Evidence for a role of cutinase in pathogenicity of Pyrenopeziza brassicaeon brassicas

Supernatants from apple cutin-induced cultures of the light lear spot patho gen Pyrenoprziza brassicae were shown to contain a single esterase with a p I of approx. 4.4 and a MW of 21 kDa. Analysis of the hydrolysis products de rived from enzymatic cleavage of H-3 labelled cutin by TLC demonstrated tha t these supernatants had cutinolytic activity. Treatment with the serine es terase inhibitors ebelactones A and B resulted in 50 % inhibition of the P. brassicae esterase activity at concentrations of 4 and 0.4 mug ml(-1), res pectively. It is therefore concluded that following induction in vitro with apple cutin, P. brassicae produces a single extracellular esterase with cu tinolytic activity. A putative cutinase gene was cloned by heterologous Southers hybridization of P. brassicae genomic DNA using the Botrytis cinerea cutinase gene as a p robe. The ORF (706 bp) is predicted to encode a polypeptide of 203 amino ac ids with a molecular weight or 20.379 kDa and a pI of 4.38. This polypeptid e shows high levels of homology with the cutinases of B. cinerea and of oth er fungi, and contains the conserved histidine, aspartic acid and serine ca talytic triad. Expression of the putative cutinase gene was shown to correl ate with induction of esterase activity in vitro. SEM analysis of Brassica napus leaves infected with P. brassicae confirmed that direct penetration of the host surface occurs with no evidence of appr essorial formation. The ebelactones A (100 mug ml(-1)) and B (10 mug ml(-1) ) had no effect on germination of P. brassicae conidia in vitro; however, a marked decrease in pathogenicity of P. brassicae was observed on B. napus plants treated with 100 mug ml(-1) ebelactone A or in mug ml(-1) ebelactone B. In these plants, failed penetration was observed and the number of acer vular conidiomata was greatly reduced. These results suggest a functional r ole for fungal cutinolytic activity in conidiomata was greatly reduced. The se pathogenicity of P. brassicae on B. napus. (C) 2000 Academic Press.