PRODUCTION AND CHARACTERIZATION OF A MONOCLONAL-ANTIBODY RAISED AGAINST SURFACE-ANTIGENS FROM MYCELIUM OF GAEUMANNOMYCES-GRAMINIS VAR TRITICI - EVIDENCE FOR AN EXTRACELLULAR POLYPHENOL OXIDASE

A murine monoclonal antibody (MAb) of immunoglobulin class M (IgM) was raised against surface antigens from Gaeumannomyces graminis var. tri tici and, by enzyme-linked immunosorbent assay, recognized isolates of G. gramminis var. tritici, G. graminis var. avenae and G. graminis va r. graminis. Characterization of the antigen by heat and protease trea tments showed that the epitope recognized by the MAb was a protein. An tigen production was detected only in live mycelia. Immunofluorescence studies showed that the antigen was associated with both the broad me lanized macrohyphae and hyaline mycelia of G. graminis var. tritici. S ecretion of antigen into an aqueous minimal medium was promoted only b y exposure of live mycelia to certain phenolic substrates, including m onophenols ortho-, para-, and meta-cresol; 3,4,5-trihydroxybenzoic aci d (gallic acid); and phenolic amino acid L-3-(3,4-dihydroxyphenyl) ala nine (L-DOPA). Antigen secretion was not promoted by 3-(4-hydroxypheny l) alanine (L-tyrosine). The MAb reacted strongly with purified enzyme laccase (polyphenol oxidase, EC 1.10.3.2) but did not recognize purif ied tyrosinase (monophenol oxidase, EC 1.14.18.1). Moreover, chemicals that bind to copper and inhibit copper-containing enzymes such as lac case completely inhibited antigen secretion in response to L-DOPA. The MAb was tested for specificity against a wide range of fungi, common yeast species, and gram positive and negative bacteria. It did not rec ognize antigens from a broad range of unrelated fungi, including Glioc ladium roseum, Fusarium sp., Phoma exigua, Phialophora fastigiata, Pen icillium crustosum, Pythiwn ultimum, Rhizopus stolonifer, Rhboctonia c arotae, R. oryzae, R. tuliparum, and Trichoderma viride, nor did it re cognize surface antigens from yeasts or bacteria. The MAb cross-reacte d with antigens from Botrytis spp., Chaetomium globosum, R. cerealis, and R. solani. However, secretion of antigen by R. solani and R. cerea lis was not promoted by L-DOPA, and secretion by C. globosum in respon se to the phenolic amino acid was significantly less compared to G. gr aminis var. tritici.