The PDA1 gene of the filamentous fungus Nectria haematococca MPVI (anamorph: Fusarium solani) encodes pisatin demethylase, a cytochrome P450. Pisatin is a fungistatic isoflavonoid produced by garden pea (Pisum sativum), a host for this fungus. Pisatin demethylase detoxifies pisatin and functions as a virulence factor for this fungus. Pisatin induces PDA1 expression both in cultured mycelia as well as during pathogenesis on pea. The regulatory element within PDA1 that provides pisatin-responsive expression was identified using a combination of in vivo functional analysis and in vitro binding analysis. The 40 bp pisatin-responsive element is located 635 bp upstream of the PDA1 transcription start site. This element was sufficient to provide strong pisatin-induced expression to a minimal promoter in vivo and was required for pisatin regulation of the PDA1 promoter. A gene encoding a DNA-binding protein specific to this 40 bp element was isolated from a N. haematococca cDNA library using the yeast one-hybrid screen. The cloned gene possesses sequence motifs found in the binuclear zinc (Cys 6-Zn 2) family of transcription factors unique to fungi. The results suggest that it is a regulator of this fungal cytochrome P450 gene and may provide pisatin-responsive regulation.
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