Skip to main content
SpringerLink
Log in

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

  • Published:
Protoplasma Aims and scope Submit manuscript

Summary

The water expulsion vacuole (WEV) in zoospores ofPhytophthora nicotianae and other members of the Oomycetes is believed to function in cell osmoregulation. We have used videomicroscopy to analyse the behaviour of the WEV during zoospore development, motility and encystment inP. nicotianae. After cleavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 ± 0.71 s. The WEV continues to pulse at this rate until approximately 4 min after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The commencement of WEV operation in sporangia coincides with the reduction of zoospore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgor pressure in the cyst. As in other organisms, the WEV inP. nicotianae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towards vacuolar H+-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma membrane. An antibody directed towards plasma membrane H+-ATPase shows the presence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in microsomal fractions fromP. nicotianae zoospores has provided information on the biochemical properties of the ATPases in these cells and has shown that they are similar to those in true fungi. Inhibition of the vacuolar H+-ATPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H+-ATPase plays an important role in water accumulation by the spongiome in oomycete zoospores, as it does in other protists.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BMM:

butyl methylmethacrylate

F:

fix 4% formaldehyde fixation

GF:

fix 4% formaldehyde and 0.2% glutaraldehyde fixation

V-ATPase:

vacuolar H+-ATPase

WEV:

water expulsion vacuole

References

  • Baumer JS, Leonard RT, Erwin DC (1987) Isolation of plasma membrane ofPhytophthora megasperma f. sp.glycinea and some properties of the associated ATPase. Exp Mycol 11: 49–59

    Google Scholar 

  • Bourett TM, Czymmek KJ, Howard RJ (1998) An improved method for affinity probe localization in whole cells of filamentous fungi. Fungal Genet Biol 24: 3–13

    Google Scholar 

  • Bowman BJ, Slayman CW (1979) The effects of vanadate on the plasma membrane ATPase ofNeurospora crassa. J Biol Chem 254: 2928–2934

    Google Scholar 

  • —, Mainzer SE, Allen KE, Slayman CW (1978) Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases ofNeurospora crassa. Biochim Biophys Acta 512: 13–28

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72: 248–254

    Google Scholar 

  • Bush J, Nolta KV, Rodriguez-Paris J, Kaufmann N, O'Halloran T, Ruscetti T, Temesvari L, Steck TL, Cardelli J (1994) A Rab4- like GTPase inDictyostelium discoideum colocalizes with V-H+-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes. J Cell Sci 107: 2801–2812

    Google Scholar 

  • —, Temesvari L, Rodriguez-Paris J, Buczynski G, Cardelli J (1996) A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function inDictyostelium discoideum. Mol Biol Cell 7: 1623–1638

    Google Scholar 

  • Cerenius L, Rennie P, Fowke LC (1988) Endocytosis of cationized ferritin by zoospores of the fungusAphanomyces euteiches. Protoplasma 144: 119–124

    Google Scholar 

  • Chen D, Zentmeyer GA (1970) Production of sporangia byPhytophthora cinnamomi in axenic culture. Mycologia 62: 397–402

    Google Scholar 

  • Cho CW, Fuller MS (1989) Observations of the water expulsion vacuole ofPhytophthora palmivora. Protoplasma 149: 47–55

    Google Scholar 

  • Cope M, Webb MC, O'Gara ET, Philip BA, Hardham AR (1996) Immunocytochemical comparison of peripheral vesicles in zoospores ofPhytophthora andPythium species. Mycologia 88: 523–532

    Google Scholar 

  • Czymmek KJ, Bourett TM, Howard RJ (1996) Immunolocalization of tubulin and actin in thick-sectioned fungal hyphae after freeze-substitution fixation and methacrylate de-embedment. J Microsc 181: 153–161

    Google Scholar 

  • de Chastellier C, Quiviger B, Ryter A (1978) Observations on the functioning of the contractile vacuole ofDictyostelium discoideum with the electron microscope. J Ultrastruct Res 62: 220–227

    Google Scholar 

  • Erwin DC, Ribeiro OK (1996) Phytophthora diseases worldwide. APS Press, St. Paul, Minn.

    Google Scholar 

  • Finbow ME, Harrison MA (1997) The vacuolar H+-ATPase: a universal proton pump of eukaryotes. Biochem J 324: 697–712

    Google Scholar 

  • Fok AK, Aihara MS, Ishida M, Nolta KV, Steck TL, Allen RD (1995) The pegs on the decorated tubules of the contractile vacuole complex ofParamecium are proton pumps. J Cell Sci 108: 3163–3170

    Google Scholar 

  • Gabor BK, O'Gara ET, Philip BA, Horan DP, Hardham AR (1993) Specificities of monoclonal antibodies toPhytophthora cinnamomi in two rapid diagnostic assays. Plant Dis 77: 1189–1197

    Google Scholar 

  • Gaertig J, Thatcher TH, McGrath KE, Callahan RC, Gorovsky MA (1993) Perspectives on tubulin isotype function and evolution based on the observation thatTetrahymena thermophila microtubules contain a single α- and β-tubulin. Cell Motil Cytoskeleton 25: 243–253

    Google Scholar 

  • Giannini JL, Gildensoph LH, Briskin DP (1987) Selective production of sealed plasma membrane vesicles from red beet (Betavulgaris L.) storage tissue. Arch Biochem Biophys 254: 621–630

    Google Scholar 

  • —, Holt JS, Briskin DP (1988) Isolation of sealed plasma membrane vesicles fromPhytophthora megasperma f. sp.glycinea I: characterization of proton pumping and ATPase activity. Arch Biochem Biophys 265: 337–345

    Google Scholar 

  • Giglione C, Gross JD (1995) Anion effects on vesicle acidification inDictyostelium. Biochem Mol Biol Int 36: 1057–1065

    Google Scholar 

  • Grove SN, Bracker CE (1978) Protoplasmic changes during zoospore encystment and cyst germination inPythium aphanidermatum. Exp Mycol 2: 51–98

    Google Scholar 

  • Gubler F, Hardham AR (1991) The fate of peripheral vesicles in zoospores ofPhytophthora cinnamomi during infection of plants. In: Mendgen K, Lesemann D-E (eds) Electron microscopy of plant pathogens. Springer, Berlin Heidelberg New York Tokyo, pp 197–210

    Google Scholar 

  • Gunderson JH, Elwood H, Ingold A, Kindle K, Sogin ML (1987) Phylogenetic relationships between chlorophytes, chrysophytes, and oomycetes. Proc Natl Acad Sci USA 84: 5823–5827

    Google Scholar 

  • Hardham AR (1985) Studies on the cell surface of zoospores and cysts of the fungusPhytophthora cinnamomi: the influence of fixation on patterns of lectin binding. J Histochem Cytochem 33: 110–118

    Google Scholar 

  • —, Gubler F (1990) Polarity of attachment of zoospores of a root pathogen and pre-alignment of the emerging germ tube. Cell Biol Int Rep 14: 947–956

    Google Scholar 

  • —, Suzaki E, Perkin JL (1986) Monoclonal antibodies to isolates-, pecies-, and genus-specific components on the surface of zoospores and cysts of the fungusPhytophthora cinnamomi. Can J Bot 64: 311–321

    Google Scholar 

  • —, Gubler F, Duniec J (1991a) Ultrastructural and immunological studies of zoospores ofPhytophthora. In: Lucas JA, Shattock RC, Shaw DS, Cooke LR (eds)Phytophthora. Cambridge University Press, Cambridge, pp 50–69

    Google Scholar 

  • Hardham AR, Gubler F, Duniec J, Elliot J (1991b) A review of methods for the production and use of monoclonal antibodies to study zoosporic plant pathogens. J Microsc 162: 305–318

    Google Scholar 

  • Heuser J, Zhu Q, Clarke M (1993) Proton pumps populate the contractile vacuoles ofDictyostelium amoebae. J Cell Biol 121: 1311–1327

    Google Scholar 

  • Hoch HC, Mitchell JE (1972a) The ultrastructure of zoospores ofAphanomyces euteiches and of their encystment and subsequent germination. Protoplasma 75: 113–138

    Google Scholar 

  • — — (1972b) The ultrastructure ofAphanomyces euteiches during asexual spore formation. Phytopathology 62: 149–160

    Google Scholar 

  • Hyde GJ, Hardham AR (1992) Confocal microscopy of microtubule arrays in cryosectioned sporangia ofPhytophthora cinnamomi. Exp Mycol 16: 207–218

    Google Scholar 

  • —, Lancelle S, Hepler PK, Hardham AR (1991) Freeze substitution reveals a new model for sporangial cleavage inPhytophthora, a result with implications for cytokinesis in other eukaryotes. J Cell Sci 100: 735–746

    Google Scholar 

  • Ishida M, Aihara MS, Allen RD, Fok AK (1993) Osmoregulation inParamecium: the locus of fluid segregation in the contractile vacuole complex. J Cell Sci 106: 693–702

    Google Scholar 

  • —, Fok AK, Aihara MS, Allen RD (1996) Hyperosmotic stress leads to reversible dissociation of the proton pump-bearing tubules from the contractile vacuole complex inParamecium. J Cell Sci 109: 229–237

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685

    Google Scholar 

  • Linder JC, Staehelin LA (1979) A novel model for fluid secretion by the trypanosomatid contractile vacuole apparatus. J Cell Biol 83: 371–382

    Google Scholar 

  • Lunney CZ, Bland CE (1976) Ultrastructural observations of mature and encysting zoospores ofPythium proliferum de Bary. Protoplasma 90: 119–137

    Google Scholar 

  • Luykx P, Hoppenrath M, Robinson DG (1997) Structure and behaviour of contractile vacuoles inChlamydomonas reinhardtii. Protoplasma 198: 73–84

    Google Scholar 

  • McKanna JA (1974) Permeability modulating membrane coats 1: fine structure of fluid segregation organelles of peritrich contractile vacuoles. J Cell Biol 63: 317–322

    Google Scholar 

  • — (1976) Fine structure of fluid segregation organelles ofParamecium contractile vacuoles. J Ultrastruct Res 54: 1–10

    Google Scholar 

  • Morsomme P, d'Exaerde AD, De Meester S, Thinès D, Goffeau A, Boutry M (1996) Single point mutations in various domains of a plant plasma membrane H+-ATPase expressed inSaccharomyces cerevisiae increase H+-pumping and permit yeast growth at low pH. EMBO J 15: 5513–5526

    Google Scholar 

  • Nelson N, Klionsky DJ (1996) Vacuolar H+-ATPase: from mammals to yeast and back. Experientia 52: 1101–1110

    Google Scholar 

  • Nolta KV, Steck TL (1994) Isolation and initial characterization of the bipartite contractile vacuole complex fromDictyostelium discoideum. J Biol Chem 269: 2225–2233

    Google Scholar 

  • Obermeyer G, Kriechbaumer R, Strasser D, Maschessnig A, Bentrup FW (1996) Boric acid stimulates the plasma membrane H+- ATPase of ungerminated lily pollen grains. Physiol Plant 98: 281–290

    Google Scholar 

  • Ohnishi T, Gall RS, Mayer ML (1975) An improved assay of inorganic phosphate in the presence of extralabile phosphate compounds: application to the ATPase assay in the presence of phosphocreatine. Anal Biochem 69: 261–267

    Google Scholar 

  • Palmgren MG, Sommarin M, Ulvskov P, Jørgensen PL (1988) Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2. Physiol Plant 74: 11–19

    Google Scholar 

  • Patterson DJ (1980) Contractile vacuoles and associated structures: their organization and function. Biol Rev 55: 1–46

    Google Scholar 

  • Roldan M, Donaire JP, Pardo JM, Serrano R (1991) Regulation of root plasma membrane H+-ATPase in sunflower seedlings. Plant Sci 79: 163–172

    Google Scholar 

  • Stevens TH, Forgac M (1997) Structure, function and regulation of the vacuolar (H+)-ATPase. Annu Rev Cell Dev Biol 13: 779–808

    Google Scholar 

  • Struck C, Hahn M, Mendgen K (1996) Plasma membrane H+- ATPase activity in spores, germ tubes, and haustoria of the rust fungusUromyces viciae-fabae. Exp Mycol 20: 30–35

    Google Scholar 

  • Temesvari LA, Rodriguez-Paris JM, Bush JM, Zhang L, Cardelli JA (1996) Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system ofDictyostelium discoideum. J Cell Sci 109: 1479–1495

    Google Scholar 

  • Tominaga T, Allen RD, Naitoh Y (1998) Electrophysiology of the in situ contractile vacuole complex ofParamecium reveals its membrane dynamics and electrogenic site during osmoregulatory activity. J Exp Biol 201: 451–460

    Google Scholar 

  • Ward JM, Reinders A, Hse HT, Sze H (1992) Dissociation and reassembly of the vacuolar H+-ATPase complex from oat roots. Plant Physiol 99: 161–169

    Google Scholar 

  • Weerakoon ND, Roberts JK, Lehnen LP, Wilkinson JM, Marshall JS, Hardham AR (1998) Isolation and characterization of the single β-tubulin gene inPhytophthora cinnamomi. Mycologia 90: 85–95

    Google Scholar 

  • Xie Y, Coukell MB, Gombos Z (1996) Antisense RNA inhibition of the putative vacuolar H+-ATPase proteolipid ofDictyostelium reduces intracellular Ca2+ transport and cell viability. J Cell Sci 109: 489–497

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, GPO Box 475, 2601, Canberra, ACT, Australia

    H. J. Mitchell & A. R. Hardham

Authors
  1. H. J. Mitchell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. R. Hardham
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H. J. Mitchell.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mitchell, H.J., Hardham, A.R. Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores. Protoplasma 206, 118–130 (1999). https://doi.org/10.1007/BF01279258

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01279258

Keywords

Search

Navigation