but high-resolution genetic mapping is necessary to define the position of the YAC ends in relation to the target locus. A prerequisite for genetic mapping of the YAC termini is a sequence polymorphism between the parental genotypes of the mapping population. We used a PCR-based approach to search for possible sequence polymorphisms. Oligonucleotides corresponding to Y30R gave rise to amplification products of different size in the resistant (Rarl) and susceptible (rarl) parents whereas the primer pair corresponding to Y113R amplified DNA fragments only in each of the resistant parents. PCR products derived from the resistant and susceptible parents for the marker loci Y18L and Y18R were analysed for DNA polymorphisms by direct sequencing. Comparative sequence analysis revealed a polymorphic Hinfl site in the case of Y18R whereas in Y18L, no DNA polymorphism was detected over about 2.7 kb. A copy of a BARE-1 retrotransposon within the Y18L sequence made it impossible to further extend this sequence by IPCR to search for polymorphisms. Genetic mapping of the polymorphic YAC ends positioned Y30R and Y113R proximal to Rarl , separated by eleven and three recombinants respectively from the target locus. Marker Y18R was found to cosegregate with Rarl .

YAC contig at Rarl

Based on information obtained by (i) presence/absence mapping on the YACs, (ii) chromosome assignment via wheat/barley addition lines and (iii) high-resolution genetic mapping, we deduced a YAC contig. YAC Y18 is likely to be the only YAC containing a non-chimeric insert which is colinear to the source DNA, since both termini map to chromosome 2HL. In contrast, YAC Y30 has probably undergone a rearrangement leading to an internal deletion including the marker Y113R. This conclusion is based on (i) the genetic mapping of Y113R between MWG87 and Y30R and (ii) absence of marker Y113R in YAC Y30 (Table 2) . A previous detailed characterisation of the YAC library by PFGE Southern analysis revealed multiple YAC-derived fragments due to instabilities of about 30% of the clones (Simons et al . 1997) . However, the recovery of an individual clone harbouring only the largest insert was usually possible. To determine if the library still contained a Y30 corresponding clone which also contained the Y113R locus, we analysed the pools of yeast DNA which were initially used to identify the YAC clones. We were unable to find Y113R specific amplicons in the pool DNA corresponding to YAC Y30, indicating that the internal deletion in YAC Y30 has occurred at an early stage during construction of the library.

Chimerism of YAC inserts was concluded for YAC Y31, Y73 and Y113. In the case of YACs Y31 and Y73 this assumption is based on assignment of Y31R and Y73R to chromosome 5HS and 6HS respectively. In contrast evidence for chimerism in YAC Y113 was based on absence of all markers distal from MWG87 and the fact that Y113L was detected in YAC Y113 only (Table 2) . Alternatively, the YAC end Y113L could be located distal from YAC terminus Y31L implying that the area between the loci Y18R and Y30L has been deleted in YAC Y113 during clone propagation.

In summary, the genomic area containing Rarl is genetically delimited by Y113R (proximal) and MWG876 (distal) . Since the presented YAC contig covers this interval physically by YAC clones Y18 and Y113 in proximal (two fold redundancy) , and YAC clones Y30 and Y31 in distal orientation (two fold redundancy), we have physically delimited the Rarl locus.

Construction of a BAC contig at Rarl

Next we established two Hindlll BAC sublibraries in vector pBelo BACH from YAC clone Y18 and YAC clone Y30. We aimed at an average insert size of 50 kb and an approximately fivefold redundancy for each sublibrary.

Five BACs, derived from YACs Y30 and Y18, were initially isolated with CAPS MWG87, cosegregating with Rarl (BAC 12, BAC 1J6, BAC 4C20, BAC 1G12 , and BAC 3H6) . PCR primers for marker Y113R were used to isolate BAC 1H1. Insert sizes of the identified BACs were determined by PFGE. End fragments of each BAC insert were isolated by inverse PCR and subsequently sequenced. Based on terminal sequences of

BAC 4C20 we derived a new co-dominant DNA marker, designated EDDA (Table 4) , detecting a sequence polymorphism between the parental genotypes of the mapping population. Analysis of the four recombinants within interval MWG876 - Y113R, positioned EDDA proximal to Rarl . Since BAC 4C20 contains each of the three loci MWG87, Y18R, and EDDA, we have physically delimited Rarl in direction to the centromere on a single BAC clone.

Subsequently, terminal sequences of BAC 12, BAC 3H6, and BAC 1B2 were employed to derive markers OKI114, OK3236, and OK5558, respectively (Table 3) . The co-dominant marker OK1114 was found to cosegregate with Rarl by inspection of genomic DNA derived from the four recombinants within target interval MWG876 - Y113R. Markers OK3236 and OK5558 detected polymorphisms between the parental genotypes Sultan5/Mla- 12 BC Pallas and Sultan5/ la-12 BC Ingrid but we failed to detect a polymorphism for this locus between Sultan5/Mla- 12 BC Siri. Therefore, only three of the four recombinants in the target MWG876 - Y113R could be tested to locate the recombination events. Analysis of the DNA of these three recombinants revealed cosegregation of markers OK3236 and OK5558 with Rarl . The insert lengths of the BACs, the presence and absence of each of the above described markers, and their genetic orientation relative to Rarl was the basis to deduce a high-resolution genetic map at the Rarl locus.

Rarl was physically delimited on the BAC level in centromeric orientation and the identification of a minimal cosegregating interval bordered by markers Y18R and OK5558.

A contiguous 66 kb DNA stretch at the Rarl locus We decided to assemble a contiguous genomic DNA sequence of the DNA interval bordered by markers MWG87 and OK5558. Inserts of BAC 1B2 and BAC 12, covering this area, were subjected to DNA sequencing by means of randomly chosen clones derived from plasmid sublibraries of each BAC into vector pBluescript II Ks+ A 49 bp gap between BAC B2 and BAC 12 was closed by using primers specific for terminal end sequences of each BAC on template DNA of BAC 3H6 and subsequent direct sequencing of the amplicon.

A search for candidate genes

Next we initiated a search for candidate genes in the contiguous 66 kb DNA stretch at the Rarl locus using the BLAST algorithm and all available data bases including anonymous ESTs and genomic as well as known gene and protein sequences. We identified three distinct regions containing highly significant homologous sequences to various entries in the databases (Table 5) . We have designated these three intervals II, 12, and 13 (corresponding to positions 43,500 to 45,000, 45,001 to 46,500, 56,000 to 61,000 in the 66 kb sequence contig) . Intervals II and 12 are sequence related to each other (59% nucleotide identity) and were identified by the same class of ESTs in the databases (Table 5) , each showing similarity to aquaporin genes [Maurel , 1997] . Thus, intervals II and 12 represent two putative barley aquaporin genes arranged in head to tail orientation. Interval 13 shows high sequence similarity ro rice EST C28356 and may represent another coding region in the 66 kb strech.

Identification of mutational events in Rarl candidate genes

Next we compared the DNA sequences of genomic amplicons from genotypes Rarl Sultan5, rarl -1 Sultan5, and rarl -2 Sultan5 each covering intervals II, 12, and 13. We failed to detect any sequence polymorphism between the three genotypes in intervals II and 12. However, two single nucleotide substitutions were discovered in interval 13 at positions 56,764 and 58,562 bp. The G->A substitutions correspond to unique sequence alterations in genotypes rarl -1 and rarl -2, respectively. The finding of mutational events in 13 then prompted us to perform reverse transcriptase-polymerse chain reactions (RT-PCR) with total leaf RNA derived from cultivar Ingrid using a series of primers deduced from the 13 sequence (both YAC and BAC recombinant clones contain genomic DNA from cultivar Ingrid) .

Sequencing of the largest RT-PCR products revealed a single extensive open reading frame of 696 bp (Figure 1) . 5' and 3' ends of the gene transcript were identified using rapid amplification of cDNA ends (RACE) technology. The deduced protein of 232 amino acids has a molecular weight of approximately 25.5 kDa . No significant homologies were found to any other characterised protein in the various databases .

A comparison of the genomic and RT-PCR-derived sequences revealed six exons, each flanked by the consensus splice site sequences (Figure 2 and Table 6) . Notable is exon 2 consisting of only three bp, flanked by consensus splice site sequences, encoding a glycine residue.

The G->A DNA substitution identified in genotype rarl - 1 Sultan5 results in a Cys²⁴ ->Tyr substitution in the putative 25.5 kDa protein (Cys²⁴ represents one of the few invariant amino acids in Rarl homologous proteins; see below) . In contrast, the G->A DNA substitution identified in genotype rarl -2 Sultan5 disrupts the 3' splice site consensus sequence of intron 2. The G nucleotide of the splice site consensus is known to be essential for effective splicing of primary mRNA transcripts in both plant and mammalian species [Goodall, 1991] . RT-PCR analysis of the rarl -2 genotype revealed that the mutation leads to utilisation of a cryptic splice site in exon 3, a phenomenon documented in numerous human herditary diseases caused by point mutations [Krawczak, 1992] [Brown, 1996] . Use of this cryptic splice site leads to a shift of the reading frame, creating a new stop codon, and consequently a truncation of the deduced 25.5 kDa protein.

The finding of single base substitutions in genotypes rarl -1 and rarl -2 is consistent with the proposed mode of action of sodiumazide, the mutagen originally utilized for mutagenesis of cultivar Sultan5. The chemically mutagenized population used to identify rarl -1 and rarl -2 revealed mutants with functional defects in single genes at a frequency of 0.5 x 10^{" 3} (Torp and Jørgensen 1986) , comparable to the average efficiency of chemical mutagenesis in barley. The probability to identify two point mutations in a single gene both compromising its function in two independent mutants is therefore approximately 0.25 x 10^"6. In conclusion, the finding of two mutational events in genotypes rarl -1 and rarl -2 which lead either to the substitution of an invariant single amino acid or splice site defect of the candidate gene in the physically delimited target interval provides good indication that we have isolated Rarl .

Homologues of barley Rarl

DNA sequencing of the anonymous rice EST C28356 revealed a single extensive open reading frame of 699 bp encoding a putative Rarl homologous protein of 233 amino acids revealing 75% DNA and 86% amino acid similarity with barley Rarl (Figure 3) (GCG program GAP with gap creation penalty = 3 and gap extension penalty = 0.1) . Furthermore, searches of the Arabidopsis thaliana genomic databases revealed a genomic interval on chromosome 5 exhibiting significant sequence relatedness to barley and rice Rarl . The sequence homologous stretches are limited to the exons identified in barley Rarl and their order in the Arabidopsis genome matches those identified in barley. Each of the sequence homologous stretches in Arabidopsis is flanked by consensus splice site sequences. This enabled us to deduce a putative homologous Arabidopsis Rarl protein of 226 amino acids sharing 67% identical and 75% similarity with barley Rarl (GCG program GAP with gap creation penalty = 3 and gap extension penalty = 0.1) . We designate the corresponding rice and Arabidopsis genes OsRarl -hl and AtRarl -hi respectively.

Primers AtRarl 5 ' - ACTCCTACCTTCTCAATTCGTCCG - and AtRarl 3 ' - TATCAGACCGCCGGATCAGG - corresponding to AtRarl-hi enabled us to isolate the cognate cDNA which confirmed the predicted intron-exon structure.

Finally, we identified a number of EST entries representing expressed genes of unknown function from man, mouse, Drosophila, Caenorabdi tis elegans, Aspergillus and the like, revealing significant homologies only at the deduced amino acid level . The relatedness found between the deduced proteins corresponding to these ESTs and barley Rarl are listed in Table 7 and an alignment of the deduced sequences is shown in Figure 3. Apparently, barley Rarl uncovers a novel protein domain shared among multicellular organisms. Interestingly Cys²⁴ of barley Rarl, substituted to Tyr in mutant rarl -1 , represents one of the few strictly conserved amino acids in all identified sequence related proteins.

DISCUSSION

We have described here the molecular isolation of barley Rarl by a map-based cloning approach. The gene is predicted to encode a novel 22.5 kDa intracellular protein. This finding needs to be interpreted in the context of genetic data indicating that Rarl is essential for a number of powdery mildew resistance reactions triggered by different race- specific resistance genes (R genes) . It is now generally accepted that plant resistance to particular pathogens involves specific recognition events, triggered by corresponding R genes in the host and avirulence genes (Avr) in the pathogen. Many effector components have been implicated in pathogen arrest of pathogens, including the generation of reactive oxygen species, phytoalexins, activation of host cell death, the accumulation of pathogenesis-related proteins, and cross-linking of the plant cell wall . Most of these responses were also shown to be activated in barley/powdery mildew interactions. The requirement of Rarl for several R-gene triggered resistance reactions and the multitude of effector components activated in a resistance response leads us to propose that the Rarl protein acts "downstream" of R gene recognition but

"upstream" of the execution of the response. Thus Rarl is likely to represent a point of convergence in the signalling of R gene triggered resistance.

A close inspection of the Rarl protein sequence and a comparison to the rice and Arabidopsis homologues reveals a striking tripartite structure (designated domain I, II, and III - Figure 6) . Interestingly domain I and III, each approximately 60 aa long and located close to the amino- and carboxy-terminal ends of Rarl respectively, are structurally related to each other. Remarkable is a strictly conserved pattern of cysteine and histidine residues in domains I and III.

The domain signature is not only conserved among plant Rarl homologues but it is also found in each of the other related peptide stretches identified in proteins from Aspergillus, Drosophila, Caenorhabdi tis , mouse, and man. We have designated this novel protein domain "CHORD" (the example in domain I of Rarl being termed "CHORDl" and the example in domain III being "CHORD2" . Apart from the characteristic string of cysteine and histidine residues, CHORD contains few other invariant amino acids, Gly²³, Phe⁴⁷, and Trp⁵⁴ as well as a negatively charged residue in position 49 and a positively charged residue at position 52 (numbering refers to the amino terminal CHORD domain in barley Rarl) . The conservation of CHORD in such diverse phyla is indicative of a selective pressure to maintain a similar three dimensional structure in which cysteine and histidine residues play a major role. Conserved strings of Cysteine and Histidine residues in intracellular protein domains have been frequently shown to be involved in binding zinc ions. However, the pattern of these residues in CHORD is distinct from any previously described zinc-binding domain in which zinc ions have a structural role to stabilize small, autonomously folding and functional protein domains (e.g. the TFIIIa zinc finger, the GAL4 zinc finger, the zinc binding domain in the oestrogen receptor, the LIM domain, the RING finger domain, and the GATA-1 finger domain) .

The CHORD domain (e.g. CHORDl and CHORD2) can be signified as

C-X₄-C-X₁₀_₁₃ -C-X₂-H-X₆-y₁-X_g.₇-C₂-X₁₅-C-X₄_₅-H and may be

C—X₄ —C -X^₃ —C— ₂ —H—X_g —H— ₇ - C₂ — ₅ -- -^₄ ^—H .

In particular, a CHORD domain according to the present invention may conform to the formula: 11

C-x₃ -G-C-x₃-A₁-x₆_₉-C-x₂-H-x₅-F-y₁-A₂ -x₁_₂-A₃-x₁-W-x₁-C-C-x₁₅-C-x₄_₅-H wherein :

C, G, F and W are the single letter code for Cys, Gly, Phe and Trp, respectively, A₁ is an aromatic amino acid, and may be selected from Phe,

Trp and Tyr,

A₂ is a negatively charged residue, and may be selected from

Glu and Asp,

A₃ is a positively charged residue, and may be selected from Arg, His and Lys, y_x is H or any amino acid, and is preferably His or Arg, and X may be any amino acid (with the numbers indicating the number of amino acids) , subject to the structural constraints on the spatial relationship of the cysteines and histidines required for zinc binding.

Domain III in plant Rarl-like proteins appears to contain another set of cysteine and histidine residues providing a domain according to the present invention: C-x₂-C-x₅-C-x₂-H.

This binds the protein encoded by the Arabidopsis homologue of the yeast SGT1 gene (Kitagawa et al (1999) Mol Cell 1: 21- 34) .

The molecular isolation of barley Rarl and the finding of related genes in rice and the dicot Arabidopsis thaliana makes it likely to be a component of all higher plant genomes. The extent of sequence conservation among plant Rarl homologues makes it likely that they share also related functions. A modest 2-3 fold overexpression of the

Arabidopsis thaliana NPR1 gene, a key regulator in systemic aquired resistance, resulted in complete resistance to the pathogens Peronospora parasi tica and Pseudomonas syringae [Cao, 1998] , providing indication that modulating steady state levels of Rarl mRNA or protein may be used to alter speed and pathogen spectrum of the resistance response.

Redirecting Rarl expression by fusing the gene to promoters from pathogenesis-related genes (PR genes) may also be used to broaden the spectrum of Rarl mediated pathogen resistance. This approach may be particularly attractive in combination with the expression of derivatives of the Rarl protein. For example, modified versions of the Rarl protein may be identified which decouple its activation from R genes and retain their activation of downstream responses (PR gene activation, HR) . The identified tripartite domain structure of the plant Rarl proteins may serve in guiding these experiments .

ADDITIONAL MATERIALS AND METHODS FOR HIGH-RESOLUTION GENETIC AND PHYSICAL MAPPING Plant Material

Seeds of doubled-haploid barley (Hordeum vulgare) line Sultan-5 and Sultan-5 derived mutants M82 (rarl- 1 ) and MlOO (rarl-2 ) were as described in Torp and Jørgensen 1986.

Sultan-5 and the mutants contain the macroscopically visible marker gene ant2. (anthocyanin deficiency in the leaf sheath) . The Mla-12 backcross (BC) lines in cultivars Siri and Pallas were as described in Kølster et al . 1986; Kølster and Stølen 1987.

The Mla -12 BC line in cultivar Ingrid was generated through seven backcrosses with H. vulgare cv Ingrid followed by at least seven selfings. Each of the mutants M82 and MlOO were pollinated with pollen derived from the Mla -12 BC line cultivars, F₁ plants from each cross were grown to maturity providing the various segregating F₂ populations.

A segregating F₂ population of 186 individuals derived from the cross Nipponbare x Kasalath (Kurata et al . 1994) was used for mapping in rice. The map position of locus MWG876 in rice was independently tested in a second segregating F₂ population of 123 individuals derived from the cross IR20 x 63-83 (Quarrie et al . 1997).

Tests for Resistance Tests for resistance were carried out as described in Freialdenhoven et al . 1994. The phenotype of the recombinants was determined after selfing and subsequent inoculation experiments in F₃ and F₄ families comprising at least 25 individuals. F₃ individuals were tested by cleavable amplifiable polymorphic sequence analysis (CAPS) to identify homozygous recombinants. These plants were again selfed and subjected to resistance tests in F₄ families. Plants were scored for resistance/susceptibility seven days after inoculation.

Pulsed- field Gel Electrophoresis (Pfge) and Southern Analysis

High molecular weight DNA of barley was isolated from leaf material of 5-7 day old seedlings using a procedure according to Siedler and Graner (1990) . DNA was digested with six rare-cutting restriction enzymes (Clal, lul, Sail, Notl , Sfil, Sgfl , Smal ) using the protocol of Ganal and Tanksley (1989). For size fractionation a 1.2% agarose gel was run in an LKB Pulsaphor™ apparatus (Pharmacia Biotech, Upsala, Sweden) at 180 V with pulse times from 10-60 s (linear interpolation) for 25 h in 0.5 x TBE (50 mM Tris-HCl, 50 mM boric acid, 1 mM EDTA, pH 8.3) at 12 °C. Capillary transfer and non-radioactive Southern hybridisation was performed as described in Lahaye et al . (1996) . AFLP /CAPS Analysis

Genomic DNA for CAPS and AFLP analysis was isolated according to Stewart and Via (1993) . Primer PCR conditions and the respective restriction enzymes used for CAPS marker display are shown in Table 1. CAPS analysis was performed in a volume of 20 μl (100 pmole of each primer, 200 μM dNTPs, 10 mM Tris-HCl pH 8.3 , 2 mM MgCl₂, 50 mM KC1₂, 0.5 U Taq Polymerase (Boehringer) using 50 ng of barley genomic template DNA. The digested PCR products were subsequently size-fractionated on 2% agarose gels. AFLP analysis (Vos et al . 1995) was performed on bulked DNA samples of resistant and susceptible plants (Giovannoni et al . 1991; Michelmore et al . 1991) using Pstl and Msel restriction enzymes, Pstl and Msel adapters, and a set of primers corresponding to the Pstl and Msel adapters with two or three selective nucleotides at the 3' -end, respectively. Utilising seven Pstl + 2 (2 selective bases) - and 56 Msel + 3 (3 selective bases) - primers 392 combinations were analysed in total.

ADDITIONAL MATERIALS AND METHODS FOR ANALYSIS OF YACS

Barley YAC Library Screening

DNA of 428 YAC pools each representing 96 yeast clones

(Simons et al . 1997) was screened using CAPS marker MWG87. YAC pools positive for the MWG87 amplicon were further analysed by colony PCR for the identification of single yeast clones. The observed amplicons were size separated on an agarose gel, excised and purified with the Quiaquick gel extraction kit (Quiagen GmbH, Dϋsseldorf, Germany) . The purified PCR products were subjected to dye terminator sequencing (Perkin Elmer Corp., Norwalk, CT, USA) to confirm that the generated amplicons correspond to the MWG87 single copy locus. The observed individual YAC clones were subsequently investigated with CAPS marker MWG876. Cycling conditions and employed primers for CAPS markers MWG87 and MWG876 were as described herein. The initial denaturation step was elongated from two to four minutes for colony PCR analysis .

Plant Material

Seeds of diteleosomic wheat/barley addition lines were as described in Shepherd and Islam 1981; Islam 1983.

CAPS Analysis

Plant DNA for PCR-based analysis was extracted according to (Stewart and Via 1993) . Primer and PCR conditions for YAC end specific markers are listed in Table 4. PCR was performed in a volume of 20 μl (100 pmole of each primer, 200 μM dNTPs, 10 mM Tris-HCl pH 8.3, 2 mM MgCl₂, 50 mM KC1₂, 0.5 U Taq Polymerase (Boehringer Mannheim, Mannheim, Germany) using 200/50 ng of wheat/barley genomic template DNA. Amplicons corresponding to the different YAC ends were cloned into pGEM-T vector (Promega, Southampton, United Kingdom) and three independent clones of each PCR product were subjected to dye terminator cycle sequencing (Perkin Elmer) .

PFGE Southern Analysis of YACS

Individual YAC clones were grown for 2 days at 30°C in 100 ml synthetic dextrose (SD) minimal medium lacking uracil and tryptophan (Rose et al . 1990). High molecular weight yeast DNA was prepared in low melting point agarose as described by Carle et al . (1985) . Separation of yeast chromosomes was performed by a 1.2% agarose gel (Seakem™ LE; FMC BioProducts, Rockland, ME, USA) in an LKB Pulsaphor™ apparatus (Pharmacia Biotech, Uppsala, Sweden) at 180 V with pulse times from 10- 80 s (linear interpolation) for 30 h in 0.5x TBE (50 mM Tris- HCI, 50 mM boric acid, 1 mM EDTA, pH 8.3) at 12 °C. MWG87 was used subsequently as a probe for Southern hybridisation as described in Lahaye et al . (1996) to determine the size of the YAC inserts .

Isolation of YAC Terminal Sequences Terminal sequences of YAC inserts were isolated by inverse PCR (IPCR) according to Silverman et al . (1989). The observed amplicons were size separated on an agarose gel, excised, purified with the Quiaquick gel extraction kit (Quiagen) and subjected to dye terminator sequencing (Perkin Elmer) . For further elongation of YAC ends by an additional IPCR new oligonucleotides have been deduced based on the sequencing information of the respective YAC end.

Database Searches

Analysis of YAC end sequences was done using programs of the Genetics Computer Group (GCG) and the STADEN software package for Unix users (fourth edition, 1994) .

EXAMPLE 2

Activation in barley of Rarl Dependent Host Cell Death

Independent of An R Gene Trigger.

The over-expression of full length and truncated Rarl gene derivatives in barley downstream of the known maize ubiquitin promoter (Ubil promoter) flanked by the Ubil-intron 1 (Wan and Lemaux 1994) is carried out by delivering a suitable construct obtained using standard gene cloning methods (Sambrook et al . 1989). Briefly, the vector pUBI-GFP (Carlsberg Research Laboratory, Copenhagen, Denmark) is modified by deleting GFP and inserting the RAR-1 gene of interest in place thereof (such as the whole RAR-1 gene, or a sequence encoding an N terminal portion or a C terminal portion thereof, e.g. as shown in Figure 5A and Figure 5C, encoded for instance by the sequences of Figure 5B and Figure 5D respectively) . Following the cloning of the constructs, they are administered to prepared 7-day old plant leaf sections from barley using a suitable transient tranformation protocol .

Plant Material for transient transformation

Seeds of Hordeum vulgare cv. Golden Promise are sterilized by incubation in 70% ethanol for one min, washing three times in Milli-Q water followed by incubation in 1.5% sodium hypochlorite for 10 min and 5 times washing in sterile Milli- Q water. Seeds are sown in magentas (15 seeds/sample) containing 2 cm vermiculite supplied with 30 ml 1/2 -strength MS basal medium (Sigma) supplemented with 2% sucrose, and cultured at 22 C (16 h light/8h darkness) .

Primary leaves of 7-day-old seedlings are excised above the coleoptile and cut into two 3cm sections. Specimens are incubated for 3h in Petri dishes on 3 ml 10% sucrose, which leaves the plant material floating. Thereafter the sucrose solution is removed and the plant material is air-dried for 5 min prior to particle bombardment.

Procedure for Transient Transformation

The transformation process uses a particle inflow gun (PIG) (Vain et al . 1992). The constructs are precipitated onto gold particles (lμm, Bio Rad) according to the method of Klein et al . 1988 introducing lμg of Quiagen-purified plasmid per bombardment. The plant material is placed 9cm below the particle outlet and covered with a steel grid with 0.4 mm pore size. Bombardment conditions are: acceleration of the particle with 2735 mbar Helium gas at an air pressure of 100 mbar. Immediately after delivery of the DNA, approx. 4ml of sterile 1/2 strength MS basal medium, supplied with 3% sucrose and ImM benzimidazol, is added to the sample which is then incubated at 24°C in the dark for 24h.

RESULTS

The appearance of cell death clusters is confirmed by trypan blue staining. Leaf specimens are stained by boiling for 8min in alcoholic lactophenol (96% ethanol -lactophenol 1:1 [v/v] containing 0. lmg/ml trypan blue (Sigma) and cleared in a chloral hydrate solution (2.5 mg/ml) overnight. Rarl constructs activating host cell death in the transient assay are selected for further modification in transgenic plants .

EXAMPLE 3

The Expression in Barley of Cell Dea th Activating Rar-1 Derivatives by Fusions to Promoters From Pathogenesis-related Genes (Pr Genes) .

PR genes are known to be activated at high levels surrounding the sites of attempted pathogen attack.

Cell death activating Rar-1 derivatives are fused to 2 kb promoter sequences of barley genes HvPRl-a and HvPRl-b (Bryngelsson et al . 1994) . These genes are known to be activated in leaf tissue in response to attack from different pathogens including powdery mildew, Drechslera teres, and Puccinia hordei (Reiss and Bryngelsson 1996) .

Fusions of HvPRl-a and HvPRl-b promoters to cell death activating Rarl derivatives as provided herein are cloned into vector pAHC25 (Wan and Lemaux 1994) by replacing both the uidA reporter gene and the maize ubiquitin promoter of pAHC25, following standard cloning procedures (Sambrook et al . 1989). Transgenic barley plants of cultivar Golden

Promise are generated using the selectable marker gene bar in pAHC25 following the procedures described in Wan and Lemaux 1994.

Transgenic lines are tested for broad spectrum resistance following inoculations with different isolates of powdery mildew, Puccinia hordei , and Drechslera teres spores. Plants displaying resistance to the described isolates are observed.

Activation of Rarl Dependent Host Cell Death Independent of An J? Gene Trigger.

The over-expression of full length and truncated Rarl gene derivatives in barley downstream of the known maize ubiquitin promoter (Ubil promoter) flanked by the Ubil-intron 1 (Wan and Lemaux 1994) is carried out by delivering constructs which are obtained using standard gene cloning methods (Sambrook et al . 1989) . Briefly, the vector pU-Mlo (constructed by replacing GFP in pUBI-GFP with the 1.8 kb Mlo cDNA (Bϋschges et al . 1997; Shirasu K. et al . , 1999, Plant J., 17(3): 293-299) is modified by deleting Mlo and inserting the RAR-1 gene of interest in place thereof (such as the whole RAR-1 gene, or a sequence encoding an N terminal portion or a C terminal portion thereof, e.g. as shown in Figure 5A and Figure 5C, encoded for instance by the sequences of Figure 5B and Figure 5D respectively) . Following the cloning of the constructs, they are administered to prepared 7 -day old plant leaf sections from barley using a suitable transient tranformation protocol (for which see Example 2) .

RESULTS

The appearance of cell death clusters is confirmed by trypan blue staining. Leaf specimens are stained by boiling for 8min in alcoholic lactophenol (96% ethanol -lactophenol 1:1 [v/v] containing 0. Img/ml trypan blue (Sigma) and cleared in a chloral hydrate solution (2.5 mg/ml) overnight.

Rarl constructs activating host cell death in the transient assay are selected for further modification in transgenic plants.

EXAMPLE 4

The Expression in Dicots of Cell Death Activating Rar-1

Derivatives

This is achieved both independently of an R gene trigger (as in Example 2 for barley) and by means of fusions to promoters from pathogenesis-related genes (Pr Genes) (as in Example 3 for barley) .

Preparation of plant material is carried out as described above for barley using surface-sterilized Arabidopsis (ecotype Columbia) and tomato seeds (cultivar Moneymaker) .

Leaves of 4 week old plants are infiltrated with Agrobacterium strain C58 containing p35S-Rarl constructs in which Rarl derivatives are driven by the 35S CaMV promoter in a T-DNA vector, pBIN19 (Bevan, 1984) .

RESULTS

The appearance of cell death clusters is confirmed by trypan blue staining. Leaf specimens are stained by boiling for 8min in alcoholic lactophenol (96% ethanol-lactophenol 1:1 [v/v] containing 0. Img/ml trypan blue (Sigma) and cleared in a chloral hydrate solution (2.5 mg/ml) overnight.

Rarl constructs activating host cell death in the transient assay are selected for further modification in transgenic plants .

EXAMPLE 5

Expression in Arabidopsis of Cell Death Activating At -Rarl Derivatives

The glucocorticoid-mediated transcriptional induction system (Aoyama and Chua, 1997, Plant Journal 11 , 605-612.) is employed in inducible over-expression of full length and truncated AtRarl gene derivatives in Arabidopsis .

The vector pTA231 (The Rockefeller University, New York, USA) is modified by inserting the AtRarl sequence of interest selected from those shown in Figures 8A to 8H, i.e. the whole AtRarl gene, a sequence encoding an N-terminal portion, an internal portion, or a C-terminal portion thereof, using the Xhol and Spel cloning sites. Primers used to amplify AtRarl gene fragments are:

OK228 5' -CCTCGAGACTCCTACCTTCTCAATTCGTCCG-3 ' and OK232 5'-AACTAGTATCAGACCGCCGGATCAGG-3 ' for whole AtRarl, OK228 and OK229 5 ' -AACTAGTCAGGCCAGAACTGGTTTCTCAGTTGT-3 ' for the N-terminal portion,

OK230 5'-AACTAGTCAAGCCTTTTGTACTGGAGGCGC-3 ' and

OK231 5'-ACTCGAGATGGCCAAATCGGTTCCAAAACATC-3 • for the internal portion, and

OK233 5'-ACTCGAGATGGCTGTGATAGACATTAATCAACCGC-3 ' and OK232 for the C-terminal portion.

Arabidopsis plants are transformed with Agrobacterium strain C58 containing above constructs by a standard Arabidopsis transformation protocol (Clough and Bent, 1998, Plant Journal 16, 735-743) . REFERENCES

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Table 1 Size of PFGE-separated restriction fragments (in kb) detected by Southern analysis in cultivar Sultan-5

probe

MWG876 WG87 MWG892

CM 350 50 110

MM 550 550 260

Notl 90 530 170

Sail 90 240 310

Sfll 120 100 140

Sgβ 580 165 ND

Smal 90 100 80

ND, not determined

Table 2 YAC overlap analysis

YACend specific markers

Y18 Y18 Y30 Y30 Y31 Y31 Y73 Y73 Y113 Y113 barleyYACs L R L R L R L R L R

Yl +

Y2 +

Y18 + + . + + . . . . +

Y30 . + + + + - + -

Y31 . + + - + + + - - -

Y73 _ + . - + . + + - -

Y113 . - . - + - - - + +

A plus sign (+) indicates that a YAC was positive by PCR analysis for the respective marker listed above. Absence of a marker is indicated by a minus sign (-).

Table 3 PCR-based YAC end specific markers

Marker Primer PCR conditions

Y18L 5 '-TCCCTCGTTCTATGGTCACGGTTG 94°C. 10s 5'-CATGCCCAGCCTGATGCATTG 60°C 20s 72°C 90s

Y18R 5'-GAATGATGTGCCCGTGCGTGC 94°C 10s 5'-GCGACGCTTCCCACCTGCAG 60°C 20s 72°C. 40s

Y30L 5'-CGATAGGTGGTAGATTTTTGACATCTTCAG 94°C 10s 5 '-GCTCATCCAGCTACAGAATGCTTTATG 60°C 20s 72°C 40s

Y30R 5 '-GGTGAGCTGCAGGAAACGGTCC 94°C 10s

5 '-AGGCCAGAGTACTCCGATCGAATGG 60°C 20s 72°C. 30s

Y3 IL 5 '-CGCCTTCTTGATCTAGCAAGAGACACG 94°C 10s 5'- GCGGAGTACATGGCTGCCTTGG 60°C 20s 72°C_: 60s

Y31R 5 '-TATGCTGACTACTGCACTCCTGATGAGG 94°C. 10s 5 '-GAGGCTGTGAGGACTGTGGTGCTG 60°C 20s 72°C 60s

Y73L 5'-TGGTATCAGAGCAGTACCGACCTGG 94°C. 10s 5 '-GAGATAACTTCAACGCTCCGGATCG 60°C 20s 72°C 90s

Y73R 5'-GCGGCAGTCGCTCGGGCGCACAGG 94°C 10s 5 '-CTCAGGAAATCAGAAAATGTTCACG 60°C 20s 72°C 90s

Yl 13L 5 '-CAGCGGCTGGACGTCCGAC 94°C. 10s 5 '-GGACGTCCAAGGGCCGGAC 60°C 20s 72°C 30s

Y113R 5'-CAGGGGAGATTTTTTACAATCACG 94°C 10s 5 '-CCATTACCTGGGCTGGCCCAT 58°C. 20s 72°C 60s

YAC ends which have been employed for high-resolution genetic mapping are shown in italics. All PCR reactions start with an initial denaturation step of 2 minutes at 94°C. Subsequently 35 cycles of the conditions indicated were performed. Table 4 CAPS marker linked to the Rarl locus

Marker Primer PCR Restriction conditions enzyme

CMWG694 5'-AGTATCAGATGCTACCATGCCTGG 94°C , 10 s Haelll 5 '-CTCTGGAGGAGCCGAGTGTC AGC 60°C , 20 s

72°C 30 s

MWG87 5'-ATCAAACCAAGCAAAGGTCCCTTG 94°C 10 s Trul 5'-CTGCAGGCGCACTTTAGGGGAAC 58°C 20 s

72°C 30 s

MWG503 5'-CGTCAGAGCCCACGCCACACGTAG 94°C 10 s Hinβl 5'-GCCGAACGTGCTCCAAGCGGCAAC 60°C 20 s

72°C 40 s

MWG876 5'-GTGGTCAAGGGCTTGTAGACTGGGTAC 94°C 10 s Mval 5'-GCCCATCGGTGGTCGCCGTAGTCGCG 60°C 20 s

72°C 30 s

MWG892 5'-GGAATCTTCCAGTGGGCTGGATGAG 94°C 10 s 5'-CAACCGGCCACTAGGCGTAAAGG 60°C, 20 s

72°C 30 s

MWG2123 5'-CTGCGGCGAGAGCTTGAGAGCAGT 94°C_; 10 s Bell 5'-GTGTGCATGGTCTCTTCCGCCCCG 60°C_; 20 s

72°C 60 s

OKI 114 5'-CCATGTCTTGTCCATGATGCACC 94°C_; 10s Hindm

5'-GCCATCTAGCTACTAACTATGGACCCG 60°C, 30s

72°C_; 90s

OK3236 5'-GACAGTAGCAGAGTGGTTGCACCG 94°C, 10s

5'-CCACATGCACACAAGTATATGCACAC 62°C, 30s

72°C, 60s

OK5558 5'-GCGATATGGAGATCAAAACCCTCA 94°C, 10s 5'-CACGAAATGCCTATGAACCATTCG 64°C, 30s

72°C, 90s

Edda 5'-ACTTTAAACTTGCTGGCGACAAGAGAC 94°C, 10s Hinfl

5 '-GGAGTTGGCTTACTTACCGTATCACATAC 64°C, 10s

72°C, 30s

For all CAPS markers after an initial denaturation step of 2 minutes at 94°C, 35 cycles were performed as indicated. Amplification products corresponding to the RFLP locus MWG892, reveal different lengths in the Rarl mutants and the Mlal 2 BC lines Ingrid, Pallas and Siri which makes an analytical digestion after PCR superfluous. RFLP loci OK 3236 and OK5558 reveal different lengths in the Rarl mutants and the Mia 12 BC lines Ingrid and Pallas only Table 5 Gene Bank accessions with sequence similarity to interval, II, 12, and 13

Interval Position Gene Bank Description Score E value accesion no. Blast

2.0

11 43500-45000 AF057183 Zea mays putative tonoplast aquaporin mRNA 496 1.00E-138

D23748 Rice cDNA, partial sequence (R0063_l A) 218 1.00E-54

AJ005078 Picea abies mRNA for aquaporin-like protein 168 1.00E-39

X80266 H. vulgare mRNA for gamma-TIP-like protein 159 1.00E-36

AF037061 Zea mays tonoplast intrinsic protein tip 1 139 1.00E-30

D40090 Rice cDNA, partial sequence (S 1835_1 A) 135 2.00E-29

D25534 Rice mRNA for gamma-Tip, complete cds 127 5.00E-27

C72141 Rice cDNA, partial sequence (E1069_3A) 127 4.00E-27

D41197 Rice cDNA, partial sequence S3526_l A) 123 6.00E-26

D39818 Rice cDNA, partial sequence (S 1424_2 A) 123 6.00E-26

12 45001-46500 D40090 Rice cDNA, partial sequence (SI 835_1 A) 90 1.00E-15

D23748 Rice cDNA, partial sequence (R0063_1A) 78 5.00E-12

D40435 Rice cDNA, partial sequence (S2414_2A) 76 2.00E-11

D41992 Rice cDNA, partial sequence (S5055_1A) 76 2.00E-11

D40210 Rice cDNA, partial sequence (S2017_2A) 76 2.00E-11

D25114 Rice cDNA, partial sequence (R3240_l A) 76 2.00E-11

D42002 Rice cDNA, partial sequence (S5071_1A) 76 2.00E-11

D41421 Rice cDNA, partial sequence (S3914_1A) 76 2.00E-11

AF057183 Zea mays putative tonoplast aquaporin mRNA 72 3.00E-10

D41197 Rice cDNA, partial sequence (S3526_l A) 70 1.00E-09

13 56000-61000 C28356 Rice cDNA, partial sequence (C60815 A) 105 2.00E-53

L37995 Brassica rapa (clone F0160) expressed sequence tag 50 0.008

Table 6 Primers used for amplifying genomic and cDNA of Rarl

Primer Sequence

OK94 5* -GTGCGCCTGCAGTTACTTGTAGC

OK96 5' -- TCTTATGCTGCTGCACTTGTGGG

OK97 5' -- GTATGTTGACAAGTGATCCTCCACTG

OK98 5' -- GCCTCCTCTCATTCTAGACCACAGTG

OK99 5' -- CTCAGAGCTCACCCAAGCAGCAG

OKI 00 5'- -CTGCAGGACCTGGATGGTAATCG

OKI 01 5' -- CATAGGCTGCGACGCCATG

OKI 02 5'- -CTGGTTTCTCAGTTGTATGCTTCCC

OKI 03 5'- -GGTAGTGGCAGGAGCATCGG

OKI 04 5'- -GCTTGCAACAGCTCCACTCTTTC

OK105 5'- -GGAGAAGGATAACCATGATGCTGC

OK106 5*- -GGGAAGCATACAACTGAGAAACCAG

OK109 5'- -GACTCAGCAGCTGTCCCGATTC

OKI 10 5'- -CAACCCCGATGGCTCCTGCCACTACCAC

OKI 11 5'- -GCAGGACCTGGATGGTAATCGCATGCAGC

OKI 12 5'- -CCTGCATTAAGATCACGGCACTC

Table 7 Gene Bank Acessions with similarity to barley Rarl

Gene GeneBank bp Organism tblastn 2.0 designation accession no.

AtRarl -hi ABO 10074 Arabidopsis thaliana 324

OsRarl-hl c28356 424 Or za sativa 158

BrRarl-hl L37995 367 Brassica rapa 92

AA134808 452 Homo sapiens 77

W92190 435 Homo sapiens 59

AA249751 291 Homo sapiens 71

AA216041 352 Homo sapiens 70

AA313760 416 Homo sapiens 42

AA346843 399 Homo sapiens 69

AA382946 314 Homo sapiens 85

AA311950 431 Homo sapiens 84

AA385686 347 Homo sapiens 82

AA313823 520 Homo sapiens 79

W20519 460 Homo sapiens 77

AA581174 385 Homo sapiens 75

AA333125 276 Homo sapiens 71

AA355517 240 Homo sapiens 61

AA333321 251 Homo sapiens 45

AA249829 370 Homo sapiens 75

AA249107 205 Homo sapiens 61

AA222387 430 Mus musculus 74

AA117998 466 Mus musculus 58

AA049028 473 Mus musculus 85

W83076 463 Mus musculus 85

AA268197 506 Mus musculus 83

AA896237 430 Mus musculus 78

AA409037 328 Mus musculus 78

AA959286 374 Mus musculus 80

AA050833 373 Mus musculus 80

W12728 439 Mus musculus 80

AA571713 349 Mus musculus 78

W70829 418 Mus musculus 78

AA052424 449 Mus musculus 77

AA240093 479 Mus musculus 77

AA171338 274 Mus musculus 77

W83689 454 Mus musculus 77

W10223 277 Mus musculus 73

W41418 336 Mus musculus 48

W98189 500 Mus musculus 80

BmRarl-hl AA509002 307 Brugia malayi 79

DmRarl-hl AA141981 566 Drosophila melanogaster 75

TgRarl-hl N81452 430 Toxoplasma gonii 74

TbRarl-hl W06674 330 Trypanosoma bruei 68

EnRarl-hl AA965935 339 Emericella nidulans 47

CeRarl-hl Y110A7.90 Caenorhabditis elegans 190

Claims

CLAIMS :

1. An isolated polynucleotide encoding a polypeptide which functions in a plant pathogen defence response signalling pathway, which polypeptide includes an amino acid sequence which has at least 70% amino acid sequence identity with the amino acid sequence shown in Figure 1.

2. An isolated polynucleotide according to claim 1 wherein the polypeptide has the amino acid sequence shown in Figure

1.

3. An isolated polynucleotide according to claim 2 wherein the polynucleotide has the coding sequence shown in Figure 1.

4. An isolated polynucleotide encoding a polypeptide which functions in a plant pathogen defence response signalling pathway, which polynucleotide selectively hybridizes under stringent conditions with a probe which is the complement of the .Rarl coding nucleotide sequence shown in Figure 1.

5. An isolated polynucleotide encoding a Barley Rarl fragment shown in Figure 5A, Figure 5C or Figure 6.

6. An isolated polynucleotide according to claim 5 wherein the polynucleotide has the coding sequence shown in Figure 5B or Figure 5D or one of those shown Figure 7.

7. An isolated polynucleotide encoding an Arabidopsis Rarl fragment shown in Figure 8D, Figure 8F or Figure 5H.

8. An isolated polynucleotide according to claim 7 wherein the polynucleotide has the coding sequence shown in Figure 8C, Figure 8E or Figure 8G.

9. An isolated polynucleotide wherein a polynucleotide according to any of claims 1 to 8 , or a polynucleotide selected from the group consisting of:

(i) a polynucleotide encoding the OsRarl-hl amino acid sequence shown in Figure 3 ; (ii) a polynucleotide encoding the AtRarl-hi amino acid sequence shown in Figure 3 ;

(iii) a polynucleotide encoding a polypeptide which functions in a plant pathogen defence response signalling pathway, which polypeptide includes an amino acid sequence which has at least 70% amino acid sequence identity with said

OsRarl-hl or AtRarl-hi amino acid sequence;

(iv) a polynucleotide encoding a polypeptide which functions in a plant pathogen defence response signalling pathway, which polynucleotide selectively hybridizes under stringent conditions with a probe which is the complement of the coding nucleotide sequence of GenBank accession c28356 encoding said OsRarl-hl or of GenBank accession AB010074 encoding said AtRarl-hi; is operably linked to a regulatory sequence for expression.

10. An isolated polynucleotide of which the nucleotide sequence is complementary to a sequence of at least 50 contiguous nucleotides of the coding sequence or sequence complementary to the coding sequence of nucleic acid according to any of claims 1 to 19 suitable for use in anti- sense or sense regulation ("co-suppression") of expression of said coding sequence and under control of a regulatory sequence for transcription.

11. An isolated polynucleotide encoding a peptide consisting of a CHORD amino acid sequence conforming to one of the following formulae :

(i)

C-x₃-G-C-X₃-A₁-x₆_₉-C-x₂-H-x₅-F-y₁-A₂-x₁_₂-A₃-x₁- -x₁-C-C-x₁₅-C-x₄_₅-H

(ii) C-x₂-C-x₅-C-x₂-H wherein:

C, G, F and are the single letter code for Cys, Gly, Phe and Trp, respectively,

A_λ is an aromatic amino acid, A₂ is a negatively charged residue,

A₃ is a positively charged residue, γ_λ is His or any amino acid, and is preferably His or Arg, and x is any amino acid with the numbers indicating the number of amino acids, and wherein peptide (i) binds zinc.

12. A nucleic acid vector suitable for transformation of a plant cell and including a polynucleotide according to any one of claims 1 to 11.

13. A host cell containing a heterologous polynucleotide or nucleic acid vector according to any one of claims 1 to 12.

14. A host cell according to claim 13 which is microbial .

15. A host cell according to claim 13 which is a plant cell.

16. A plant cell according to claim 15 having heterologous said polynucleotide within its chromosome.

17. A plant cell according to claim 16 having more than one said polynucleotide per haploid genome.

18. A plant cell according to any of claims 15 to 17 which is comprised in a plant, a plant part or a plant propagule, or an extract or derivative of a plant.

19. A method of producing a cell according to any of claims 13 to 17, the method including incorporating said polynucleotide or nucleic acid vector into the cell by means of transformation.

20. A method according to claim 19 which includes recombinmg the polynucleotide with the cell genome nucleic acid such that it is stably incorporated therein.

21. A method according to claim 19 or claim 20 which includes regenerating a plant from one or more transformed cells .

22. A plant comprising a plant cell according to any of claims 15 to 17.

23. A part or propagule of a plant comprising a plant cell according to any of claims 15 to 17.

24. A method of producing a plant, the method including incorporating a polynucleotide or nucleic acid vector according to any of claims 1 to 12 into a plant cell and regenerating a plant from said plant cell .

25. A method according to claim 24 including sexually or asexually propagating or growing off-spring or a descendant of the plant regenerated from said plant cell .

26. A method of influencing a characteristic of a plant, the method including causing or allowing transcription from a heterologous polynucleotide according to any of claims 1 to 11 within cells of the plant.

27. Use of a polynucleotide according to any of claims 1 to 11 in the production of a transgenic plant.

28. A method of identifying or obtaining a polynucleotide encoding a polypeptide which functions in a plant pathogen defence response signalling pathway, the method including screening candidate nucleic acid using a nucleic acid molecule which selectively hybridises under stringent conditions with a polynucleotide according to claim 4 or the complement thereof.

29. A nucleic acid probe or primer which has a nucleotide sequence of at least about 20-30 nucleotides, which sequence is shown in, or is complementary to, the coding region of Figure 1.

30. A pair of primers according to claim 29 suitable for amplification of a fragment of the coding region of a polynucleotide according to any one of claims 1 to 9.

31. A method for identifying in or obtaining from a plant or plant cell a polynucleotide encoding a polypeptide which functions in a plant pathogen defence response signalling pathway, which method employs a nucleic acid probe or primer according to claim 29 or claim 30.

32. A method as claimed in claim 31 including:

(a) providing a nucleic acid preparation from a plant cell; (b) providing a probe according to claim 29;

(c) contacting the nucleic acid preparation with the probe under conditions for selective hybridisation of the probe to any nucleic acid encoding a said polypeptide;

(d) identifying said nucleic acid encoding a said polypeptide if present by its hybridisation with the probe; and optionally

(e) confirming the identity of said polypeptide encoded by the nucleic acid by expression in an expression system and determination of ability to function in a plant pathogen defence response signalling pathway.

33. A method according to claim 31 including:

(a) providing a preparation of nucleic acid from a plant cell ; (b) providing a pair of primers according to claim 30;

(c) contacting nucleic acid in said preparation with said primers under conditions for performance of PCR;

(d) performing PCR and determining the presence or absence of an amplified PCR product; and optionally

(e) confirming the identity of the amplified PCR product by expression in an expression system to produce a polypeptide and determination of ability of the produced polypeptide to function in a plant pathogen defence response signalling pathway.

34. An isolated polypeptide encoded by a polynucleotide according to any of claims 1 to 9.

35. An isolated antibody including an antigen-binding site with specific binding affinity for the polypeptide according to claim 34.

_*

36. A polypeptide including the antigen-binding site of an antibody according to claim 35.

37. A method of identifying or obtaining a polypeptide according to claim 34, the method including screening candidate polypeptides with an antibody or polypeptide according to claim 35 or claim 36.

38. An isolated peptide encoded by a polynucleotide according to claim 11.