Evidence for a role of cutinase in pathogenicity of Pyrenopeziza brassicae on brassicas

Supernatants from apple cutin-induced cultures of the light lear spot pathogen Pyrenoprziza brassicae were shown to contain a single esterase with a pI of approx. 4.4 and a MW of 21 kDa. Analysis of the hydrolysis products derived from enzymatic cleavage of H-3 labelled cutin by TLC demonstrated that these supernatants had cutinolytic activity. Treatment with the serine esterase 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), respectively. It is therefore concluded that following induction in vitro with apple cutin, P. brassicae produces a single extracellular esterase with cutinolytic activity. A putative cutinase gene was cloned by heterologous Southers hybridization of P. brassicae genomic DNA using the Botrytis cinerea cutinase gene as a probe. The ORF (706 bp) is predicted to encode a polypeptide of 203 amino acids with a molecular weight or 20.379 kDa and a pI of 4.38. This polypeptide shows high levels of homology with the cutinases of B. cinerea and of other fungi, and contains the conserved histidine, aspartic acid and serine catalytic triad. Expression of the putative cutinase gene was shown to correlate 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 appressorial 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 acervular conidiomata was greatly reduced. These results suggest a functional role for fungal cutinolytic activity in conidiomata was greatly reduced. These pathogenicity of P. brassicae on B. napus. (C) 2000 Academic Press.