Production of species-specific monoclonal antibodies that react with surface components on zoospores and cysts of Phytophthora nicotianae

Abstract

Monoclonal antibodies were generated against components on the surface of zoospores and cysts of the Oomycete, Phytophthora nicotianae, with the aim of obtaining antibodies diagnostic for this species of plant pathogen. A dipstick version of an enzyme-linked immunosorbent assay was used to screen hybridoma cell lines produced by following a coimmunization protocol in which a mouse was immunized with Phytophthora nicotianae cysts mixed with murine antisera raised against cysts of Phytophthora cinnamomi and Phytophthora cryptogea. Of the nine hybridoma cells lines which remained positive, five produced antibodies that reacted with species-specific epitopes on the surface of the spores. Immunofluorescence, immunogold, and immunoblot labelling showed that three of the five species-specific antibodies reacted with a polypeptide of relative molecular mass greater than 205 kDa which was distributed over the entire zoospore surface, including that of the two flagella. These antibodies also labelled the surface of cysts to varying degrees. The other two species-specific antibodies bound to the shaft of tubular mastigonemes that form two rows on the anterior flagellum. In immunoblots, one of these antibodies recognised a 40-kDa glycoprotein. Antibodies produced by the other four hybridoma cell lines reacted with all Phytophthora and Pythium species tested. The results (i) showed that the coimmunization technique effectively produced antibodies directed towards components specific for Phytophthora nicotianae in the presence of antigens common to many Phytophthora species, and (ii) revealed for the first time the biochemical nature of molecular constituents of flagellar mastigonemes in the Oomycetes.Key words: cell surface, flagella, immunodiagnostics, mastigonemes, monoclonal antibodies.