Metabolism of Pisatin Stereoisomers byAscochyta rabiei Strains Transformed with the PisatinDemethylase Gene of Nectria haematococca MP VI. Klaus-Michael Weltring . Institut fur Biochemie und Biotechnologie der Pflanzen, Wesfalische Wilhelms-Universitat Munster, Hinden-burgplatz 55, 48143 Munster, Germany. Hans-Peter Schaub, and Wolfgang Barz. Institut fur Biochemie und Biotechnologie der Pflanzen, Wesfalische Wilhelms-Universitat Munster, Hinden-burgplatz 55, 48143 Munster, Germany. Current address of K.-M. Weltring: Institut fur Botanik und Botanischer Garten, Westfalische Wilhelms-Universitat Munster, SchloBgarten 3, 48149 Munster, Germany. MPMI 8:499-505. Accepted 28 April 1995 . Copyright 1995 The American Phytopathological Society.

The chickpea pathogen Ascochyta rabiei was transformed with plasmid pUCHl/PDA containing the hygromycin B resistance gene as a selectable marker and the pisatin demethylase gene of Nectria haematococca mating population VI, a pathogen of pea. The rate of transformation was approximately 2 transformants per ug of DNA. While un-transformed A. rabiei is inhibited by hygromycin B at 50 (g/ml, transformants were able to grow on 300 (g of this antibiotic. Transformants demethylated the pea phytoa-lexin (+)pisatin to (+)6a-hydroxymaackiain (6a-HMK) within 2 to 4 h. The product was not further metabolized and accumulated in the medium. Untransformed strains failed to show any significant metabolism of (+)pisatin to (+)6a-HMK within a given interval of 24 h. One of the transformants, when tested for metabolism of (-)pisatin, produced a demethylation product that only transiently accumulated in the medium. One subsequent degradation product was tentatively identified as 3,7,2'-trihydroxy-4',5'-methylenedioxy isoflavan. These results support the hypothesis that the enzymes of A. rabiei involved in the metabolism of pterocarpan phytoalexins are specific for (-) isomers such as (-)maackiain and (-)medicarpin produced by the natural host of this fungus.