ADHESION OF PHYTOPHTHORA PALMIVORA ZOOSPORES: ELECTRON MICROSCOPY OF CELL ATTACHMENT AND CYST WALL FIBRIL FORMATION

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1 J. Cell Sci. 18, (1975) 123 Printed in Great Britain ADHESION OF PHYTOPHTHORA PALMIVORA ZOOSPORES: ELECTRON MICROSCOPY OF CELL ATTACHMENT AND CYST WALL FIBRIL FORMATION V. O. SING AND S. BARTNICKI-GARCIA Department of Plant Pathology, University of California, Riverside, California, 92502, U.S.A. SUMMARY Zoospores of Phytophthora palmivora adhered to a plastic film surface were examined by electron microscopy. Three stages of adhesion were compared: (1) non-adhesive, unencysted zoospores, (2) adhered incipient cysts, and (3) adhered mature cysts. Thin sections of incipient cysts revealed cells attached to thefilmsurface through the partially discharged contents of the so-called peripheral vesicles; this seems to be the first step in cell adhesion. In mature cysts, the adhesive appeared to have been compacted into an electron-dense deposit binding the cyst wall to the plastic surface. The adhesion zone was also examined in face view after lysing attached incipient cysts with sodium dodecyl sulphate. Cyst wall microfibrils were seen together with an amorphous substance (presumably the adhesive material). The microfibrils were in various stages of formation. Seemingly, adhesion and microfibril formation take place concurrently. The possibility was considered that the material contained in the peripheral vesicles serves in both cell adhesion and microfibril elaboration. INTRODUCTION After a period of active swimming, zoospores of Phytophthora palmivora become sluggish, settle down and encyst. Zoospores have a tendency to become firmly attached to any substratum on to which they settle. The adhesive phase is brief; it takes place during the early stages of encystment, before a recognizable (alkaliresistant) cyst wall is formed (Sing & Bartnicki-Garcia, 1972). Thus, if a zoospore encysts before reaching a solid surface, it will probably not adhere to it. This communication is part of a series dealing with cytological, physiological, and biochemical aspects of adhesion of zoospores of P. palmivora to living or inanimate surfaces. Presently, we describe ultrastructural details of the adhesion phenomenon and the genesis of the cyst wall. Both events appear to be intimately related. MATERIALS AND METHODS Zoospores of Phytophthora palmivora, isolate Pi 13, were obtained as previously described (Tokunaga & Bartnicki-Garcia, 1971a). Electron microscopy of spores attached to plastic surfaces. Samples (5 ml) of a swimming zoospore suspension, at room temperature, were transferred to 2 precooled 30-ml beakers held at 1 C. Several thin plastic disks of Epon 812 had been placed at the bottom of the beakers.

2 124 V O- ^"W an d S. Bartnicki-Garcia After 20 min of incubation, one sample was fixed immediately with 5 ml of 2 % glutaraldehyde in o-i M sodium cacodylate buffer, ph 7-2; under these conditions, most zoospores were able to initiate the processes of encystment and adhesion but did not develop further (incipient cysts). The other sample was incubated at room temperature for an additional 30 min - to allow zoospores to complete their encystment and adhesion - and then fixed with glutaraldehyde for 1 h (mature cysts). The plastic disks with the attached cells were washed with cacodylate buffer, postfixed with 1 % osmium tetroxide in cacodylate buffer for 1 h, washed, dehydrated with an ethanol series and embedded in Epon 812. Thin sections were prepared and stained with uranyl acetate and lead citrate. The specimens were viewed in a Hitachi HU-12 electron microscope. Samples of unencysted, unattached zoospores were fixed with glutaraldehyde, centrifuged and prepared for electron microscopy by a similar procedure. Electron microscopy of fibrillar cyst wall formation. Zoospores were allowed to adhere to electron-microscope grids (coated with Formvar film) placed at the bottom of a precooled beaker as described above. After 20 min incubation, the attached incipient cysts were lysed with 1 % sodium dodecyl sulphate (SDS) and washed extensively with a jet of distilled water. The grids were dried and shadow cast with palladium at a 19 0 angle. Mature cyst walls were isolated by sonication (Tokunaga & Bartnicki-Garcia, 19716) and shadow cast as above. RESULTS Thin sections of zoospores and attached cysts Fig. 1 shows the typical appearance of an unencysted zoospore of Phytophthora palmivora. This appearance is closely similar to that described previously for this organism (Hemmes & Hohl, 1971), other species of Phytophthora (Ho, Zachariah & Hickman, 1968; Reichle, 1969a) and Pythium (Grove, 1971). The plasmalemma is the outermost boundary of the cell. Conspicuous under the plasmalemma are numerous spheroidal vesicles with a homogeneous finely granular content. These vesicles have been called 'peripheral' vesicles (Grove, 1971), vesicles with 'granular to fibrous' content (Reichle, 1969a), or 'fibrillar' vacuoles (Hemmes & Hohl, 1971). In addition, there are also many flattened vesicles, with mostly electron-transparent contents, in close proximity to the plasmalemma. Fig. 2 shows an incipient cyst beginning to adhere to an inanimate surface. The cell has lost most of its peripheral and flattened vesicles, but has not yet rounded off. The prominent groove of the zoospore is still conspicuous. The incipient cyst lacks a well defined cell wall and shows only a fine fuzz (perhaps nascent wall) covering the plasmalemma surface (Fig. 3). In these fortuitous sections, we can see that the cell is anchored to the plastic surface through the partially secreted contents of a peripheral vesicle (Figs. 2, 3). Fig. 4 shows a mature cyst attached to a solid surface. The cell has completely rounded off and a thin, electron-transparent wall is evident. Essentially all the peripheral and flattened vesicles have disappeared by this time. Electron-dense cementing material can be seen in the area between the cyst wall and the plastic surface. The zone of attachment is rather small; sections which failed to cut through this zone were commonly found. They showed a cell 'suspended' above the plastic film with empty space in between.

3 Zoospore adhesion and wall formation 125 Face view of the adhesion zone Zoospores were allowed to attach themselves to electron-microscope grids, treated with ionic detergent to lyse the cells, mainly incipient cysts, washed extensively to eliminate loose material and shadow cast. Figs. 5-7 show the materials that remained attached to the grids. They consist mainly of microfibrils with some amorphous material. From the overall appearance, it was concluded that in most instances each deposit corresponded to the remnants of an individual cyst. The appearance of these remnants varied considerably, reflecting different stages of cyst wall formation (Figs. 5-7); some contained only short microfibrils while others were meshworks of long microfibrils, interwoven into loose or tight fabrics. For comparison, the appearance of a broken fragment of a mature cyst wall isolated by sonication (Tokunaga & Bartnicki-Garcia, 1971 A) is shown in Fig. 8. DISCUSSION By a mild cold shock, zoospore populations were induced to adhere to plastic disks placed at the bottom of a small beaker. This cold treatment also triggered the onset of encystment. Both processes, encystment and adhesion, are intimately related (see below). Seemingly, the gradual drop in temperature caused the zoospores to (a) settle down on to the plastic disks, (b) become adhesive and (c) begin wall formation. In samples kept at low temperature, the cells remained attached to the plastic disks but did not complete their encystment (incipient cysts). Presumably the enzymes responsible for wall synthesis were slowed down and wall formation ceased prematurely. In such samples, we found cyst walls in widely different stages of development (Figs. 5-7). Most of the cells that were brought back to room temperature after the cold shock resumed and completed the synthesis of a cyst wall and remained firmly adhered to the plastic surface (mature cysts). Our present results on the fine structure of incipient and mature cysts of P. palmivora attached to plastic films agree with the previous conclusion that zoospores adhere to surfaces early in the encystment process (Sing & Bartnicki-Garcia, 1972), i.e. before an alkali-resistant cyst wall is formed. They also show that adhesion is probably established through the newly discharged contents of the peripheral vesicles. These vesicles occur abundantly next to the cell surface and disappear early during encystment by fusing with the plasmalemma and discharging their contents to the outside. This secretory process was described earlier by Hemmes & Hohl (1971) for P.palmivora and by Grove (1971) for Pythium aphanidermatum. The secreted material forms an irregular amorphous coat on the cyst surface (Sing & Bartnicki-Garcia, 1975 a). Apparently, if the zoospore is in contact with a solid surface as it initiates encystment, the peripheral vesicle secretion at the point of contact would cement the cell to the solid surface (Figs. 2, 3). After the cyst wall is fully developed, the cell remains firmly attached by an electron-dense substance located between the wall and the plastic surface. The higher electron density of the adhesive in the mature cyst, as compared with the incipient

4 126 V. O. Sing and S. Bartnicki-Garcia cyst (Figs. 2, 3 vs 4), could be largely due to compaction of the adhesive material originally discharged by the peripheral vesicles. To obtain face views of the adhesion zone we took advantage of two distinct actions of SDS on encysting zoospores. First, this detergent causes complete cell disintegration of incipient cysts of P. palmivora (mature cysts are also lysed by SDS but the wall prevents complete dispersal of cell particles) (S. Bartnicki-Garcia, unpublished). Secondly, SDS cannot detach adhered cysts of P. palmivora, probably because the cement is unaffected by the detergent (Sing & Bartnicki-Garcia, 19756). It follows then that SDS treatment of incipient cysts adhered to a film surface would remove all cell components, leaving behind only the cementing material attached to the film surface together with any other cell structures associated with the adhesive. The latter proved to be microfibrils in various stages of formation. The zoospore of P. palmivora lacks preformed microfibrils; these must be assembled at the cell surface during encystment (Bartnicki-Garcia, 1973). It has been proposed that peripheral vesicles (fibrillar vacuoles) play a role in cyst wall elaboration (Hemmes & Hohl, 1971; Grove, 1971). Conceivably these vesicles and/or the flattened vesicles may deliver enzymes and/or precursors for microfibril synthesis. We have evidence that the peripheral vesicles contain a glycoprotein with receptor sites for concanavalin A; the former substance is probably involved in adhesion (Sing & Bartnicki- Garcia, 1975 a). We have therefore conjectured that the materials discharged by these vesicles may serve for both microfibril synthesis and cell adhesion. Conceivably, the main role of the adhesive glycoprotein would be that of a matrix for wall neogenesis, binding nascent microfibrils to the cell surface and to one another. Additionally, the adhesive would serve to anchor the cell to any external surface with which the zoospore might be in contact at the time of encystment. Our present findings together with other recent electron-microscopic studies (Hemmes & Hohl, 1971; Grove, 1971; Tokunaga & Bartnicki-Garcia, 19716) permit a deeper understanding of the process of encystment of an oomycetous zoospore. They indicate the crucial role played by preformed vesicles in wall formation and development of adhesiveness - two key events in encystment. The classical description of events in zoospore encystment (Waterhouse, 1962; Hickman, 1970) may thus be updated to include~the following: (1) cessation of motility; (2) loss of flagella either by retraction or shedding (see Reichle, 19696); (3) discharge of peripheral and flattened vesicles and formation of an amorphous cyst wall coat; (4) acquisition of adhesiveness; (5) microfibril synthesis and development of an alkali-resistant cyst wall; and (6) change from pyriform to spherical shape (see Desjardins, Wang & Bartnicki-Garcia, 1973)- This investigation was supported in part by a research grant from the National Science Foundation and by a predoctoral fellowship from the University of California to V. O. Sing. We are much indebted to D. E. Hemmes and W. Van Der Woude for their valuable advice.

5 Zoospore adhesion and wall formation 127 REFERENCES BARTNICKI-GARCIA, S. (1973). Cell wall genesis in a natural protoplast: the zoospore of Phytophtliora palmivora. Jn Yeast, Mould and Plant Protoplasts (ed. J. R. Villanueva, I. Garcia-Acha, S. Gascon & F. Uruburu), pp London: Academic Press. DESJARDINS, P. R., WANG, M. C. & BARTNICKI-GARCIA, S. (1973). Electron microscopy of zoospores and cysts of Phytophtliora palmivora: morphology and surface texture. Arch. Mikrobiol. 88, GROVE, S. N. (1971). Protoplasmic Correlates of Hyfilial 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 Phytophtliora parasitica. J. Cell Sci. 9, HICKMAN, C. J. (1970). Biology of Phytophtliora zoospores. Phytopathology 60, Ho, H. H., ZACHARIAH, K. & HICKMAN, C. J. (1968). The ultrastructure of zoospores of Phytophtliora megasperma var. sojae. Can.J. Bot. 46, REICHLE, R. E. (1969a). Fine structure of Phytophtliora parasitica zoospores. Mycologia 61, 3O-5 ' REICHLE, R. E. (19696). Retraction of nagella of Phytophtliora parasitica var. nicotiana zoospores. Arch. Mikrobiol. 66, SING, V. O. & BARTNICKI-GARCIA, S. (1972). Adhesion of zoospores of Phytophthora palmivora to solid surfaces. Phytopathology 62, 790. SING, V. O. & BARTNICKI-GARCIA, S. (19750). Adhesion of zoospores of Phytophthora palmivora. Detection and ultrastructural visualization of concanavalin A receptor sites appearing during encystment. J. Cell Sci. (in press.) SlNC, V. O. & BARTNICKI-GARCIA, S. (:975ft). Adhesion of Phytophthora palmivora zoospores: detachment by enzymic and chemical treatments. J. gen. Microbiol. (Submitted for publication.) TOKUNACA, J. & BARTNICKI-GARCIA, S. (1971a). Cyst wall formation and endogenous carbohydrate utilization during synchronous encystment of Phytophthora palmivora zoospores. Arch. Mikrobiol. 79, TOKUNACA, J. & BARTNICKI-GARCIA, S. (19716). Structure and differentiation of the cell wall of Phytophthora palmivora: cysts, hyphae and sporangia. Arch. Mikrobiol. 79, WATERHOUSE, G. M. (1962). The zoospore. Trans. Br. mycol. Soc. 45, (Received 29 November 1974)

6 128 V. O. Sing and S. Bartnicki-Garcia pm Fig. i. Cross-section of an. unencysted zoospore of Phytophtiiora palmivora. fp, finger-print vacuoles; fv, flattened vesicles; /, lipid-like body; m, mitochondria; n, nucleus; pm, plasma membrane; pv, peripheral vesicles, x

7 Zoospore adhesion and wall formation 129 Figs. 2, 3. Cross-sections of an encysting zoospore (incipient cyst) as it initiates adhesion to a plastic surface. Fig. 2. Overall view of the zoospore anchored to a plastic surface through the newly discharged contents of a peripheral vesicle (pv). The encysting zoospore has not yet rounded off, and a prominent groove (g) is still present, x, microbody-like structures with electron-dense cortex and core. Other symbols as in Fig. 1. x Fig. 3. Details of the adhesion area in a different serial section of the same zoospore. x Q CEL IS

8 130 V. 0. Sing and S. Bartnicki-Garcia pm cm Fig. 4. Cross-section of a mature cyst adhered to a plastic surface. A thin, electrontransparent cyst wall (ca. 60 nm thick) covers the cell but it is not visible on this reproduction. Electron-dense adhesive material (cm) cements the cyst to the plastic surface. Other symbols as in Fig. 1. x

9 Zoospore adhesion and wall formation Figs, s, 6. Face views of the adhesion zone showing early stages in cyst wall assembly. From a sample of incipient cysts of P. palmivora lysed in situ with i % SDS and shadow cast, x

10 V. O. Sing and S. Bartnicki-Garcia Fig. 7. A more advanced stage in cyst wall.assembly"(cf. Figs. 5, 6). x Fig. 8. Fragment of a fully assembled cyst wall of P. palmivora prepared by sonication. x