Regulation of Pisatin Demethylation in Nectria haematococca and its Influence on Pisatin Tolerance and Virulence

Abstract

Nectria haematococca, a fungal pathogen of pea, demethylates the pea isoflavonoid phytoalexin pisatin to yield the less inhibitory product, 3,6a-dihydroxy-8,9-methylenedioxypterocarpan. Among naturally occurring isolates that demethylate pisatin (PDA+ isolates), some can be induced to do so at a high rate by pretreatment with the substrate (PDA1 isolates), while others demethylate pisatin only slowly, regardless of pretreatment (PDAn isolates). Genetic analysis of these pisatin demethylation phenotypes has indicated that one gene confers the PDA1 phenotype and that two distinct genes at different loci each confer the PDAn phenotype. We report here the relationship of these PDA phenotypes to pisatin tolerance and to virulence toward pea. The contribution of each PDA gene to these traits was also evaluated. In initial crosses between tolerant, PDA+ parents, recombinant progeny lacking demethylating activity (PDA- isolates) occurred. These PDA1 progeny were uniformly more sensitive to pisatin than the PDA* parents and all PDA+ progeny. Progeny having PDA1 phenotypes were more tolerant to pisatin than PDAn progeny, which in turn were more tolerant than PDA1 phenotype. Further genetic analyses confirmed that a characteristic pattern of pisatin tolerance was associated with each PDA gene. The gene conferring the PDA1 phenotype was associated with the highest level of tolerance, while the two genes conferring PDAn phenotypes were each associated with an intermediate level of pisatin tolerance. Thus relative tolerance to pisatin appears to depend upon both the presence and amount of pisatin demethylase activity. Virulence toward pea may also be determined by the ability to demethylate pisatin rapidly. In numerous crosses between vV. haematococca isolates with different pisatin demethylation phenotypes, high virulence in progeny was always correlated to the PDA1 phenotype. AH PDAn and PDA1 progeny were low in virulence. Thus the gene that confers high pisatin demethylase activity in N. haematococca is, or is closely linked to, a gene for virulence to pea.