Isolation and characterization ofCryphonectria parasiticamutants that mimic a specific effect of hypovirulence-associated dsRNA on laccase activity

Hypovirulent, double-stranded (ds) RNA-containing strains of the chestnut blight fungus Cryphonectria parasitica were previously shown to produce less laccase activity than virulent strains when grown on malt extract agar containing tannic acid (Bavendamm reaction). Three mutants that lacked this specific laccase activity were selected after UV mutagenesis of a dsRNA-free, wild-type strain. Complementation tests and sexual crosses showed that all mutations were recessive, two were allelic (lacR1-1 and lacR1-2), and one (lacR2) was nonallelic. No linkage was detected between the two loci. None of the known C. parasitica laccases (LAC1, LAC2, and LAC3) was substantially reduced in the lacR1 mutants. The lacR2 mutant, in contrast, produced about 10-fold less extracellular LAC1 and LAC3 activities than the wild-type strain. Intracellular LAC2 was reduced to about 50% in this mutant. These results suggest a role for both LAC1 and LAC3 in the Bavendamm reaction. The three mutations had no significant effect on fungal virulence, pigmentation, and sporulation, all phenotypes that were suppressed in an isogenic dsRNA-containing strain.