Monoclonal antibodies to the adhesive cell coat secreted by Pythium aphanidermatum zoospores recognise 200x103 Mr glycoproteins stored within large peripheral vesicles

M. TERESA ESTRADA-GARCIA 0 1 JAMES A. CALLOW 0 1 JONATHAN R. GREEN 0 1 0 School of Biological Sciences, University of Birmingham , PO Box 363, Birmingham BIS 2TT , UK 1 (Received I August 1989 - Accepted , in revised form, 3 November 1989) within large peripheral vesicles Author for correspondence Summary 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 - Pythium aphanidermatum (Edson) Fitz is a soil-borne plant pathogen of warm regions and it attacks a wide variety of plants (Van der Plaats-Niterink, 1981). During asexual reproduction, motile, biflagellate zoospores are produced by the fungus: these wall-less cells are attracted to roots and after initial recognition and binding to the root, the zoospores adhere strongly and differentiate into cysts, which then germinate and penetrate the host plant (Longman and Callow, 1987). Evidence suggests that initial recognition and encystment triggering is effected by specific saccharide residues of the root-surface mucilage polysaccharides (Longman and Callow, 1987; Estrada-Garcia et al. 1990). During encystment, pythiaceous zoospores secrete an adhesive cell coat that binds cells to root surfaces (Sing and Bartnicki-Garcia, 1972) and this is followed by the formation of a fibrillar cyst 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. wall (Bartnicki-Garcia and Wang, 1983). Previous studies have indicated that the adhesive material produced by zoospores of Phytophthora palmivora and Pythium aphanidermatum is stored within large peripheral vesicles just beneath the plasma membrane of these cells, and that this material is probably a glycoprotein (Sing and BartnickiGarcia, 1975a,6; Grove and Bracker, 1978). In contrast, recent work on Phytophthora cinnamomi, using two monoclonal antibodies (MAbs) that recognise large and small peripheral vesicles of zoospores of this fungus, has shown that the secreted adhesive glycoproteins of this organism are stored within the small vesicles (Gubler and Hardham, 1988). The large peripheral vesicles seem to migrate towards the centre of the cell during encystment. A set of MAbs (designated PA 1-8) has been raised against a variety of cell surface components of zoospores and cysts of P. aphanidermatum (Estrada-Garcia et al. 1989). Immunofluorescence studies showed that four of the antibodies (PA3-6) bound to patches of material on the surface of some zoospores and to the material occurring in aggregates of encysting zoospores and cysts; they were judged to be binding to the adhesive cell coat secreted by zoospores (Estrada-Garcia et al. 1989). In the present paper the MAbs PA3-6 were used to study the formation of the adhesive cell coat of P. aphanidermatum and to determine the molecular nature of this material. Materials and methods Production of zoospores and cysts Zoospores were released from cultures of P. aphanidermatum (C.M.I. 104926) as described previously (Estrada-Garcia et al. 1989). Suspensions containing zoospores at approximately 107 cells cm"3 in distilled water were obtained. Cysts were produced either by agitation of a zoospore suspension in a vortex mixer for 2min (Tokunaga and Bartnicki-Garcia, 1971), or by centrifuging zoospores at 800 for 5 min at 4C: the resuspended pellet contained approx. 95 % cysts. Monoclonal antibodies MAbs were raised against glutaraldehyde-fixed zoospores as described previously (Estrada-Garcia et al. 1989). Immunofluorescence studies showed that four antibodies, designated PA3 (IgGl), PA4 (lgG3), PA5 (IgGl) and PA6 (IgM), bound to some zoospores exhibiting patches of fluorescence distributed irregularly over their surface. In addition, these antibodies bound to the surface of cysts in an irregular manner and they labelled material occurring in aggregates of zoospores and cysts. A MAb (FS5) that binds to Fitcus sperm was used as a control antibody (Jones et al. 1988). All MAbs were used in the form of tissue culture supernatant (TCS). hnmunogold labelling Zoospores and cysts were embedded in LR White resin (London Resin Co. Surrey, UK) or Lovvicryl K4M-resin (Chemische Werke Lowi, Waldkraiburg, FRG) at low temperature. Cells were fixed with 4 % (w/v) glutaraldehyde in 100 mMPipes for 10 min, then washed three times with phosphatebuffered saline (PBS). The samples were dehydrated through increasing concentrations of ethanol (up to 70 % ethanol for LR White and 100% ethanol for Lowicryl K4M), at progressively lower temperatures down to 15C (2h per step). Samples were infiltrated with increasing concentrations of the resins for nine days at 20C and were finally embedded in a fresh batch of resin by polymerisation under ultraviolet (u.v.) light at 20C overnight. The blocks were slowly warmed to room temperature and further polymerised under u.v. light for 3 days. Sections were cut with a Reichert-Jung MU-4 Ultracut microtome and applied to Formvar-coated copper grids. The samples were washed three times in SOmM-Tris-HCl, pH7.5, containing 1 % (w/v) bovine serum albumin (BSA) and 0.2 % (v/v) Tween 20 (buffer A). Sections were incubated with MAbs for 45 min, rinsed three times in buffer A and incubated with gold-conjugated rabbit anti-mouse immunoglobulins (RAMIG-Auis, Jansens Life Science Products) diluted 1:20 in buffer A for a further 45 min. After washing the samples five times in buffer A and finally (...truncated)