Analysis of Determinants of Binding and Transcriptional Activation of the Pisatin-Responsive DNA Binding Factor ofNectria haematococca

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Abstract
Pisatin is a fungistatic isoflavonoid produced by garden pea. Field isolates of the ascomycete Nectria haematococca mating population VI (anamorph: Fusarium solani) that are highly virulent on pea have been found to possess the PDA1 gene encoding a pisatin detoxifying activity. Expression of PDA1 is specifically and highly induced by exposure of mycelia to pisatin. A pisatin-responsive DNA-binding activity has previously been identified with properties suggestive of a transcriptional regulator of PDA1. In this study, the sequence determinants for binding this pisatin-responsive factor (PRF) were localized to a 14-bp region through analysis of sequence alterations that reduced PRF binding. Using a homologous in vitro transcription system, a transcriptional activator of PDA1 was shown to be present in mycelial extracts that shared the sequence specificity characteristic of the PRF, indicating function of the DNA-binding protein in transcriptional control. A 70-kDa protein was shown to be a DNA-binding component of PRF by three independent assays for DNA-binding proteins: Southwestern (DNA-protein) blotting, UV-crosslinking, and binding to immobilized DNA. These results characterize a transcriptional activator acting on the PDA1 promoter that is responsive to a host-specific compound and provide insight into the regulation of fungal genes in response to plant flavonoids.

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