UC Riverside

Phytophthora infestans, an oomycete within the phylum Heterokontophyta is one of the most devastating phytopathogens, causing late blight in potato and tomato. Its pathogenic success depends on the formation of different asexual spores such as sporangia and zoospores. Our goal was to identify what regulates transition between each of the five different asexual stages viz. hyphae, spores, cleaving sporangia, swimming zoospores and germinating cysts, by understanding what determines stage-specific transcription. To help accomplish this, we have identified potential binding sites for regulatory elements within the promoters of stage-specific, co-expressed Phytophthora infestans genes, by integrating bioinformatics and traditional molecular biology techniques. Promoter sets, of co-expressed genes identified from expression data, were searched for over-represented motifs using motif discovery algorithms. We were able to detect approximately 20 to 30 over-represented motifs for each of the five stages. Phylogenetic footprinting and positional bias analyses increased the robustness of Transcription Factor Binding Site (TFBS) predictions. Some of the over-represented motifs, which were evolutionarily conserved and showed bias for a certain position within the promoters, were tested for their functionality. Putative TFBSs related to each of the above-mentioned asexual stages, other than the short and unstable swimming zoospore stage, were shown to be biologically active. Molecular biology techniques like promoter-reporter fusion assay, serial deletion, target-specific mutation, RNA-blotting and electromobility shift assay, were used for the functional validation of these potential TFBSs, all of which acted as proximal promoter elements. In addition to these elements, we also looked at the core promoter elements for genes, which had EST evidences for their transcription start sites. A novel core promoter element, specific to the group Pythiales and named DPEpyth, was identified. We have also come up with better Phytophthora specific definitions (consensus sequence), for `FPR' that has been previously detected within the oomycete core promoters and also for the very well known Initiator element (`Inr'). I believe that the identification of these binding sites should lead to a better understanding of signaling pathways regulating spore development and provide insight into new disease control strategies in the future.