In vitro transcription and binding analysis of promoter regulation by a host-specific signal in a phytopathogenic fungus.

Abstract

The PDA1 promoter of the phytopathogenNectria haematococca MPVI (anamorph Fusarium solani) offers a model for regulation of a fungal virulence gene in response to plant host-specific signals. Expression of the PDA1 gene, encoding pisatin demethylase, is induced in culture by pisatin, the isoflavanoid phytoalexin of pea. This pisatin induction is suppressed by nutritional factors. We have been studying the mechanism of pisatin induction through in vitro identification of regulatory factors and regulatory elements of the PDA1 promoter. We have developed an in vitro transcription system for N. haematococca which accurately initiates at the PDA1 promoter and reflects the pisatin induction of PDA1 mRNA observed in vivo. This in vitro activity allowed a functional test of a limited set of 5' upstream deletions in the PDA1 promoter. In vitro binding studies have identified a DNA binding factor which is appears in mycelial extract after treatment of the mycelium with pisatin. This pisatin-responsive factor binds to a minimum size region of 35 bp approximately 500 bp upstream of the transcription initiation site. Tests using the in vitro transcription assay and in vivo competition both indicate a role for this binding region in the high expression of PDA1 under pisatin-induced conditions. Southwestern blotting has identified one component of this binding activity to be a approximately 35 kDa protein. The availability of these functional and structural tests of function, in conjunction with complementary in vivo tests, allow the detailed dissection of the signal pathway leading from exposure of the cell to pisatin towards the activation of PDA1 transcription.