PRIMARY STRUCTURE AND EXPRESSION OF A PATHOGEN-INDUCED PROTEASE (PR-P69) IN TOMATO PLANTS - SIMILARITY OF FUNCTIONAL DOMAINS TO SUBTILISIN-LIKE ENDOPROTEASES

A 69-kDa proteinase (P69), a member of the pathogenesis-related protei ns, is induced and accumulates in tomato (Lycopersicon esculentum) pla nts as a consequence of pathogen attack, We have used the polymerase c hain reaction to identify and clone a cDNA from tomato plants that rep resent the pathogenesis-related P69 proteinase. The nucleotide sequenc e analysis revealed that P69 is synthesized in a preproenzyme form, a 745-amino acid polypeptide with a 22-amino acid signal peptide, a 92-a mino acid propolypeptide, and a 631-amino acid mature polypeptide. Wit hin the mature region the most salient feature was the presence of dom ains homologous to the subtilisin serine protease family, The amino ac id sequences surrounding Asp-146, His-203, and Ser-532 of P69 are clos ely related to the catalytic sites (catalytic triad) of the subtilisin -like proteases, Northern blot analysis revealed that the 2.4-kb P69 m RNA accumulates abundantly in leaves and stem tissues from viroid-infe cted plants, whereas the mRNA levels in tissues from healthy plants we re undetectable, Our results indicate that P69, a secreted calcium-act ivated endopeptidase is a plant pathogenesis-related subtilisin-like p roteinase that may collaborate with other defensive proteins in a gene ral mechanism of active defense against attacking pathogens.