Characterization of pisatin-inducible cytochrome P450s in fungal pathogensof pea that detoxify the pea phytoalexin pisatin

Many fungi that are pathogenic on pea have the ability to demethylate and t hus detoxify the pea phytoalexin pisatin. This detoxification reaction has been studied most thoroughly in Nectria haematococca MP VI where it functio ns as a virulence trait. The enzyme catalyzing this reaction [pisatin demet hylase (pda)] is a cytochrome P450. In the current study, the induction of whole-cell pda activity and the biochemical properties of pda in microsomal preparations from the pea pathogens Ascochyta pisi, Mycosphaerella pinodes , and Phoma pinodella are compared to the pda produced by N. haematococca. Based on cofactor requirements and their inhibition by carbon monoxide, cyt ochrome P450 inhibitors, and antibodies to NAD-PH:cytochrome P450 reductase , we conclude that the pdas from the other pea pathogens also are cytochrom e P450s. All of the enzymes show a rather selective induction by pisatin, h ave a low K-m toward pisatin, and have a fairly high degree of specificity toward pisatin as a substrate, suggesting that each pathogen may have a spe cific cytochrome P450 for detoxifying this plant antibiotic. Since the pdas in these fungi differ in their pattern of sensitivity to P450 inhibitors a nd display other minor biochemical differences, we suggest that these fungi may have independently evolved a specialized cytochrome P450 as a virulenc e trait for a common host. (C) 2001 Academic Press.