Expression of the pisatin detoxifying genes (PDA) of Nectria haematococca in vitro and in planta.

Abstract

The phytopathogenic fungus Nectria haematococca detoxifies pisatin, a phytoalexin produced by pea. Pisatin demethylating ability (a phenotype called Pda) is due to pisatin demethylase (pdm) and the genes encoding this enzyme are called PDA. Some isolates rapidly acquire a high to moderate rate of pisatin demethylating activity culture in response to pisatin (phenotypes PdaSH and PdaSM), while other isolates only slowly demethylate pisatin (phenotype PdaLL). Here we report that PDA-specific RNA levels increased more quickly in response to pisatin in isolates with PDA genes confering a PdaSH or PdaSM phenotype than in isolates with gene conferring a PdaLL phenotype. In addition, the pdm activity of transformants of N. haematococca containing chimeric constructs of PDASH and PDALL genes in which the 5' regulatory regions of these genes had been switched supports the conclusion that differential expression of PDA genes is responsible for the different Pda phenotypes detected in vitro. Northern analysis of pea tissue infected with isolates carrying PDASH or PDALL genes indicated that differential induction of these genes also occurred in planta. Only PDASH-specific RNA is readily detected in tissue infected with isolates containing PDASH and PDALL genes. Recently a pisatin biosynthetic gene, isoflavone reductase (IFR), has been identified. Using the polymerase chain reaction, qualitative detection of IFR and PDASH transcripts in infected tissue were made to assess the relative timing of these genes' expression. No transcripts were detected 6 h after inoculation, but transcripts of both genes were detected a 12 h, suggesting an interplay between the regulatory systems controlling the plants's defense response and the pathogen's counter response.