Purification of a glycoprotein elicitor of phytoalexin formation from Verticillium dahliae

A glycoprotein, isolated from culture fluids of Verticillium dahliae was 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 H₂O₂ formation 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.