CA2216406A1 - Plant pathogen resistance genes and uses thereof - Google Patents

Abstract

The Arabidopsis RPP5 gene has been cloned and its sequence provided, along with the encoded amino acid sequence. DNA encoding the polypeptide, and alleles, mutants and derivatives thereof, may be introduced into plant cells and the encoded polypeptide expressed, conferring pathogen resistance on plants comprising such cells and descendants thereof. The RPP5 sequence comprises leucine rich repeats and the presence of such repeats enables identification of other plant pathogen resistance genes. Homologies between RPP5 and other pathogen resistance genes reveal motifs useful in identification of other pathogen resistance genes.

Description

W 096/31608 PCT/GB9~'~CU19 PLANT PATHOGEN RESISTANOE GENES AND USES ~l~REOF
The present invention relates to pathogen resistance in plants and more particularly to the identification and use of pathogen resistance genes.
It is based on cloning o~ the Arabi~opsis RPP5 gene.
Plants are constantly challenged by potentially pathogenic microorganisms. ~rop plants are particularly vulnerable, because they are usually grown as genetically uni~orm monocultures; when disease strikes, losses can be severe. However, most plants are resistant to most plant pathogens. To defend themselves, plants have evolved an array of both preexisting and inducible de~ences. Pathogens must specialize to circumvent the de~ence mechanisms o~ the host, especially those biotrophic pathogens that derive their nutrition ~rom an intimate association with living plant cells. I~ the pathogen can cause disease, the interaction is said to be compatible, but i~ the plant is resistant, the interaction is said to he incompatible. Race speci~ic resistance is strongly correlated with the hypersensitive response (HR), an induced response by which (it is hypothesized) the plant deprives the pathogen o~ living host cells by localized cell death at sites o~ attempted pathogen ingress.
It has long been known that HR-associated disease resistance is often (though not exclusively) speci~ied by dominant genes (R genes). Flor showed that when W O96~1608 PCT/~b5~ 9 pathogens mutate to overcome such R genes, these mutations are recessive. Flor concluded that for R
genes to function, there must also be corresponding genes in the pathogen, denoted avirulence genes (Avr genes). To become virulent, pathogens must thus stop making a product that activates R gene-dependent de~ence mechanisms (Flor, 1971). A broadly accepted working hypothesis, o~ten termed the elicitor/ _ receptor model, is that R genes encode products t~at enable plants to detect the presence o~ pathogens, provided said pathogens carry the corresponding Avr gene (Gabriel and Rol~e, 1990). This recognition is then transduced into the activation o~ a de~ence response.
Some interactions exhibit di~erent genetic properties. Helminthospo~ium carbonum races that express a toxin (Hc toxin) in~ect maize lines that lack the Hml resistance gene Mutations to loss o~ Hc toxin expression are recessive, and correlated with 2~ loss o~ virulence, in contrast to gene-~or-gene interactions in which mutations to virulence are recessive. A major accomplishment was reported in 1992, with the isolation by tagging o~ the Hml gene-(Johal and Briggs, 1992). Plausible arguments have been made ~or how gene-~or-gene interactions could evolve ~rom toxin-dependent virulence. For example, plant genes whose products were the target o~ the toxin might mutate to con~er even greater sensitivity W 096/31608 PCTtGB9GI'~ 9 to the toxin, leading to HR, and the conversion of a sensitivity gene to a resistance gene. However, this does not seem to be the mode o~ action of ~ml, whose gene product inactivates Hc toxin.
Pathogen avirulence genes are still poorly understood. Several bacterial Avr genes encode hydrophilic proteins with no homology to other classes of protein, while others carry repeating units whose number can he modified to change the range of plants on which they exhibit avirulence (Keen, 1992; Long and Staskawicz, 1993). Additional bacterial genes (hrp genes) are required for bacterial Avr genes to induce HR, and also for pathogenicity (Keen, 1992; Long and Staskawicz, 1993). It is not clear why pathogens make products that enable the plant to detect them. It is widely believed that certain easily discarded Avr genes contribute to but are not re~uired for pathogenicity, whereas other Avr genes are less dispensable (Keen, 1992; Long, et al, 1993). The characterization of two fungal avirulence genes has also been reported. The Avr9 gene of Cladosporium fulvum, which confers avirulence on C. fulvum races that attempt to attack tomato varieties that carry the Cf-9 gene, encodes a secreted cysteine-rich peptide with a final processed size of 28 amino acids but its role in compatible interactions is not clear (De Wit, 1992). The Avr4 gene of C. fulvum encodes a secreted peptide that is processed to a ~inal size of amino W 096131608 PCTIG~9~ 9 acids 106 (Joosten et al, 1994 ) The technology for gene isolation based primarily on genetic criteria has improved dramatically :Ln recent years, and many workers are currently attempting to clone a variety of R genes.
The map based cloning of the tomato Pto gene that confers "gene-for-gene" resistance to the bacterial speck pathogen Pseudomonas syringae pv tomato (Pst) has been reported (Martin et al, 1993). A YAC (yeast artificial chromosome) clone was identified that carried restriction fragment length polymorphism (RFLP) markers that were very tightly linked to the gene. This YAC was used to isolate homologous cDNA
clones. Two of these cDNAs were fused to a stro~g promoter, and after transformation of a disease sensitive tomato variety, one of these gene fusions was shown to confer resistance to Pst strains that carry the corresponding avirulence gene, Av~Pto. These two cDNAs show homology to each other. Indeed, the Pto cDNA probe reveals a small gene family of at least six members, 5 of which can be found on the YAC from which Pto was isolated, and which thus comprise exactly the kind of local multigene family inferred from genetic analysis of other R gene loci.
The Pto gene cDNA sequence is puzzling for proponents of the simple elicitor/receptor model. It reveals unambiguous homology to serine/threonine kinases, consistent with a role in signal W 096/31608 P~l/~br~00~19 transduction. Intriguingly, there is strong homology to the kinases associated with sel~ incompatibility in Brassicas, which carry out an analogous role, in that they are required to prevent the growth of genotypically defined incompatible pollen tubes.
However, in contrast to the Brassica SRK kinase (Stein et al, 1991), the Pto gene appears to code for little more than the kinase catalytic domain and a potential N-terminal myristoylation site that could promote association with membranes. It would be surprising i~
such a gene product could act alone to accomplish the speci~ic recognition required to initiate the de~ence response only when the AvrPto gene is detected in invading microorganisms. The race-speci~ic elicitor molecule made by Pst strains that carry AvrPto is still unknown and needs to he characterized be~ore possible recognition o~ this molecule by the Pto gene product can be investigated.
Since the isolation o~ the Pto gene a number o~
other resistance genes have been isolated. The isolation o~ the tobacco mosaic resistance gene N ~rom tobacco was reported by Whitham et al ( 1994) The isolation o~ the ~lax rust resistance gene ~6 ~rom ~lax was reported by Lawrence et al (1995). The isolation of two Arabidopsis thaliana genes ~or resistance to PSe77~J~mQn~ syringae has been reported.
The isolation o~ RPS2 was reported by Bent et al (1994) and by Mindrinos et al (1994) and the isolation W 096/31G08 PCT/GB9"~C~~9 o~ RPM1 was reported by Grant et al (1995). These genes probably encode cytoplasmic proteins that carry a nucleotide binding site (NBS) and a leucine-rich repeat (LRR). The ligands with which they interact are S uncharacterised and it is not known what other plant proteins they interact with to accomplish the de~ence response. Our own laboratory has reported the isolation o~ the tomato Cf-9 gene which con~ers resistance against the ~ungus Cladosporium fulvum.
This is disclosed in WO95/18230 and has been reported in Jones et al (1994). We have also cloned the tomato Cf-2 gene, which con~ers resistance against ~l adosporium f ul vum; this is disclosed in an International patent application filed by us on 1 lS April 1996 claiming priority from GB 9506658.5 filed 31 March 1995 and has been reported in Dixon et al ~ (1996). Its structure resembles the Cf-9 gene in that the.DNA sequence predicts a protein which is predominantly extracellular, with many leucine-rich 2~ repeats and which carries a C-terminal putative membrane anchor. The Xa21 gene of rice has also been cloned recently (Song et al ., 1995). The predicted protein product of this gene exhibits an N-terminal, presumably extracellular, domain composed principally o~ leucine rich repeats similar to those of Cf-9 and ~-2, a predicted transmembrane domain, and a presumably cytoplasmic domain with strong similarities to serine-threonine protein kinases, particularly W O 96/31608 P~ll~b~'oo819 that encoded by Pto.
The subject-matter o~ the present invention relates to "pathogen resistance genes" or "disease resistance genesn and uses thereo~. A pathogen S resistance gene (R) enables a plant to detect the presence o~ a pathogen expressing a corresponding avirulence gene (Avr). When the pathogen is detected, a de~ence response such as the hypersensitive response (HR) is activated. By such means a plant may deprive the pathogen of living cells by localised cell death at sites o~ attempted pathogen ingress. Other genes, including the PGIP gene o~ WO93/11241 (~or example), are induced in the plant de~ence response resulting ~rom detection of a pathogen by an R gene.
A pathogen resistance gene may be envisaged as encoding a receptor to a pathogen-derived and Avr dependent molecule In this way it may be likened to the RADAR o~ a plant ~or detection o~ a pathogen.
Genes involved in the de~ence the plant mounts to~the pathogen once detected are not pathogen resistance genes. Expression o~ a pathogen resistance gene in a plant causes activation o~ a de~ence response in the plant. This may be upon contact o~ the plant with a pathogen or a corresponding elicitor molecule, though the possibility o~ causing activation by over-- expression o~ the resistance gene in the absence o~

elicitor has been reported. The de~ence response may be activated locally, e.g. at a site o~ contact o~ the W 096/31608 PCTrCB~G/C~49 plant with pathogen or elicitor molecule, or systemically. Activation of a de~ence response in a plant expressing a pathogen resistance gene may be caused upon contact o~ the plant with an appropriate, corresponding elicitor molecule. The elicitor may be contained in an extract o~ a pathogen such as Peronospora parasitica, or may be wholly or partially puri~ied and may be wholly or partially synthetic. An elicitor molecule may be said to "correspond" i~ it is a suitable ligand ~or the R gene product to ellcit activation o~ a de~ence response.
We have now isolated the Arabidopsis RPP5 gene which con~ers resistance against the downy mildew ~ungus (Peronospora parasi tica) . We have sequenced the DNA and deduced the most likely amino acid sequence ~rom this gene. The DNA se~uence o~ the Arabi~opsis RPP5 genomic gene is shown in Figure 1 (SEQ ID NO. 1) and the deduced amino acid sequence is shown in Figure 2 (SEQ ID NO. 2). The prediction o~
the Amino acid sequence is based on the identi~ication o~ introns by reverse transcriptase polymerase chain reaction using primers designed to the determined genomic sequence. The part o~ the DNA sequence that is presumed to be spliced into exons and encoding the 25 RPP5 polypeptide is shown in capital letters in Figure 1. Figure 4 (SEQ ID NO 5) shows a contiguous nucleotide sequence coding ~or the amino acid sequence o~ Figure 2, made by joining together the exons o~ the -WO96/31608 P~~ G~00~9 sequence of Figure 1.
As described in more detail below, the Arabidopsis RPP5 gene was isolated by map-based cloning. In this technique the locus that con~ers resistance is mapped at high resolution relative to restriction fragment length polymorphism (RFLP) markers that are linked to the resistance gene. We identi~ied a marker that appeared to be absolutely linked to the resistance gene and used probes corresponding to this marker to isolate binary vector cosmid clones ~rom a library made with DNA o~ an Arabldopsis landrace Landsberg erecta that carried the RPP5 gene. A binary vector cosmid clone designated 29L17, on trans~ormation into disease sensitive Arabidopsis, con:Eerred disease resistance. DNA
sequence analysis o~ the cloned DNA identi~ied a gene with leucine-rich repeats. A subclone o~ 29L17, designated pRPP5-1, containing 6304 bp o~ DNA
including 1298 bp 5' to the probable initiation codon (Figure 1) and ~58 bp 3' to the probable termination codon was constructed in a binary vector. The subclone was used to trans~orm Arabidopsis ecotype Columbia and shown to con~er disease resistance Analysis o~ a ~ast neutron induced mutation o~ Landsberg that had become disease sensitive revealed rearrangement o~ the DNA structure o~ this gene. Taken together these data provide the necessary evidence that the sequences as shown in Figures 1 and 2 correspond to the RPP5 gene.

W 096/31608 P~l/~r~/00819 According to one aspect, the present invention provides a nucleic acid isolate encoding a pathogen resistance gene, the gene being characterized in that it encodes the amino acid sequence shown in SEQ ID NO
2, or a fragment thereof, or an amino acid sequence showing a significant degree of homology thereto. N
and L6 may be excluded.
For instance, embodiments of nucleic acid according to the invention, e.g. encoding a polypeptide comprising an amino acid sequence that is a mutant, derivative, allele or variant of the sequence shown in Figure 2 (as discussed further herein), may be distinguished from other pathogen resistance genes such as N, L6 by optionally having any one or more of the following features:
the encoded polypeptide has less than 30~
homology with the amino acid sequence o~ the tobacco N
protein, shown in Figure 3 and less than 25~ homology with the amino acid sequence of the flax L6 protein, 2~ shown in Figure 3i its expression does not activate said defence response upon contact of the plant with a molecule that is an elicitor of the tobacco N protein;
its expression does not activate said defence response upon contact of the plant with a molecule that is an elicitor of the ~1ax L6 protein;
its expression does not when in a tobacco plant activate said de~ence response upon contact of the W 096J31608 P~ll~5GJ~ 9 tobacco plant with Tobacco Mosaic Virus;
its expression does not when in a flax plant activate said defence response upon contact of the flax plant with Melampsora lini;
its expression does not activate said de~ence response upon contact of the plant with a molecule that is an elicitor of the Arabidopsis RPS2 protein;
its expression does not activate said defence response upon contact of the plant with a molecule that is an elicitor of the Arabidopsis RPM1 protein;
its expression does not when in Arabidopsis thaliana activate said defence response upon contact of the plant with Pseudomonas syringae;
the encoded polypeptide shows less than 20~
homology with the amino acid sequence of the tomato Cf-9 protein and less than 20~ homology with the amino acid sequence of a tomato Cf-2 protein;
its expression does not activate said defence response upon contact of the plant with a molecule that is an elicitor of the tomato Cf-9 protein nor with a molecule that is an elicitor of the tomato Cf-2 protein;
its expression does not when in a tomato plant activate said defence response upon contact of the 2~ tomato plant with Cladosporium fulvum expressing an Avr2 molecule nor Cladosporium fulvum expressing an Avr9 molecule;
the encoded polypeptide comprises a putative W O96/31608 PCTI~b~ 9 nucleotide binding site;
the encoded polypeptide is a cytoplasmic protein;
the encoded polypeptide comprises a region having homology to the cytoplasmic domain o~ the Drosophil a S Toll protein.
Another way of distinguishing nucleic acid according to the present invention from other pathogen resistance genes such as N and L6 may be for the encoded polypeptide to comprise an N-terminal domain that has greater than 60~ homology with the amino acid sequence o~ the N-terminal domain o~ RPP5 shown in Figure 2 (encoded by exon 1 o~ Figure 1), and/or comprise a nucleotide binding site domain that has greater than 40~ homology with the amino acid sequence o~ the domain of RPP5 shown in Figure 2 encoded by exon 2 of Figure 1, and/or comprise a domain that has greater than 30~ homology with the amino acid sequence of the domain of RPP5 shown in Figure 2 encoded by exon 3 of Figure 1, and/or comprise a domain that has greater than 30~ homology with the amino acid sequence of the leucine-rich repeat (LRR) domain of RPP5 shown in Figure 2 encoded by exons 4, S and 6 of Figure 1.
Table 2 shows ~ amino acid identity between putative domains of RPP5 and N, and RPP5 and L6, as encoded by exons of the genomic sequences.
The nucleic acid may comprise a sequence of nucleotides encoding an amino acid sequence showing at least about 60~ homology, preferably at least about -WO 96/31608 PCTIGB~GJ'~ 9 70~ homology, at least about 80~ homology, or more preferably at least about 90~ or greater homology to the amino acid sequence shown in SEQ ID NO 2.
Generally, "~ amino acid homology" is used to refer to ~ amino acid identity. High homology may be indicated by ability of complementary nucleic acid to hybridise under appropriate conditions, for instance conditions stringent enough to exclude hybridisation to sequences not encoding a pathogen resistance gene. Thus, the words allele, derivative or mutant may in context be used in respect of any sequence of nucleotides capable of hybridising with any of the nucleotide sequences encoding a polypeptide comprising the relevant sequence of amino acids.
Most preferably the nucleic acid encodes the amino acid sequence shown in SEQ ID No 2 in which case the nucleic acid may comprise DNA with an encoding sequence shown in SEQ ID NO 1 or sufficient part to encode the desired polypeptide (eg ~rom the initiating methionine codon to the first in frame downstream stop codon o~ the mRNA). In one embodiment, DNA comprises a sequence of nucleotides which are the nucleotides 1966 to 6511 of SEQ ID NO 1, or a mutant, derivative or allele thereo~, for instance lacking introns.
Figure 4 provides a contiguous sequence encoding the amino acid sequence o~ Figure 2.
A further aspect of the invention provides a nucleic acid isolate encoding a pathogen resistance WO 96131608 PCr/GB~6/0084g gene, or a fragment thereo~, obtainable by screening a nucleic acid library with a probe comprising nucleotides 1966 to 6511 o~ SEQ ID NO 1, nucleotides complementary thereto, or a ~ragment, derivative, S mutant or allele thereo~, and isolating nucleic acid which encodes a polypeptide able to con~er pathogen resistance to a plant. Suitable techniques are well known in the art. Thus, the present invention also provides a method of identi~ying and/or isolating nucleic acid encoding a pathogen resistance gene comprising probing candidate (or "target") nucleic acid with nucleic acid which has a sequence o~
nucleotides which encodes the amino acid sequence shown in Figure 2, which is complementary to an encoding sequence or which encodes a ~ragment of either an encoding sequence or a sequence complementary to an encoding sequence. The candidate nucleic acid (which may be, ~or instance, cDNA or genomic DNA) may be derived from any cell or organism which may contain or is suspected o~ containing nucleic acid encoding a pathogen resistance gene. A
pre~erred nucleotide sequence appears in Figure 1.
Sequences complementary to the sequence shown, and ~ragments thereo~, may be used.
Pre~erred conditions ~or probing are those which are stringent enough ~or there to be a simple pattern with a small number o~ hybridisations identi~ied as positive which can be investigated ~urther. It is W O96/31608 PCTI~~'~'~0~9 well known in the art to lncrease stringency of hybridisation gradually until only a few positive clones remain.
Nucleic acid according to the present invention may encode the amino acid sequence shown in SEQ ID NO
2 or a mutant, derivative or allele of the sequence provided. Preferred mutants, derivatives and alleles are those which retain a functional characteristic of the protein encoded by the wild-type gene, especially the ability to confer pathogen resistance. Changes to a sequence, to produce a mutant or derivative, may be by one or more of insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the insertion, deletion or substitution of one or more amino acids. Of course, changes to the nucleic acid which make no difference to the encoded amino acid sequence are included.
The nucleic acid may be DNA or RNA and may be synthetic, eg with optimised codon usage for expression in a host organism of choice. Nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of nucleic acid or genes of the species of interest or origin other than the sequence encoding a polypeptide with the required function. Nucleic acid according to the present invention may comprise cDNA, RNA, genomic CA 022l6406 l997-09-24 WO 96/31608 P~lt~5G~O~q9 DNA and may be wholly or partially synthetic. The term "isolate" encompasses all these possibilities.
Also provided by an aspect o~ the present invention is nucleic acid comprising a sequence o~
nucleotides complementary to a nucleotide sequence hybridisable with any encoding sequence provided herein. Another way of looking at this would be for nucleic acid according to this aspect to be hybridisable with a nucleotide sequence complementary to any encoding sequence provided herein O~ course, DNA is generally double-stranded and blotting techniques such as Southern hybridisation are often per~ormed following separation of the strands without a distinction being drawn between which o~ the strands is hybridising. Pre~erably the hybridisable nucleic acid or its complement encode a polypeptide ab]e to con~er pathogen resistance on a host, i.e includes a pathogen resistance gene. Preferred conditions ~or hybridisation are familiar to those skilled in the art, but are generally stringent enough ~or there to be positive hybridisation between the sequences of interest to the exclusion of other sequences, i.e sequences not encoding polypeptides able to con~er pathogen resistance on a host.
The nucleic acid may be in the ~orm o~ a recombinant vector, ~or example a phage or cosmid vector. The nucleic acid may be under the control of an appropriate promoter and regulatory elements ~or CA 022l6406 l9W 096/31608 PCT/GB96/00849 expression in a host cell, for example a plant cell.
In the case of genomic DNA, this may contain its own promoter and regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter and regulatory elements for expression in the host cell.
Those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, 1~ Molecular Cloning: a ~aboratory 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 Short 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.
~ 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 W 096/31608 PCTIG~G~49 nucleic acid to be inserted may be assembled within a construct which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the construct into S the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material may or may not occur according to dif~erent embo~;m~n~s o~ the invention. Finally, as ~ar as plants are concerned the target cell type must be such that cells can be regenerated into whole plants.
Plants transformed with a DNA segment containing pre-sequence may be produced by standard techniques which are already known ~or the genetic manipulation of plants. DNA can be transformed into plant cells lS using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene trans~er ability (EP-A-270355, EP-A-0116718, NAR 12(22) 8711 - 87215 1984), particle or microprojectile bombardment (US 5100792, 20 EP-A-444882, EP-A-434616) microinjection (WO 92/09696, 3, EP 331083, EP 175966), electroporation (EP 290395, WO 8706614) or other forms of direct DNA
uptake (DE 4005152, WO 9012096, US 4684611).
Agrobacterium trans~ormation is widely used by those skilled in the art to trans~orm dicotyledonous species. Although Agrobacterium has been reported to be able to transform foreign DNA into some monocotyledonous species (WO 92/14828), W O96131608 PCT/~b5G/Q0~9 microproiectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to S enhance the e~ficiency of the transformation process, eg. bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).
The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology o~ 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.
The RPP5 gene, modified versions thereof and related genes encoding a protein showing a significant degree of homology to the protein product o~ the RPPS
gene, alleles, mutants and derivatives thereof, may be used to con~er pathogen resistance, e.g. to downy mildews, in plants. For this purpose nucleic acid as described above may be used for the production of a transgenic plant. Such a plant may possess pathogen resistance conferred by the RPP5 gene.
The invention thus further encompasses a host W O96131608 PCT/~-B9~'00849 cell transformed with a vector as disclosed, especially a plant or a microbial cell. Thus, a host cell, such as a plant cell, comprising nucleic acid according to the present invention is provided. Within the cell, the nucleic acid may be incorporated within the chromosome.
A vector comprising nucleic acid according to the present invention need not include a promoter, _ particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.
Also according to the invention there is provided a plant cell having incorporated into its genome a sequence o~ nucleotides as provided by the present invention, under operative control o~ a promoter ~or control of expression o~ the encoded polypeptide. A
further aspect o~ the present invention provides a method o~ making such a plant cell involving introduction of a vector comprising the sequence o~
nucleotides into a plant cell. Such introduction may be ~ollowed by recombination between the vector and the plant cell genome to introduce the sequence o~
nucleotides into the genome. The polypeptide encoded by the introduced nucleic acid may then be expressed.
A plant which comprises a plant cell according to the invention is also provided, along with any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part o~ any of these, such as -W O96131608 PC~IGB96100849 cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on.
The invention further provides a method comprising expression from nucleic acid encoding the amino acid sequence SEQ ID NO 2, or a mutant, allele or derivative thereof, or a signi~icantly homologous amino acid sequence, within cells of a plant (thereby producing the encoded polypeptide), following an earlier step o~ introduction of the nucleic acid into a cell of the plant or an ancestor thereof. Such a method may con~er pathogen resistance on the plant.
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. Pathogen resistance may be determined by assessing compatibility of a pathogen such as Peronospora parasi tica or Bremia lactucae .
Sequencing of the RPP5 gene has shown that like the Cf-9 gene and the C~-2 gene it includes DNA
sequence encoding leucine-rich repeat ( LRR) regions and homology searching has revealed strong homologies to other genes containing LRRs. As discussed in WO95/18230, and ~urther validated in this discovery, the presence of LRRs may be characteristic of many pathogen resistance genes and the presence of LRRs can W O96131608 PCT/~TB9rll~q9 thus be used in a method of identifying further pathogen resistance genes.
Furthermore, there are some striking homologies between RPP5 and the tobacco mosaic virus resistance S gene N and the flax rust resistance gene L6. (Figure 3). (As can be derived from Figure 3, the overall homology between RPP5 and N is 33~ amino acid identity, while the figure for RPP5 and L6 is 2.7~.) These homologies may also be used to identify further resistance genes, for example using oligonucleoti~es (e.g. a degenerate pool) designed on the basis o~
sequence conservation, preferably conservation o~
amino acid secluence. In particular, primers may be designed that ampli~y DNA between the regions of the gene that encode the amino acid sequence F Y D V D P
(SBQ ID No 6) o~ RPP5 and N and where in L~ it encodes F Y M v D P (SEQ ID NO 7), and the region I A C F F
(SEQ ID NO 8) of RPP~, where the sec~uence is identical in L6 and in N is I A C F L (SEQ ID NO 9).
2~ According to a further aspect, the present invention provides a method of identifying a plant pathogen resistance gene comprising use of an oligonucleotide(s) which comprise(s) a sequence or sec~uences that are conserved between pathogen resistance genes such as ~PP5, N and L6 to searc:h for new resistance genes. Thus, a method of obtaining nucleic acid comprising a pathogen resistance gene (encoding a polypeptide able to confer pathogen W 096/31608 PCTIGB96/0084g 23 resistance) is provided, comprising hybridisation of an oligonucleotide (details o~ which are discussed herein) or a nucleic acid molecule comprising such an oligonucleotide to target/candidate nucleic acid.
Target or candidate nucleic acid may, for example, comprise a genomic or cDNA library obtainable from an organism known to encode a pathogen resistance gene.
Successful hybridisation may be identified and target/candidate nucleic acid isolated for further investigation and/or use.
Hybridisation may involve probing nucleic acid and identifying positive hybridisation under suitably stringent conditions (in accordance with known techniques) and/or use of oligonucleotides as primers in a method of nucleic acid amplification, such as PCR. For probing, preferred conditions are those which are stringent enough ~or there to be a simple pattern with a small number o~ hybridisations identified as positive which can be investigated further. It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain.
As an alternative to probing, though still employing nucleic acid hybridisation, oligonucleotides designed to amplify DNA sequences may be used in PCR
reactions or other methods involving amplification of nucleic acid, using routine procedures. See ~or instance "PCR protocols; A Guide to Methods and W O 96/31608 P~1/~96/00849 Applications", Eds. Innis et al, 1990, Academic Press, New York.
Preferred amino acid sequences suitable for use in the design of probes or PCR primers are se~lences conserved (completely, substantially or partly) between at least two polypeptides able to confer pathogen resistance such as those encoded by R~'P5 and N and/or L6.
On the basis of amino acid sequence information oligonucleotide probes or primers may be designed, taking into account the degeneracy o~ the genetic code, and, where appropriate, codon usage of the organism from the candidate nucleic acid is derived.
Pre~erred nucleotide sequences may include those comprising or having a sequence encoding amino acids (i) F Y D V D P (SEQ ID NO 6); (ii) I A C F F (SEQ ID
NO 8) or a sequence complementary to these encoding sequences. Suitable fragments of these may be employed. For example, the oligonucleotide TTC/T
TAC/T GAC/T GTX GAT/C CC (SEQ ID NO 10) can be derived ~rom the amino acid sequence F Y D V D P. Such an oligonucleotide primer could be used in PCR in combination with the primer A A G/A AA G/A CA XGC
T/G/A AT (SEQ ID NO 11), derived from the bottom strand o~ the sequence that encodes I A C F F. (All sequences given 5' to 3'; see Figure 3). X indicates A, G, C or T.
Preferably an oligonucleotide in accordance with W O 96/31608 PCT/G~96/~3~9 the invention, e.g. ~or 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).
Assessment of whether or not such a PCR product corresponds to resistance genes may be conducted in various ways. A PCR band from such a reaction might contain a complex mix of products. Individual ~roducts may be cloned and each one individually screened for linkage to known disease resistance genes that are segregating in progeny that showed a polymorphism for this probe. Alternatively, the PCR product may be treated in a way that enables one to display the polymorphism on a denaturing polyacrylamide gel and specific bands that are linked to the resistance gene may be preselected prior to cloning. Once a candidate PCR band has been cloned and shown to be linked to a known resistance gene, it may then be used to isolate cDNA clones which may be inspected for other features and homologies to either RPP5, N or L6. 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.
A further method of using the RPP5 sequence to identify other resistance genes is to use computer CA 022l6406 l997-09-24 W 096J31608 P~l/~b~ 19 searches of expressed sequence tag (EST) and other DNA
sequence databases to identify genes in other species that encode proteins with signi~icant RPP5 homology.
For example, a homology score of at least 60 using one of the BLAST algorithms (Altschul e~ al, 1990) would indicate a candidate resistance gene.
Having obtained nucleic acid using any of these approaches, a nucleic acid molecule comprising_all or part of the sequence of the obt~ained nucleic acid ~ay be used in the production of a transgenic plant, ~or example in order to con~er pathogen resistance on the plant.

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

Figure 1 shows the genomic DNA sequence o:E the RPP5 gene (SEQ ID NO. 1). Introns are shown in this Figure in non-capitalised letters. Features: Nucleic acid sequence - Translation start at nucleotide 1966;
translation stop at nucleotide 6512.
2~ Figure 2 shows predicted RPP5 protein amino acid sequence (SEQ ID NO 2).
- Figure 3 shows a comparison of the predicted amino acid sequence o~ the RPP5 (SEQ ID NO 2), N (SFQ

WO 96/31608 PCII~D, ~ 8 ~9 ID NO 3) and L6 (SEQ ID NO ~) genes. The protein sequences are aligned according to predicted protein ~ domains. Figure 3 was produced using the PRETTYBOX
and PileUp programs of the University of Wisconsin Genetics Computer Group Sequence Analysis So~tware Package Version 7.2.
Figure 4 shows a contiguous nucleotide sequence (SEQ ID NO 5) encoding the amino acid sequence_shown in Figure 2 (SEQ ID NO 2), and made by joining together the sequences o~ the exons o~ the sequence o~
Figure 1 (SEQ ID NO 1).

Cloning o~ the Arabidopsis RPP5 gene The RPP5 gene was cloned using a map-based cloning strategy similar in principle to that used ~or the isolation o~ the tomato Pto gene, described brie~ly earlier.

(i) Assignment o~ RPP5 gene map locations The map location o~ RPP~ on the Arabidopsis RFLP
map has been reported earlier (Parker et al, 1993).
This paper describes in detail how two landraces o~
Arabidopsis, designated Columbia and TIAn~qherg erecta, showed a di~erential response to a race o~
25 Peronospora parasitica designated NoCo-2; T~n~.qherg erecta is resistant, and Columbia is sensitive.
Recombinant inbred lines (Lister and Dean, 1993) had been constructed, derived ~rom carrying out single =

W 096/31608 PCT/GB~ OM9 seed descent on F2 seed derived ~rom an F1 between Landsberg and Columbia, and these recombinant inbred lines were tested ~or resistance or sensitivity to NoCo-2. This analysis showed that the RPP5 gene lay on Chromosome 4 between the RFLP markers m226 and g4539. The DNA o:E r~n~qherg and ColurrJ~ia was analysed using the RAPD (randomly ampli~ied polymorphic DNA) techni~ue (Williams et al, 1990) and polymorphisms between T,~n~.~herg and Columbia were analysed ~or linkage to RPP5. One polymorphism derived using the operon primer OPC18, which ampli~ied a band in Columbia but not in ~.~n~.~herg was absolutely linked to RPP5. ThiS DNA band, o~ 540 bp (rei~erred to as OPC18540 in Parker et al., 1993) was cloned and the resulting probe was designated the C18 probe.

(ii) Establishment of a physical map between marker m226 and marker g4539 The Arabidopsis genome project has as an objective the establishment o~ a physical map of Chromosome 4, and ultimately o~ the entire Arabidopsis genome. The C18 probe was used to identify hybridising yeast artificial chromosome (YAC) clones. This ~acilitated the establishment o~ a physical contig 25 between 4539 and 226 incorporating other linked markers, such as gl3683. The C18 RAPD band was cloned and used as a probe on Columbia and Landsberg genomic DNA. Hybridisation o~ this probe revealed a very CA 022l6406 l997-09-24 W O 96/31608 PCT/~ 0849 polymorphic small mu~ti-gene family in these two genotypes. Hybridisation to recombinant inbred lines ~ (Lister and Dean, 1993) showed that all members of this multi-gene family were absolutely linked to the resistance gene locus. Using the QPS procedure (Konieczny and Ausubel, 1993) the individuals in an F2 population derived ~rom sel~ing an Fl o~ a Columbia and Landsberg cross were screened for recombinants between the linked markers Ara-l and 4539. The primers used ~or the Ara-1 locus were Ara-l 5' TCG ACG ACT CTC AAG AAC CC 3' Ara-2 5' CAC AAG CTA TAC GAT GCT CAC C 3' This gave a 700 bp band in Columbia and T~n~herg which, after digestion with Acc-1, cut T,~n~.~herg DNA
giving a 360 bp and a 340 bp band. The primers used ~or the 4539 locus were 4539 F 5' GGT CAT CCG TTC CCA GGT AAA G 3' 4539 R 5' GGA CGT AGA ATC TGA GAG CTC 3' A~ter Hind III digestion, the Columbia 600 bp band remained uncut, whilst the ~andsberg band was cut to give 480 bp and 120 bp ~ragments. In this way twenty-~our additional recombinants were derived in this interval. Analysis o~ these recombinants showed that again all members o~ the Cl8 multi-gene ~amily co-~ segregated exactly with ~PP5. Since linked multi-gene ~amilies are a characteristic o~ disease resistance genes (Martin et al, 1993; Jones et al, 1994; Whitham et al, 1994) we tested the hypothesis that the C18 band might hybridise to the RPP5 gene Cosmids were identi~ied from a T,~n~.~herg binary vector cosmid library in the vector pCLD04541 (C. Dean, pers. com.;
S Bent et al, 1994) and cosmid clones that hybridised to the C18 probe were identi~ied. Table 1 lists the hybridising clones. Each o~ these were used in trans~ormation experiments with the readily trans~ormable Ara~idopsis landrace, No-O,which is sensitive to NoC0-2 A trans~ormant was identi~iea derived ~rom trans~ormation with cosmid 29L17, and sel~-progeny o~ this trans~ormant segregated ~or resistance to P. parasitica NoC0-2. This demonstrated that the clone 29L17, which carries a band that hybridises to the C18 RAPD probe, carries a ~unctional Peronospora parasitica resistance gene.

- (iii) DNA sequence analysis of the 29L17 plant DNA
insert Cosmid DNA was prepared ~rom 29L17, sonicated and cloned into pUC18 vector and randomly sequenced. Two hundred and ~orty (2~0) DNA sequencing reactions were per~ormed on random clones that were identi~ied as clones that hybridised to 29L17 insert DNA, i.e.
clones that carried inserts o~ plant DNA. From a computer analysis of this DNA sequence data, a DNA
sequence contig could be established comprising 14.3 kb o~ DNA. This DNA sequence was inspected ~or the WO 96/31608 P~ D~G~e49 presence of sequences that encoded leucine-rich repeats. One such region, nucleotide 3000 to nucleotide 6138 in SEQ ID NO. 1, was found.

(iv) Analysis o~ a DNA rearrangement associated with an RPP5 mutation One criterion for establishing whether or not a characterised region of plant DNA corresponds to the gene of interest is to inspect whether mutations in the corresponding gene, caused by ionizing radiation, are associated with DNA rearrangements in the region of interest. Fast neutron mutagenised Landsberg seed were screened with Peronospora parasitica ~or mutants to disease sensitivity. Three mutations were found and analysed by Southern blots for perturbations or rearrangements in DNA corresponding to the gene, carrying leucine rich repeats. One mutant line, FNB387, showed an altered pattern of Southern blot hybridisation. More detailed analysis showed that the perturbation consists of an insertion of 270 bp of DNA
in the C-terminus of the reading frame that carries leucine-rich repeats. Sequence analysis of this region showed that an insertion o~ 270 bp had arisen from the duplication o~ several LRRs within the gene carried on cosmid 29L~7. This provides very strong evidence that the RPP5 gene corresponds to the reading frame that carries leucine-rich repeats.

W O96/31608 PCT/~b'''~C~49 (v) Demonstration that a subclone o~ 29Ll7 contains RPP~
To confirm that the gene identified by mutagenesis is not only necessary but also su~ficient to confer disease resistance, a subclone of 29L17 was constructed in binary vector SLJ7292. The subclone, designated pRPP5-1, contained a 6304 bp DNA ~ragment de~ined by a BglII restriction enzyme site 5' to the gene (nucleotide 668 in SEQ ID NO. 1) and a Ps~I
restriction enzyme site 3' to the gene (nucleotide 6971 in SEQ ID NO. 1). pRPP5-1 was used to transform Arabidopsis ecotype Columbia and shown to con~er disease resistance.

(vi) RT-PCR analysis of the RPP5 transcript First strand cDNA was prepared ~rom seedling lea~
messenger RNA and PCR ampli~ication ~rom this cDNA was per~ormed using intron ~lanking primers The primers were: for intron 1, 5'-GAGTTCGCTCTATCATCTCC and 5'-TTATTGCATTCGA~ACATCATTG; for introns 2 and 3, 5'-AAATTGATCGTGCAAAGTCC and 5'-AAGATTCGCATTCTTCAAGATT;

for intron 4, 5'-GAAGATGGATTTGTATAATTCC and 5' -TCA~ATTCGGGCATCCAGTG. For intron 5 a nested PCR

strategy was employed, an aliquot of the products of the first amplification being used as the template for the second. The primers used were: for the first amplification, 5'-TGGTGACACTTCCTTCCTCG and 5~- CCA~ACTTTTGCAGTTGTTG; ~or the second ampli~ication CA 022l6406 l997-09-24 W 096/31608 PCT/GB~ 49 5'-TCTCAATGTGAGCGGCTGCAAGC and 5'-AACTTGAGCAACCACTGAGATCG. Cloned PCR products were sequenced using a combination of vector-speci~ic and insert-speci~ic primers. Intron sequences are shown in lower case in SEQ ID NO. 1 (Figure 1) between the exon sequences shown in upper case.

(vii ) Comparlson of the RPP5 gene se~uence wi th the se~uences of other resistance genes Comparison o~ the RPP5 sequence to the genes N
and L6 reveals very strong homologies throughout the N-terminal region. These regions are highlighted in Figure 3. They include regions involved in nucleotide binding, designated Kinase-la, Kinase-2, Kinase-3a.
Kinase-la is o~ten re~erred to as the P-loop. Also, regions N-terminal to the nucleotide binding domain show conspicuous homologies. Primers were designed particularly to the conserved regions carrying the amino acid sequence F Y D V D P (amino acids 104 to 109 of ~PP5~ and to amino acids I A C F F (437-441 o~
RPP5). When degenerate oligonucleotide primers based on amino acid sequence were used in PCR reactions, both on Arabidopsis genomic DNA and on cDNA made ~rom RNA o~ other species, products were observed o~ the size consistent with the potential to encode resistance genes.
These primers could alone, or in combination with W O96/31608 PCT/G-B~''0~649 other primers encoding conserved and non-conserved regions of the identi~ied resistance genes, be used to isolate other homologous gene sequences which could include previously uncharacterized resistance genes.
-W 096/31608 PCT/~b~G/00819 Table 1:
Binary vector cosmid clones hybridising to C18 Binary vector: 04541 Transformed into No-o Subsequently identi~ied:

W 096/31608 P~l/~G~ B19 Table 2:
amino acid identity between RPP5 and N; RPP5 and L6 N exonl N exon 2 N exon 3 N exon 4+5 exon 1 55 exon 2 36 exon 3 26 exon 4,5+6 26 L6 exon 1 L6 exon 2 L6 exon 3 L6 exon 4 exon 1 37 exon 2 30 exon 3 17 exon 4,5+6 26 W 096/31608 PCT/GB96/~49 RE:FERENCES
1. Bent AF, et al., (1994). RPS2 of Arabidopsis thaliana: Science 265:1856.

2. De Wit PJGM (1992). Ann.Rev.Phytopathol.
30:391-418.

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

4. Flor HH (1971). Ann.Rev.Phytopathol. 9:275-296.

5. Gabriel DW, et al., (1990). Ann.Rev.Phytopathol.
28:365-391.

6. Grant, MR, et al. Science 269: 843-846 (1995) 7. Johal GS, et al., (1992). Science (Wash.).
258:985-987.

8. Jones DA, et al., (1994). Science 266:789-793.

9. Joosten MHAJ, et al., (1994). Nature 367:384.

10. Keen NT, (1992). Ann.Rev.Gen. 24:447-463.

11. Konieczny A, et al., (1993). Plant Journal 4:403-407.

W 096/31608 P~l/~r./r~849 12. Lawrence, GJ et al. Plant Cell 7: 1195-1206 (1995) 13. Lister C, et al., (1993). Plant Journal (in S press).

14. Long SR, et al., (1993). Cell 73:921-935.

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Claims (27)

39

1. A nucleic acid isolate encoding a pathogen resistance gene whose expression in a plant can cause activation of a defence response in the plant, comprising a sequence of nucleotides encoding a polypeptide comprising the sequence of amino acids shown in Figure 2

2. Nucleic acid according to claim 1 wherein said activation is upon contact of the plant with a pathogen or corresponding elicitor molecule.

3. Nucleic acid according to claim 1 wherein the sequence of nucleotides comprises an encoding sequence shown in Figure 1.

4. Nucleic acid according to claim 1 wherein the sequence of nucleotides comprises an allele, derivative or mutant, by way of addition, insertion, deletion or substitution of one or more nucleotides, of an encoding sequence shown in Figure 1.

5. Nucleic acid encoding a pathogen resistance gene whose expression in a plant can cause activation of a defence response in the plant, comprising a sequence of nucleotides encoding a polypeptide, the polypeptide comprising an amino acid sequence which comprises an allele, derivative or mutant, by way of addition, insertion, deletion or substitution of one or more amino acids, of the amino acid sequence shown in Figure 2;
with the proviso that the encoded polypeptide has at least about 60% homology with the amino acid sequence shown in Figure 2.

6. Nucleic acid encoding a pathogen resistance gene whose expression in a plant can cause activation of a defence response in the plant, comprising a sequence of nucleotides encoding a polypeptide, the polypeptide comprising an amino acid sequence which comprises an allele, derivative or mutant, by way of addition, insertion, deletion or substitution of one or more amino acids, of the amino acid sequence shown in Figure 2;
with the proviso that expression of the nucleic acid can cause said activation of a defence response upon contact of the plant with an Oomycete fungus, such as Peronospora parasitica, or an extract thereof.

7. Nucleic acid according to claim 5 or claim 6 wherein said activation is upon contact of the plant with a pathogen or corresponding elicitor molecule.

8. Nucleic acid which is a vector comprising nucleic acid according to any one of claims 1 to 7.

9. Nucleic acid according to claim 8 further comprising regulatory sequences for expression of said polypeptide.

10. Use of nucleic acid according to any one of the precedings claims in production of a transgenic plant.

11 A host cell comprising exogenous nucleic acid according to any one of claim 1 to 9.

12. A host cell according to claim 11 which is microbial.

13. A host cell according to claim 11 which is a plant cell.

14. A plant or any part thereof comprising a cell according to claim 13.

15, Seed, selfed or hybrid progeny or a descendant or derivative or extract of a plant according to claim 14, or any part thereof.

16. A method which comprises introduction of nucleic acid according to any one of claim 1 to 9 into a host cell.

17. A method according to claim 16 wherein the host cell is a plant or microbial cell.

18. A method of conferring pathogen resistance on a plant, comprising expression from nucleic acid according to any one of claims 1 to 9, within cells of the plant, following an earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof.

19. A method according to claim 18 wherein the nucleic acid encodes an amino acid sequence shown in Figure 2.

20. An oligonucleotide of about 18 nucleotides encoding an amino acid sequence conserved between RPP5 of Arabidopsis (the sequence of which is shown in Figure 2) and the N gene of tobacco or L6 gene of flax, or a sequence complementary to a nucleotide sequence encoding a said amino acid sequence.

21. An oligonucleotide according to claim 20 comprising a nucleotide sequence encoding one of the amino acid sequences:

(i) F Y D/M V D P; and (ii) I A C F F/L
or comprising a nucleotide sequence complementary to a said encoding sequence.

22. An oligonucleotide according to claim comprising a sequence selected from:
(i) T T C/T T A C/T G A C/T G T X G A T/C C C;
(ii) A A G/A A A G/A C A X G C T/G/A A T; and (iii ) a sequence complementary to (i) or (ii).

23. A method of obtaining nucleic acid comprising a pathogen resistance gene comprising hybridisation of an oligonucleotide according to any one of claims 20 to 22, or a nucleic acid molecule comprising a said oligonucleotide, to target nucleic acid.

24. A method according to claim 23 involving use of nucleic acid amplification.

25. A method according to claim 23 or claim 2 wherein the hybridisation is followed by identification of successful hybridisation and isolation of target nucleic acid.

26. A method wherein following the obtaining of nucleic acid using the method of any of claim 23 to 25 a nucleic acid molecule comprising all or part of the sequence of the obtained nucleic acid is used in the production of a transgenic plant.

27. A method according to claim 26 wherein said nucleic acid molecule is used to confer pathogen resistance on said plant.