Characterization of a 60 kDa phytotoxic glycoprotein produced by Phoma tracheiphila and its relation to malseccin

An extracellular glycoprotein (Pt60), produced in concentrations of about 1 0 mR l(-1) in stationary cultures by Phoma tracheiphila, the causal agent o f mal secco disease of citrus, was purified by affinity and ion exchange ch romatography. Its molecular mass by SDS-PAGE was 60 kDa. Injections of 1-5 mu g Pt60 into the mesophyll of sour orange or lemon leaves induced chloros is and necrosis of tissues within 4-6 days. These symptoms closely resemble d those caused by foliar inoculation with the pathogen. Results from carboh ydrate analysis, following NaOH and N-glycosidase F treatments, indicated t hat Pt60 is a highly glycosylated protein (55 % carbohydrate). Recognition by polyclonal antibodies, raised in rabbit against the whole glycoprotein, was strongly affected by the above treatments, indicating that the protein is enclosed by the antigenic glycosidic moiety. This carbohydrate sheath ef ficiently protects the protein moiety from degradation by proteolytic enzym es. The amino acid sequence of six peptides resulting from CNBr treatment o f Pt60 followed by trypsin digestion did not reveal sequence homology with any known protein. Pt60 is thought to be the most toxic component of the ma lseccin complex which probably results from a mixture of several compounds, each differing from the others in the extent of glycosylation. (C) 1998 Ac ademic Press.