Purification of a glycoprotein elicitor of phytoalexin formation fromVerticillium dahliae

Abstract

A glycoprotein, isolated from culture fluids ofVerticillium dahliaewas found to be a heat stable elicitor of phytoalexin biosynthesis in cotton cell suspension cultures. The elicitor was purified utilizing anion exchange, size exclusion, and Concanavalin-A (Con-A) chromatography in addition to electroelution from SDS-PAGE. The 65 kDa protein could be deglycosylated to a protein of molecular mass 53 kDa as determined by SDS-PAGE. Elicitor activity from the culture fluids as well as the Con-A chromatography purified elicitor was completely abolished by protease treatment, but not by periodate treatment or peptide N-glycosidase F (PNGase-F) digestion, indicating that only protein components are responsible for the elicitation of phytoalexins in cotton cells. The purified 65 kDa protein did not elicit H2O2formation in contrast to the crude elicitor, which elicited both phytoalexin accumulation and the oxidative burst. The 65 kDa species was found to aggregate to a high molecular weight protein which retained some of its phytoalexin-inducing activity. The glycoprotein elicitor should prove useful in identifying second messengers leading to induction of phytoalexin biosynthesis.