Cloning and characterization of the PDA6-1 gene encoding a fungal cytochrome P-450 which detoxifies the phytoalexin pisatin from garden pea.

The ability to detoxify pisatin, a phytoalexin produced by garden pea (Pisum sativum), is controlled by a family of PDA (pisatin demethylating ability) genes in the phytopathogenic fungus Nectria haematococa, MP (mating population) VI. Six known PDA genes each encode characteristic levels of inducible enzyme activity and are associated with different degrees of virulence on pea. To elucidate the phenotypic differences associated with these genes, we have cloned and characterized the PDA6-1 gene which encodes a pisatin-detoxifying enzyme and we compare it to another PDA gene, PDAT9. Pisatin demethylation was measured in PDA6-1 transformants of Aspergillus nidulans and shown to be regulated by glucose. The deduced amino acid (aa) sequence of PDA6-1 was 90% identical to that of the cytochrome P-450 encoded by PDAT9, but lacked nine aa at the C terminus, which has been postulated to be a site involved in substrate binding. A 35-bp sequence present upstream of a third PDA gene, PDA1, which appears to be important for induction of PDA1 by pisatin, was conserved in PDAT9, but not in PDA6-1. We conclude that PDA6-1, which does not appear to contribute to the virulence of N. haematococa on pea, differs significantly from PDAT9, which is associated with high virulence.