Characterization of the PDA1 Promoter of Nectria haematococca and Identification of a Region That Binds a Pisatin-Responsive DNA Binding Factor. David C. Straney. (1)Department of Plant Pathology, Cornell University, Ithaca, NY, 14853 U.S.A.; (2)Department of Botany and the Maryland Agriculture Experiment Station, University of Maryland, College Park, MD 20742 U.S.A. Hans D. VanEtten(1,3). (1)Department of Plant Pathology, Cornell University, Ithaca, NY, 14853 U.S.A; (3)Department of Plant Pathology, University of Arizona, Tucson, AZ 85721 U.S.A. MPMI 7:256-266. Accepted 10 December 1993. Copyright The American Phytopathological Society.

Isolates of Nectria haematococca (anamorph: Fusarium solani) are able lo detoxify the pea Phytoalexin pisatin through expression of pisatin demethylase (pda). This enzyme is a substrate-inducible cytochronie P450 monooxy-enase that is encoded by the PDA gene family. In the current study, PDA1, a highly inducible PDA gene, was cloned and the 5' untranslated region was sequenced. The PDA mRNA levels were measured in pisatin-treated mycelium and found to increase by 20-fold over untreated control. Gel shift assays identified a 35-bp region, -514 to -480 bp relative to the first mRNA start site, that binds a factor found in extracts of pisatin-treated mycelium and absent in untreated mycelium. The function of the binding site in pisatin regulation of the PDA1 gene was tested in an in vivo competition assay by introduction of multiple ectopic copies of the binding site inio N. haematococca through transformation. In such transformants, induction of pda activity by pisatin was delayed and reduced, consistent with the titration of a trans-acting factor which responds lo pisatin. These results suggest the 35-bp region is functioning as a pisatin-responsive activator binding site for PDA1. Additional controls were characterized that act on PDA1 expression. Induction of pda by pisatin was suppressed by the addition of 0.8% Casamino Acids or 5% glucose lo the suspended mycelium. A unique DNA binding factor was detected only in extracts from mycelia treated with the Casamino Acids that bind to the same 35-bp region of the PDA1 gene as the pisatin-responsive factor.