Proteomic analysis of secreted proteins from Phytophthora infestans
In order to identify potential avirulence factors of Phytophthora infestans, secreted protein profiles of six strains (IPO-0, CBS431.90, IPO-655-2A, IPO-428-2, 88069 and 90128) with different avirulence phenotypes were analysed by proteomics. The proteins from culture filtrates were visualised on 2D gels with a total number between 350 and 1000. PiAVR X was found only in avirulent strains and considered therefore as a possible avirulence determinant. Its expression increased in all strains tested when grown in Modified Henniger medium. Single nucleotide polymorphisms (SNPs) were detected within a 735 nt fragment of PiAvrX but none was linked to avirulence characteristics. The consensus EST database facilitated identification of approximately 50% of 45 most abundant proteins excised from strain 88069. Beta-glucosidase, Pi-NIP2 (Phytophthora infestans necrosis inducing protein-like protein), enoyl CoA hydratase, glutathione S transferase, peptidylprolyl isomerase, acidic chitinase and Pi-PR1 (Phytophthora infestans homologue of plant PR-1 protein) were among the identified proteins with a recognisable signal sequence. Enolase, quinine-oxido reductase, nucleoside di-phosphate kinase, actin depolymerisation factor, thioredoxin, ubiquitin and 14-3-3 protein were also identified but without a signal peptide. The occurrence and expression of Pi-nip2, Pi-chi1 and Pi-pr1 were confirmed in the strains tested. Transformants were obtained from P. infestans strain T30-4 via a biolistic particle delivery approach using a single plasmid vector containing Pi-pr1. Detailed analysis of these transformants did not demonstrate induction of homology-dependent gene silencing. It was found that transformation rates were different among the tested P. infestans strains.