Evidence for the requirement of extracellular protease in the pathogenic interaction of Pyrenopeziza brassicae with oilseed rape

Using a detached cotyledon test for pathogenicity, a UV-induced, non-pathogenic mutant of Pyrenopeziza brassicae was isolated which was also deficient in extracellular protease production in vitro . The proteolytic activity in the wild type was apparently due to a single cysteine protease with a mol. wt of 34 k, a temperature optimum of 40°C and a pH optimum of 8. When the mutant was crossed with a wild-type isolate of P. brassicae , the non-proteolytic and non-pathogenic traits co-segregated in the resulting progeny. The protease − mutant was transformed with clones from a genomic library of P. brassicae and a transformant obtained which had a single cosmid insert and showed concomitant restoration of pathogenicity and proteolytic activity in vitro . These results suggest that extracellular protease is a pathogenicity determinant of P. brassicae and possible functions for this protease in the disease process are discussed.