Monoclonal antibodies to the adhesive cell coat secreted by Pythium aphanidermatum zoospores recognise 200×103Mr glycoproteins stored within large peripheral vesicles

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Abstract
During encystment the motile zoospores of the plant pathogen Pythium aphanidermatum secrete an adhesive cell coat that is involved in their attachment to roots. Previous ultrastructural studies have indicated that the adhesive material is pre-packaged within large peripheral vesicles underlying the zoospore plasma membrane. In the present study, four monoclonal antibodies (MAbs) designated PA3-6, which were raised against zoospores and cysts of P. aphanidermatum, were used to re-examine the formation of the adhesive cell coat and to study its molecular nature. Immunogold labelling of zoospores and cysts shows that all the antibodies recognise material contained within the large peripheral vesicles of zoospores. These structures are morphologically distinct from the microbodies, which also underly the plasma membrane, and the latter are not labelled by the antibodies. During encystment the material recognised by the MAbs is secreted to form a cell coat around the zoospores and cysts and this can be seen to be separated from the cyst plasma membrane by a distinct layer (cyst wall). The MAbs also label material within vesicles that are located towards the centre of the cyst cytoplasm. Western blotting and antigen-modification techniques have shown that all the MAbs bind to carbohydrate epitopes of a set of high molecular weight glycoproteins (> 200xl03Mr). One of the antibodies, PA6, also binds to several lower molecular weight components. Overall, the results show that the adhesive material secreted by P. aphanidermatum zoospores is stored within large peripheral vesicles and is composed of several glycoproteins. The results are discussed in the context of studies on the secretion of adhesive material by zoospores of related oomycete fungi.

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