1887

Microbiology Society logo

Volume 155, Issue 9

Uptake of the fluorescent probe FM4-64 by hyphae and haemolymph-derived hyphal bodies of the entomopathogenic fungus Free

Abstract

The entomopathogenic fungus is under intensive study as a pest biological control agent. produces several distinct single-cell types that include aerial conidia, blastospores and submerged conidia. Under appropriate nutrient conditions these cells can elaborate germ tubes that form hyphae, which in turn lead to the formation of a fungal mycelium. In addition, displays a dimorphic transition, producing specific yeast-like hyphal bodies during growth in the arthropod haemolymph. The amphiphilic styryl dye FM4-64 was used to investigate internalization and morphological features of and insect haemolymph-derived cells. blastospores and submerged conidia displayed a punctate pattern of internal labelling, whereas aerial conidia failed to internalize the dye under the conditions tested. FM4-64 was also taken up into both apical and subapical compartments of living hyphae in a time-dependent manner, with clearly observable vesicle labelling. Internalization, where occurring, was reversibly disrupted by lowering the temperature of the assay or by treatment with azide/fluoride and latrunculin A. Treatment with cytochalasin D and monensin also caused abnormal vesicle trafficking, although some staining of vesicles was noted. Fungal cells derived from infected haemolymph ( cells) actively internalized FM4-64. The blastospores or hyphal bodies displayed bright membrane and internal vesicle staining, although diffuse staining of internal structures was also visible. These results suggest active uptake by different developmental stages of , including haemolymph-derived cells that can evade the insect immune system.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): CDCFDA, 5,6-carboxy-2′,7′-dichlorofluorescein diacetate , DAPI, 4′,6-diamidino-2-phenylindole , DIC, differential interference contrast , LYCH, Lucifer Yellow carbohydrazide and PI, propidium iodide
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.029165-0
2009-09-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/9/3110.html?itemId=/content/journal/micro/10.1099/mic.0.029165-0&mimeType=html&fmt=ahah

References

  1. Araujo-Bazan L., Penalva M. A., Espeso E. A. 2008; Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans . Mol Microbiol 67:891–905
     [Google Scholar]
  2. Atkinson H. A., Daniels A., Read N. D. 2002; Live-cell imaging of endocytosis during conidial germination in the rice blast fungus, Magnaporthe grisea . Fungal Genet Biol 37:233–244
     [Google Scholar]
  3. Ayscough K. R. 2005; Defining protein modules for endocytosis. Cell 123:188–190
     [Google Scholar]
  4. Cho E. M., Boucias D., Keyhani N. O. 2006a; EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. II. Fungal cells sporulating on chitin and producing oosporein. Microbiology 152:2855–2864
     [Google Scholar]
  5. Cho E. M., Liu L., Farmerie W., Keyhani N. O. 2006b; EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria ( Cordyceps) bassiana. I. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia. Microbiology 152:2843–2854
     [Google Scholar]
  6. Cole L., Hyde G. J., Ashford A. E. 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
     [Google Scholar]
  7. Dijksterhuis J. 2003; Confocal microscopy of Spitzenkorper dynamics during growth and differentiation of rust fungi. Protoplasma 222:53–59
     [Google Scholar]
  8. Dimbi S., Maniania N. K., Lux S. A., Ekesi S., Mueke J. K. 2003; Pathogenicity of Metarhizium anisopliae (Metsch.) Sorokin and Beauveria bassiana (Balsamo) Vuillemin, to three adult fruit fly species: Ceratitis capitata(Weidemann), C. rosa var. fasciventris Karsch and C. cosyra (Walker) (Diptera: Tephritidae). Mycopathologia 156:375–382
     [Google Scholar]
  9. Eley K. L., Halo L. M., Song Z. S., Powles H., Cox R. J., Bailey A. M., Lazarus C. M., Simpson T. J. 2007; Biosynthesis of the 2-pyridone tenellin in the insect pathogenic fungus Beauveria bassiana . ChemBioChem 8:289–297
     [Google Scholar]
  10. Fischer-Parton S., Parton R. M., Hickey P. C., Dijksterhuis J., Atkinson H. A., Read N. D. 2000; Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae. Journal of Microscopy 198:246–259
     [Google Scholar]
  11. Fuchs U., Hause G., Schuchardt I., Steinberg G. 2006; Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis . Plant Cell 18:2066–2081
     [Google Scholar]
  12. Gachet Y., Hyams J. S. 2005; Endocytosis in fission yeast is spatially associated with the actin cytoskeleton during polarised cell growth and cytokinesis. J Cell Sci 118:4231–4242
     [Google Scholar]
  13. Gurunathan S., Marash M., Weinberger A., Gerst J. E. 2002; t-SNARE phosphorylation regulates endocytosis in yeast. Mol Biol Cell 13:1594–1607
     [Google Scholar]
  14. Harris S. D., Read N. D., Roberson R. W., Shaw B., Seiler S., Plamann M., Momany M. 2005; Polarisome meets Spitzenkorper: microscopy, genetics, and genomics converge. Eukaryot Cell 4:225–229
     [Google Scholar]
  15. Hegedus D. D., Bidochka M. J., Miranpuri G. S., Khachatourians G. G. 1992; A comparison of the virulence, stability, and cell-wall-surface characteristics of three spore types produced by the entomopathogenic fungus Beauveria bassiana . Appl Microbiol Biotechnol 36:785–789
     [Google Scholar]
  16. Hickey P. C., Jacobson D. J., Read N. D., Glass N. L. 2002; Live-cell imaging of vegetative hyphal fusion in Neurospora crassa . Fungal Genet Biol 37:109–119
     [Google Scholar]
  17. Higuchi Y., Shoji J. Y., Arioka M., Kitamoto K. 2009; Endocytosis is crucial for cell polarity and apical membrane recycling in the filamentous fungus Aspergillus oryzae . Eukaryot Cell 8:37–46
     [Google Scholar]
  18. Holder D. J., Keyhani N. O. 2005; Adhesion of the entomopathogenic fungus Beauveria ( Cordyceps) bassiana to substrata. Appl Environ Microbiol 71:5260–5266
     [Google Scholar]
  19. Holder D. J., Kirkland B. H., Lewis M. W., Keyhani N. O. 2007; Surface characteristics of the entomopathogenic fungus Beauveria ( Cordyceps) bassiana . Microbiology 153:3448–3457
     [Google Scholar]
  20. Inglis G. D., Goettel M. S., Butt T. M., Strasser H. 2001; Use of hyphomycetous fungi for managing insect pests. In Field Manual of Techniques in Invertebrate Pathology pp 651–679 Edited by Lacey L. A., Kaya H. K. Dordrecht, The Netherlands: Kluwer Academic;
     [Google Scholar]
  21. Kaksonen M., Sun Y., Drubin D. G. 2003; A pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell 115:475–487
     [Google Scholar]
  22. Kassa A., Stephan D., Vidal S., Zimmermann G. 2004; Production and processing of Metarhizium anisopliae var. acridum submerged conidia for locust and grasshopper control. Mycol Res 108:93–100
     [Google Scholar]
  23. Kirkland B. H., Westwood G. S., Keyhani N. O. 2004; Pathogenicity of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to Ixodidae tick species Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis . J Med Entomol 41:705–711
     [Google Scholar]
  24. Lecuona R. E., Turica M., Tarocco F., Crespo D. C. 2005; Microbial control of Musca domestica (Diptera: Muscidae) with selected strains of Beauveria bassiana . J Med Entomol 42:332–336
     [Google Scholar]
  25. Lee S. C., Schmidtke S. N., Dangott L. J., Shaw B. D. 2008; Aspergillus nidulans ArfB plays a role in endocytosis and polarized growth. Eukaryot Cell 7:1278–1288
     [Google Scholar]
  26. Lomer C. J., Bateman R. P., Johnson D. L., Langewald J., Thomas M. 2001; Biological control of locusts and grasshoppers. Annu Rev Entomol 46:667–702
     [Google Scholar]
  27. Mazet I., Hung S. Y., Boucias D. G. 1994; Detection of toxic metabolites in the hemolymph of Beauveria bassiana infected Spodoptera exigua larvae. Experientia 50:142–147
     [Google Scholar]
  28. McCoy C. W. 1990; Entomogenous fungi as microbial pestidides. In New Directions in Biological Control pp 139–159 Edited by Baker R. R., Dunn P. E. New York: A.R. Liss;
     [Google Scholar]
  29. Murphy A. S., Bandyopadhyay A., Holstein S. E., Peer W. A. 2005; Endocytotic cycling of PM proteins. Annu Rev Plant Biol 56:221–251
     [Google Scholar]
  30. Pathan A. A. K., Devi K. U., Vogel H., Reineke A. 2007; Analysis of differential gene expression in the generalist entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin grown on different insect cuticular extracts and synthetic medium through cDNA-AFLPs. Fungal Genet Biol 44:1231–1241
     [Google Scholar]
  31. Penalva M. A. 2005; Tracing the endocytic pathway of Aspergillus nidulans with FM4-64. Fungal Genet Biol 42:963–975
     [Google Scholar]
  32. Pendland J. C., Hung S. Y., Boucias D. G. 1993; Evasion of host defense by in vivo-produced protoplast-like cells of the insect mycopathogen Beauveria bassiana . J Bacteriol 175:5962–5969
     [Google Scholar]
  33. Pringle J. R., Preston R. A., Adams A. E. M., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. 1989; Fluorescence microscopy methods for yeast. Methods Cell Biol 31:357–435
     [Google Scholar]
  34. Read N. D., Kalkman E. R. 2003; Does endocytosis occur in fungal hyphae?. Fungal Genet Biol 39:199–203
     [Google Scholar]
  35. Rehner S. A., Buckley E. 2005; A Beauveria phylogeny inferred from nuclear ITS and EF1-alpha sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97:84–98
     [Google Scholar]
  36. Samson R. A., Evans C. S., Latgé J. P. 1988 Atlas of Entomopathogenic Fungi Berlin: Springer-Verlag;
     [Google Scholar]
  37. Scholte E. J., Knols B. G. J., Samson R. A., Takken W. 2004; Entomopathogenic fungi for mosquito control: a review. J Insect Sci 4:19
     [Google Scholar]
  38. Shoji J. Y., Arioka M., Kitamoto K. 2006; Vacuolar membrane dynamics in the filamentous fungus Aspergillus oryzae . Eukaryot Cell 5:411–421
     [Google Scholar]
  39. Smythe E., Ayscough K. R. 2006; Actin regulation in endocytosis. J Cell Sci 119:4589–4598
     [Google Scholar]
  40. Steinberg G. 2007; On the move: endosomes in fungal growth and pathogenicity. Nat Rev Microbiol 5:309–316
     [Google Scholar]
  41. Tartar A., Boucias D. G. 2004; A pilot-scale expressed sequence tag analysis of Beauveria bassiana gene expression reveals a tripeptidyl peptidase that is differentially expressed in vivo . Mycopathologia 158:201–209
     [Google Scholar]
  42. Thomas K. C., Khachatourians G. G., Ingledew W. M. 1987; Production and properties of Beauveria bassiana conidia cultivated in submerged culture. Can J Microbiol 33:12–20
     [Google Scholar]
  43. Toret C. P., Drubin D. G. 2006; The budding yeast endocytic pathway. J Cell Sci 119:4585–4587
     [Google Scholar]
  44. Toret C. P., Lee L., Sekiya-Kawasaki M., Drubin D. G. 2008; Multiple pathways regulate endocytic coat disassembly in Saccharomyces cerevisiae for optimal downstream trafficking. Traffic 9:848–859
     [Google Scholar]
  45. Torralba S., Heath I. B. 2002; Analysis of three separate probes suggests the absence of endocytosis in Neurospora crassa hyphae. Fungal Genet Biol 37:221–232
     [Google Scholar]
  46. Vida T. A., Emr S. D. 1995; A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128:779–792
     [Google Scholar]
  47. Virag A., Harris S. D. 2006; The Spitzenkorper: a molecular perspective. Mycol Res 110:4–13
     [Google Scholar]
  48. Wanchoo A., Lewis M. W., Keyhani N. O. 2009; Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and haemolymph-derived cells of the entomopathogenic fungus Beauveria bassiana . Microbiology 155:3121–3133
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.029165-0
Loading
Uptake of the fluorescent probe FM4-64 by hyphae and haemolymph-derived in vivo hyphal bodies of the entomopathogenic fungus Beauveria bassiana
Microbiology 155, 3110 (2009); https://doi.org/10.1099/mic.0.029165-0
/content/journal/micro/10.1099/mic.0.029165-0
/content/journal/micro/10.1099/mic.0.029165-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error