PURIFICATION AND CHARACTERIZATION OF AN ASPARTIC PROTEINASE SECRETED BY BOTRYTIS-CINEREA PERS EX PERS IN CULTURE AND IN INFECTED CARROTS

A single spore isolate of Botrytis cinerea, obtained from carrot tissue, when grown in a liquid medium containing gelatin as sole nitrogen source, secreted an aspartic proteinase (E.C. 3.4.23) with many similarities to the one extracted from B. cinerea-infected carrot tissue. This enzyme was not detected in healthy tissue. The enzyme from both culture and infected tissue had a pH optimum of 3·5 and a pI of 4·0; its action was inhibited by pepstatin and diazoacetyl-norleucine methyl ester, known inhibitors of aspartic proteinases, but not by compounds known to inhibit serine-, thiol- or metalloproteinases. The enzyme existed as a single unit with a molecular mass of 38-39 kD on sodium dodecylsulphate polyacrylamide gels. It was present in ungerminated spores and was produced during germination before pectic enzyme components, both in culture and in host tissue. Cell death was detected in B. cinerea-infected carrot root tissue before pectic enzyme or necrosis-inducing glycoprotein or polysaccharide production, supporting the view that the aspartic proteinase may be primarily responsible for phytotoxicity. © 1990.