Proteinase inhibitor-inducing activity of the prohormone prosystemin resides exclusively in the C-terminal systemin domain

Prosystemin is the 200-amino acid precursor of the 18-amino acid polypeptid e defense hormone, systemin. Herein, we report that prosystemin was found t o be as biologically active as systemin when assayed for proteinase inhibit or induction in young tomato plants and nearly as active in the alkalinizat ion response in Lycopersicon esculentum suspension-cultured cells. Similar to many animal prohormones that harbor multiple signals, the systemin precu rsor contains five imperfect repetitive domains N-terminal to a single syst emin domain. Whether the five repetitive domains contain defense signals ha s not been established. N-terminal deletions of prosystemin had little effe ct on its activity in tomato plants or suspension-cultured cells. Deletion of the C-terminal region of prosystemin containing the 18-amino acid system in domain completely abolished its proteinase inhibitor induction and alkal inization activities. The apoplastic fluid from tomato leaves and the mediu m of cultured cells were analyzed for proteolytic activity that could proce ss prosystemin to systemin. These experiments showed that proteolytic enzym es present in the apoplasm and medium could cleave prosystemin into large f ragments, but the enzymes did not produce detectable levels of systemin. Ad ditionally, inhibitors of these proteolytic enzymes did not affect the biol ogical activity of prosystemin. The cumulative data indicated that prosyste min and/or large fragments of prosystemin can be active inducers of defense responses in both tomato leaves and suspension-cultured cells and that the only region of prosystemin that is responsible for activating the defense response resides in the systemin domain.