Molecular analysis of a fungal virulence gene regulation by host signals during pathogenesis

Plant-pathogen interactions provide particularly interesting systems to study gene regulation in response to environmental signals. Each organism in a pathogenic interaction usually responds to cues derived from the other organism to trigger gene expression necessary for either pathogenesis or defense. One well-defined model for the study of gene regulation among eukaryotic plant pathogens is the PDA1 gene of the filamentous fungus Nectria haematococca MPVI (anamorph: Fusarium solani), a pathogen of garden pea. This gene is induced by pisatin, an antimicrobial compound produced by garden pea. The PDA1 gene encodes pisatin demethylase, a cytochrome P450 monooxygenase, which detoxifies pisatin. Correlative evidence links high virulence of the fungus to the ability of the PDA1 gene to be induced by pisatin.;This dissertation is directed at elucidating the mechanisms by which pisatin, glucose, and amino acids regulate the expression of PDA1. A GUS reporter gene system was used to measure the effects of promoter alterations. Regions of the PDA1 promoter which respond to specific signals were delineated. The results suggest that the PDA1 gene is under combinatorial mode of regulation; pisatin and nutritional signals act through independent pathways.;Given the regulation of the PDA1 gene by two different signals, a detailed analysis of PDA1 regulation in the plant was conducted to determine which of the two signals was inducing the gene during pathogenesis. The results indicate that the host-specific signal, pisatin, is the key stimulus for PDA regulation during pea pathogenesis.;Considering the importance of pisatin-mediated PDA1 regulation, the pisatin-responsive element was extensively characterized, and the region narrowed down to a 40 bp sequence using in vitro techniques. This sequence harbors the binding site of a putative pisatin-responsive transcriptional activator. Attempts to clone the gene mediating pisatin-induction of PDA1, by one-hybrid assay, were unsuccessful. In summary, these results are the first of its kind to correlate the regulation of a virulence gene in culture to its expression during pathogenesis, and is a significant step forward towards understanding specificity of gene regulation during plant-microbe interactions.