PURIFICATION OF A GLYCOPROTEIN ELICITOR OF PHYTOALEXIN FORMATION FROMVERTICILLIUM-DAHLIAE

A glycoprotein, isolated from culture fluids of Verticillium dahliae w as found to be a heat stable elicitor of phytoalexin biosynthesis in c otton cell suspension cultures. The elicitor was purified utilizing an ion exchange, size exclusion, and Concanavalin-A (Con-A) chromatograph y in addition to electroelution from SDS-PAGE. The 65 kDa protein coul d be deglycosylated to a protein of molecular mass 53 kDa as determine d by SDS-PAGE. Elicitor activity from the culture fluids as well as th e Con-A chromatography purified elicitor was completely abolished by p rotease treatment, but not by periodate treatment or peptide N-glycosi dase F (PNGase-F) digestion, indicating that only protein components a re responsible for the elicitation of phytoalexins in cotton cells. Th e purified 65 kDa protein did nor 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 hi gh molecular weight protein which retained some of its phytoalexin-ind ucing activity. The glycoprotein elicitor should prove useful in ident ifying second messengers leading to induction of phytoalexin biosynthe sis. (C) 1998 Academic Press.