Skip to main content
SpringerLink
Log in

Brefeldin A-induced structural changes in the endomembrane system of a filamentous fungus,Magnaporthe grisea

  • Published:
Protoplasma Aims and scope Submit manuscript

Summary

Subcellular compartments comprising the endomembrane system in filamentous fungi are poorly characterized with most showing significant morphological differences from eukaryotic cells. For example, many filamentous fungi lack stacked Golgi-body cisternae, but contain “Golgi equivalents” — single cisternae or tubules which appear to serve the same functions. To help identify fungal endomembrane compartments and interrelationships between them we used a pharmacological agent, brefeldin A, known to affect specific endomembrane organelles in other organisms, most prominently the Golgi apparatus. At 10 μg/ml brefeldin A, radial hyphal growth of the rice blast pathogenMagnaporthe grisea on solid agar medium was reduced by 96% over an initial 48 h, but recovered and was reduced by only 20% over a subsequent 72 h exposure. Light microscopic examination of individual living hyphae showed that apical elongation generally halted within 1 min after exposure to brefeldin A. Acute effects of 14 μg/ml brefeldin A were characterized ultrasiructurally in cells prepared by freeze substitution. These included the appearance of two types of cisternae with unusual morphology, associated with ca. 45 nm diameter vesicles, as well as the unexpected persistence and increase in complexity of the Golgi equivalents. Also observed were (1) reduced numbers of apicale vesicles and disruption of Spitzenkörper organization, (2) apical clusters of 30–35 nm diameter microvesicles and associated tubular arrays, (3) dilation of rough endoplasmic reticulum, (4) packets of membrane-bounded electron-opaque cell wall inclusions, and (5) altered morphology of some vacuolar compartments. The distribution of concanavalin A binding sites, previously mapped to particular endomembrane compartments, was documented to aid the interpretation of these results. We conclude that brefeldin A effects on cells ofM. grisea differ from those reported with plant and animal cells, perhaps reflecting underlying differences in the endomembrane systems among these eukaryotes.

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

BFA:

brefeldin A

ConA:

concanavalin A

ER:

endoplasmic reticulum

PDA:

potato dextrose agar

RER:

rough endoplasmic reticulum

References

  • Aist JR (1969) The mitotic apparatus in fungi,Ceratocystis fagacearum andFusarium oxysporum. J Cell Biol 40: 120–135

    PubMed  Google Scholar 

  • Baba M, Osumi M (1987) Transmission and scanning electron microscopic examination of intracellular organelles in freeze-substitutedKloeckera andSaccharomyces cerevisiae yeast cells. J Electron Microsc Tech 5: 249–261

    Google Scholar 

  • —, Baba N, Ohsumi Y, Kanaya K, Osumi M (1989) Three-dimensional analysis of morphogenesis induced by mating pheromone a factor inSaccharomyces cerevisiae. J Cell Sci 94: 207–216

    PubMed  Google Scholar 

  • Baráthová H, Betina V, Ulicky L (1977) Regulation of differentiation of the dimorphic fungusPaecilomyces viridis by nitrogen sources, antibiotics and metabolic inhibitors. Folia Microbiol 22: 222–231

    Google Scholar 

  • Betina V, Janstová D, Spišiaková J (1975) Effects of antibiotics on the life cycle ofNeurospora crassa. Folia Microbiol 20: 340–345

    Google Scholar 

  • Bourett TM, Howard RJ (1991) Ultrastructural immunolocalization of actin in a fungus. Protoplasma 163: 199–202

    Google Scholar 

  • — — (1993) Use of a post-embedding, indirect, double-sided labeling procedure to identify ConA binding sites within apical vesicles of a filamentous fungus. In: Bailey GW, Rieder CL (eds) Proceedings of the 51st Annual Meeting of the Microscopy Society of America. San Francisco Press, San Francisco, pp 282–283

    Google Scholar 

  • — — (1994) Enhanced labelling of concanavalin A binding sites in fungal endomembranes using a double-sided, indirect method. Mycol Res 98: 769–775

    Google Scholar 

  • —, Picollelli MA, Howard RJ (1993) Postembedment labeling of intracellular concanavalin A-binding sites in freeze-substituted fungal cells. Exp Mycol 17: 223–235

    Google Scholar 

  • Dargent R, Touzé-Soulet J-M, Rami J, Montant C (1982) Cytochemical characterization of Golgi apparatus in some filamentous fungi. Exp Mycol 6: 101–114

    Google Scholar 

  • Donaldson JG, Lippincott-Schwartz J, Bloom GS, Kreis TE, Klausner RD (1990) Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in bre-feldin A action. J Cell Biol 111: 2295–2306

    PubMed  Google Scholar 

  • Driouich A, Zhang GF, Staehelin LA (1993) Effects of brefeldin A on the structure of the Golgi apparatus and on the synthesis and secretion of proteins and polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells. Plant Physiol 101: 1363–1373

    PubMed  Google Scholar 

  • Girbardt M (1969) Die Ultrastruktur der Apikalregion von Pilzhyphen. Protoplasma 67: 413–441

    Google Scholar 

  • Grove SN, Bracker CE (1970) Protoplasmic organization of hyphal tips among fungi: vesicles and Spitzenkörper. J Bacteriol 104: 989–1009

    PubMed  Google Scholar 

  • — —, Morré DJ (1968) Cytomembrane differentiation in the endoplasmic reticulum-Golgi apparatus-vesicle complex. Science 161: 171–173

    PubMed  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

    PubMed  Google Scholar 

  • Hoch HC, Howard RJ (1980) Ultrastructure of freeze-substituted hyphae of the basidiomyceteLaetisaria arvalis. Protoplasma 103: 281–297

    Google Scholar 

  • —, Staples RC (1983) Ultrastructural organization of the non-differentiated uredospore germling ofUromyces phaseoli variety typica. Mycologia 75: 795–824

    Google Scholar 

  • Howard RJ (1981) Ultrastructural analysis of hyphal tip cell growth in fungi: Spitzenkörper, cytoskeleton and endomembranes after freeze-substitution. J Cell Sci 48: 89–103

    PubMed  Google Scholar 

  • —, Aist JR (1979) Hyphal tip cell ultrastructure of the fungusFusarium: improved preservation by freeze-substitution. J Ultrastruct Res 66: 224–234

    PubMed  Google Scholar 

  • — — (1980) Cytoplasmic microtubules and fungal morphogenesis: ultrastructural effects of methyl benzimidazole-2-ylcarbamate determined by freeze-substitution of hyphal tip cells. J Cell Biol 87: 55–64

    Google Scholar 

  • —, Ferrari MA (1989) Role of melanin in appressorium function. Exp Mycol 13: 403–418

    Google Scholar 

  • —, O'Donnell KL (1987) Freeze substitution of fungi for cytological analysis. Exp Mycol 11: 250–269

    Google Scholar 

  • Khan SR (1978) The Golgi cisternae ofCunninghamella echinulata. Can J Bot 56: 432–439

    Google Scholar 

  • Klausner RD, Donaldson JG, Lippincott-Schwartz J (1992) Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol 116: 1071–1080

    Google Scholar 

  • Lippincott-Schwartz J, Yuan LC, Bonifacino JS, Klausner RD (1989) Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56: 801–813

    PubMed  Google Scholar 

  • — —, Tipper C, Amherdt M, Orci L, Klausner RD (1991) Brefeldin A's effects on endosomes, lysosomes and TGN suggests a general mechanism for regulating organelle structure and membrane traffic. Cell 67: 601–616

    PubMed  Google Scholar 

  • López-Franco R, Howard RJ, Bracker CE (1995) Satellite Spitzenkörper in growing hyphal tips. Protoplasma 188: 85–103

    Google Scholar 

  • Morré DJ (1990) Endomembrane system of plants and fungi. In: Heath IB (ed) Tip growth in plant and fungal cells. Academic Press, San Diego, pp 183–210

    Google Scholar 

  • Newhouse JR, Hoch HC, MacDonald WL (1983) The ultrastructure ofEndothia parasitica. Comparison of a virulent with a hypovirulent isolate. Can J Bot 61: 389–399

    Google Scholar 

  • Orci L, Tagaya M, Amherdt M, Perrelet A, Donaldson JG, Lippincott-Schwartz J, Klausner RD, Rothman JE (1991) Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae. Cell 64: 1183–1195

    PubMed  Google Scholar 

  • Pearse BMF, Robinson MS (1990) Clathrin, adaptors, and sorting. Annu Rev Cell Biol 6: 151–171

    PubMed  Google Scholar 

  • Powell MJ (1979) The structure of microbodies and their associations with other organelles in zoosporangia ofEndophlyctis variabilis. Protoplasma 98: 177–198

    Google Scholar 

  • Rambourg A, Clermont Y, Képés F (1993) Modulation of the Golgi apparatus inSaccharomyces cerevisiae sec 7 mutants as seen by three-dimensional electron microscopy. Anat Rec 237: 441–452

    PubMed  Google Scholar 

  • — —, Jackson CL, Képés F (1995) Effects of brefeldin A on the three-dimensional structure of the Golgi apparatus in a sensitive strain ofSaccharomyces cerevisiae. Anat Rec 241: 1–9

    PubMed  Google Scholar 

  • Roberson RW, Fuller MS (1988) Ultrastructural aspects of the hyphal tip ofSclerotium rolfsii preserved by freeze substitution. Protoplasma 146: 143–149

    Google Scholar 

  • Rossman AY, Howard RJ, Valent B (1990)Pyricularia grisea, the correct name for the rice blast disease fungus. Mycologia 82: 509–512

    Google Scholar 

  • Rothman JE (1994) Mechanisms of intracellular protein transport. Nature 372: 55–63

    PubMed  Google Scholar 

  • —, Orci L (1992) Molecular dissection of the secretory pathway. Nature 355: 409–415

    PubMed  Google Scholar 

  • Rowley JC III, Moran DT (1975) A simple procedure for mounting wrinkle-free sections on Formvar-coated slot grids. Ultramicroscopy 1: 151–155

    PubMed  Google Scholar 

  • San-Blas G, Yegres FJ (1978) Effects of cyanein and ramihyphin A on the dimorphism ofParacoccidioides brasiliensis. Folia Microbiol 23: 198–201

    Google Scholar 

  • Satiat-Jeunemaitre B, Hawes C (1992) Redistribution of a Golgi glycoprotein in plant cells treated with brefeldin A. J Cell Sci 103: 1153–1166

    Google Scholar 

  • — — (1993) The distribution of secretory products in plant cells is affected by brefeldin A. Cell Biol Int 17: 183–193

    Google Scholar 

  • Schindler T, Bergfeld R, Hohl M, Schopfer P (1994) Inhibition of Golgi-apparatus function by brefeldin A in maize coleoptiles and its consequences on auxin-mediated growth, cell-wall extensibility and secretion of cell-wall proteins. Planta 192: 404–413

    Google Scholar 

  • Sewall TC, Roberson RW, Pommerville JC (1989) Identification and characterization of Golgi equivalents fromAllomyces macrogynus. Exp Mycol 13: 239–252

    Google Scholar 

  • Taylor JW, Fuller MS (1981) The Golgi apparatus, zoosporogenesis, and development of the zoospore discharge appratus ofChytridium confervae. Exp Mycol 5: 35–59

    Google Scholar 

  • Ulmer JB, Palade GE (1989) Targeting and processing of glycophorins in murine erythroleukemia cells: use of brefeldin A as a perturbant of intracellular traffic. Proc Natl Acad Sci USA 86: 6992–6996

    PubMed  Google Scholar 

  • Wainright PO, Hinkle G, Sogin ML, Stickel SK (1993) Monophyletic origins of the metazoa: an evolutionary link with the fungi. Science 260: 340–342

    PubMed  Google Scholar 

  • Wessels JGH (1994) Developmental regulation of fungal cell wall formation. Annu Rev Phytopathol 32: 413–437

    Google Scholar 

  • Wood SA, Park JE, Brown WJ (1991) Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endo-somes. Cell 67: 591–600

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Central Research and Development, DuPont Company, P.O. Box 80402, 19880-0402, Wilmington, DE, USA

    T. M. Bourett & R. J. Howard

Authors
  1. T. M. Bourett
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. J. Howard
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bourett, T.M., Howard, R.J. Brefeldin A-induced structural changes in the endomembrane system of a filamentous fungus,Magnaporthe grisea . Protoplasma 190, 151–163 (1996). https://doi.org/10.1007/BF01281314

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01281314

Keywords

Search

Navigation