Molecular evolution of the disease resistance gene Rx in Solanum

Potato (Solanum tuberosum ssp. tuberosum) is the fourth most important food crop with an annual yield of about 300 million tons over the world. The history of the domestication of potato shows that disease-causing agents followed the tracks of potato cultivation in temperate climates across continents, resulting in substantial crop losses. Plants including potato have evolved defence mechanisms against pathogens, of which the pathotype-specific system involving resistance genes (R genes) is very effective. In search for durable resistance in crop plants, an increasing number of R genes has been identified and characterized, providing valuable information on their genomic organisation and evolutionary dynamics. They are often located in R gene clusters on complex loci or so called ‘hotspots’ for resistance in the plant genome. The evolution of host pathogen-interactions based on gene-for-gene interactions has been described in two prevailing theories. The arms race hypothesis assumes that new R gene alleles continuously emerge in a plant population and replace resistance alleles that are overcome by the pathogen. A short lifespan is postulated for a particular R gene allele. More recent, the ‘trench warfare’-model is proposed to explain the dynamics of R genes and Avr genes as repeated advances and retreats of resistance and virulence alleles. This frequency dependent selection pressure results in the maintenance of resistance alleles as balanced polymorphisms in natural populations over long periods of time (long lifespan). The aim of the research described in this thesis was to study the molecular mechanisms underlying the evolution of R gene recognition specificities using the virus resistance gene Rx1 from potato. Rx1 and its close homologue Gpa2 are both located in a single R gene cluster of about 110 kB on chromosome XII in potato and share an overall homology in the amino acid sequence of about 87%. Interestingly, these two R genes confer resistance to taxonomically unrelated pathogens, a virus and a nematode. However, Rx1 results in extreme resistance to potato virus X (PVX), whereas Gpa2 gives a much slower and milder resistance response to the potato cyst nematode Globodera pallida. The Rx1/Gpa2 resistance cluster was analysed on three S. tuberosum ssp. tuberosum haplotypes derived from the diploid potato clones SH and RH, homeologous to the resistant S. tuberosum ssp. andigena haplotype of SH, which resulted in the identification of nine additional homologues of Rx1 and Gpa2 in this region. In postulating a long lifespan for Rx, the findings of functional orthologues from distant related Solanum species give support for the trench warfare hypothesis. A survey across 35 species from the genus Solanum resulted in the identification of two novel functional orthologues of the disease resistance gene Rx1 in the wild potato species S. lepthophyes/sparsipilum and S. albicans, Rx3 and Rx4. By functional assays it was shown, that Rx3 and Rx4 confer Rx-mediated resistance to PVX in the corresponding S. lepthophyes/sparsipilum and S. albicans genotypes. Our finding of Rx3 and Rx4, together with Rx1 from S. tuberosum ssp. andigena and Rx2 from S. acaule, represents the first example of functional orthologues from the class of NBS-LRR resistance genes in four different plant species. Subsequent screening of about 5000 genotypes derived from 1000 accessions across 200 Solanum species resulted in the identification of 14 additional distantly related Solanum species harbouring Rx candidate genes, which shows that the Rx orthologues are of ancient origin and derived from one ancestral gene. This indicates that Rx mediated PVX recognition arose before the groups of Tuberosa (Peru) and Acaule/Demissum diverged. To gain more insight in the structural and evolutionary relationship of the resistance gene cluster a set of 75 Rx1/Gpa2 homologues was obtained from 10 different Solanum species, including the four functional orthologous virus resistance genes Rx1-4, the truncated Rxedn that harbours Rx resistance specificity and the nematode resistance gene Gpa2. About one third of the homologues were found to encode for a protein harbouring complete CC-, NBS- and LRR- domains analogous to Rx and Gpa2, indicating the presence of functional R genes with unknown specificities. We identified a local structural variability in the sequence region encoding LRR10, dividing the homologues in 3 structural subclasses. The occurrence of similar sequence exchange tracks in homologues isolated from different Solanum species indicates that interallelic exchanges took place before speciation. Analysis for positive selection revealed very significant positive selection on codon sites within the LRR domain, but also within the CC- and NBS- domain. In addition, the genomic organisation of the Rx1/Gpa2 cluster was analysed. Therefore, sequence analysis was carried out on several BAC clones derived from S. tuberosum ssp. andigena, S. tuberosum ssp. tuberosum and S. lepthophyes/sparsipilum harbouring in total 17 Rx1/Gpa2 homologues, including the functional resistance genes Rx1 and Gpa2 from potato. The homeologous chromosomal regions derived from S. tuberosum ssp. andigena and S. tuberosum ssp. tuberosum were assembled and aligned based on syntenic regions. However, comparative sequence analysis revealed large intergenic sequence divergence in this region, which was increasing for more distant related Solanum species. The occurrence of a family of conserved genes with homology to RING zink-finger protein encoding genes in vicinity of the Rx/Gpa2 homologues suggests that tandem duplication of these genes has occurred at this locus. The major results described in this thesis are discussed and an outlook on structure-function analyses of Rx1 and Gpa2 is presented.