IDENTIFICATION AND FIRST STEPS TOWARDS CLONING THE PLASMODIOPHORA BRASSICAE RESISTANCE GENE, RPB1, IN ARABIDOPSIS THALIANA
J SIEMENS, A ARBEITER, P KOBELT, C KÖHN, H LÜRßEN and MD SACRISTAN
FU Berlin, Berlin, Germany
A stable, race-specific resistance of A. thaliana to the obligate biotrophic pathogen P. brassicae (isolate eH), the causal agent of clubroot, is reported. By genetic analysis of the ecotypes Tsu-0 (resistant) and Cvi (susceptible) we found that resistance to P. brassicae isolate eH is conferred by a dominant allele of a single nuclear gene (RPB1) located on chromosome 1 between the markers th1 and tt1. With the purpose of isolating the RPB1 gene by positional cloning, a backcross population from the cross Tsu (RPB1/RPB1) x Cvi (rpb1/rpb1) of about 900 individuals has been built up. This population was pre-screened for recombination events around the RPB1 region using two RAPD markers flanking the gene at distances of 9.1 and 3.6 cM, respectively. In a second step we mapped the resistance locus against 13 published molecular markers. We could show that the RFLP markers m253 and AIG1 flank the RPB1 locus on the left and right sides, respectively. An additional marker, rpHS-1, shows absolute linkage. These three markers serve as starting points for the chromosomal walk. Corresponding YACs were used for establishing a YAC contig for this region. The IPCR products of one YAC clone mapped on the left and the right side of RPB1. Using this YAC we will screen a BAC library to achieve a BAC contig spanning the region defined by the YAC insert ends. These BACs will serve as tools for a high-resolution mapping around the RPB1 gene.
The second strategy to examine resistance is microscopical analysis of the compatible and incompatible interactions using several ecotypes, mutant and transgenic lines. Immunohistological staining of cytoskeletal elements has been used to detect the pathogen during the very early steps of the infection, when the pathogen succeeded in invasion of the host root or when the resistant lines were able to build up a necrotic barrier. Further antibodies as well as conventional staining techniques were used to demonstrate the changes during the early stages of pathogenesis and the primary resistance reaction.