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Synthesis of zoospore peripheral vesicles during sporulation of Phytophthora cinnamomi
39
Mycol. Res. 100 (1):39-48 (1996) Printed in Great Britain
Synthesis of zoospore peripheral vesicles during sporulation of Phytophthora cinnamorni
J. D. W. DEARNALEY, J. MALESZKA A N D A. R. HARDHAM Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, Canberra, A.C.T. 2601, Australia
Three types of spherical vesicles (dorsal, ventral and large peripheral vesicles) are found in the peripheral cytoplasm of Phytophfhora c~nnamomizoospores. Each is believed to participate in the infection of host plants through formation of a cyst coat, deposition of an adhesive pad or supply of proteins during early germling growth. In the present study, the occurrence of these three vesicles was determined during the asexual life cycle of P. cinnamomi. The vesicles were found to be absent from rapidly growing, vegetative hyphae, but to appear in the hyphae under conditions that induced sporulation. Vesicle formation was correlated with sporulation via the production of sporangia or chlamydospores. In mycelia transferred to nutrient-poor medium, large peripheral and ventral vesicles first appeared about 5 h after transfer; dorsal vesicles appeared about 30 min later. The first sporangia were detected at 75 h. Large peripheral and ventral vesicles were also produced before dorsal vesicles in mycelia growing on nutrient agar and in the hyphae formed by germinating cysts. On nutrient agar, the three vesicle types appeared about 10 h in advance of the development of chlamydospores.
Phytophthora cinnamomi Rands, the Dieback fungus, is an Oomycete pathogen with a broad host range (Zentmyer, 1980). Motile biflagellate zoospores are the major infective agent and are produced by the cleavage of multinucleate sporangia. Invasion of hosts is mediated by chemotaxis of the zoospores and the subsequent encystment of these cells on plant roots (Carlile, 1983). Under conditions not suitable for the development of sporangia, mycelia will form chlamydospores which can perpetuate the pathogen for long periods of time in the absence of hosts (Mircetich & Zentmyer, 1966). When favourable conditions return, chlamydospores germinate and form mycelia, sporangia or new chlamydospores (Weste, 1983). Three types of immunologically distinct vesicles localized in discrete zones in the peripheral cytoplasm of P. cinnamomi zoospores appear to play important roles in zoospore encystment and early germ tube growth (Gubler & Hardham, 1990; Hardham & Gubler, 1990; Hardham ef al., 1991). Large peripheral vesicles occur predominantly beneath the dorsal surface of the zoospores (Gubler & Hardham, 1988). They contain glycoproteins with a relative molecular weight of over 300 kDa. They become randomly distributed during encystment and appear to serve as a source of proteins that are used to support the first few hours of growth of the germinated cyst (Gubler & Hardham, 1990). Ventral vesicles are concentrated along the ridges of the groove that runs along the ventral surface of the zoospores. They contain a protein whose relative molecular weight is over 220 kDa (Hardham & Gubler, 1990). These vesicles are rapidly secreted during encystment, and their contents form a plaque of
material believed to function in the adhesion of the cyst to the adjacent surface (Hardham & Gubler, 1990). Dorsal vesicles occur predominantly under the dorsal surface of the zoospores. They contain glycoproteins over 300 kDa in relative molecular weight. Their contents are secreted during encystment and form a coating of material that surrounds the cysts (Gubler & Hardham, 1988). The available evidence indicates that the three zoospore peripheral vesicles function during zoospore encystment and early germling growth, and suggests that they might be synthesised exclusively during zoosporogenesis. There is, however, little information on the presence or absence of these vesicles at other stages of the life cycle of P. cinnamomi. In the present study we have used monoclonal antibodies Lpv-1, Vsv-1 and Cpa-2 which are specific for the large peripheral, ventral and dorsal vesicles, respectively, to determine the site and timing of vesicle synthesis throughout the asexual life cycle of P. cinnamomi. W e have discovered that the vesicles are absent from rapidly growing vegetative hyphae but are synthesized when the production of sporangia or chlamydospores is induced. The vesicles are thus sporulation specific components.
MATERIALS A N D METHODS Zoospore, cyst and germling production Culturing and production of zoospores from Phyfophfhora cinnamomi (6BR; DAR 52646) followed methods described in Hardham ef al. (1991). Seven small squares (5 mm x 5 mm) of
Vesicle production during P. cinnamomi sporulation
Figs 1-12. For captions see facing page
J. D. W. Dearnaley, J. Maleszka and A. R. Hardham
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sterile 100 ml Schott bottle (Duran, Mainz, Germany). To produce germlings, approximately 2-5 x lo6 germinated cysts were inoculated into 10 ml of 5 % V8 broth and incubated at 25' in 15 ml culture tubes (Disposable Products, Adelaide, Australia). In some experiments, after 6 h the V8 broth was replaced with mineral salts solution and samples taken after 0, 6, 18 and 30 h incubation.
Chlamydospore production Chlamydospores were produced via the method of Englander & Tubitt (1979). A small piece of V8 agar with mycelium was Lpv- 1
Fig. 13. Immunodot blot of cell extracts used to detect changes in the relative abundance of the three antigens in germlings growing in 5 % V8 broth for 10 h. The level of ventral vesicle antigen did not change during the first 2 h after encystment, but decreased between 2 and 10 h. No changes in the level of dorsal vesicle antigen were detected over the 10 h period. A decline in large peripheral vesicle antigen was observed between 2 h and 4 h.
mycelia were cut from the edge of a colony of P. cinnamomi growing on V8 nutrient agar containing 10% V8 juice (Campbell's Soups Pty Ltd, Lemnos, Australia), 0.002 % Psitosterol (Sigma Chemical Co., St Louis, MO, U.S.A.), 0.01% CaCO, and 1.7% Bacto agar (Difco, Detroit, MI, U.S.A.). The squares were inoculated onto a sterile miracloth disc overlying V8 nutrient agar and incubated for 5 d at 25 'C in the dark. Each disc was then transferred to 100 ml of 5 % V8 broth (5 % cleared V8 juice, 0.01 % CaCO,, 0.002 % p-sitosterol) and shaken overnight at 150 rpm at 22'. The cultures were washed three times in mineral salts solution (10 mM Ca (NO,),, 5 mM KNO,, 4 mM MgSO, and 2 ml1-I of a solution containing 10 mM FeSO, and 10 mM Na, EDTA) and incubated at 22' for 24 h in 100 ml of this solution, during which time sporangia developed. Cleavage of sporangia was induced by rinsing miracloth cultures three times in cold double-distilled water and incubating them at 4' for 13 min in 10 ml of distilled water. The discs were then transferred to a light box at 18'. Zoospores were released 60-75 min later. Zoospores were induced to encyst by agitation for 20 s in a
placed onto a V8 agar in a Petri plate and incubated in the light at 25'. Samples were taken at intervals over the next 80 h and the presence or absence of chlamydospores noted.
Sporangium production Small (5 mm x 5 mm) pieces of agar with mycelium were taken from the growing edge of a colony on V8 agar, placed in 5 % V8 broth and incubated in the dark at 25' for 17 h. The samples were then washed three times in mineral salts solution and incubated in the light in a final volume of 15 ml of mineral salts at 22'. Samples were taken at intervals over the next 11 h.
Immunofluorescent labelling Cysts, germlings and hyphae were fixed in 4% paraformaldehyde in 50 mM Pipes (piperazine-N, N-bis[2-ethanesulphonic acid]) buffer (pH 7.0) for 1h at room temperature. After rinsing twice in Pipes buffer the cells were frozen in Tissue Tek embedding compound (Miles Inc., Elkhart, IN, U.S.A.) in plastic moulds by plunging in liquid nitrogen. Ten urn thick sections of cysts and germlings and 12 pm thick sections of hyphae were cut on a Reichert-Jung 2800 Frigocut E cryotome and dried at room temperature onto poly-L-lysine coated slides. Immunostaining was carried out in 10 cm square dishes lined with moist filter paper. The sections were incubated for 45 min at 37' in one of three monoclonal antibodies raised against P. cinnamomi zoospore vesicle antigens. Monoclonal antibody Vsv-1 reacts with the contents of ventral vesicles and was purified and used at 1 pg ml-'. Cpa-2 reacts with the contents of dorsal vesicles and was purified and used at 0.5 pg ml-l. Lpv-1 reacts with the
Figs 1-12. Zoospore peripheral vesicles in Phytophthora cinnamomi cysts and gerrnlings growing in 5 % V8 broth. In each case observations are paired, with the immunofluorescence micrograph above and differential interference contrast micrograph of the same field of view below. Figs 1-4. Vsv-1; Figs 5-8. Cpa-2; Figs 9-12. Lpv-1. Bar = 20 pm.Figs 1-4. At 5 min (Fig. I), 2 h (Fig. 2) and 4 h (Fig. 3) after encystment, Vsv-1 labelling showed that a few ventral vesicles (arrowheads) could be observed in the cyst cytoplasm and the germ tubes. The vesicles had disappeared by 10 h (Fig. 4). Most of the Vsv-1 antigen was secreted onto the cyst surface (Figs 3, 4) during the first few minutes after the onset of encystment. This material is out of the plane of focus in Figs 1 and 2. Figs 5-8. Labelling of 5 rnin old cyst with Cpa-2 showed that the contents of the dorsal vesicles were completely secreted during encystment to form a coat on the outside of the cyst (Fig. 5). This coat was still present on the surface of the cyst at 2 h (Fig. 6), 4 h (Fig. 7) and 10 h (Fig. 8) after encystment. Figs 9-12. Labelling of 5 min old cysts with Lpv-1 showed that the large peripheral vesicles were not secreted during encystment (Fig. 9). These vesicles remained in the cyst and were present in germ tubes at 2 h (Fig. 10). By 4 h large peripheral vesicles had disappeared from germlings (Fig. 11) and they were still absent at 10 h (Fig. 1.2). At no stage in the 10 h period was the Lpv-1 antigen observed on the surface of the germlings. Photographic exposures for Figs 11 and 12 are the same as those for Figs 9 and 10.
Vesicle production during P. cinnamomi sporulation
Figs 14-25. For captions see facing page
42
J. D. W. Dearnaley, J. Maleszka and A. R. Hardham contents of large peripheral vesicles and was used as hybridoma supernatant diluted 1 : r O in phosphate buffered saline (PBS; 20 mM sodium phosphate, 150 mM NaC1) containing - 1 % bovine serum albumin (BSA). After three washes in PBS, the sections were incubated in sheep antimouse immunoglobulin-fluorescein isothioc~anate (SAMFITC [Silenus, Australia]) diluted 1:60 in 1 % BSA-PBS, for 45 min at 37'. Sections were subsequently washed three times in PBS and once in distilled water. They were then dried and mounted on glass slides in mowiol containing 0.1% p-phenylenediamine. The samples were examined and photographed with a Zeiss Axioplan microscope equipped with epifluorescence optics with an F 1 filter cube (excitation 450-490 nm; dichroic mirror 510 nm; barrier 515-565 nm). For quantitative analysis of the occurrence of the three types of vesicles, triplicate samples (blocks) of the mycelium were taken from different parts of the colony or at different times during the culturing protocols. Cryosections were cut from at least two of the blocks at two different depths within the mycelium, giving a total of four different regions of sampling. The sections at each region were examined after immunolabelling for fluorescence microscopy and the presence or absence of each vesicle type was determined in 100 hyphal fragments for each group of sections. The average percentage of hyphae containing each vesicle type was determined from the four samples for each time or location point. Experiments analysing the appearance of vesicles following transfer of the mycelium to mineral salts solution were repeated five times. Experiments examining the presence of vesicles across colonies growing on V8 agar or during chlamydospore formation were each conducted four times.
ande en on^.
Immunodot blot analysis Cysts and germlings were frozen in liquid nitrogen, lyophilized, then ground in eppendorf tubes using a glass rod. The ground material was extracted for 20 min with 6 M guanidine hydrochloride, then diluted 50 times with Tris buffered saline (TBS; 10 mM Tris-HC1, 150 mM NaC1, pH 7.4). The samples were incubated on ice for 10 min before centrifuging at 13000 rpm for 10 min. Solubilized material was transferred to nitrocellulose in an immunodot blot apparatus (Biorad, Nth Ryde, NSW, Australia). The efficacy of the transfer was monitored by staining with 0.2% Ponceau S in 3 % trichloroacetic acid for 5 min. After blocking nonspecific binding sites for 1h in 5 % skim milk powder in TBS and washing three times in TBS-0.1% Tween-20, the nitrocellulose was incubated for 45 min in undiluted supernatants of Vsv-1
43 or Lpv-1, or 3 pg ml-' purified Cpa-2 in 1% BSA-PBS. Bound antibody was visualized with peroxidase-conjugated SAM (Silenus) using 4-chloro-1-napthol as substrate.
RESULTS Zoospore peripheral vesicles in germlings
growing in 5%
V8 broth Labelling of 5 min old cysts (cysts fixed 5 min after the induction of encystment) with Vsv-1 showed that most of the ventral vesicle antigen was secreted and coated part of the surface of the cysts (see Fig. 5 in Hardham & Gubler, 1990), although some ventral vesicles remained in the cytoplasm of the cyst (Fig. I). At 2 h (Fig. 2) and 4 h (Fig. 3) after encystment, ventral vesicles were still observed in the germlings but by 10 h after encystment (Fig. 4), all ventral vesicles had disappeared. The secreted Vsv-I antigen remained associated with the cyst surface during this period (Figs 3, 4). Labelling of 5 min old cysts with Cpa-2 showed that the contents of the dorsal vesicles were completely secreted during encystment to form a coat on the outside of the cyst (Fig. 5). This coat remained on the surface of the cyst during the next 10 h of germling g o w t h (Figs 6-8). No dorsal vesicles were seen in germlings during the 10h period. Labelling of 5 min old cysts with Lpv-1 showed that the contents of the large peripheral vesicles were not secreted during encystment (Fig. 9). These vesicles remained in the cyst and were present in the germlings at 2 h (Fig. lo), but by 4 h they had completely disappeared (Fig. 11). They were also absent at 10 h (Fig. 12). At no stage was the Lpv-1 antigen observed on the surface of the germlings. Changes in the relative abundance of the three antigens in germlings growing in 5 % V8 broth were analysed using immunodot blotting of cell extracts (Fig. 13). The level of ventral vesicle antigen did not change during the first 2 h after encystment, but decreased during subsequent growth. No changes in the level of dorsal vesicle antigen were detected over the 10 h period. A decline in large peripheral vesicle antigen was observed between 2 h and 4 h.
Peripheral vesicles in germlings transferred to mineral salts solution At the time of transfer to mineral salts solution (6 h after encystment), the germlings did not contain any ventral, dorsal or large peripheral vesicles (Figs 14, 18, 22) although secreted ventral and dorsal vesicle material coated the cyst surface (Figs 14, 18). Both ventral (Fig. 15) and large peripheral (Fig. 23)
Figs 14-25. Zoospore peripheral vesicles in P. cinnamomi germlings transferred to mineral salts solution. In each case observations are paired, with the immunofluorescence micrograph above and differential interference contrast micrograph of the same field of view below. Figs 14-17. Vsv-1; Figs 18-21. Cpa-2; Figs 22-25. Lpv-1. Bar = 20 pm. Figs 14-17. Ventral vesicles were absent at the time of transfer to mineral salts solution (6 h germlings, Fig. 14) but after 6 h in the mineral salts solution, they had reappeared (Fig. 15) and they increased in number over the next 12 h (Fig. 16) and 24 h (Fig. 17). Figs 18-21. Dorsal vesicles were not present at the time of transfer (Fig. 18) or after 6 h (Fig. 19) or 18 h (Fig. 20) in the mineral salts solution. After 30 h in the mineral salts solution dorsal vesicles were present (Fig. 21) and at this time sporangia (s) had appeared. Figs 22-25. Large peripheral vesicles were absent at the time of transfer to mineral salts solution (6 h germlings, Fig. 22) but after 6 h in the mineral salts solution they had reappeared (Fig. 23) and they increased in number over the next 12 h (Fig. 24) and 24 h (Fig. 25).
..
Vesicle production during P. cinnamami sporulation 0
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cyst germination V8 broth 2 h
Lpv- 1
Fig. 26. Immunodot blot of cell extracts used to determine changes in the relative abundance of the three antigens after germlings were transferred to mineral salts solution after 6 h in 5 % V8 broth. After 6 h incubation in the mineral salts solution the Vsv-1 antigen was detected and levels of the antigen increased over the next 24 h. No changes were evident in the level of Cpa-2 antigen over the 30 h period. Lpv-1 antigen was first detected 18 h after transfer to mineral salts solution.
vesicles were present 6 h after transfer to mineral salts solution. The number of ventral (Figs 16, 17) and large peripheral (Figs 24, 25) vesicles in the hyphae increased over the following 24 h. Dorsal vesicles appeared between 1 8 h and 3 0 h after transfer to mineral salts solution (Figs 19-21). Sporangia were observed in the germling culture at 30 h (Fig. 21). Immunodot blotting of extracts from germlings incubated in mineral salts solution detected the appearance of Vsv-1 antigen after 6 h and its increase over the next 12 h (Fig. 26). No changes in the level of Cpa-2 antigen were evident. Lpv-1 antigen was first detected 1 8 h after transfer to mineral salts solution. The presence or absence of the three types of vesicles in zoospores, cysts and germlings is summarized diagrammatically in Fig. 27.
Zoospore peripheral vesicles in mycelia, sporangia and chlamydospores Samples of P. cinnamomi mycelia were immunofluorescently labelled and examined for the presence or absence of the three types of vesicles. The occurrence of the vesicles was analysed by counting the percentage of hyphal fragments containing each vesicle type in cryosections of samples taken during a culture protocol used to produce sporangia. The experiment was repeated five times and in each case the results were similar to those shown in Fig. 28. The initial sample was mycelium that had been growing on V8 agar for 5 d. All three
Fig. 27. Diagram illustrating the presence or absence of ventral (dark hexagons), dorsal (light circles) and large peripheral (ovals) vesicles in zoospores, cysts and germlings of P. cinnamomi at different stages of growth in nutrient broth or mineral salts solution.
4
Time in mineral salts solution (h)
Fig. 28. Vesicle occurrence during culture regime for production of sporangia. Percentage of P. cinnamomi hyphal fragments containing large peripheral vesicles labelled with Lpv-1 (01,ventral vesicles and dorsal vesicles labelled with Cpa-2 (0). labelled with Vsv-1 (0) Samples of mycelia growing on V8 agar were transferred to V8 broth and incubated for 18 h (0 h time point). Cultures were then transferred to mineral salts solution and sampled over the next 11 h. Sporangia were first observed at 7.5 h.
vesicles were present in these samples, with ventral and large peripheral vesicles being much more abundant than dorsal vesicles (Fig. 28). When mycelia were transferred to 5% V8 broth and incubated for 1 8 h, all . peripheral vesicles disappeared. The mycelia were then transferred to mineral salts solution. Ventral and large peripheral vesicles first appeared 5 h after transfer to mineral salts solution and rapidly increased in number to a plateau level b y about 7 h. Dorsal vesicles began to appear about 30 min after formation of ventral and large peripheral vesicles. They increased in number at a slower rate than the latter, reaching a plateau frequency at 11-12 h after transfer to mineral salts solution. In -
J. D. W. Dearnaley, J. Maleszka and A. R. Hardham
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0
1 2 Radial distance (cm)
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Figs 29, 30. Vesicle occurrence in mycelia growing across VS agar. Percentage of P. cinnamomi hyphal fragments containing large peripheral vesicles labelled with Lpv-1 (O), ventral vesicles labelled with Vsv-1 (0) and dorsal vesicles labelled with Cpa-2 (0). Samples were taken every 5 mm from the centre of the plate. Fig. 29. Samples taken 3 d after inoculation. Fig. 30. Samples taken 5 d after inoculation.
this and the other four experiments, sporangia first appeared at 7.5 h after transfer to mineral salts solution. Another set of experiments examined the occurrence of the three vesicle types in hyphae at different distances from the centre of a colony growing on V8 agar plates. In plates inoculated 2 d (data not shown) or 3 d (Fig. 29) before sampling, most hyphae at the centre of the colony, but very few at the leading edge, contained vesicles. However, samples plates, taken 5 d (or more) after inoculation onto the V8 agar . . showed the presence of all three vesicle types throughout the colony (Fig. 30). These data are summarized diagrammatically in Fig. 31. Although sporangia do not normally form in mycelia growing on nutrient agar, after growing on V8 agar plates for 2-3 d, P. cinnamomi mycelia produced chlamydospores. The relationship between chlamydospore formation and the
Fig. 31. Diagram illustrating the presence or absence of ventral (dark hexagons), dorsal (light circles) and large peripheral (ovals) vesicles in hyphae of P. cinnamorrli growing on V8 agar plates. A, sample taken from a confluent culture; B, sample taken from the leading edge of a colony growing across a fresh V8 agar plate; C, samples taken from the edge or the centre of a colony.
appearance of the three vesicles was examined by sampling .. expanding colonies at intervals after subculture onto V8 agar plates. Both thin (2.5 mm) and thick (5 mm) layers of V8 agar were used. The results showed that the abundance of vesicles decreased after inoculation of the sample onto the fresh agar plates. O n thin plates, the vesicles were not detectable for about 20 h and then ventral and large peripheral vesicles rapidly reappeared between 32 and 40 h after subculture (Fig. 32). Dorsal vesicles began to appear at about 40 h and the first chlamydospores were observed at 48 h. The same trend was seen on the thick plates but the time of both vesicle reappearance and chlamydospore formation was delayed by about 8 h (Fig. 33). All three vesicles were observed within chlamydospores (Figs 34-37). In these experiments, at 40 h and thereafter the expanding colony was sampled at 5 mm intervals from the inoculation site at the centre of the colony. For clarity, only data from the central sample are shown. Vesicle occurrence in other positions displayed the same trends shown in Figures 32 and 33. Chlamydospores were first observed at the centre of the colony. The presence or absence of vesicles in P.cinnamomi hyphae under different g o w t h conditions is summarized diagrammatically in Figure 38. -
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Vesicle production during P. cinnamomi sporulation
'chlamydospores
Figs 34-37. The presence of ventral, dorsal and large peripheral vesicles in chlamydospores of P. cinnamomi. Fig. 34. Light micrograph of chlamydospore from 2-day mycelial colony. Bar = 23 vm. Figs 35-37. Ventral (Fig. 35), dorsal (Fig. 36) and large peripheral (Fig. 37) vesicles in cryosectioned chlamydospores immunofluorescently labelled with Vsv-I, Cpa-2 and Lpv-1 respectively. Bar = 16 pm.
Time since subculture (h) Figs 32, 33. Vesicle occurrence in mycelia growing on V8 agar during chlamydospore production. Percentage of P. cinnamomi hyphal fragments containing large peripheral vesicles labelled with Lpv-1 (O), ventral vesicles labelled with Vsv-1 (0) and dorsal vesicles labelled with Cpa-2 (0). At 0 h, a plug of mycelium was inoculated in the centre of an agar plate. Samples were taken over the next 72-80 h. As the mycelia grew, samples were taken every 5 mm across the colony, however only the values for the sample at the centre are shown. Fig. 32. Values for mycelia growing on a thin layer (2.5 mp) of V8 agar. Chlamydospores were first observed at 48 h. Fig. 33. Values for mycelia growing on a thick layer (5 mm) of V8 agar. Chlamydospores were first observed at 56 h.
DISCUSSION The major outcome of this study is the demonstration of the close association between the induction of sporulation of P. cinnamomi and the appearance of three types of vesicles destined for specific zones within the zoospore peripheral cytoplasm. The three types of vesicles were not present in rapidly growing vegetative hyphae, but appeared under conditions that induced sporulation through the production of either sporangia or chlamydospores. The three vesicles and their Lpv, Cpa and Vsv contents may thus be considered to be sporulation specific components. The unambiguous identification of these three types of vesicles has been made possible by the generation of monoclonal antibodies directed towards molecules within them (Hardham e f al., 1991). Immunolabelling of zoospores and young cysts has revealed the distribution of the vesicles within zoospores and their fate during zoospore encystment and cyst germination, information which has given clues as to
their function during plant infection. Dorsal and ventral vesicles are secreted within the first two minutes after the induction of encystment (Hardham & Gubler, 1990): large peripheral vesicles are degraded during the first few hours after cyst germination (Gubler & Hardham, 1990). The fates of the three vesicles suggest that they may be made exclusively for and function only in zoospores and cysts, however, there has been little information on whether or not they occur at other stages of the life cycle. The three types are present in mature sporangia (Hyde & Hardham, 1993) and vesicles with a morphology similar to the large peripheral vesicles occur in developing and germinating chlamydospores of P. cinnamomi (Hemmes & Wong, 1975). The aim of the present study was to determine the presence or absence of the three vesicles in all asexual stages of the life cycle of this organism and thereby to uncover spatial and temporal aspects of their synthesis and assembly, and further evidence of their function. The results of this study have revealed that the vesicles were all absent in germlings from about 2-4 h after cyst germination in hyphae growing rapidly on fresh nutrient medium. The vesicles reappeared rapidly in mycelia when the production of sporangia was induced by replacement of the nutrient medium with a mineral salts solution. Sporulation (via formation of chlamydospores) and the production of vesicles occurred more slowly when the mycelium was growing on nutrient agar. In mineral salts solution, large peripheral and ventral vesicles first appeared 5 h after nutrient removal, and dorsal vesicles about 30 min later. The first sporangia were recognizable through examination of the colony under a dissecting microscope at about 7.5 h but, since sporangial development takes about 3 h (Christen & Hohl, 1972).
47
J. D. W. Dearnaley, J. Maleszka and A. R. Hardham
be more resistant to disruption, or other components in the cells might bind to the Lpv glycoproteins and render them insoluble. The formation of large peripheral and ventral vesicles before dorsal vesicles was observed in all situations in which sporulation was induced. The reason for this lag in dorsal vesicle appearance is not clear. Once formation of dorsal vesicles has begun, production of all three vesicles occurs together. Cpa and Lpv antigens are processed within a single Golgi stack and occur together within a single cisterna (Dearnaley & Hardham, 1994). Observations of the presence of the three types of vesicles in cultures growing on nutrient agar initially raised doubts V8 agar about their association with sporulation. However, it soon became clear that their appearance was correlated with the formation of chlamydospores, thereby reinforcing evidence of the close relationship between vesicle synthesis and sporulation. One might ask why these vesicles would form during chlamydospore development if they function specifically in zoospores and germinated cysts. Chlamydospores are resting spores that perpetuate the organism through adverse conditions. They may germinate to form a small number of sporangia (Weste, 1983). The presence of the three types of Fresh nutrient al suggests that the chlamydospores zoospore ~ e r i ~ h e rvesicles medium contain the necessary complement of organelles to produce motile zoospores in the minimum amount of time when favourable conditions return. Previous studies of a range of Oomycetes have also Mineral salts solution searched for the origin of vesicles that occur in motile and Fig. 38. Diagram illustrating the presence or absence of ventral (dark encysted spores. Some organelles, such as nuclei and hexagons), dorsal (light circles) and large peripheral (ovals)vesicles mitochondria, are common to both vegetative and sporulating in hyphae, chlamydospores (top right) or sporangia (lower right) of cells, and are believed to flow into the developing sporangia P. cinnamomi growing in different media. Vesicles and chlamydospores or chlamydospores from the subtending hyphae (Hemmes & appear in cultures grown for an extended period on V8 agar; vesicles Wong, 1975; Hemmes, 1983). Other components are found and sporangia appear in cultures transferred to mineral salts solution. only in spores. In addition to the vesicles destined for the zoospore peripheral cytoplasm, dense body vesicles (fingerswelling of the hyphal apices and initiation of sporanguium print vesicles) (Gay & Greenwood, 1966), mastigonemes (the formation will have commenced at some time prior to this, tubular hairs on the anterior flagellum) (Holloway & Heath, possibly as early as about 5 h after transfer. In cultures 1977; Cope & Hardham, 1994) and other flagellar surface growing on nutrient agar, there was a larger time difference components (Cope & Hardham, 1994) appear during sporu(of the order of 4-8 h) between the first detection of vesicles lation. Evidence from Phyfophfhora (Cope & Hardham, 1994) Chlamydospores may develop more and Pyfhium (Lunney & Bland, 1976) suggests that these and chlamyd~s~ores. slowly than sporangia, or vesicle formation may precede the components also flow into the developing sporangium before onset of chlamydospore formation by several hours. septum formation. Sporangia form earlier in mycelia than they do in germlings, Analysis of vesicle origin is complicated in the diplanetic perhaps because the mycelium has acquired the necessary genera by the production of primary and secondary zoospores 'competence' (Griffin, 1981) to respond to stimuli that induce and cysts. Vesicles present in the secondary zoospores, which sporulation. In Aspergillus nidulans, for example, a minimum are equivalent to the reniform zoospores of the Peronosporales, amount of submerged vegetative growth is necessary before may be absent or morphologically distinct from their conidia will form (Champe ef al., 1981). counterparts in primary zoospores. Thus, primary encystment The inability of the immunodot blots to detect any changes vesicles (bar bodies), which are apparently homologous to in Cpa-2 antigen levels in the experiments involving germlings dorsal vesicles in P. cinnamomi, have been observed in is an indication of the masking effects of the secreted cell coat sporangia either before (Armbruster, 1982; Beakes, 1983) or which is still highly immunoreactive even 36 h after after (Holloway & Heath, 1977; Schnepf, Deichgraber & encystment (not shown). The immunodot blots also failed to Drebes, 1978) insertion of the basal septum. Primary K bodies detect initial large peripheral vesicle synthesis in germlings (K, bodies), which we believe are homologous to ventral growing in mineral salts solution. It is possible that the vesicles, have also been observed in sporangia (Holloway & extraction of Lpv antigen is not as efficient from older Heath, 1977). The saprolegnian organelles apparently homgermlings as it is from younger germlings. The hyphae may ologous to the large peripheral vesicles of the peronosporalean - -
Vesicle production during P. cinnamomi sporulation species are the fibrous vesicles. A 'precursor' organelle for fibrous vesicles has not been seen in primary zoospores, but fibrous vesicles, along with secondary encystment vesicles and secondary K bodies (K, bodies) develop in primary cysts (Holloway & Heath, 1977; Beakes, 1983; Lehnen & Powell, 1991). Sporulation of fungal colonies is favoured or induced by nutritional conditions that restrict growth (Dahlberg & Van Etten, 1982; Ribeiro, 1983; Hohl, 1990). There is considerable interest in the phenomenon of sporulation in fungi. One of the reasons for this is that a number of fungi constitute good model systems for studies of the mechanisms underlying processes of differentiation and development. Changes in patterns of proteins (e.g. Springer ef a/., 1992) or mRNAs (e.g. Marshall & Timberlake, 1991; Sato ef al., 1994) have been observed during sporulation but in most cases, the identity and function of the proteins or gene products are unknown. We have shown in this study that the three zoospore peripheral vesicles are sporulation specific components, and that the monoclonal antibodies, Lpv-1, Cpa-2 and Vsv-1 provide a rare opportunity for studies of the synthesis of sporulation specific molecules whose nature, and putative function are known. Future molecular genetic analysis will elucidate details of the transcriptional or translational mechanisms that control their synthesis.
48
Gay. J. L. & Greenwood. A. D. (1966). Structural aspects of zoospore production in Saprolegnia ferax with particular reference to the cell and vacuolar membranes. In Tke Fungus Spore (ed. M. F. Madelin), pp. 95-108. Butterworths: London. U.K. Grifin, D. H. (1981). Fungal Physiology. John Wiley & Sons: New York, U.S.A. Gubler, F. & Hardham, A. R. (1988). Secretion of adhesive material during encystment of Phytophthora cinrrarnomi zoospores, characterized by immunogold labelling with monoclonal antibodies to components of peripheral vesicles. Journal of Cell Science 90, 225-235. Gubler, F. & Hardham. A. R. (1990). Protein storage in large peripheral vesicles in Phytophthora zoospores and its breakdown after cyst germination. Experimental Mycology 14, 393-404. Hardham, A. R. & Gubler, F. (1990). Polarity of attachment of zoospores of a root pathogen and pre-alignment of the emerging germ tube. Cell Biology I~rternationnlReports 14, 947-956. Hardham, A. R., Gubler. F., Duniec, J. &Elliott, J. (1991). A review of methods for the production and use of monoclonal antibodies to study zoosporic plant pathogens. Journal of Microscopy 162. 305-318. Hemmes, D. E. & Wong, L. D. S. (1975). Ultrastructure of chlamydospores of Pkytophthorn cinnarnomi during development and germination. Carladinn Journal of Botany 53, 294552957, Hemmes. D. E. (1983). Cytology of Phytopktkora. In Phytophthora. Its B~ology. Taxonomy, Ecology, and Patitology (ed. D. C. Erwin, S. Bartn~cki-Garcia& P. H. Tsao), pp. 9-40. American Phytopathological Society: St Paul, MN, U.S.A. Holloway, S. A. & Heath, I. B. (1977). An ultrastructural analysis of the changes in organelle arrangement and structure between the various spore types of Saprolegnia. Canadian Journal of Botany 55. 1328-1339. Hyde, G. J. & Hardham, A. R. (1993).Microtubules regulate the generation of polarity in zoospores of Phytophthora cintlamomi. Europea~rJournal of Cell Biology 62, 75-85. Lehnen, L. P. Jr. & Powell, M. J. (1991). Formation of K,-bodies in prtmary REFERENCES cysts of Saprolegnia ferax. Mycologia 83, 163-179. Lunney, C. Z. & Bland, C. E. (1976). An ultrastructural study of zoosporoArmbruster, B. L. (1982). Sporangiogenesis in three genera of the Saprolegniaceae. I. Pre-sporangium hyphae to early primary spore initial stage. genesis in Pythinm proliferrrm de Bary. Protoplasma 88, 85-100. Mycologia 74, 433-459. Marshall, M. A. & Timberlake, W. E. (1991). Aspergillus nidulans wetA Beakes, G. W. (1983). A comparative account of cyst coat ontogeny in activates spore-specific gene expression. Molecrrlar and Cellular Biology 11, 5542. saprophytic and fish-lesion (pathogenic) isolates of the Saprolegnia diclinaMircetich, S. M. & Zentmyer, G. A. (1966). Production of oospores and parasitica complex. Canadian Journal of Botany 61, 603-625. Carlile, M. J. (1983). Motility, taxis, and tropism in Phytophthora. In chlamydospores of Phytophthora cinnamomi in roots and soil. Pkytopatkology Phytophthora. Its Biology, Taxonomy, Ecology, and Pathology (ed. D. C. Erwin, 56, 1076-1078. S. Bartnicki-Garcia & P. Tsao), pp. 95-107. American Phyt~patholo~ical Ribeiro, 0.K. (1983).Physiology of asexual sporulation and spore germination in Pl~~tophthora.In Phytophthora. Its Biology, Taxonomy, Ecology, and Society: St Paul, MN, U.S.A. Pathology (ed. D. C. Erwin, S. Bartnicki-Garcia & P. Tsao), pp. 55-70. Champe, S. P., Kurtz, M. B., Yager, L. N., Butnick, N. J. & Axelrod, D. E. American Phytopathological Society: St Paul, MN, U.S.A. (1981). Spore formation in Aspergillns nidulans: competence and other Widyastuti, U., Hotta, Y. & Tabata, S. (1994). Identification Sato, S., Suzuki, H., developmentai processes. In 7he Fungal Spore: Morphogenetic Controls (ed. and characterization of genes induced during sexual differentiation in G. Turian & H. R. Hohl), pp. 255-276. Academic Press: London, U.K. Schizosaccharomyces pombe. Current Genetics 26, 31-37. Christen, J. & Hohl, H. R. (1972). Growth and ultrastructural differentiation of sporangia in Phytophthora palmiuora. Canadian Journal of Microbrology 18, Schnepf, E., Deichgraber, G. & Drebes, G. (1978). Development and ultrastructure of the marine, parasitic Oomycete, Lagenisma coscinodisci 1959-1964. Cope, M. & Hardham, A. R. (1994). Synthesis and assembly of flagellar Drebes (Lagenidiales): Formation of the primary zoospores and their release. Protoplasma 94, 263-280. surface antigens during zoosporogenesis in Phytophthora cinnamomi. Protoplasma 180, 158-168. Springer, M. L., Hager, K. M., Garrett-Engele, C. & Yanofsky, C. (1992). Dahlberg, K. R. & Van Etten, J. L. (1982). Physiology and biochemistry of Timing of synthesis and cellular localization of two conidiation-specific proteins of Neurospora crassa. Developmental Biology 152, 255-262. fungal sporulation. Annual Reuiew of Phytopathology 20, 281-301. Dearnaley, I. D. W. & Hardham, A. R. (1994). The Golgi apparatus of Weste, G. (1983). Population dynamics and survival of Phytophthora. In Phytophthora. Its Biology, Taxonomy, Ecology, and Pathology (ed. D. C. Erwin, Phytophthora cinnamomi. makes three types of secretory or storage vesicles concdrrently. Protoplasm 182, 75-79. S. Bartnicki-Garcia & P. Tsao), pp. 237-257. American Phytopathological Society: St Paul, MN, U.S.A. Englander, L. & Turbitt, W. (1979). Increased chlamydospore production by Phytophthora cinnamomi using sterols and near-ultraviolet light. PhytoZentmyer, G. A. (1980). Phytophthora cinnamomi and the Diseases rt Causes. The pathology 69, 813-817. American Phytopathological Society: St Paul, MN, U.S.A.
(Accepted 22 M a y 1995)
Mycol. Res. 100 (1):39-48 (1996) Printed in Great Britain
Synthesis of zoospore peripheral vesicles during sporulation of Phytophthora cinnamorni
J. D. W. DEARNALEY, J. MALESZKA A N D A. R. HARDHAM Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, Canberra, A.C.T. 2601, Australia
Three types of spherical vesicles (dorsal, ventral and large peripheral vesicles) are found in the peripheral cytoplasm of Phytophfhora c~nnamomizoospores. Each is believed to participate in the infection of host plants through formation of a cyst coat, deposition of an adhesive pad or supply of proteins during early germling growth. In the present study, the occurrence of these three vesicles was determined during the asexual life cycle of P. cinnamomi. The vesicles were found to be absent from rapidly growing, vegetative hyphae, but to appear in the hyphae under conditions that induced sporulation. Vesicle formation was correlated with sporulation via the production of sporangia or chlamydospores. In mycelia transferred to nutrient-poor medium, large peripheral and ventral vesicles first appeared about 5 h after transfer; dorsal vesicles appeared about 30 min later. The first sporangia were detected at 75 h. Large peripheral and ventral vesicles were also produced before dorsal vesicles in mycelia growing on nutrient agar and in the hyphae formed by germinating cysts. On nutrient agar, the three vesicle types appeared about 10 h in advance of the development of chlamydospores.
Phytophthora cinnamomi Rands, the Dieback fungus, is an Oomycete pathogen with a broad host range (Zentmyer, 1980). Motile biflagellate zoospores are the major infective agent and are produced by the cleavage of multinucleate sporangia. Invasion of hosts is mediated by chemotaxis of the zoospores and the subsequent encystment of these cells on plant roots (Carlile, 1983). Under conditions not suitable for the development of sporangia, mycelia will form chlamydospores which can perpetuate the pathogen for long periods of time in the absence of hosts (Mircetich & Zentmyer, 1966). When favourable conditions return, chlamydospores germinate and form mycelia, sporangia or new chlamydospores (Weste, 1983). Three types of immunologically distinct vesicles localized in discrete zones in the peripheral cytoplasm of P. cinnamomi zoospores appear to play important roles in zoospore encystment and early germ tube growth (Gubler & Hardham, 1990; Hardham & Gubler, 1990; Hardham ef al., 1991). Large peripheral vesicles occur predominantly beneath the dorsal surface of the zoospores (Gubler & Hardham, 1988). They contain glycoproteins with a relative molecular weight of over 300 kDa. They become randomly distributed during encystment and appear to serve as a source of proteins that are used to support the first few hours of growth of the germinated cyst (Gubler & Hardham, 1990). Ventral vesicles are concentrated along the ridges of the groove that runs along the ventral surface of the zoospores. They contain a protein whose relative molecular weight is over 220 kDa (Hardham & Gubler, 1990). These vesicles are rapidly secreted during encystment, and their contents form a plaque of
material believed to function in the adhesion of the cyst to the adjacent surface (Hardham & Gubler, 1990). Dorsal vesicles occur predominantly under the dorsal surface of the zoospores. They contain glycoproteins over 300 kDa in relative molecular weight. Their contents are secreted during encystment and form a coating of material that surrounds the cysts (Gubler & Hardham, 1988). The available evidence indicates that the three zoospore peripheral vesicles function during zoospore encystment and early germling growth, and suggests that they might be synthesised exclusively during zoosporogenesis. There is, however, little information on the presence or absence of these vesicles at other stages of the life cycle of P. cinnamomi. In the present study we have used monoclonal antibodies Lpv-1, Vsv-1 and Cpa-2 which are specific for the large peripheral, ventral and dorsal vesicles, respectively, to determine the site and timing of vesicle synthesis throughout the asexual life cycle of P. cinnamomi. W e have discovered that the vesicles are absent from rapidly growing vegetative hyphae but are synthesized when the production of sporangia or chlamydospores is induced. The vesicles are thus sporulation specific components.
MATERIALS A N D METHODS Zoospore, cyst and germling production Culturing and production of zoospores from Phyfophfhora cinnamomi (6BR; DAR 52646) followed methods described in Hardham ef al. (1991). Seven small squares (5 mm x 5 mm) of
Vesicle production during P. cinnamomi sporulation
Figs 1-12. For captions see facing page
J. D. W. Dearnaley, J. Maleszka and A. R. Hardham
@
@
vsv- 1
dD
sterile 100 ml Schott bottle (Duran, Mainz, Germany). To produce germlings, approximately 2-5 x lo6 germinated cysts were inoculated into 10 ml of 5 % V8 broth and incubated at 25' in 15 ml culture tubes (Disposable Products, Adelaide, Australia). In some experiments, after 6 h the V8 broth was replaced with mineral salts solution and samples taken after 0, 6, 18 and 30 h incubation.
Chlamydospore production Chlamydospores were produced via the method of Englander & Tubitt (1979). A small piece of V8 agar with mycelium was Lpv- 1
Fig. 13. Immunodot blot of cell extracts used to detect changes in the relative abundance of the three antigens in germlings growing in 5 % V8 broth for 10 h. The level of ventral vesicle antigen did not change during the first 2 h after encystment, but decreased between 2 and 10 h. No changes in the level of dorsal vesicle antigen were detected over the 10 h period. A decline in large peripheral vesicle antigen was observed between 2 h and 4 h.
mycelia were cut from the edge of a colony of P. cinnamomi growing on V8 nutrient agar containing 10% V8 juice (Campbell's Soups Pty Ltd, Lemnos, Australia), 0.002 % Psitosterol (Sigma Chemical Co., St Louis, MO, U.S.A.), 0.01% CaCO, and 1.7% Bacto agar (Difco, Detroit, MI, U.S.A.). The squares were inoculated onto a sterile miracloth disc overlying V8 nutrient agar and incubated for 5 d at 25 'C in the dark. Each disc was then transferred to 100 ml of 5 % V8 broth (5 % cleared V8 juice, 0.01 % CaCO,, 0.002 % p-sitosterol) and shaken overnight at 150 rpm at 22'. The cultures were washed three times in mineral salts solution (10 mM Ca (NO,),, 5 mM KNO,, 4 mM MgSO, and 2 ml1-I of a solution containing 10 mM FeSO, and 10 mM Na, EDTA) and incubated at 22' for 24 h in 100 ml of this solution, during which time sporangia developed. Cleavage of sporangia was induced by rinsing miracloth cultures three times in cold double-distilled water and incubating them at 4' for 13 min in 10 ml of distilled water. The discs were then transferred to a light box at 18'. Zoospores were released 60-75 min later. Zoospores were induced to encyst by agitation for 20 s in a
placed onto a V8 agar in a Petri plate and incubated in the light at 25'. Samples were taken at intervals over the next 80 h and the presence or absence of chlamydospores noted.
Sporangium production Small (5 mm x 5 mm) pieces of agar with mycelium were taken from the growing edge of a colony on V8 agar, placed in 5 % V8 broth and incubated in the dark at 25' for 17 h. The samples were then washed three times in mineral salts solution and incubated in the light in a final volume of 15 ml of mineral salts at 22'. Samples were taken at intervals over the next 11 h.
Immunofluorescent labelling Cysts, germlings and hyphae were fixed in 4% paraformaldehyde in 50 mM Pipes (piperazine-N, N-bis[2-ethanesulphonic acid]) buffer (pH 7.0) for 1h at room temperature. After rinsing twice in Pipes buffer the cells were frozen in Tissue Tek embedding compound (Miles Inc., Elkhart, IN, U.S.A.) in plastic moulds by plunging in liquid nitrogen. Ten urn thick sections of cysts and germlings and 12 pm thick sections of hyphae were cut on a Reichert-Jung 2800 Frigocut E cryotome and dried at room temperature onto poly-L-lysine coated slides. Immunostaining was carried out in 10 cm square dishes lined with moist filter paper. The sections were incubated for 45 min at 37' in one of three monoclonal antibodies raised against P. cinnamomi zoospore vesicle antigens. Monoclonal antibody Vsv-1 reacts with the contents of ventral vesicles and was purified and used at 1 pg ml-'. Cpa-2 reacts with the contents of dorsal vesicles and was purified and used at 0.5 pg ml-l. Lpv-1 reacts with the
Figs 1-12. Zoospore peripheral vesicles in Phytophthora cinnamomi cysts and gerrnlings growing in 5 % V8 broth. In each case observations are paired, with the immunofluorescence micrograph above and differential interference contrast micrograph of the same field of view below. Figs 1-4. Vsv-1; Figs 5-8. Cpa-2; Figs 9-12. Lpv-1. Bar = 20 pm.Figs 1-4. At 5 min (Fig. I), 2 h (Fig. 2) and 4 h (Fig. 3) after encystment, Vsv-1 labelling showed that a few ventral vesicles (arrowheads) could be observed in the cyst cytoplasm and the germ tubes. The vesicles had disappeared by 10 h (Fig. 4). Most of the Vsv-1 antigen was secreted onto the cyst surface (Figs 3, 4) during the first few minutes after the onset of encystment. This material is out of the plane of focus in Figs 1 and 2. Figs 5-8. Labelling of 5 rnin old cyst with Cpa-2 showed that the contents of the dorsal vesicles were completely secreted during encystment to form a coat on the outside of the cyst (Fig. 5). This coat was still present on the surface of the cyst at 2 h (Fig. 6), 4 h (Fig. 7) and 10 h (Fig. 8) after encystment. Figs 9-12. Labelling of 5 min old cysts with Lpv-1 showed that the large peripheral vesicles were not secreted during encystment (Fig. 9). These vesicles remained in the cyst and were present in germ tubes at 2 h (Fig. 10). By 4 h large peripheral vesicles had disappeared from germlings (Fig. 11) and they were still absent at 10 h (Fig. 1.2). At no stage in the 10 h period was the Lpv-1 antigen observed on the surface of the germlings. Photographic exposures for Figs 11 and 12 are the same as those for Figs 9 and 10.
Vesicle production during P. cinnamomi sporulation
Figs 14-25. For captions see facing page
42
J. D. W. Dearnaley, J. Maleszka and A. R. Hardham contents of large peripheral vesicles and was used as hybridoma supernatant diluted 1 : r O in phosphate buffered saline (PBS; 20 mM sodium phosphate, 150 mM NaC1) containing - 1 % bovine serum albumin (BSA). After three washes in PBS, the sections were incubated in sheep antimouse immunoglobulin-fluorescein isothioc~anate (SAMFITC [Silenus, Australia]) diluted 1:60 in 1 % BSA-PBS, for 45 min at 37'. Sections were subsequently washed three times in PBS and once in distilled water. They were then dried and mounted on glass slides in mowiol containing 0.1% p-phenylenediamine. The samples were examined and photographed with a Zeiss Axioplan microscope equipped with epifluorescence optics with an F 1 filter cube (excitation 450-490 nm; dichroic mirror 510 nm; barrier 515-565 nm). For quantitative analysis of the occurrence of the three types of vesicles, triplicate samples (blocks) of the mycelium were taken from different parts of the colony or at different times during the culturing protocols. Cryosections were cut from at least two of the blocks at two different depths within the mycelium, giving a total of four different regions of sampling. The sections at each region were examined after immunolabelling for fluorescence microscopy and the presence or absence of each vesicle type was determined in 100 hyphal fragments for each group of sections. The average percentage of hyphae containing each vesicle type was determined from the four samples for each time or location point. Experiments analysing the appearance of vesicles following transfer of the mycelium to mineral salts solution were repeated five times. Experiments examining the presence of vesicles across colonies growing on V8 agar or during chlamydospore formation were each conducted four times.
ande en on^.
Immunodot blot analysis Cysts and germlings were frozen in liquid nitrogen, lyophilized, then ground in eppendorf tubes using a glass rod. The ground material was extracted for 20 min with 6 M guanidine hydrochloride, then diluted 50 times with Tris buffered saline (TBS; 10 mM Tris-HC1, 150 mM NaC1, pH 7.4). The samples were incubated on ice for 10 min before centrifuging at 13000 rpm for 10 min. Solubilized material was transferred to nitrocellulose in an immunodot blot apparatus (Biorad, Nth Ryde, NSW, Australia). The efficacy of the transfer was monitored by staining with 0.2% Ponceau S in 3 % trichloroacetic acid for 5 min. After blocking nonspecific binding sites for 1h in 5 % skim milk powder in TBS and washing three times in TBS-0.1% Tween-20, the nitrocellulose was incubated for 45 min in undiluted supernatants of Vsv-1
43 or Lpv-1, or 3 pg ml-' purified Cpa-2 in 1% BSA-PBS. Bound antibody was visualized with peroxidase-conjugated SAM (Silenus) using 4-chloro-1-napthol as substrate.
RESULTS Zoospore peripheral vesicles in germlings
growing in 5%
V8 broth Labelling of 5 min old cysts (cysts fixed 5 min after the induction of encystment) with Vsv-1 showed that most of the ventral vesicle antigen was secreted and coated part of the surface of the cysts (see Fig. 5 in Hardham & Gubler, 1990), although some ventral vesicles remained in the cytoplasm of the cyst (Fig. I). At 2 h (Fig. 2) and 4 h (Fig. 3) after encystment, ventral vesicles were still observed in the germlings but by 10 h after encystment (Fig. 4), all ventral vesicles had disappeared. The secreted Vsv-I antigen remained associated with the cyst surface during this period (Figs 3, 4). Labelling of 5 min old cysts with Cpa-2 showed that the contents of the dorsal vesicles were completely secreted during encystment to form a coat on the outside of the cyst (Fig. 5). This coat remained on the surface of the cyst during the next 10 h of germling g o w t h (Figs 6-8). No dorsal vesicles were seen in germlings during the 10h period. Labelling of 5 min old cysts with Lpv-1 showed that the contents of the large peripheral vesicles were not secreted during encystment (Fig. 9). These vesicles remained in the cyst and were present in the germlings at 2 h (Fig. lo), but by 4 h they had completely disappeared (Fig. 11). They were also absent at 10 h (Fig. 12). At no stage was the Lpv-1 antigen observed on the surface of the germlings. Changes in the relative abundance of the three antigens in germlings growing in 5 % V8 broth were analysed using immunodot blotting of cell extracts (Fig. 13). The level of ventral vesicle antigen did not change during the first 2 h after encystment, but decreased during subsequent growth. No changes in the level of dorsal vesicle antigen were detected over the 10 h period. A decline in large peripheral vesicle antigen was observed between 2 h and 4 h.
Peripheral vesicles in germlings transferred to mineral salts solution At the time of transfer to mineral salts solution (6 h after encystment), the germlings did not contain any ventral, dorsal or large peripheral vesicles (Figs 14, 18, 22) although secreted ventral and dorsal vesicle material coated the cyst surface (Figs 14, 18). Both ventral (Fig. 15) and large peripheral (Fig. 23)
Figs 14-25. Zoospore peripheral vesicles in P. cinnamomi germlings transferred to mineral salts solution. In each case observations are paired, with the immunofluorescence micrograph above and differential interference contrast micrograph of the same field of view below. Figs 14-17. Vsv-1; Figs 18-21. Cpa-2; Figs 22-25. Lpv-1. Bar = 20 pm. Figs 14-17. Ventral vesicles were absent at the time of transfer to mineral salts solution (6 h germlings, Fig. 14) but after 6 h in the mineral salts solution, they had reappeared (Fig. 15) and they increased in number over the next 12 h (Fig. 16) and 24 h (Fig. 17). Figs 18-21. Dorsal vesicles were not present at the time of transfer (Fig. 18) or after 6 h (Fig. 19) or 18 h (Fig. 20) in the mineral salts solution. After 30 h in the mineral salts solution dorsal vesicles were present (Fig. 21) and at this time sporangia (s) had appeared. Figs 22-25. Large peripheral vesicles were absent at the time of transfer to mineral salts solution (6 h germlings, Fig. 22) but after 6 h in the mineral salts solution they had reappeared (Fig. 23) and they increased in number over the next 12 h (Fig. 24) and 24 h (Fig. 25).
..
Vesicle production during P. cinnamami sporulation 0
vsv- 1
6
18
30
.....
zoospore
germling
. a @
encystment 5 min
cyst germination V8 broth 2 h
Lpv- 1
Fig. 26. Immunodot blot of cell extracts used to determine changes in the relative abundance of the three antigens after germlings were transferred to mineral salts solution after 6 h in 5 % V8 broth. After 6 h incubation in the mineral salts solution the Vsv-1 antigen was detected and levels of the antigen increased over the next 24 h. No changes were evident in the level of Cpa-2 antigen over the 30 h period. Lpv-1 antigen was first detected 18 h after transfer to mineral salts solution.
vesicles were present 6 h after transfer to mineral salts solution. The number of ventral (Figs 16, 17) and large peripheral (Figs 24, 25) vesicles in the hyphae increased over the following 24 h. Dorsal vesicles appeared between 1 8 h and 3 0 h after transfer to mineral salts solution (Figs 19-21). Sporangia were observed in the germling culture at 30 h (Fig. 21). Immunodot blotting of extracts from germlings incubated in mineral salts solution detected the appearance of Vsv-1 antigen after 6 h and its increase over the next 12 h (Fig. 26). No changes in the level of Cpa-2 antigen were evident. Lpv-1 antigen was first detected 1 8 h after transfer to mineral salts solution. The presence or absence of the three types of vesicles in zoospores, cysts and germlings is summarized diagrammatically in Fig. 27.
Zoospore peripheral vesicles in mycelia, sporangia and chlamydospores Samples of P. cinnamomi mycelia were immunofluorescently labelled and examined for the presence or absence of the three types of vesicles. The occurrence of the vesicles was analysed by counting the percentage of hyphal fragments containing each vesicle type in cryosections of samples taken during a culture protocol used to produce sporangia. The experiment was repeated five times and in each case the results were similar to those shown in Fig. 28. The initial sample was mycelium that had been growing on V8 agar for 5 d. All three
Fig. 27. Diagram illustrating the presence or absence of ventral (dark hexagons), dorsal (light circles) and large peripheral (ovals) vesicles in zoospores, cysts and germlings of P. cinnamomi at different stages of growth in nutrient broth or mineral salts solution.
4
Time in mineral salts solution (h)
Fig. 28. Vesicle occurrence during culture regime for production of sporangia. Percentage of P. cinnamomi hyphal fragments containing large peripheral vesicles labelled with Lpv-1 (01,ventral vesicles and dorsal vesicles labelled with Cpa-2 (0). labelled with Vsv-1 (0) Samples of mycelia growing on V8 agar were transferred to V8 broth and incubated for 18 h (0 h time point). Cultures were then transferred to mineral salts solution and sampled over the next 11 h. Sporangia were first observed at 7.5 h.
vesicles were present in these samples, with ventral and large peripheral vesicles being much more abundant than dorsal vesicles (Fig. 28). When mycelia were transferred to 5% V8 broth and incubated for 1 8 h, all . peripheral vesicles disappeared. The mycelia were then transferred to mineral salts solution. Ventral and large peripheral vesicles first appeared 5 h after transfer to mineral salts solution and rapidly increased in number to a plateau level b y about 7 h. Dorsal vesicles began to appear about 30 min after formation of ventral and large peripheral vesicles. They increased in number at a slower rate than the latter, reaching a plateau frequency at 11-12 h after transfer to mineral salts solution. In -
J. D. W. Dearnaley, J. Maleszka and A. R. Hardham
0
0
1 2 Radial distance (cm)
3
Figs 29, 30. Vesicle occurrence in mycelia growing across VS agar. Percentage of P. cinnamomi hyphal fragments containing large peripheral vesicles labelled with Lpv-1 (O), ventral vesicles labelled with Vsv-1 (0) and dorsal vesicles labelled with Cpa-2 (0). Samples were taken every 5 mm from the centre of the plate. Fig. 29. Samples taken 3 d after inoculation. Fig. 30. Samples taken 5 d after inoculation.
this and the other four experiments, sporangia first appeared at 7.5 h after transfer to mineral salts solution. Another set of experiments examined the occurrence of the three vesicle types in hyphae at different distances from the centre of a colony growing on V8 agar plates. In plates inoculated 2 d (data not shown) or 3 d (Fig. 29) before sampling, most hyphae at the centre of the colony, but very few at the leading edge, contained vesicles. However, samples plates, taken 5 d (or more) after inoculation onto the V8 agar . . showed the presence of all three vesicle types throughout the colony (Fig. 30). These data are summarized diagrammatically in Fig. 31. Although sporangia do not normally form in mycelia growing on nutrient agar, after growing on V8 agar plates for 2-3 d, P. cinnamomi mycelia produced chlamydospores. The relationship between chlamydospore formation and the
Fig. 31. Diagram illustrating the presence or absence of ventral (dark hexagons), dorsal (light circles) and large peripheral (ovals) vesicles in hyphae of P. cinnamorrli growing on V8 agar plates. A, sample taken from a confluent culture; B, sample taken from the leading edge of a colony growing across a fresh V8 agar plate; C, samples taken from the edge or the centre of a colony.
appearance of the three vesicles was examined by sampling .. expanding colonies at intervals after subculture onto V8 agar plates. Both thin (2.5 mm) and thick (5 mm) layers of V8 agar were used. The results showed that the abundance of vesicles decreased after inoculation of the sample onto the fresh agar plates. O n thin plates, the vesicles were not detectable for about 20 h and then ventral and large peripheral vesicles rapidly reappeared between 32 and 40 h after subculture (Fig. 32). Dorsal vesicles began to appear at about 40 h and the first chlamydospores were observed at 48 h. The same trend was seen on the thick plates but the time of both vesicle reappearance and chlamydospore formation was delayed by about 8 h (Fig. 33). All three vesicles were observed within chlamydospores (Figs 34-37). In these experiments, at 40 h and thereafter the expanding colony was sampled at 5 mm intervals from the inoculation site at the centre of the colony. For clarity, only data from the central sample are shown. Vesicle occurrence in other positions displayed the same trends shown in Figures 32 and 33. Chlamydospores were first observed at the centre of the colony. The presence or absence of vesicles in P.cinnamomi hyphae under different g o w t h conditions is summarized diagrammatically in Figure 38. -
-
Vesicle production during P. cinnamomi sporulation
'chlamydospores
Figs 34-37. The presence of ventral, dorsal and large peripheral vesicles in chlamydospores of P. cinnamomi. Fig. 34. Light micrograph of chlamydospore from 2-day mycelial colony. Bar = 23 vm. Figs 35-37. Ventral (Fig. 35), dorsal (Fig. 36) and large peripheral (Fig. 37) vesicles in cryosectioned chlamydospores immunofluorescently labelled with Vsv-I, Cpa-2 and Lpv-1 respectively. Bar = 16 pm.
Time since subculture (h) Figs 32, 33. Vesicle occurrence in mycelia growing on V8 agar during chlamydospore production. Percentage of P. cinnamomi hyphal fragments containing large peripheral vesicles labelled with Lpv-1 (O), ventral vesicles labelled with Vsv-1 (0) and dorsal vesicles labelled with Cpa-2 (0). At 0 h, a plug of mycelium was inoculated in the centre of an agar plate. Samples were taken over the next 72-80 h. As the mycelia grew, samples were taken every 5 mm across the colony, however only the values for the sample at the centre are shown. Fig. 32. Values for mycelia growing on a thin layer (2.5 mp) of V8 agar. Chlamydospores were first observed at 48 h. Fig. 33. Values for mycelia growing on a thick layer (5 mm) of V8 agar. Chlamydospores were first observed at 56 h.
DISCUSSION The major outcome of this study is the demonstration of the close association between the induction of sporulation of P. cinnamomi and the appearance of three types of vesicles destined for specific zones within the zoospore peripheral cytoplasm. The three types of vesicles were not present in rapidly growing vegetative hyphae, but appeared under conditions that induced sporulation through the production of either sporangia or chlamydospores. The three vesicles and their Lpv, Cpa and Vsv contents may thus be considered to be sporulation specific components. The unambiguous identification of these three types of vesicles has been made possible by the generation of monoclonal antibodies directed towards molecules within them (Hardham e f al., 1991). Immunolabelling of zoospores and young cysts has revealed the distribution of the vesicles within zoospores and their fate during zoospore encystment and cyst germination, information which has given clues as to
their function during plant infection. Dorsal and ventral vesicles are secreted within the first two minutes after the induction of encystment (Hardham & Gubler, 1990): large peripheral vesicles are degraded during the first few hours after cyst germination (Gubler & Hardham, 1990). The fates of the three vesicles suggest that they may be made exclusively for and function only in zoospores and cysts, however, there has been little information on whether or not they occur at other stages of the life cycle. The three types are present in mature sporangia (Hyde & Hardham, 1993) and vesicles with a morphology similar to the large peripheral vesicles occur in developing and germinating chlamydospores of P. cinnamomi (Hemmes & Wong, 1975). The aim of the present study was to determine the presence or absence of the three vesicles in all asexual stages of the life cycle of this organism and thereby to uncover spatial and temporal aspects of their synthesis and assembly, and further evidence of their function. The results of this study have revealed that the vesicles were all absent in germlings from about 2-4 h after cyst germination in hyphae growing rapidly on fresh nutrient medium. The vesicles reappeared rapidly in mycelia when the production of sporangia was induced by replacement of the nutrient medium with a mineral salts solution. Sporulation (via formation of chlamydospores) and the production of vesicles occurred more slowly when the mycelium was growing on nutrient agar. In mineral salts solution, large peripheral and ventral vesicles first appeared 5 h after nutrient removal, and dorsal vesicles about 30 min later. The first sporangia were recognizable through examination of the colony under a dissecting microscope at about 7.5 h but, since sporangial development takes about 3 h (Christen & Hohl, 1972).
47
J. D. W. Dearnaley, J. Maleszka and A. R. Hardham
be more resistant to disruption, or other components in the cells might bind to the Lpv glycoproteins and render them insoluble. The formation of large peripheral and ventral vesicles before dorsal vesicles was observed in all situations in which sporulation was induced. The reason for this lag in dorsal vesicle appearance is not clear. Once formation of dorsal vesicles has begun, production of all three vesicles occurs together. Cpa and Lpv antigens are processed within a single Golgi stack and occur together within a single cisterna (Dearnaley & Hardham, 1994). Observations of the presence of the three types of vesicles in cultures growing on nutrient agar initially raised doubts V8 agar about their association with sporulation. However, it soon became clear that their appearance was correlated with the formation of chlamydospores, thereby reinforcing evidence of the close relationship between vesicle synthesis and sporulation. One might ask why these vesicles would form during chlamydospore development if they function specifically in zoospores and germinated cysts. Chlamydospores are resting spores that perpetuate the organism through adverse conditions. They may germinate to form a small number of sporangia (Weste, 1983). The presence of the three types of Fresh nutrient al suggests that the chlamydospores zoospore ~ e r i ~ h e rvesicles medium contain the necessary complement of organelles to produce motile zoospores in the minimum amount of time when favourable conditions return. Previous studies of a range of Oomycetes have also Mineral salts solution searched for the origin of vesicles that occur in motile and Fig. 38. Diagram illustrating the presence or absence of ventral (dark encysted spores. Some organelles, such as nuclei and hexagons), dorsal (light circles) and large peripheral (ovals)vesicles mitochondria, are common to both vegetative and sporulating in hyphae, chlamydospores (top right) or sporangia (lower right) of cells, and are believed to flow into the developing sporangia P. cinnamomi growing in different media. Vesicles and chlamydospores or chlamydospores from the subtending hyphae (Hemmes & appear in cultures grown for an extended period on V8 agar; vesicles Wong, 1975; Hemmes, 1983). Other components are found and sporangia appear in cultures transferred to mineral salts solution. only in spores. In addition to the vesicles destined for the zoospore peripheral cytoplasm, dense body vesicles (fingerswelling of the hyphal apices and initiation of sporanguium print vesicles) (Gay & Greenwood, 1966), mastigonemes (the formation will have commenced at some time prior to this, tubular hairs on the anterior flagellum) (Holloway & Heath, possibly as early as about 5 h after transfer. In cultures 1977; Cope & Hardham, 1994) and other flagellar surface growing on nutrient agar, there was a larger time difference components (Cope & Hardham, 1994) appear during sporu(of the order of 4-8 h) between the first detection of vesicles lation. Evidence from Phyfophfhora (Cope & Hardham, 1994) Chlamydospores may develop more and Pyfhium (Lunney & Bland, 1976) suggests that these and chlamyd~s~ores. slowly than sporangia, or vesicle formation may precede the components also flow into the developing sporangium before onset of chlamydospore formation by several hours. septum formation. Sporangia form earlier in mycelia than they do in germlings, Analysis of vesicle origin is complicated in the diplanetic perhaps because the mycelium has acquired the necessary genera by the production of primary and secondary zoospores 'competence' (Griffin, 1981) to respond to stimuli that induce and cysts. Vesicles present in the secondary zoospores, which sporulation. In Aspergillus nidulans, for example, a minimum are equivalent to the reniform zoospores of the Peronosporales, amount of submerged vegetative growth is necessary before may be absent or morphologically distinct from their conidia will form (Champe ef al., 1981). counterparts in primary zoospores. Thus, primary encystment The inability of the immunodot blots to detect any changes vesicles (bar bodies), which are apparently homologous to in Cpa-2 antigen levels in the experiments involving germlings dorsal vesicles in P. cinnamomi, have been observed in is an indication of the masking effects of the secreted cell coat sporangia either before (Armbruster, 1982; Beakes, 1983) or which is still highly immunoreactive even 36 h after after (Holloway & Heath, 1977; Schnepf, Deichgraber & encystment (not shown). The immunodot blots also failed to Drebes, 1978) insertion of the basal septum. Primary K bodies detect initial large peripheral vesicle synthesis in germlings (K, bodies), which we believe are homologous to ventral growing in mineral salts solution. It is possible that the vesicles, have also been observed in sporangia (Holloway & extraction of Lpv antigen is not as efficient from older Heath, 1977). The saprolegnian organelles apparently homgermlings as it is from younger germlings. The hyphae may ologous to the large peripheral vesicles of the peronosporalean - -
Vesicle production during P. cinnamomi sporulation species are the fibrous vesicles. A 'precursor' organelle for fibrous vesicles has not been seen in primary zoospores, but fibrous vesicles, along with secondary encystment vesicles and secondary K bodies (K, bodies) develop in primary cysts (Holloway & Heath, 1977; Beakes, 1983; Lehnen & Powell, 1991). Sporulation of fungal colonies is favoured or induced by nutritional conditions that restrict growth (Dahlberg & Van Etten, 1982; Ribeiro, 1983; Hohl, 1990). There is considerable interest in the phenomenon of sporulation in fungi. One of the reasons for this is that a number of fungi constitute good model systems for studies of the mechanisms underlying processes of differentiation and development. Changes in patterns of proteins (e.g. Springer ef a/., 1992) or mRNAs (e.g. Marshall & Timberlake, 1991; Sato ef al., 1994) have been observed during sporulation but in most cases, the identity and function of the proteins or gene products are unknown. We have shown in this study that the three zoospore peripheral vesicles are sporulation specific components, and that the monoclonal antibodies, Lpv-1, Cpa-2 and Vsv-1 provide a rare opportunity for studies of the synthesis of sporulation specific molecules whose nature, and putative function are known. Future molecular genetic analysis will elucidate details of the transcriptional or translational mechanisms that control their synthesis.
48
Gay. J. L. & Greenwood. A. D. (1966). Structural aspects of zoospore production in Saprolegnia ferax with particular reference to the cell and vacuolar membranes. In Tke Fungus Spore (ed. M. F. Madelin), pp. 95-108. Butterworths: London. U.K. Grifin, D. H. (1981). Fungal Physiology. John Wiley & Sons: New York, U.S.A. Gubler, F. & Hardham, A. R. (1988). Secretion of adhesive material during encystment of Phytophthora cinrrarnomi zoospores, characterized by immunogold labelling with monoclonal antibodies to components of peripheral vesicles. Journal of Cell Science 90, 225-235. Gubler, F. & Hardham. A. R. (1990). Protein storage in large peripheral vesicles in Phytophthora zoospores and its breakdown after cyst germination. Experimental Mycology 14, 393-404. Hardham, A. R. & Gubler, F. (1990). Polarity of attachment of zoospores of a root pathogen and pre-alignment of the emerging germ tube. Cell Biology I~rternationnlReports 14, 947-956. Hardham, A. R., Gubler. F., Duniec, J. &Elliott, J. (1991). A review of methods for the production and use of monoclonal antibodies to study zoosporic plant pathogens. Journal of Microscopy 162. 305-318. Hemmes, D. E. & Wong, L. D. S. (1975). Ultrastructure of chlamydospores of Pkytophthorn cinnarnomi during development and germination. Carladinn Journal of Botany 53, 294552957, Hemmes. D. E. (1983). Cytology of Phytopktkora. In Phytophthora. Its B~ology. Taxonomy, Ecology, and Patitology (ed. D. C. Erwin, S. Bartn~cki-Garcia& P. H. Tsao), pp. 9-40. American Phytopathological Society: St Paul, MN, U.S.A. Holloway, S. A. & Heath, I. B. (1977). An ultrastructural analysis of the changes in organelle arrangement and structure between the various spore types of Saprolegnia. Canadian Journal of Botany 55. 1328-1339. Hyde, G. J. & Hardham, A. R. (1993).Microtubules regulate the generation of polarity in zoospores of Phytophthora cintlamomi. Europea~rJournal of Cell Biology 62, 75-85. Lehnen, L. P. Jr. & Powell, M. J. (1991). Formation of K,-bodies in prtmary REFERENCES cysts of Saprolegnia ferax. Mycologia 83, 163-179. Lunney, C. Z. & Bland, C. E. (1976). An ultrastructural study of zoosporoArmbruster, B. L. (1982). Sporangiogenesis in three genera of the Saprolegniaceae. I. Pre-sporangium hyphae to early primary spore initial stage. genesis in Pythinm proliferrrm de Bary. Protoplasma 88, 85-100. Mycologia 74, 433-459. Marshall, M. A. & Timberlake, W. E. (1991). Aspergillus nidulans wetA Beakes, G. W. (1983). A comparative account of cyst coat ontogeny in activates spore-specific gene expression. Molecrrlar and Cellular Biology 11, 5542. saprophytic and fish-lesion (pathogenic) isolates of the Saprolegnia diclinaMircetich, S. M. & Zentmyer, G. A. (1966). Production of oospores and parasitica complex. Canadian Journal of Botany 61, 603-625. Carlile, M. J. (1983). Motility, taxis, and tropism in Phytophthora. In chlamydospores of Phytophthora cinnamomi in roots and soil. Pkytopatkology Phytophthora. Its Biology, Taxonomy, Ecology, and Pathology (ed. D. C. Erwin, 56, 1076-1078. S. Bartnicki-Garcia & P. Tsao), pp. 95-107. American Phyt~patholo~ical Ribeiro, 0.K. (1983).Physiology of asexual sporulation and spore germination in Pl~~tophthora.In Phytophthora. Its Biology, Taxonomy, Ecology, and Society: St Paul, MN, U.S.A. Pathology (ed. D. C. Erwin, S. Bartnicki-Garcia & P. Tsao), pp. 55-70. Champe, S. P., Kurtz, M. B., Yager, L. N., Butnick, N. J. & Axelrod, D. E. American Phytopathological Society: St Paul, MN, U.S.A. (1981). Spore formation in Aspergillns nidulans: competence and other Widyastuti, U., Hotta, Y. & Tabata, S. (1994). Identification Sato, S., Suzuki, H., developmentai processes. In 7he Fungal Spore: Morphogenetic Controls (ed. and characterization of genes induced during sexual differentiation in G. Turian & H. R. Hohl), pp. 255-276. Academic Press: London, U.K. Schizosaccharomyces pombe. Current Genetics 26, 31-37. Christen, J. & Hohl, H. R. (1972). Growth and ultrastructural differentiation of sporangia in Phytophthora palmiuora. Canadian Journal of Microbrology 18, Schnepf, E., Deichgraber, G. & Drebes, G. (1978). Development and ultrastructure of the marine, parasitic Oomycete, Lagenisma coscinodisci 1959-1964. Cope, M. & Hardham, A. R. (1994). Synthesis and assembly of flagellar Drebes (Lagenidiales): Formation of the primary zoospores and their release. Protoplasma 94, 263-280. surface antigens during zoosporogenesis in Phytophthora cinnamomi. Protoplasma 180, 158-168. Springer, M. L., Hager, K. M., Garrett-Engele, C. & Yanofsky, C. (1992). Dahlberg, K. R. & Van Etten, J. L. (1982). Physiology and biochemistry of Timing of synthesis and cellular localization of two conidiation-specific proteins of Neurospora crassa. Developmental Biology 152, 255-262. fungal sporulation. Annual Reuiew of Phytopathology 20, 281-301. Dearnaley, I. D. W. & Hardham, A. R. (1994). The Golgi apparatus of Weste, G. (1983). Population dynamics and survival of Phytophthora. In Phytophthora. Its Biology, Taxonomy, Ecology, and Pathology (ed. D. C. Erwin, Phytophthora cinnamomi. makes three types of secretory or storage vesicles concdrrently. Protoplasm 182, 75-79. S. Bartnicki-Garcia & P. Tsao), pp. 237-257. American Phytopathological Society: St Paul, MN, U.S.A. Englander, L. & Turbitt, W. (1979). Increased chlamydospore production by Phytophthora cinnamomi using sterols and near-ultraviolet light. PhytoZentmyer, G. A. (1980). Phytophthora cinnamomi and the Diseases rt Causes. The pathology 69, 813-817. American Phytopathological Society: St Paul, MN, U.S.A.
(Accepted 22 M a y 1995)