ADHESION OF PHYTOPHTHORA ZOOSPORES: DETECTION AND ULTRASTRUCTURAL VISUALIZATION OF CONCANAVALIN A-RECEPTOR SITES APPEARING DURING ENCYSTMENT

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1 J, Cell Sci. 19, (i975) 11 Printed in Great Britain ADHESION OF PHYTOPHTHORA PALMIVORA ZOOSPORES: DETECTION AND ULTRASTRUCTURAL VISUALIZATION OF CONCANAVALIN A-RECEPTOR SITES APPEARING DURING ENCYSTMENT V. O. SING AND S. BARTNICKI-GARCIA Department of Plant Pathology, University of California, Riverside, California 92502, U.S.A. SUMMARY The binding of concanavalin A (Con A) to the cell surface of zoospores and cysts of Phytophthora palmivora was studied by radiometry ( lis I-Con A), ultraviolet microscopy (fluorescein- Con A) and electron microscopy (peroxidase-diaminobenzidine technique). Zoospores were found to secrete during the early stages of encystment a Con A-binding material susceptible to trypsin digestion. This glycoprotein is contained in the so-called peripheral vesicles and is probably responsible for the adhesion of the encysting zoospores to solid surfaces. INTRODUCTION The zoospores of Phytophthora palmivora, a plant pathogenic fungus, have the ability to adhere strongly to the solid surfaces upon which they settle down to encyst. The adhesive phase is short (ca. 1 min or less) and is concomitant with the early stages of cyst wall formation (Sing & Bartnicki-Garcia, 1972). Mature cysts lack the ability to attach themselves to a surface but they retain some adhesiveness as evidenced by the formation of tenacious clumps on centrifugation. Cytological studies (Sing & Bartnicki-Garcia, 1975) indicated that the adhesive material was derived from 'peripheral' vesicles and was secreted immediately before, or at the beginning of, cyst wall synthesis. Past studies of isolated cyst walls of P. palmivora showed that this wall is essentially a microfibrillar envelope (Tokunaga & Bartnicki-Garcia, 197ib) and gave no indication of any amorphous material, on either inner or outer wall surfaces, that could be regarded as a potential cementing substance for adhesion of the cell to an external surface. However, the presumptive adhesive material was probably lost during wall isolation procedures (sonication or alkaline extraction) as deduced from carbon replicas of intact cysts where patches of amorphous material were found partially covering the fibrillar texture of the cyst wall (Desjardins, Wang & Bartnicki-Garcia, 1973)- Preliminary observations indicated the presence of a substance capable of binding concanavalin A (Con A) on the surface of encysting zoospores of P. palmivora.

2 12 V. O. Sing and S. Bartnicki-Garcia The time and mode of appearance of this surface material, as described herein, support the possibility that it may be the suspected cell adhesive. In this study, we examined the binding of m I-Con A to zoospores and cysts of P. palmivora, and the location of the Con A-receptor sites on the cell surface by light microscopy with fluorescein-conjugated Con A and by electron microscopy using the peroxidasediaminobenzidine technique employed by Bernhard & Avrameas (1971) for the localization of Con A-binding sites in animal cells. MATERIALS AND METHODS The method for obtaining zoospores of P. palmivora has been described previously (Tokunaga & Bartnicki-Garcia, 1971a). Con A was purchased from Miles-Yeda Ltd, Israel; horseradish peroxidase, lactoperoxidase and fluorescein isothiocyanate from Calbiochem., La Jolla, California; 3,3'-diaminobenzidine tetrahydrochloride from Sigma Corp., St. Louis, Missouri; Na 116 I (in o-i N NaOH) from ICN, Irvine, California. The method of Tkacz, Cybulska & Lampen (1971) for conjugating fluorescein to Con A was used. The procedure for the iodination of Con A was a modification of the method employed by Phillips & Morrison (1971) for iodination of surface proteins of the plasma membrane. The reaction mixture contained: 2 ml Con A (54 mg), 2 ml of 01 M sodium phosphate buffer (ph 7-4), 2 ml lactoperoxidase (0-3 mg/ml in phosphate buffer) and 0-75 mci of m I, as Nal, in 20 fi\. The reaction was started by adding 20 fil of 1 % H,O a ; 20 such additions were made at 15-s intervals. The reaction mixture was allowed to stand for 2 h and then dialysed against phosphate buffer in 1 M NaCl at 1 C with changes of the dialysing solution until no radioactivity appeared outside the dialysis bag. m I-Con A with specific activity from 1-5 x io* to 6 x 10' cpm/mg was thus obtained. Zoospore suspensions were agitated in a Vortex mixer for s to induce synchronous encystment (Tokunaga & Bartnicki-Garcia, 1971a). Glutaraldehyde, in 01 M sodium phosphate buffer (ph 7'4), was added immediately after each agitation period to a final concentration of 2-5 %. The cells were kept for 1 h in the glutaraldehyde solution and washed 3 times with phosphate buffer containing 1 M NaCl. Binding of li6 I-Con A to zoospores and cysts The reaction mixture contained 10' cells, 1 mg Con A in i-o ml of phosphate buffer with 1 M NaCl. As controls, similar mixtures were prepared but with methyl-a-d-mannoside added at a final concentration of 02 M. One drop of 1 % sodium azide was added, and the suspensions were incubated in a rotary shaker for 8 h at 60 rev/min at room temperature. The cells were washed twice by centrifugation with phosphate buffer in IM NaCl, collected on a Millipore filter (3-fim pore size), and washed with 20 ml phosphate buffer. The filters with the cells were placed in 10 ml of liquid scintillation counting solution [60 g of naphthalene, 100 ml of methanol, 20 ml of ethylene glycol, 0-2 g of i,4-bis[2-(4-methyl-5-phenyloxazolyl)] benzene, and 4 g of 2,5-diphenyloxazole made to 1000 ml with />-dioxane]. Labelling of zoospores and cysts withfluorescein-conjugatedcon A The cells were incubated in the same manner as for the binding of m I-Con A, except that fluorescein-conjugated Con A was used. Methyl-a-D-mannoside (0-2 M) was present in the controls. After washing with phosphate buffer in 1 M NaCl, the cells were examined under a dark-field ultraviolet Reichert microscope. Ultrastructural localization of Con A-receptor sites Cells were incubated with unlabelled Con A as above and, after 8 h, they were washed twice with sodium phosphate buffer in 1 M NaCl. The cells were then incubated with 1 mg of horseradish peroxidase in a total volume of 1 ml for 1 h, washed twice with phosphate buffer

3 Con A binding and zoospore adhesion 13 and once with 0-05 M Tris-HCl buffer, ph 7-6. In the controls, 0-2 M methyl-a-d-mannoside was present during the binding of Con A and also during the peroxidase treatment. The diaminobenzidine reagent of Graham & Karnovsky (1966) was applied for 15 min and the cells were washed twice with distilled water and postfixed with 1 % osmium tetroxide in 01 M cacodylate buffer, ph 7-2. Specimens were embedded in Epon 812. Thin sections, stained with both uranyl acetate and lead citrate, were observed in an Hitachi HU-12 electron microscope. The action of trypsin on the Con A-binding material of the cell surface was tested on young cysts (obtained by agitating a zoospore suspension for 45 s) fixed with 2 % glutaraldehyde for 1 h. The cysts (2 x io 7 ) were thoroughly washed and suspended in 3 ml of 005 M Tris-HCl buffer, ph 8, containing 05 mg/ml of trypsin and a drop of 1 % sodium azide. After incubation for 8 h, the cells were centrifuged, washed 3 times with o-i M phosphate buffer, ph 72, containing 1 M NaCl, and exposed to Con A (1 mg/ml), peroxidase and diaminobenzidine as described above. RESULTS The ability of encysting zoospores to bind Con A was determined in samples agitated for various short times, i.e. at various consecutive stages of encystment (young cysts < 60 s agitation; mature cysts > 120 s) Agitation time, s Fig. 1. Kinetics of appearance of Con A-binding sites in zoospores of P. palmivora during encystment. Zoospore suspensions were agitated for the times shown,fixedwith glutaraldehyde to stop development and treated with 1 mg of 1M I-Con A/10 7 cells. Values shown are means of duplicates after subtracting values of control samples incubated in the presence of 0-2 M methyl-a-d-mannoside. Control samples had about io 3 cpm. 120 With zoospores agitated for only 10 s, the amount of 125 I-Con A bound to the cells was nearly doubled over that bound to unencysted (un-agitated) zoospores (Fig. 1). A maximal, approximately 3-fold increase in binding capacity was observed after 30 s of agitation. The amount of Con A bound to the cells in this plateau area was about 10 /tg/10 7 cells. Unencysted zoospores incubated with fluorescein-conjugated Con A exhibited only a faint diffuse fluorescence. In young cysts (45 s), the overallfluorescencewas markedly

4 14 V. O. Sing and S. Bartnicki-Garcia increased by the appearance of small fluorescent patches. Mature cysts (agitated for 120 s plus 10 min of stationary incubation) showed large fluorescent patches together with small protuberant areas of intense fluorescence (Fig. 2). Control cells, incubated with Con A in the presence of methyl-a-d-mannoside, displayed only faint fluorescence. Unencysted zoospores incubated with Con A and subsequently treated with peroxidase-diaminobenzidine exhibited a uniform deposit of electron-dense material on the plasma membrane (Figs. 3, 9). In young cysts (agitated for 45 s), there was also a uniform deposit of electron-dense material lining the entire cell plus some heavy irregular accumulations of electron-dense material on the cell surface. The latter correspond to the discharged contents of 'peripheral' vesicles. These vesicles appear to have coalesced with the plasma membrane and their contents have been secreted on to the cell surface (Figs. 5, io, 11). There was no such staining in control cells incubated in the presence of methyl-a-d-mannoside (Fig. 4, 6). In mature cysts, the heavy irregular deposits of electron-dense substance persist outside the cell wall. The walls themselves were slightly stained by this procedure (Figs. 7, 12). Mature cysts, treated as above but in the presence of methyl-a-d-mannoside, do not show comparable staining (Fig. 8). The Con A-binding material on the surface of young cysts can be largely removed by trypsin digestion. This was shown by the peroxidase diamino-benzidine technique. Trypsin-treated cysts exhibit only remnants of the highly electron-dense material found around the cell surface and in the cavities of discharged peripheral vesicles (see Figs. 13, 14 vs 15, 16). The contents of undischarged vesicles were not affected (Fig. 16, arrow), probably because trypsin did not penetrate into the cell. DISCUSSION Major changes occur in rapid succession at the surface of a zoospore of P. palniivora (or related fungi) undergoing encystment. In zoospore populations induced to encyst synchronously, the following sequence of events can be detected (Sing & Bartnicki- Garcia, 1975): within 30 s, most zoospores shed their flagella and acquire a surface coat of amorphous material secreted by peripheral vesicles (Hemmes & Hohl, 1971; Grove, 1971). Cell wall fibrils probably begin to be synthesized at this time. After 30 s, alkali-resistant cyst walls can be recognized in the population. By 120 s agitation, % of the cells have well defined cyst walls; the cysts continue to incorporate alkali-insoluble glucan into the wall up to 10 min (Bartnicki-Garcia, 1973) when they seemingly reach maturity. The binding of Con A to the surface of zoospores and cysts of P. palmivora was conclusively demonstrated by 3 different procedures: binding of radiolabelled Con A, fluorescence microscopy, and electron microscopy. In all cases, the binding of Con A to the cells was effectively suppressed by methyl-a-d-mannoside, a potent haptenic binder of Con A (Goldstein, Reichert & Misaki, 1974). By these criteria, we showed that the plasma membrane of unencysted zoospores can bind small amounts of Con A. Since at this time the cells are not adhesive, the binding is probably due to some component of the plasma membrane, which probably has no role in cell adhesion.

5 Con A binding and zoospore adhesion 15 With the onset of encystment, there is a sharp increase in binding capacity for 126 I-Con A. The new binding sites are on the cell surface. Our electron micrographs show convincingly that the amorphous cyst coat secreted by the peripheral vesicles binds Con A intensely. No staining was found in internal (undischarged) peripheral vesicles but this was expected due to the probable lack of penetration of Con A into the cell. Essentially all the Con A-binding material is secreted within 30 s after encystment is initiated. Both light and electron microscopy indicate that the Con A-binding material does not spread uniformly on the zoospore surface but may accumulate in irregular deposits that persist after the cyst wall is formed. These probably correspond to the patches of amorphous material covering the microfibrils of the cyst wall in carbon replicas of intact cysts (Desjardins et al. 1973) and probably account for the residual adhesiveness of mature cysts (Sing & Bartnicki-Garcia, 1972). Its susceptibility to trypsin indicates that the Con A-binding material is most likely a glycoprotein. Whether this glycoprotein is the sole or main constituent of the peripheral vesicle remains to be determined. We previously (Sing & Bartnicki-Garcia, 1972) showed that adhesiveness of zoospores to solid surfaces is greatest in the earliest stages of encystment, i.e. during the secretion of the amorphous coat and immediately preceding the formation of a well defined cyst wall. Thus the Con. A-binding glycoprotein of the peripheral vesicles appears on the cell surface at the right time to be regarded as a prime candidate for the adhesive role. Electron micrographs of encysting zoospores attached to plastic films support the claim that the contents of the peripheral vesicles are involved in cell adhesion (Sing & Bartnicki-Garcia, 1975). In zoospores of the marine alga, Enteromorpha, there is also evidence suggesting that the cell adhesive is a vesicle-secreted glycoprotein (Evans & Christie, 1970; Christie, Evans & Shaw, 1970; Callow & Evans, 1974). This investigation was supported in part by a pre-doctoral fellowship from the University of California to V. O. Sing and by a research grant from the National Science Foundation. REFERENCES BARTNICKI-GARCIA, S. (1973). Cell wall genesis in a natural protoplast: the zoospore of Phytophthorapalmivora. InYeast, Mould and Plant Protoplasts (ed. J.R.Villanueva, I. Garcia- Acha, S. Gascon & F. Uruburu), pp London: Academic Press. BERNHARD, W. & AVRAMEAS, S. (1971). Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A. Expl Cell Res. 64, CALLOW, M. E. & EVANS, L. V. (1974). Studies on the ship-fouling alga Enteromorplux. III. Cytochemistry and autoradiography of adhesive production. Protoplasma 80, CHRISTIE, A. O., EVANS, L. V. & SHAW, M. (1970). Studies on the ship-fouling alga Enteromorpha. II. The effect of certain enzymes on the adhesion of zoospores. Ann. Bot. 34, DESJARDINS, P. R., WANG, M. C. & BARTNICKI-GARCIA, S. (1973). Electron microscopy of zoospores and cysts of Phytophthora palmivora: morphology and surface texture. Arch. Mikrobiol. 88, EVANS, L. V. & CHRISTIE, A. O. (1970). Studies on the ship-fouling alga Enteromorpha. I. Aspects of the fine-structure and biochemistry of swimming and newly settled zoospores. Ann. Bot. 34,

6 16 V. O. Sing and S. Bartnicki-Garcia GOLDSTEIN, I. J., REICHERT, C. M. & MISAKI, A. (1974). Interaction of concanavalin A with model substrates. Ann. N.Y. Acad. Sci. 234, GRAHAM, R. C. & KARNOVSKY, M. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, GROVE, S. N. (1971). Protoplasmic Correlates of Hyplial Tip Initiation and Development in Fungi. Ph.D. Dissertation. Purdue University. HEMMES, D. E. & HOHL, H. R. (1971). Ultrastructural aspects of encystation and cyst-germination in Phytophthora parasitica.j. Cell Sci. 9, PHILLIPS, D. R. & MORRISON, M. (1971). Exposed protein on the intact human erythrocyte. Biochemistry, N.Y. 10, SING, V. O. & BARTNICKI-GARCIA, S. (1972). Adhesion of zoospores oi Phytophthora palmivora to solid surfaces. Phytopathology 62, 790. SING, V. O. & BARTNICKI-GARCIA, S. (1975). Adhesion of Phytophthora palmivora zoospores: electron microscopy of cell attachment and cyst wall fibril formation. J. Cell Sci. 18, TKACZ, J. S., CYBULSKA, B. & LAMPEN, J. O. (1971). Specific staining of wall mannan in yeast cells with fluorescein-conjugated concanavalin A._7- Bad. 105, 1-5. TOKUNAGA, J. & BARTNICKI-GARCIA, S. (1971a). Cyst wall formation and endogenous carbohydrate utilization during synchronous encystment of Phytophthora palmivora zoospores. Arch. Mikrobiol. 79, TOKUNAGA, J. & BARTNICKI-GARCIA, S. (19716). Structure and differentiation of the cell wall of Phytophthora palmivora: cysts, hyphae and sporangia. Arch. Mikrobiol. 79, {Received 29 November 1974)

7 Con A binding and zoospore adhesion Fig. 2. Binding of fluorescein-conjugated Con A to the surface of mature cysts of P. palmivora. Note the irregular accumulation of Con A-binding material in large diffuse patches and small, intensely fluorescent protuberances, x E 1, 19

8 i8 V. O. Sing and S. Bartnicki-Garcia Figs Localization of Con A-binding sites on the surface of zoospores and cysts by the peroxidase-diaminobenzidine reaction. Fig. 3, unencysted zoospores, x4000. Fig. 5, young cyst (45 s of agitation), x Fig. 7, mature cyst (120 s agitation + 10 min stationary incubation), x The control samples were incubated with Con A in the presence of 02 M methyl-a-d-mannoside (Fig. 4, unencysted zoospores, X4000; Fig. 6, young cysts, x 10000; Fig. 8, mature cyst, x 7500). d, discharged peripheral vesicle.

9 Con A binding and zoospore adhesion Figs Ultrastrucrural details of Con A-binding sites during zoospore encystment as revealed by the peroxidase-diaminobenzidine reaction. Fig. 9, binding of Con A to the plasma membrane of an unencysted zoospore, x Fig. 10, young cyst (from Fig. s) showing Con A-binding material discharged by the peripheral vesicles, X Fig. 11, young cyst at a more advanced stage from that in Fig. 5 with Con A- binding material spread over a wider area, x Fig. 12, mature cyst from Fig. 7 showing some binding of Con A to the cyst wall but considerably more binding to the cyst coat, x cc, cyst coat; civ, cyst wall; d, discharged peripheral vesicle; fp,' finger-print' vesicle; m, mitochondrion; mt, microtubules; pm, plasma membrane; t>v, peripheral vesicle. '»

10 20 V. 0. Sing and S. Bartnicki-Garcia o 15 Figs Effect of trypsin on the Con A-binding material discharged on to the surface of young cysts. Figs. 13 and 14 show the highly electron-dense material discharged or being discharged by peripheral vesicles. Figs. 15 and 16 show the removal of this Con A-binding material upon digestion with trypsin. Con A-binding material was stained by the peroxidase-diaminobenzidine technique, d, discharged contents of peripheral vesicles; e, empty peripheral vesicle; m, mitochondria. Arrow points at a peripheral vesicle seemingly unaffected by either trypsin or the peroxidase-diaminobenzidine reaction. Fig. 13, x 18000; Fig. 14, x 38000; Fig. 15, x 20000; Fig. 16, x