BSPP Presidential Meeting 2000

Plant-pathogen Interactions:
Understanding Mechanisms of Resistance and Pathogenicity for Disease Control

Session III: Resistance genes, structure and evolution

Natural and artificial evolution of resistance gene clusters, particularly of the pto and Dm3 loci
Richard Michelmore
University of California, Davis, CA 95616, USA

Molecular studies and classical genetics have shown that resistance genes tend to be clustered in the genome. Resistance genes seem to be members of multigene families encoding receptors with individual members of these families having diverged to acquire novel recognition specificities. Classical genetics also indicated that clusters of resistance genes were unstable. This led to the idea that clusters of resistance genes are dynamic regions of the genome in which new specificities are frequently being generated by unequal crossing-over. Characterization of the Pto and Dm3 resistance gene clusters at the molecular level confirms that unequal crossing-over and gene conversions can occur. We have sequenced multiple haplotypes of the Pto locus in tomato that contains duplicated Pto homologs and a single NBS-LRR gene, Prf . There is greater sequence similarity between Pto homologs in comparable genomic positions in different genotypes than between homologs within the same haplotype. There is some evidence for gene conversion events but the overall structure of three haplotypes was similar, indicating that the Pto locus is not highly unstable. This is similar to the situation that we have observed for the major cluster of NBS-LRR containing resistance genes in lettuce. The major cluster of disease resistance genes in lettuce comprises of over 30 NBS-LRR encoding RGC2 genes and spans at least 4 Mb. Only one member encodes Dm3 specificity. We have evidence for point mutations, recombination, gene conversions, and unequal crossing-over within and between homologs at this complex locus. We have fully sequenced 22 RGC2 genes and partially sequenced an additional six from genomic clones of the cultivar Diana. The 3 ends of RGC2 homologs were amplified using PCR from two genotypes each of L. saligna, L. serriola, and three genotypes of L. sativa. Phylogenetic analysis indicated two groups of homologs. One group comprised of obvious chimeras; the other showed little or no evidence of chimeras. These two groups were non-randomly distributed in the cluster. The latter group was located peripherally and at one end. These RGC2 sequences are not evolving rapidly. The primary evolutionary force seems to be divergent evolution on orthologs with a birth-and-death process responsible for changes in copy number.

In addition, we are using in vitro gene shuffling to study the domains determining specificity of Pto. This has provided a range of molecules that are allowing us to dissect the early molecular interactions that determine specificity.

Michelmore, R.W. and Meyers, B.C. (1998). Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Research 8:1113-1130.

Chin, D.B., Arroyo-Garcia, R., Ochoa, O., Kesseli, R.V., Lavelle, D.O., Michelmore, R.W. Recombination and spontaneous mutation at the major cluster of resistance genes in lettuce (Lactuca sativa). Genetics in press.

Function and evolution of the RPP13disease resistance locus from Arabidopsis
Jim Beynon, Ian Crute, Eric Holub and Peter Bittner-Eddy
Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK.

We have been developing the interaction between Arabidopsis and the downy mildew pathogen Peronospora parasitica (AT) as a model system for studying plant/oomycete interactions. As part of that analysis we initially identified over 20 plant genes (RPP Recognition of Peronospora parasitica) that were involved in isolate specific interaction with P.parasitica (AT). Following this analysis we have cloned genes from four of these loci, RPP1, RPP2, RPP13 and RPP28. To complement this study we are developing resources that will allow us to clone the complementary pathogen genes identified by the interaction with the RPP1 and RPP13 genes and others. Each of the cloned RPP loci has revealed a different evolutionary strategy. Here I will describe our analysis of the RPP13 locus. RPP13 is a LZ:NBS:LRR class disease resistance gene and forms a functional allelic series that shows a high level of sequence diversification in the LRR domain. We have analysed the structure of the locus in Arabidopsis and compared this to the structure in Brassica oleracea. We also demonstrate that RPP13 signal transduction is completely independent of the eds1 and ndr1 pathways and does not require salycilic acid accumulation for a disease resistance response.

Arabidopsis mildew resistance
Shunyuan Xiao, Simon Ellwood, Ozer Calis, Elaine Patrick, Tianxian Li, Daniel Jaggard, Paul Beckett, Richard Oliver, Mark Coleman and John G. Turner
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, England.

We have recently isolated the Arabidopsis thaliana RPW8 locus, which confers resistance to a broad spectrum of powdery mildew fungi. The locus contains two functional genes (designated RPW8.1 and RPW8.2) and these encode novel protein products, unrelated to the products of other plant disease resistance genes and not closely related to any other characterised proteins. RPW8.1 and RPW8.2 encode small, basic proteins that are 45% identical; both encode products containing predicted coiled-coils, implying the formation of homotypic or heterotypic complexes. RPW8.1 and RPW8.2 are members of a small, linked gene family. In at least some susceptible Arabidopsis accessions, genes encoding proteins very closely related to RPW8.1 and RPW8.2 have been identified.

Despite differing in their spectrum of activity, primary structure and degree of polymorphism, the RPW8 proteins are functionally similar to the products of race-specific disease resistance (R) genes. RPW8-mediated resistance thus involves typical R-gene mediated defence responses such as the accumulation of hydrogen peroxide and pathogenesis related proteins. It is abolished by the expression of nahG, indicating a salicylate requirement, is partially suppressed by mutations in NPR1, and exhibits an absolute requirement for functional EDS1.

Resistance mediated by RPW8 will be compared to that mediated by other disease resistance genes and will be discussed with particular reference to the somewhat unusual, polyphagous nature of the fungi against which it is effective.