, Volume 216, Issue 1–2, pp 101–112 | Cite as

The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria ofMagnaporthe grisea

  • Roland W. S. Weber
  • Gavin E. Wakley
  • Eckhard Thines
  • Nicholas J. Talbot
Original Article


Histochemical and ultrastructural studies were carried out on a wild-type strain (Guyll) and a melanin-deficient mutant(büβ) of the rice-blast pathogen,Magnaporthe grisea (=Pyricularia oryzae), in order to investigate the destination of lipid storage reserves during appressorium development. Lipid droplets were abundant in conidia and were mobilised upon germination, accumulating in the appressorial hook which developed at the tip of each germ tube. Following the formation of a septum at the base of the nascent appressorium, one or a few closely appressed central vacuoles became established and were observed to enlarge in the course of appressorium maturation. On unyielding artificial surfaces such as glass or plastic, appressoria matured to completion within 36–48 h, by which time the enlarged vacuole filled most of the inside volume of the appressorium. Light and transmission electron microscopical observations revealed that the lipid droplets entered the vacuole by autophagocytosis and were degraded therein. Histochemical approaches confirmed the vacuole as the key lytic element in maturing appressoria. Endocytosis of a vital dye, Neutral Red, progressed via endosomes which migrated into the vacuole and lysed there, releasing their dye content into the vacuolar lumen. Furthermore, activity of the lysosomal marker enzyme, acid phospho-monoesterase, was strongly localised in the vacuole at all stages of appressorium maturation. It is therefore envisaged that vacuoles are involved in the degradation of lipid storage reserves which may act as sources of energy and/or osmotically active metabolites such as glycerol, which generate the very high turgor pressure known to be crucial for penetration of hard surfaces. On softer surfaces such as onion epidermis, appressoria ofM. grisea were able to penetrate before degradation of lipid droplets had been completed.


Acid phosphatase Autophagocytosis Endocytosis Neutral Red Turgor Magnaporthe grisea


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adachi K, Hamer JE (1998) Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea. Plant Cell 10:1361–1373PubMedCrossRefGoogle Scholar
  2. Allison AC, Young MR (1969) Vital staining and fluorescence microscopy of lysosomes. In: Dingle JT, Fell HB (ed) Lysosomes in biology and pathology, vol 2. North-Holland, Amsterdam, pp 600–628Google Scholar
  3. Baba M, Takeshige K, Baba N, Ohsumi Y (1994) Ultrastructural analysis of the autophagic process in yeast: detection of auto-phagosomes and their characterization. J Biol Chem 124:903–913Google Scholar
  4. Banuett F (1998) Signalling in the yeasts: an informational cascade with links to the filamentous fungi. Microbiol Mol Biol Rev 62: 249–274PubMedGoogle Scholar
  5. Bonfante P, Balestrini R, Mendgen K (1994) Storage and secretion processes in the spore of Gigaspora margarita Becker & Hall as revealed by high-pressure freezing and freeze-substitution. New Phytol 128: 93–101CrossRefGoogle Scholar
  6. Bourett TM, Howard RJ (1990) In vitro development of penetration structures in the rice blast fungus Magnaporthe grisea. Can J Bot 68: 329–342CrossRefGoogle Scholar
  7. Chayen J, Bitensky L (1991) Practical histochemistry, 2nd edn. Wiley, ChichesterGoogle Scholar
  8. Chumley FG, Valent B (1990) Genetic analysis of melanin-deficient, nonpathogenic mutants of Magnaporthe grisea. Mol Plant Microbe Interact 3:135–143Google Scholar
  9. Cole L, Hyde GJ, Ashford AE (1997) Uptake and compartmentalisation of fluorescent probes by Pisolithus tinctorius hyphae: evidence for an anion transport mechanism at the tonoplast but not for fluid-phase endocytosis. Protoplasma 199:18–29CrossRefGoogle Scholar
  10. — Orlovich DA, Ashford AE (1998) Structure, function, and motility of vacuoles in filamentous fungi. Fungal Genet Biol 24:86–100PubMedCrossRefGoogle Scholar
  11. Davis DJ, Burlak C, Money NP (2000a) Biochemical and biomechanical aspects of appressorial development in Magnaporthe grisea. In: Tharreau D, Lebrun M-H, Talbot NJ, Notteghem JL (eds) Advances in rice blast research. Kluwer, Dordrecht, pp 248–256Google Scholar
  12. ———(2000b) Osmotic pressure of fungal compatible osmolytes. Mycol Res 104: 800–804CrossRefGoogle Scholar
  13. Dean RA (1997) Signal pathways and appressorium morphogenesis. Annu Rev Phytopathol 35: 211–234PubMedCrossRefGoogle Scholar
  14. de Jong JC, McCormack BJ, Smirnoff N, Talbot NJ (1997) Glycerol generates turgor in rice blast. Nature 389:244–245CrossRefGoogle Scholar
  15. de Zwaan TM, Carroll AM, Valent B, Sweigard JA (1999) Magnaporthe grisea Pthllp is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues. Plant Cell 11: 2013–2030CrossRefGoogle Scholar
  16. Ebata Y, Yamamoto H, Uchiyama T (1998) Chemical composition of the glue from Magnaporthe grisea. Biosci Biotechnol Biochem 62:672–674CrossRefGoogle Scholar
  17. Gilbert RD, Johnson AM, Dean RA (1996) Chemical signals responsible for appressorium formation in the rice blast fungus Magnaporthe grisea. Physiol Mol Plant Pathol 48: 335–346CrossRefGoogle Scholar
  18. Glauert AM (1974) Fixation, dehydration and embedding of biological specimens. In: Glauert AM (ed) Practical methods in electron microscopy, vol 3. North-Holland, Amsterdam, pp 1–207Google Scholar
  19. Gomori G (1950) An improved histochemical technic for acid phosphatase. Stain Technol 25: 81–85Google Scholar
  20. Greenspan P, Mayer EP, Fowler SD (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100: 965–973PubMedCrossRefGoogle Scholar
  21. Hamer JE, Holden DW (1997) Linking approaches in the study of fungal pathogenesis. Fungal Genet Biol 21:11–16PubMedCrossRefGoogle Scholar
  22. — Howard RJ, Chumley FG, Valent B (1988) A mechanism for surface attachment in spores of a plant pathogenic fungus. Science 239:288–290PubMedCrossRefGoogle Scholar
  23. Hänssler G, Maxwell DP, Maxwell MD (1975) Demonstration of acid phosphatase-containing vacuoles in hyphal tip cells of Sclerotium rolfsii. J Bacteriol 124: 997–1006PubMedGoogle Scholar
  24. ————Barczewski H, Bernhardt E (1977) Cytochemische Lokalisation der sauren Phosphatase in Hyphen von Pythium paroecandrum, Botrytis cinerea und Rhizoctonia solani. Phytopathol Z 88: 289–298Google Scholar
  25. Henry SA, Patton-Vogt JL (1998) Genetic regulation of phospholipid metabolism: yeast as a model eukaryote. Prog Nucleic Acid Res Mol Biol 61:133–179PubMedCrossRefGoogle Scholar
  26. Hoch HC, Howard RJ (1980) Ultrastructure of freeze-substituted hyphae of the basidiomycete Laetisaria arvalis. Protoplasma 103: 281–297CrossRefGoogle Scholar
  27. Holtzman E (1989) Lysosomes. Plenum, New YorkGoogle Scholar
  28. Howard RJ (1981) Ultrastructural analysis of hyphal tip cell growth in fungi: Spitzenkörper, cytoskeleton and endomembranes after freeze-substitution. J Cell Sci 48: 89–103PubMedGoogle Scholar
  29. — (1997) Breaching the outer barriers: cuticle and cell wall penetration. In: Carroll GC, Tudzynski P (ed) The Mycota, vol 5A. Springer, Berlin Heidelberg New York Tokyo, pp 43–60Google Scholar
  30. — Aist JR (1979) Hyphal tip cell ultrastructure of the fungus Fusarium: improved preservation by freeze-substitution. J Ultrastruct Res 66:224–234CrossRefGoogle Scholar
  31. — Ferrari MA (1989) Role of melanin in appressorium formation. Exp Mycol 13:403–418CrossRefGoogle Scholar
  32. — O’Donnell KL (1987) Freeze-substitution of fungi for cytological analysis. Exp Mycol 11: 250–269CrossRefGoogle Scholar
  33. — Valent B (1996) Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea. Annu Rev Microbiol 50: 491–512CrossRefGoogle Scholar
  34. — Ferrari MA, Roach DH, Money NP (1991) Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc Natl Acad Sci USA 88:11281–11284CrossRefGoogle Scholar
  35. Lee Y-H, Dean RA (1994) Hydrophobicity of contact surface induces appressorium formation in Magnaporthe grisea. FEMS Microbiol Lett 115: 71–76CrossRefGoogle Scholar
  36. Leung H, Borromeo ES, Bernardo MA, Notteghem JL (1988) Genetic analysis of virulence in the rice blast fungus Magnaporthe grisea. Phytopathology 78:1227–1233CrossRefGoogle Scholar
  37. Mendgen K, Hahn M, Deising H (1996) Morphogenesis and mechanisms of penetration by plant pathogenic fungi. Annu Rev Phytopathol 34: 367–386PubMedCrossRefGoogle Scholar
  38. Mims CW, Roberson RW, Richardson EA (1988) Ultrastructure of freeze-substituted and chemically fixed basidiospores of Gym-nosporangium juniperi-virginianae. Mycologia 80: 356–364CrossRefGoogle Scholar
  39. — Richardson EA, Clay RP, Nicholson RL (1995) Ultrastructure of conidia and the conidium aging process in the plant pathogenic fungus Colletotrichum graminicola. Int J Plant Sci 156: 9–18CrossRefGoogle Scholar
  40. Mitchell TK, Dean RA (1995) The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast fungus Magnaporthe grisea. Plant Cell 7:1869–1878PubMedCrossRefGoogle Scholar
  41. Money NP (1997) Mechanism linking cellular pigmentation and pathogenicity in the rice blast fungus: a commentary. Fungal Genet Biol 22:151–152PubMedCrossRefGoogle Scholar
  42. — (1998) Mechanics of invasive fungal growth and the significance of turgor in plant infection. In: Kohmoto K, Yoder OC (eds) Molecular genetics of host-specific toxins in plant disease. Kluwer, Dordrecht, pp 261–271Google Scholar
  43. — Howard RJ (1996) Confirmation of a link between fungal pigmentation, turgor pressure, and pathogenicity using a new method of turgor measurement. Fungal Genet Biol 20:217–227CrossRefGoogle Scholar
  44. — That TCCT, Frederick B, Henson JM (1998) Melanin synthesis is associated with changes in hyphopodial turgor, permeability, and wall rigidity in Gaeumannomyces graminis var. graminis. Fungal Genet Biol 24: 240–251PubMedCrossRefGoogle Scholar
  45. Pearse AGE (1968) Histochemistry: theoretical and applied, vol 1, 3rd edn. Churchill, LondonGoogle Scholar
  46. Pitt D (1968) Histochemical demonstration of certain hydrolytic enzymes within cytoplasmic particles of Botrytis cinerea Fr. J Gen Microbiol 52: 67–75Google Scholar
  47. — (1975) Lysosomes and cell function. Longman, LondonGoogle Scholar
  48. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17: 208–212PubMedCrossRefGoogle Scholar
  49. Rost FWD, Shepherd VA, Ashford AE (1995) Estimation of vacuolar pH in actively growing hyphae of the fungus Pisolithus tinctorius. Mycol Res 99: 549–553Google Scholar
  50. Schadeck RJG, Buchi DF, Leite B (1998a) Ultrastructural aspects of Colletotrichum graminicola conidium germination, appressorium formation and penetration on cellophane membranes: focus on lipid reserves. J Submicrosc Cytol Pathol 30: 555–561Google Scholar
  51. — Leite B, Buchi DF (1998b) Lipid mobilization and acid phosphatase activity in lytic compartments during conidium dormancy and appressorium formation of Colletotrichum graminicola. Cell Struct Funct 23: 333–340PubMedCrossRefGoogle Scholar
  52. Spurr AR (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26: 31–43PubMedCrossRefGoogle Scholar
  53. Takano Y, Kikuchi T, Kubo Y, Hamer JE, Mise K, Furusawa I (2000) The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis. Mol Plant Microbe Interact 13: 374–383PubMedCrossRefGoogle Scholar
  54. Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y (1992) Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J Cell Biol 119: 301–311PubMedCrossRefGoogle Scholar
  55. Talbot NJ (1999) Forcible entry. Science 285:1860–1861CrossRefGoogle Scholar
  56. — Ebbole DJ, Hamer JE (1993) Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5:1575–1590PubMedCrossRefGoogle Scholar
  57. Thines E, Eilbert F, Sterner O, Anke H (1997a) Glisoprenin A, an inhibitor of the signal transduction pathway leading to appressorium formation in germinating conidia of Magnaporthe grisea on hydrophobic surfaces. FEMS Microbiol Lett 151: 219–224CrossRefGoogle Scholar
  58. ———(1997b) Signal transduction leading to appressorium formation in germinating conidia of Magnaporthe grisea: effects of second messengers diacylglycerols, ceramides and sphingomyelin. FEMS Microbiol Lett 156: 91–94CrossRefGoogle Scholar
  59. -Thines E, Eilbert F, Sterner O, Anke H Weber RWS, Talbot NJ (1999) Cellular and biochemical analysis of appressorium turgor generation by the rice blast fungus Magnaporthe grisea. In: Molecular Plant-Microbe Interactions, 9th International Congress, Book of Abstracts, p 121Google Scholar
  60. ——— (2000) MAP kinase and protein kinase A-dependent mobilisation of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea. Plant Cell 12: 1703–1718PubMedCrossRefGoogle Scholar
  61. Uchiyama T, Okuyama K (1990) Participation of Oryza sativa leaf wax in appressoria formation by Pyricularia oryzae. Phytochemistry 29: 91–92CrossRefGoogle Scholar
  62. Weber RWS, Pitt D (1997) Acid phosphatase secretion by Botrytis cinerea. Mycol Res 101: 349–356CrossRefGoogle Scholar
  63. — Wakley GE, Pitt D (1999) Histochemical and ultrastructural characterization of vacuoles and spherosomes as components of the lytic system in hyphae of the fungus Botrytis cinerea. Histochem J 31:293–301PubMedCrossRefGoogle Scholar
  64. Wilson CL (1973) A lysosomal concept for plant pathology. Annu Rev Phytopathol 11: 247–272CrossRefGoogle Scholar
  65. Xu JR, Staiger CJ, Hamer JE (1998) Inactivation of the mitogen-activated protein kinase Mpsl from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses. Proc Natl Acad Sci USA 95:12713–12718PubMedCrossRefGoogle Scholar
  66. Yatsu LY, Jacks TJ (1972) Spherosome membranes: half-unit membranes. Plant Physiol 49: 937–943PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2001

Authors and Affiliations

  • Roland W. S. Weber
    • 1
  • Gavin E. Wakley
    • 2
  • Eckhard Thines
    • 1
  • Nicholas J. Talbot
    • 2
  1. 1.Lehrbereich BiotechnologieUniversität KaiserslauternKaiserslautern
  2. 2.School of Biological SciencesUniversity of ExeterExeter

Personalised recommendations