Characterization of an EST collection from potato genotypes resistant and susceptible to Ralstonia solanacearum

Host resistance is the only control system available for the bacterial wilt of potato, caused by Ralstonia solanacearum. This resistance is horizontal, polygenic and its genetic basis is not understood yet. In order to identify genes involved into this plant-pathogen interaction, two different transcriptomic approaches have been used. In particular both a cDNA-AFLP-TP analysis and microarray experiments were performed on the resistant wild S. commersonii and the susceptible S. tuberosum cv. Blondy for monitoring gene expression during infection. A total of 32 primer combinations were used to amplify cDNA-AFLP fragments and PCR products were sequenced resulting into 124 differentially expressed ESTs. Most of them (45.8%) were specific of the S. commersonii genotype after bacterial inoculation, whereas the remaining were obtained from both resistant and susceptible genotypes after inoculum (4.2%), or were over- or under-expressed in one of the two genotypes (36.7%). The EST electropherograms were processed to trim out the low quality bases from the ends of the reads. Vector contaminations were identified and removed and sequences that were < 25 nucleotides in length were discarded. This process reduced the original dataset to 36 sequences, which were fed into the assembling process resulting into 5 tentative consensus sequences and 25 singletons. The microarray analysis was performed on a Combimatrix 4X2k chip on which around 650 oligos were synthesized based on an EST collection obtained from a PCR select experiment carried out on the resistant tomato cv. Hawaii 7996. After chip hybridization with RNA extracted from both resistant and susceptible genotypes, before and after inoculum, 59 differentially expressed sequences were selected. Both differentially expressed sequences from the AFLP sequencing and the tomato EST collection used to design oligos for the synthesis of the Combimatrix chip were functionally annotated. Automated annotation was performed by BLASTx searches against the UniProtKB/TrEMBL database and the Arabidopsis thaliana protein complement. Association to Gene Ontology terms (GO) was electronically inferred by exploiting TrEMBL entries. Interestingly, among the 89 differentially expressed sequences the majority were related to metabolism, plant defence and signalling and transcription regulation. Further investigations will focus on understanding the exact role of these sequences in protecting plants against pathogen attacks.