Freeze substitution reveals a new model for sporangial cleavage in Phytophthora, a result with implications for cytokinesis in other eukaryotes

From the great number of ideas that have been put forward to explain the genesis of the additional plasma membranes required by cells formed during cytokinesis (Pickett-Heaps, 1972a; Rappaport, 1971, 1986), two principal models have emerged. The first involves a two-stage process in which vesicles are aligned along some part or all of the future plane of cleavage, this part then being established by fusion of the vesicles. The second model involves the progressive extension or stretching of the partitioning membranes along some part or all of the developing cleavage plane, without any observed prealignment of vesicles. Examples in support of both mechanisms have been reported in animal (Thomas, 1968; Rappaport, 1986), plant (Gunning, 1982; Volker, 1972), fungal (Hawker and Gooday, 1967; Cole, 1986) and protoctistan (sensu Margulis et al. 1989: Rawlence, 1973; Lokhorst and Segaar, 1989) systems.

In a previous study of chemically fixed sporangia of the notorious plant pathogen, Phytophthora cinnamomi, we employed electron microscopy to examine the process by which the infectious, motile zoospores of this organism are formed. We concluded that formation of the zoosporic plasma membranes during sporangial cleavage followed the first of the models described above, that is, fusion of prealigned vesicles (Hyde et al. 1991a). This process has also been described for P. palmivora (formerly P. parasitica, Hohl and Hamamoto, 1967) and all other species of Phytophthora for which the mechanism of zoosporic plasma membrane genesis has been described (review by Hemmes, 1983). In our previous study, however, we noted that the appearance of elements of the cleavage system of P. cinnamomi showed some marked variations that did not conform to the vesicle alignment model and that were related to the osmolarity of the fixative (Hyde et al. 1991a). These variations led us to test our conclusions by examining sporangia prepared by rapid freezing, a procedure which is considered to provide better preservation of membrane morphology than chemical fixation.

In this study we re-investigate zoospore formation in P. cinnamomi and P. palmivora using rapid freezing, at both ambient and high pressure, followed by freeze substitution (RF-FS). Our studies have been assisted by the use of a monoclonal antibody that labels cleavage elements in P. cinnamomi and P. palmivora. This is the first time, to our knowledge, that rapid freezing has been used to study cytokinesis in any eukaryotic organism for which vesicle alignment has been proposed to play a role in partitioning membrane formation. The results indicate that cleavage of the sporangium follows not from the fusion of prealigned vesicles, as suggested by chemical fixation but rather from the progressive extension of partitioning membranes. We believe that the apparent misinterpretation of the cytokinetic process that has occurred in Phytophthora may reflect a much wider problem, involving studies of a great variety of eukaryotic organisms. We also report other novel observations of sporangial structure, and partially characterize an extensive extracellular matrix that derives from the contents of the cleavage system and that may play a role in zoospore release.

The cultures of P. cinnamomi and P. palmivora used in this study were induced to produce sporangia and zoospores by the methods of Hardham and Suzaki (1986). Briefly, sporangial formation was induced by transfer of mycelium to a nutrient-poor medium; sporangial cleavage was induced by treatment with cold distilled water. Zoospores were released at about 50 min (P. cinnamomi) and 20 min (P. palmivora) after a cold shock. Samples were taken before induction of cleavage and at intervals between induction and release.

Wet tufts of mycelium with attached sporangia were placed in gold specimen holders (Balzers BB113142-1) with mineral salts solution (Hardham and Suzaki, 1986), (for pre-induction) or distilled water (post-induction) filling the remaining space. Pairs of holders were clamped together and frozen in a Balzers HPM 010 hyperbaric freezer. After freezing, the holders were snapped apart under liquid nitrogen and transferred to the substitution medium.

Tissue was frozen on Formvar-covered loops following the procedures of Lancelle et al. (1986). To place tissue on the loop, a novel approach was used. The loop was held in place on the platform of a dissecting microscope by plasticine. About 8-10 μl of distilled water or mineral salts solution was placed on the upper face of the loop. A tiny tuft of lightly blotted mycelium was then placed in this drop and spread out using fine forceps. The water was then wicked off and the loop rapidly transferred to the plunging device.

Tissue frozen by the above methods was freeze substituted using the procedures of Lancelle et al. (1986). The method was modified by the inclusion of 0.05% uranyl acetate in the substitution medium, and after 36h at -80°C, the vials were first warmed to −30 °C for 10 h before being brought to room temperature. Tissue was then rinsed in acetone several times, and stained en bloc in 5% uranyl acetate in methanol for 2h. After rinsing with acetone, the tissue was infiltrated with Epon resin and polymerized. Sections were stained for 3-5 min in Reynold’s lead citrate.

For material destined for immu-nogold-labelling, the freeze substitution, infiltration and polymerization procedures of Lancelle and Hepler (1989) were followed with the inclusion of a 10 h stage at −30 °C prior to bringing the samples to room temperature during freeze substitution. The material was then embedded in LR White resin. Infiltration and polymerization with UV light were carried out at room temperature. Immunolabelling of sections on gold grids followed the methods of Gubler and Hardham (1988). Monoclonal antibody (mAb) Cpw-1 was used. This mAb has previously been shown to have a strong affinity for elements of the cleavage system and weaker binding to dictyosomes and peripheral cistemae in sporangia of P. cinnamomi (Hyde et al. 1991a). After immunolabelling, sections were stained with 2% aqueous uranyl acetate for 20-30 min, followed by 2 min in lead citrate.

Proteins from freeze dried samples were solubilized in SDS sample buffer (63 mM Tris-HCl, pH 6.8, containing 2% SDS, 50 mM dithiothreitol, 0.001% bromphenol blue and 10% glycerol) for 5 min at 100 °C. After heating, samples were centrifuged and the supernatant kept on ice until use. Solubilized proteins were separated by homogeneous (7% or 12% acrylamide) or gradient (5% to 20% acrylamide) SDS-PAGE (Laemmli, 1970) and transferred to nitrocellulose (Towbin et al. 1979). After transfer, the nitrocellulose sheets were stained with 0.2% Ponceau S in 3% trichloroacetic acid, cut into strips and blocked with 5% skimmed milk powder in Tris-buffered saline (TBS: 10 mM Tris-HCl, pH 7.5 containing 150 mM NaCl). After 1-2 h, the strips were washed with TBS containing 0.5% Tween 20 (TBST). They were then incubated with antibodies in ascites fluid (diluted 1/800 in TBST containing 3% bovine serum albumin) for 1 h, washed five times with TBST for 5 min each, and then incubated for 45 min with sheep anti-mouse IgG antibodies conjugated to alkaline phosphatase. After washing twice in TBST, twice in TBS and once in enzyme substrate buffer (ESB: 100 mM Tris-HCl, pH 9.5 containing 100 mM NaCl and 50 mM MgCl), bound phosphatase was detected by immersing the strips in enzyme substrate containing 0.44% Nitro Blue Tétrazolium (Sigma, St. Louis, USA) and 0.33% 5-brom-4-chloro-3-indolylphosphate (Boehringer Mannheim, Germany) in ESB. Prior to antibody incubation, some strips were treated for 1 h with either Pronase E (Sigma, 1 mg ml⁻¹ in 50 mM Tris-HCl, pH 7.5) or sodium periodate (20 mM in sodium acetate buffer, pH 4.5).

The most notable feature of sporangia rapidly frozen before the induction of cleavage and during early postinduction stages was that these were the only sporangia in which large discrete cleavage vesicles were observed. These vesicles were most evident in regions of the cytoplasm that were close to the basal body-associated pole of a nucleus and relatively free of other major organelles (Figs 1 and 3). In P. cinnamomi, cleavage vesicles were especially numerous in these regions (Fig. 1). In both species the basal body-associated poles of nuclei in the sporangial cortex pointed towards the wall (Figs 1 and 3). During early post-induction, irregularly shaped cleavage elements were often seen around the nuclear pole (Fig. 2).

A significant difference between the two species was the presence of a single large vacuole in the centre of the sporangium of P. palmivora (Fig. 3). The vacuole was evident before induction, and was often ringed by a multilayered array of rough endoplasmic reticulum (Fig. 3). Such arrays have not previously been described in Phytophthora and nothing comparable was seen in P. cinnamomi, which has a series of smaller central vacuoles (Hyde et al. 1991a). In these and later stages microtubules were seen radiating from the basal bodies of both species (Fig. 1). In P. palmivora, limited flagellar (Fig. 3) and cleavage plane development was sometimes evident prior to induction.

The conformation of elements of the developing cleavage system, at these and later times, was directly comparable in high pressure- and plunge-frozen sporangia. With the exception of the large peripheral vesicles, other sporangial components were equally well preserved by both techniques. The large peripheral vesicles were well preserved in plunge-frozen sporangia but not in high pressure-frozen material (Fig. 3; Hyde et al. 1991b).

Subsequent stages of cleavage involved the formation of a single, cortical cleavage plane parallel to the sporangial wall and a series of internal cleavage planes separating uninucleate domains. In both species, organization of the cortical plane proceeded more rapidly than that of the internal planes.

The first indications of the development of the cortical plane were extended cleavage elements which lay mainly parallel to the sporangial wall (Fig. 4). These cleavage elements were frequently irregularly arranged (Fig. 4), especially at the apex of the sporangium, and were apparently of greater surface area than would ultimately be required. Several sporangial structures appeared to be associated with the development of the cleavage elements. The contents of elongated cisternae at the trans face of the Golgi apparatus often appeared similar to those of cleavage elements (Figs 5 and 2). A irans-Golgi network sometimes appeared to interconnect these cisternae with cleavage elements (Fig. 5). Small coated and uncoated vesicles were often numerous adjacent to cleavage elements and dictyosomes, and blebs were frequently seen on the surface of the elements (Figs 5 and 6). Discrete cleavage vesicles of the type seen earlier (Fig. 1) were not evident at this or any later stages. Serial section analysis revealed that occasional circular profiles suggestive of such vesicles (Fig. 4) were continuous with the extended cleavage elements.

By the time the cortical cleavage plane was fully developed (Fig. 7), most of the irregular and superfluous elements of the cortical plane had disappeared. This reduction occurred more rapidly in P. cinnamomi than in P. palmivora. Flagella were evident within the developing (Fig. 4) and completed (Fig. 7) cortical cleavage plane, which is part of the future extracellular space of the cleaved sporangium. The fully developed cortical cleavage plane cut off a shell of cytoplasm between itself and the sporangial plasma membrane (Fig. 7). This shell occasionally had connections to the main cytoplasm. Apparent cytoplasmic islands (Figs 7 and 8) which were evident in the cortical cleavage plane often proved to be projections of the shell or of the main cytoplasm when checked by serial sectioning.

The cleavage planes which will surround the internal nuclei of P. cinnamomi appeared to develop initially as membranous sheets extending back behind the nucleus from the narrow pole region (Fig. 9). The distal edges of these sheets were commonly dilated (Fig. 9). As in the case of the developing cortical cleavage plane, there were often very irregular arrangements of cleavage elements around the narrow poles of internal ‘"nuclei. These irregularities disappeared as cleavage progressed. In P. palmivora, there was no direct evidence of a polar basis to cleavage element extension in the sporangial interior. This may, however, reflect the difficulty of capturing intermediate stages of development in this species due to the rapidity of its cleavage process (Hohl and Hamamoto, 1967). In both species, cleavage progressed as the partitioning membranes continued to extend throughout the cytoplasm, interconnecting with each other to delineate the future zoospores (Figs 10-12). Just prior to release, the zoospores rounded up and the intercellular spaces became more obvious (Fig. 13).

The central vacuoles of both species disappeared during cleavage; there was no indication of their fate. The cortical shell of cytoplasm described earlier (Fig. 7) had also disappeared in both species by the completion of cleavage. This occurred at a later stage of internal cleavage in P. palmivora than in P. cinnamomi (compare Figs 10 and 11). Prior to its disappearance the shell became thinner and often fragmented (Fig. 8). Our evidence suggests that this fragmentation involved localized fusion of the outer membrane of the cortical cleavage plane with the plasma membrane of the sporangium (Fig. 8). Serial sectioning shows that the fragments were often interconnected and were occasionally continuous with the main cytoplasm. The inner membrane of the cortical plane was retained and formed part of the plasma membrane of the zoospores.

Apart from the cleavage system and an extracellular matrix described below, two other novel features of sporangial structure were evident in this study. First, sinuous projections of cytoplasm were observed within the water expulsion vacuoles of both species and the membrane surrounding these vacuoles had numerous vesicles and blebs associated with it (Figs 11 and 12). Second, peripheral cisternae (flattened organelles previously described in sporangia of P. cinnamomi at late stages of cleavage only; Hyde et al. 1991a) were evident in both species from very early cleavage stages (Fig. 5) onwards and they were much more extensive in the zoospore initials (Figs 11 and 12) than previously observed.

A notable and previously undescribed feature of sporangia was the dark, grainy appearance of material within the cleavage system (Figs 1, 2, 4-12). When cleavage was complete, this material was external to the zoospore initials, forming an extracellular matrix (Figs 11-13). Variability in the apparent density of this material was marked in high pressure-frozen material (e.g. Figs 3 and 8). In plunge-frozen material, a consistent decrease in density was evident in sporangia sampled just prior to zoospore release (Fig. 13).

The extracellular matrix of both species was labelled by mAb Cpw-1 (Figs 14-16). A thick layer of extracellular matrix was typically seen at the sporangial apex and showed particularly strong binding of mAb Cpw-1 (Fig. 15). The sporangial wall also showed some binding of mAb Cpw-1, especially near the sporangial apex (Fig. 15), suggesting that the antigen is leaking outwards. As in P. cinnamomi (Hyde et al. 1991a), the central vacuole of P. palmivora was not labelled by mAb Cpw-1 (Fig. 16). The antigen(s) recognized by Cpw-1 were identified on immunoblots of sporangial extracts. In P. cinnamomi, Cpw-1 bound to several broad bands with apparent molecular weights between 60 and 330 kDa (Fig. 17). Similar results were obtained for P. palmivora (data not shown). The cleavage state of sporangia had no bearing on the number of bands detected. Pronase treatment of the transferred proteins of P. cinnamomi abolished antibody binding; treatment with periodate did not (Fig. 17).

The results of this study describe a significantly different process of partitioning membrane formation to that previously reported during zoosporogenesis in P. palmivora and P. cinnamomi (Hohl and Hamamoto, 1967; Hyde et al. 1991a). There was no evidence that cleavage follows from the fusion of prealigned vesicles, but rather the data indicate that subdivision of the sporangium results from the progressive extension and eventual interconnection of membranous sheets. These new findings are summarized and compared with the previous model in Fig. 18. We consider that the discrepancy between this and previous reports arises from the different techniques used to preserve sporangia, namely chemical fixation and RF-FS.

RF-FS is considered superior to chemical fixation for the preservation of cell structure generally and membranous components particularly (Lancelie et al. 1985, 1986; Gilkey and Staehelin, 1986; Cresti et al. 1987; Howard and O’Donnell, 1987). Chemical fixation may cause artefactual alterations in the morphology of membranous components. In a comparative study of wall-destined vesicles in freeze-substituted and chemically fixed hyphae of the Oomycete Saprolegnia, for example, Heath et al. (1985) observed in frozen material densely staining tubular elements that became partially vesiculated and lost their contents following chemical fixation. Also, studies by McCully and co-workers have shown that in petiolar hairs of certain plants the membranous canalicular system, which is visible as elongated strands in living cells, becomes highly vesiculated during chemical fixation (O’Brien et al. 1973; Mersey and McCully, 1978) but retains its in vivo morphology when rapidly frozen (McCully and Canny, 1985). We propose that the data from sporangia preserved by RF-FS also represent more faithfully the structure of the living cell, and that the apparent alignment of vesicles seen in previous studies of cleavage in Phytophthora probably resulted from artefac-tual vesiculation during chemical fixation of membranous sheets similar to those seen in this study. Since the cleavage membranes of chemically fixed sporangia do not vesiculate during the final stages of zoosporogenesis, the network of aligned vesicles seen earlier (Fig. 18) can readily be misinterpreted as an intermediate stage in the cleavage process. In addition, rather than occurring within specialized ‘axonemal vacuoles’ (Hohl and Hama-moto, 1967; Hyde et al. 1991a; Hemmes, 1983), flagellar development is shown by RF-FS to occur within the general system of cleavage planes.

The present study provides evidence that development of the cleavage planes, at least in P. cinnamomi, begins in regions near the basal body-associated nuclear poles where dictyosomes are concentrated and cleavage vesicles are initially clustered. The disappearance of the cleavage vesicles coincides with the formation of the first expanses of cleavage membranes, indicating that cleavage vesicles may be incorporated into the developing cleavage planes. Irregular cleavage elements that are seen in early postinduction stages may be transitional stages in the transformation of cleavage vesicles into more extended forms. Further expansion of the partitioning membranes appears to involve the contribution of membrane from dictyosomes, some of whose cisternae appear interconnected with developing cleavage elements through a irans-Golgi network. This is the first report of a transGolgi network in Phytophthora; the network may be disrupted during chemical fixation. Coated and uncoated vesicles, often seen near dictyosomes and cleavage elements, may also be involved in membrane augmentation during cleavage element extension. These observations are in agreement with previous proposals of a dictyosomal origin for cleavage elements in Phytophthora (Hohl and Hamamoto, 1967; Elsner et al. 1970; Hyde et al. 1991a). Vesicles may be transported to the edge of the expanding system, which is characteristically dilated, or could be incorporated close to the nuclear pole. The vesicles and blebs could also play a role in membrane retrieval during development of the cleavage system.

The absence of any structural interconnection between the large vacuoles and the cleavage system and the lack of labelling of the vacuolar contents with mAb Cpw-1 indicate that the large vacuoles do not play a role in the cleavage process.

To our knowledge, this is the first published study to employ rapid freezing instead of chemical fixation to study cytokinesis in any system in which partitioning membrane formation would be expected to involve the fusion of prealigned vesicles. As such it represents the fairest test yet of this model, given the unpredictable preservation of membrane form by chemical fixation. The apparent failure of the alignment/fusion model in this study of zoosporogenesis in P. cinnamomi and P. palmivora has wide-ranging implications for our understanding of cytokinesis in other eukaryotes. It has already been proposed (Schroeder, 1970; Rappaport, 1971, 1986) that the small body of studies which report vesicle alignment during cytokinesis in animal cells suffers from the use of a chemical fixation protocol that leads to vesiculation of the furrowing membranes. To our knowledge, there has been no consideration of similar vesiculation during cytokinesis in non-animal systems, although many studies have expressed concern over the difficulty of discerning whether apparent vesicles, seen in single cross-sections, might actually be part of a continuous system (Burr and West, 1970; Marchant and Pickett-Heaps, 1971; Zaar and Kleinig, 1975). Wilson et al. (1990) have recently outlined a theoretical basis for the general case of vesiculation of membranes by chemical fixatives. For reasons given below we believe that artefactual vesiculation of partitioning membranes may have occurred in many studies of a wide variety of eukaryotes, especially protoctists.

Many of the studies that report vesicle alignment in protoctistan systems (e.g. see Goodman and Rusch, 1970; Porter, 1972; Mims, 1973) have one, possibly critical, feature in common with the suspect animal reports and the chemical fixation studies of zoosporogenesis in Phytophthora. In all cases, a stage is described where vesicles are arranged in a row corresponding to the future plane of cleavage, but there is, mostly, no prior stage described where the vesicles are loosely arranged in this region. A loose arrangement of vesicles is, however, typically described during early cell plate formation in plants (Hepler and Jackson, 1968; Gunning, 1982). It is commonly believed that this loose arrangement in plant cells arises because the vesicles are in the process of moving towards the future zone of cleavage from either side of it (Hepler and Jackson, 1968; Gunning, 1982). The absence of such a stage in some protoctistan systems might be explained by Heath’s (1975) proposal that cleavage vesicles in the Saprolegniales move along the future plane of cleavage before coming to rest in that plane and fusing. In other cases, insufficient sampling of cells from different stages of cleavage may have resulted in the loose arrangement of vesicles having gone undetected. Alternatively, if the neatly arranged stage is itself an artefact of preparation, then a prior loose stage may not be required for whatever is the true process of membrane genesis. The present study, when viewed in the light of previous studies of zoosporogenesis in Phytophthora, suggests that the absence of a loosely arranged stage may well be a good indicator that the neatly arranged stage is an artefact.

The taxonomic distribution of organisms in which vesicle alignment/fusion has been described is perplexing. Since this process is commonly considered to be an evolution-arily advanced feature of cytokinesis (Pickett-Heaps, 1972a; Rawlence, 1973), one might expect to trace clear lines of ancestry for this character back through the taxa in which it appears towards some primitive organism in which it first evolved. The impossibility of doing this is best illustrated by considering the protoctista. The vesicle alignment model has been proposed for a large number of phylogenetically distant protoctistan taxa, in many of which there have also been reports of the more primitive mechanism, namely progressive extension of the partitioning membranes (Table 1). In a number of cases the two mechanisms have been proposed in reports describing cytokinesis at different (e.g. Labyrinthula sp., Plasmodio-phora brassicae, Oedogonium cardiacum, Table 1) or even the same {Physarumpolycephalum, Spirogyra sp., Table 1) stage of the life cycle in the one genus or species.

While taxonomic heterogeneity such as this may have a variety of natural sources (Fowke and Pickett-Heaps, 1969; Coss and Pickett-Heaps, 1973; Watson et al. 1985) another possibility is that some variability has arisen from methodological complications such as those seen in this study. It is interesting that, with limited exception (Lucarotti and Federici, 1984), apparently the only eukaryotes that exhibit, during cytokinesis, a loose arrangement of vesicles similar to that seen in higher plant cell plate formation are certain green algae (Phyla Conjugaphyta and Chlorophyta, sensuMargulis et al. 1989), e.g. reports by Fowke and Pickett-Heaps (1969), Pickett-Heaps and Fowke (1970), Floyd et al. (1972), Pickett-Heaps (1973), Marchant and Pickett-Heaps (1973). The green algae and higher plants are commonly considered to be phylogenetically related (Pickett-Heaps, 1972a) and these two groups may represent the true evolutionary lineage of cytokinesis involving vesicle alignment/fusion. Cell plate formation has been proposed to occur in some brown algae (Rawlence, 1973; Markey and Wilce, 1975) but these descriptions do not include the loosely arranged stage and may be cases of artefactual vésiculation.

We believe that descriptions of cell plate formation in plants and certain green algae have features that make them distinct from, and more credible than, reports of vesicle alignment/fusion in other eukaryotes. Nevertheless, the possiblity remains that cell plate membranes which have formed in vivo from the fusion of aligned vesicles may exhibit a temporary labile phase, similar to that shown by cleavage membranes in Phytophthora, during which they are susceptible to vesiculation by chemical fixation.

The results describe, for the first time in Phytophthora, the presence of a dense, grainy material filling elements of the developing cleavage system and forming an extracellular matrix which surrounds the zoospores in the fully cleaved sporangium (Fig. 18). The passage of this material during cleavage was traced, in both species, by immunogold-labelling using mAb Cpw-1. In chemically fixed material of P. cinnamomi, mAb Cpw-1 binds to flocculent material inside the cleavage system (Hyde et al. 1991a). This material probably represents all that remains after chemical fixation of the dense matrix seen in freeze-fixed sporangia. Polyacrylamide gel electrophoresis indicates that Cpw-1 binds to one or more proteins with apparent molecular weights between 60 and 330 kDa. Although antibody binding was not abolished by periodate treatment, the smearing of the bands is suggestive of extensive glycosylation of the antigen (Goldkorn et al. 1989).

Extracellular material, often referred to as mucilage, has been described surrounding the spores, sperms and similar structures of many protoctists (Moore, 1965; Pickett-Heaps, 1972b; Scott and Dixon, 1973; Toth, 1974; Lunney and Bland, 1976; Franke et al. 1977; Duckett and Peel, 1978) and fungi (Ingold, 1968) with several reports noting its origin from cleavage elements (Moore, 1965; Scott and Dixon, 1973; Lunney and Bland, 1976; Franke et al. 1977). The major significance of this material is its proposed capacity to act as a swelling gel (de Bary, 1887; Pickett-Heaps, 1972b; Toth, 1974; Lunney and Bland, 1976; Duckett and Peel, 1978; Ingold, 1968) or as an osmoticum (Scott and Dixon, 1973; Ingold, 1971) which may be involved in events leading to rupture of the storage organ and release of its contents. Although it has been considered that the swelling gel model is theoretically plausible to explain sporangial discharge in Phytophthora (MacDonald and Duniway, 1978; Gisi and Zentmyer, 1980) there has never, until now, been evidence of any extracellular material extensive enough to be seriously considered. There is indirect evidence that the extracellular matrix described herein might swell, since the volume occupied by it apparently increases just prior to zoospore release. If swelling does occur this may account for some or all of the force needed for bursting the sporangium.

Alternatively, the extracellular matrix material may operate as an osmoticum. Numerous studies of Phytophthora have proposed that sporangial discharge results from an osmotically driven increase in hydrostatic pressure (reviewed by Gisi, 1983). One deficiency of the hydrostatic model is that it relies on the existence of a semipermeable barrier across which the hypothetical osmoticum exerts its influence (Gisi and Zentmyer, 1980). The present study indicates that the sporangial membrane disappears too early (at least in P. cinnamomi) for it to have any role in this process. A similar dilemma in other systems has been addressed by examining the possibility that the sporangial wall itself may act as a semipermeable barrier (Money and Webster, 1988, 1989; Money, 1990). While further work is needed to assess the applicability of this proposal in Phytophthora, if swelling of an extracellular matrix is responsible for sporangial discharge, then no semipermeable barrier is required.

In conclusion we point out that this study is another dramatic example of the superiority of rapid freezing procedures over chemical fixation for the preservation of cell structure. In particular, the maintenance of the apparent true form of the developing cleavage planes and the irans-Golgi network in Phytophthora demonstrates again the effectiveness of rapid freezing in bringing to light extensive membrane systems which have gone undetected in chemically fixed material.

We thank Dale Callaham and Frank Sek for excellent technical assistance, Hans Hohl, Frank Gubler, Larry Lehnen, Les Watson and Geoff Wasteneys for helpful suggestions, James Whitehead for the illustration and Margaret Wigney for typing the manuscript. This research was supported by a Cooperative Research Grant from the Australian Department of Industry, Technology and Commerce to G.H. for a visit to Amherst to use the high-pressure freezer; an Australian Postgraduate Research Award (G.H.) and NSF grants DCB-90-04191 (P.K.H.) and BBS-87-14235 (to the University of Massachusetts Microscopy Center).