Identification of pathogenesis-related ESTs in the crucifer downy mildew oomycete Hyaloperonospora parasitica by high-throughput differential display analysis of distinct phenotypic interactions with Brassica oleracea.

Crucifer downy mildew is caused by the obligatory biotrophic oomycete Hyaloperonospora parasitica (formerly Peronospora parasitica). So far, isolates infecting Arabidopsis thaliana have proven to be non-pathogenic on other crucifers and, despite its unequivocal merit as a research model, the pathosystem A. thaliana-H. parasitica by itself will not provide all the answers onto crucifer downy mildew genetics and biology. In this report, we present the development of a differential display (DD)-based strategy, suitable for high-throughput analysis of expressed sequence tags (ESTs) in plant-pathogen interactions, in this work applied to the analysis of the pathosystem Brassica oleracea-H. parasitica interaction transcriptome. Our purpose was the mining for pathogen-specific ESTs that can be used in future research for virulence factors and Avr genes. A total of 743 specific cDNAs showing differential expression in B. oleracea seedlings infected with H. parasitica, as opposed to healthy seedlings, were isolated by DD-PCR. We found 21 exclusively H. parasitica cDNAs from 433 sequenced DD clones, 18 encoding for potential new genes. Our results reinforce the abilities of DD-PCR for differential screening of pathosystems transcriptomes, leading to the finding of more new potential genes than the previously used techniques. Both the improved DD-based methodology and the graphical representations based on Venn diagrams from polyominoes are appropriate for large-scale analysis of multiple interaction transcriptomes. The obtained data are also innovative since this is the first approach to study the interaction of H. parasitica with its natural host.