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

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Summary

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.

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References

  • Adachi K, Hamer JE (1998) Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea. Plant Cell 10:1361–1373

    Article  PubMed  CAS  Google Scholar 

  • 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 600–628

  • 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 124:903–913

    CAS  Google Scholar 

  • Banuett F (1998) Signalling in the yeasts: an informational cascade with links to the filamentous fungi. Microbiol Mol Biol Rev 62: 249–274

    PubMed  CAS  Google Scholar 

  • 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: 93–101

    Article  Google Scholar 

  • Bourett TM, Howard RJ (1990) In vitro development of penetration structures in the rice blast fungus Magnaporthe grisea. Can J Bot 68: 329–342

    Article  Google Scholar 

  • Chayen J, Bitensky L (1991) Practical histochemistry, 2nd edn. Wiley, Chichester

    Google Scholar 

  • Chumley FG, Valent B (1990) Genetic analysis of melanin-deficient, nonpathogenic mutants of Magnaporthe grisea. Mol Plant Microbe Interact 3:135–143

    CAS  Google Scholar 

  • 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 199:18–29

    Article  CAS  Google Scholar 

  • — Orlovich DA, Ashford AE (1998) Structure, function, and motility of vacuoles in filamentous fungi. Fungal Genet Biol 24:86–100

    Article  PubMed  Google Scholar 

  • 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 248–256

    Google Scholar 

  • ———(2000b) Osmotic pressure of fungal compatible osmolytes. Mycol Res 104: 800–804

    Article  CAS  Google Scholar 

  • Dean RA (1997) Signal pathways and appressorium morphogenesis. Annu Rev Phytopathol 35: 211–234

    Article  PubMed  CAS  Google Scholar 

  • de Jong JC, McCormack BJ, Smirnoff N, Talbot NJ (1997) Glycerol generates turgor in rice blast. Nature 389:244–245

    Article  CAS  Google Scholar 

  • 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: 2013–2030

    Article  Google Scholar 

  • Ebata Y, Yamamoto H, Uchiyama T (1998) Chemical composition of the glue from Magnaporthe grisea. Biosci Biotechnol Biochem 62:672–674

    Article  CAS  Google Scholar 

  • 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: 335–346

    Article  CAS  Google Scholar 

  • 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 1–207

  • Gomori G (1950) An improved histochemical technic for acid phosphatase. Stain Technol 25: 81–85

    CAS  Google Scholar 

  • Greenspan P, Mayer EP, Fowler SD (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100: 965–973

    Article  PubMed  CAS  Google Scholar 

  • Hamer JE, Holden DW (1997) Linking approaches in the study of fungal pathogenesis. Fungal Genet Biol 21:11–16

    Article  PubMed  CAS  Google Scholar 

  • — Howard RJ, Chumley FG, Valent B (1988) A mechanism for surface attachment in spores of a plant pathogenic fungus. Science 239:288–290

    Article  PubMed  CAS  Google Scholar 

  • Hänssler G, Maxwell DP, Maxwell MD (1975) Demonstration of acid phosphatase-containing vacuoles in hyphal tip cells of Sclerotium rolfsii. J Bacteriol 124: 997–1006

    PubMed  Google Scholar 

  • ————Barczewski H, Bernhardt E (1977) Cytochemische Lokalisation der sauren Phosphatase in Hyphen von Pythium paroecandrum, Botrytis cinerea und Rhizoctonia solani. Phytopathol Z 88: 289–298

    Google Scholar 

  • Henry SA, Patton-Vogt JL (1998) Genetic regulation of phospholipid metabolism: yeast as a model eukaryote. Prog Nucleic Acid Res Mol Biol 61:133–179

    Article  PubMed  CAS  Google Scholar 

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

    Article  Google Scholar 

  • Holtzman E (1989) Lysosomes. Plenum, New York

    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  CAS  Google Scholar 

  • — (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 43–60

    Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

  • — Valent B (1996) Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea. Annu Rev Microbiol 50: 491–512

    Article  Google Scholar 

  • — Ferrari MA, Roach DH, Money NP (1991) Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc Natl Acad Sci USA 88:11281–11284

    Article  Google Scholar 

  • Lee Y-H, Dean RA (1994) Hydrophobicity of contact surface induces appressorium formation in Magnaporthe grisea. FEMS Microbiol Lett 115: 71–76

    Article  Google Scholar 

  • Leung H, Borromeo ES, Bernardo MA, Notteghem JL (1988) Genetic analysis of virulence in the rice blast fungus Magnaporthe grisea. Phytopathology 78:1227–1233

    Article  Google Scholar 

  • Mendgen K, Hahn M, Deising H (1996) Morphogenesis and mechanisms of penetration by plant pathogenic fungi. Annu Rev Phytopathol 34: 367–386

    Article  PubMed  CAS  Google Scholar 

  • Mims CW, Roberson RW, Richardson EA (1988) Ultrastructure of freeze-substituted and chemically fixed basidiospores of Gym-nosporangium juniperi-virginianae. Mycologia 80: 356–364

    Article  Google Scholar 

  • — 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: 9–18

    Article  Google Scholar 

  • 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 7:1869–1878

    Article  PubMed  CAS  Google Scholar 

  • Money NP (1997) Mechanism linking cellular pigmentation and pathogenicity in the rice blast fungus: a commentary. Fungal Genet Biol 22:151–152

    Article  PubMed  CAS  Google Scholar 

  • — (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 261–271

    Google Scholar 

  • — Howard RJ (1996) Confirmation of a link between fungal pigmentation, turgor pressure, and pathogenicity using a new method of turgor measurement. Fungal Genet Biol 20:217–227

    Article  Google Scholar 

  • — 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: 240–251

    Article  PubMed  CAS  Google Scholar 

  • Pearse AGE (1968) Histochemistry: theoretical and applied, vol 1, 3rd edn. Churchill, London

    Google Scholar 

  • Pitt D (1968) Histochemical demonstration of certain hydrolytic enzymes within cytoplasmic particles of Botrytis cinerea Fr. J Gen Microbiol 52: 67–75

    CAS  Google Scholar 

  • — (1975) Lysosomes and cell function. Longman, London

    Google Scholar 

  • Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17: 208–212

    Article  PubMed  CAS  Google Scholar 

  • Rost FWD, Shepherd VA, Ashford AE (1995) Estimation of vacuolar pH in actively growing hyphae of the fungus Pisolithus tinctorius. Mycol Res 99: 549–553

    Google Scholar 

  • 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: 555–561

    Google Scholar 

  • — 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: 333–340

    Article  PubMed  CAS  Google Scholar 

  • Spurr AR (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26: 31–43

    Article  PubMed  CAS  Google Scholar 

  • 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: 374–383

    Article  PubMed  CAS  Google Scholar 

  • 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: 301–311

    Article  PubMed  CAS  Google Scholar 

  • Talbot NJ (1999) Forcible entry. Science 285:1860–1861

    Article  CAS  Google Scholar 

  • — Ebbole DJ, Hamer JE (1993) Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5:1575–1590

    Article  PubMed  CAS  Google Scholar 

  • 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: 219–224

    Article  CAS  Google Scholar 

  • ———(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: 91–94

    Article  CAS  Google Scholar 

  • -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 121

  • ——— (2000) MAP kinase and protein kinase A-dependent mobilisation of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea. Plant Cell 12: 1703–1718

    Article  PubMed  CAS  Google Scholar 

  • Uchiyama T, Okuyama K (1990) Participation of Oryza sativa leaf wax in appressoria formation by Pyricularia oryzae. Phytochemistry 29: 91–92

    Article  CAS  Google Scholar 

  • Weber RWS, Pitt D (1997) Acid phosphatase secretion by Botrytis cinerea. Mycol Res 101: 349–356

    Article  CAS  Google Scholar 

  • — 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 31:293–301

    Article  PubMed  CAS  Google Scholar 

  • Wilson CL (1973) A lysosomal concept for plant pathology. Annu Rev Phytopathol 11: 247–272

    Article  Google Scholar 

  • 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 95:12713–12718

    Article  PubMed  CAS  Google Scholar 

  • Yatsu LY, Jacks TJ (1972) Spherosome membranes: half-unit membranes. Plant Physiol 49: 937–943

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Lehrbereich Biotechnologie, Universität Kaiserslautern, Kaiserslautern

    Roland W. S. Weber & Eckhard Thines

  2. School of Biological Sciences, University of Exeter, Exeter

    Gavin E. Wakley & Nicholas J. Talbot

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  1. Roland W. S. Weber
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  2. Gavin E. Wakley
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Weber, R.W.S., Wakley, G.E., Thines, E. et al. The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria ofMagnaporthe grisea . Protoplasma 216, 101–112 (2001). https://doi.org/10.1007/BF02680137

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