1887

Journal of General Virology header logo

Volume 93, Issue 11

Recombinant expression of the coat protein of and development of an immunofluorescence detection method to study its intracellular distribution in Free

Abstract

is infected by many mycoviruses with varying phenotypical effects on the fungal host, including (BVX), a mycovirus that has been found in several isolates worldwide with no obvious effects on growth. Here we present results from serological and immunofluorescence microscopy (IFM) studies using antiserum raised against the coat protein of BVX expressed in fused to maltose-binding protein. Due to the high yield of recombinant protein it was possible to raise antibodies that recognized BVX particles. An indirect ELISA, using BVX antibodies, detected BVX in partially purified virus preparations from fungal isolates containing BVX alone and in mixed infection with . The BVX antiserum also proved suitable for IFM studies. Intensely fluorescing spots (presumed to be virus aggregates) were found to be localized in hyphal cell compartments and spores of natural and experimentally infected isolates using IFM. Immunofluorescently labelled sections through fungal tissue, as well as fixed mycelia grown on glass slides, showed aggregations of virions closely associated with fungal cell membranes and walls, next to septal pores, and in hyphal tips. Also, calcofluor white staining of mature cell walls of virus-transfected clones revealed numerous cell wall areas with increased amounts of chitin/glycoproteins. Our results indicate that some BVX aggregates are closely associated with the fungal cell wall and raise the question of whether mycoviruses may be able to move through the wall and therefore not be totally dependent on intracellular routes of transmission.

  • Received:
  • Accepted:
  • Published Online:
© 2012 SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.043869-0
2012-11-01
2024-05-20
Loading full text...

Full text loading...

/deliver/fulltext/jgv/93/11/2502.html?itemId=/content/journal/jgv/10.1099/vir.0.043869-0&mimeType=html&fmt=ahah

References

  1. Arthur K. 2007 Detection and incidence of the mycoviruses BVX and BCVF in the plant pathogenic fungus Botrytis cinerea MSc thesis The University of Auckland; New Zealand:
     [Google Scholar]
  2. Baneyx F. 1999; Recombinant protein expression in Escherichia coli . Curr Opin Biotechnol 10:411–421 [View Article][PubMed]
     [Google Scholar]
  3. Banks G. T., Buck K. W., Chain E. B., Darbyshire J. E., Himmelweit F., Ratti G., Sharpe T. J., Planterose D. N. 1970; Antiviral activity of double stranded RNA from a virus isolated from Aspergillus foetidus . Nature 227:505–507 [View Article][PubMed]
     [Google Scholar]
  4. Beever R. E., Weeds P. L. 2007; Taxonomy and genetic variation of Botrytis and Botryotinia . In Botrytis: Biology, Pathology and Control pp. 29–52 Edited by Elad Y, Williamson B, Tudzynski P, Delen N. Dordrecht, The Netherlands: Kluwer Academic Publishers; [View Article]
     [Google Scholar]
  5. Boine B. 2012 A study of the interaction between the plant pathogenic fungus Botrytis cinerea and the filamentous ssRNA mycoviruses Botrytis virus X and Botrytis virus F PhD thesis The University of Auckland; New Zealand:
     [Google Scholar]
  6. Buck K. W., Chain E. B., Darbyshire J. E. 1969; High cell wall galactosamine content and virus particles in Penicillium stoloniferum . Nature 223:1273–1273 [View Article][PubMed]
     [Google Scholar]
  7. Casadevall A., Nosanchuk J. D., Williamson P., Rodrigues M. L. 2009; Vesicular transport across the fungal cell wall. Trends Microbiol 17:158–162 [View Article][PubMed]
     [Google Scholar]
  8. Castro M., Kramer K., Valdivia L., Ortiz S., Benavente J., Castillo A. 1999; A new double-stranded RNA mycovirus from Botrytis cinerea . FEMS Microbiol Lett 175:95–99 [View Article][PubMed]
     [Google Scholar]
  9. Castro M., Kramer K., Valdivia L., Ortiz S., Castillo A. 2003; A double-stranded RNA mycovirus confers hypovirulence-associated traits to Botrytis cinerea . FEMS Microbiol Lett 228:87–91 [View Article][PubMed]
     [Google Scholar]
  10. Cerovska N., Moravec T., Rosecka P., Dedic P., Filigarova M. 2003; Production of polyclonal antibodies to a recombinant coat protein of Potato mop-top virus . J Phytopathol 151:195–200 [View Article]
     [Google Scholar]
  11. Chanda A., Roze L. V., Linz J. E. 2010; A possible role for exocytosis in aflatoxin export in Aspergillus parasiticus . Eukaryot Cell 9:1724–1727 [View Article][PubMed]
     [Google Scholar]
  12. Coley-Smith J. R., Verhoeff K., Jarvis W. R. 1980 The Biology of Botrytis London: Academic Press;
     [Google Scholar]
  13. Faretra E., Antonacci E., Pollastro S. 1988; Sexual behaviour and mating system of Botryotinia fuckeliana, teleomorph of Botrytis cinerea. . J Gen Microbiol 134:2543–2550
     [Google Scholar]
  14. Goodman R. M., McDonald J. G., Horne R. W., Bancroft J. B. 1976; Assembly of flexuous plant viruses and their proteins. Philos Trans R Soc Lond B Biol Sci 276:173–179 [View Article][PubMed]
     [Google Scholar]
  15. Gull K., Trinci A. P. J. 1971; Fine structure of spore germination in Botrytis cinerea . J Gen Microbiol 68:207–220 [View Article]
     [Google Scholar]
  16. Gull K., Trinci A. P. J. 1974; Detection of areas of wall differentiation in fungi using fluorescent staining. Arch Mikrobiol 96:53–57[PubMed] [CrossRef]
     [Google Scholar]
  17. Howitt R., Beever R., Pearson M. N., Forster R. L. S. 1995; Presence of double-stranded RNA and virus-like particles in Botrytis cinerea . Mycol Res 99:1472–1478 [View Article]
     [Google Scholar]
  18. Howitt R. L., Beever R. E., Pearson M. N., Forster R. L. S. 2001; Genome characterization of Botrytis virus F, a flexuous rod-shaped mycovirus resembling plant ‘potex-like’ viruses. J Gen Virol 82:67–78[PubMed]
     [Google Scholar]
  19. Howitt R. L., Beever R. E., Pearson M. N., Forster R. L. S. 2006; Genome characterization of a flexuous rod-shaped mycovirus, Botrytis virus X, reveals high amino acid identity to genes from plant ‘potex-like’ viruses. Arch Virol 151:563–579 [View Article][PubMed]
     [Google Scholar]
  20. Hughes R. C. 1975; The complex carbohydrates of mammalian cell surfaces and their biological roles. Essays Biochem 11:1–36[PubMed]
     [Google Scholar]
  21. Jacob-Wilk D., Turina M., Van Alfen N. K. 2006; Mycovirus Cryphonectria hypovirus 1 elements cofractionate with trans-Golgi network membranes of the fungal host Cryphonectria parasitica . J Virol 80:6588–6596 [View Article][PubMed]
     [Google Scholar]
  22. Johnston P. R., Sutherland P. W., Joshee S. 2006; Visualising endophytic fungi within leaves by detection of (1→3)-β-d-glucans in fungal cell walls. Mycologist 20:159–162 [View Article]
     [Google Scholar]
  23. Kapust R. B., Waugh D. S. 1999; Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8:1668–1674 [View Article][PubMed]
     [Google Scholar]
  24. Kim Y. J., Kim J. Y., Kim J. H., Yoon S. M., Yoo Y. B., Yie S. W. 2008; The identification of a novel Pleurotus ostreatus dsRNA virus and determination of the distribution of viruses in mushroom spores. J Microbiol 46:95–99 [View Article][PubMed]
     [Google Scholar]
  25. Kingston R. L., Baase W. A., Gay L. S. 2004; Characterization of nucleocapsid binding by the measles virus and mumps virus phosphoproteins. J Virol 78:8630–8640 [View Article][PubMed]
     [Google Scholar]
  26. Ling Q., Wu J. X., Qi Y. J., Zhou X. P., Li D. B. 2000; Production of monoclonal antibodies to broad bean wilt virus and application in virus detection. Wei Sheng Wu Xue Bao 40:166–173 (in Chinese)
    [Google Scholar]
  27. Maeda H., Ishida N. 1967; Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener. J Biochem 62:276–278[PubMed]
     [Google Scholar]
  28. Martelli G. P., Adams M. J., Kreuze J. F., Dolja V. V. 2007; Family Flexiviridae: a case study in virion and genome plasticity. Annu Rev Phytopathol 45:73–100 [View Article][PubMed]
     [Google Scholar]
  29. McCabe P. M., Pfeiffer P., Van Alfen N. K. 1999; The influence of dsRNA viruses on the biology of plant pathogenic fungi. Trends Microbiol 7:377–381 [View Article][PubMed]
     [Google Scholar]
  30. Medina V., Tian T. Y., Wierzchos J., Falk B. W. 1998; Specific inclusion bodies are associated with replication of lettuce infectious yellows virus RNAs in Nicotiana benthamiana protoplasts. J Gen Virol 79:2325–2329[PubMed]
     [Google Scholar]
  31. Newhouse J. R., Hoch H. C., MacDonald W. L. 1983; The ultrastructure of Endothia parasitica comparison of a virulent with a hypovirulent isolate. Can J Bot 61:389–399 [View Article]
     [Google Scholar]
  32. Nolan R. A., Clovis C. J., Davidson W. S. 1988; Microbodies and a virus-like particle in Entomophaga aulicae . Trans Br Mycol Soc 90:315–318 [View Article]
     [Google Scholar]
  33. Pearson M. N., Beever R. E., Boine B., Arthur K. 2009; Mycoviruses of filamentous fungi and their relevance to plant pathology. Mol Plant Pathol 10:115–128 [View Article][PubMed]
     [Google Scholar]
  34. Riggs P. 2000; Expression and purification of recombinant proteins by fusion to maltose-binding protein. Mol Biotechnol 15:51–63 [View Article][PubMed]
     [Google Scholar]
  35. Ro H. S., Lee N. J., Lee C. W., Lee H. S. 2006; Isolation of a novel mycovirus OMIV in Pleurotus ostreatus and its detection using a triple antibody sandwich-ELISA. J Virol Methods 138:24–29 [View Article][PubMed]
     [Google Scholar]
  36. Rodrigues M. L., Nakayasu E. S., Oliveira D. L., Nimrichter L., Nosanchuk J. D., Almeida I. C., Casadevall A. 2008; Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence. Eukaryot Cell 7:58–67 [View Article][PubMed]
     [Google Scholar]
  37. Rubinson E., Galiakparov N., Radian S., Sela I., Tanne E., Gafny R. 1997; Serological detection of grapevine virus A using antiserum to a nonstructural protein, the putative movement protein. Phytopathology 87:1041–1045 [View Article][PubMed]
     [Google Scholar]
  38. Salonen A., Ahola T., Kääriäinen L. 2005; Viral RNA replication in association with cellular membranes. Curr Top Microbiol Immunol 285:139–173 [View Article][PubMed]
     [Google Scholar]
  39. Sasaki A., Kanematsu S., Onoue M., Oyama Y., Yoshida K. 2006; Infection of Rosellinia necatrix with purified viral particles of a member of Partitiviridae (RnPV1-W8). Arch Virol 151:697–707 [CrossRef]
     [Google Scholar]
  40. Segers G. C., Zhang X., Deng F., Sun Q., Nuss D. L. 2007; Evidence that RNA silencing functions as an antiviral defense mechanism in fungi. Proc Natl Acad Sci U S A 104:12902–12906 [View Article][PubMed]
     [Google Scholar]
  41. Urayama S., Kato S., Suzuki Y., Aoki N., Le M. T., Arie T., Teraoka T., Fukuhara T., Moriyama H. 2010; Mycoviruses related to chrysovirus affect vegetative growth in the rice blast fungus Magnaporthe oryzae . J Gen Virol 91:3085–3094 [View Article][PubMed]
     [Google Scholar]
  42. Vilches S., Castillo A. 1997; A double-stranded RNA mycovirus in Botrytis cinerea . FEMS Microbiol Lett 155:125–130 [View Article][PubMed]
     [Google Scholar]
  43. Vogel H. J. 1956; A convenient growth medium for Neurospora (medium N). Microb Genet Bull 13:42–43
     [Google Scholar]
  44. Yaegashi H., Sawahata T., Ito T., Kanematsu S. 2011; A novel colony-print immunoassay reveals differential patterns of distribution and horizontal transmission of four unrelated mycoviruses in Rosellinia necatrix . Virology 409:280–289 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.043869-0
Loading
Recombinant expression of the coat protein of Botrytis virus X and development of an immunofluorescence detection method to study its intracellular distribution in Botrytis cinerea
J. Gen. Virol. 93, 2502 (2012); https://doi.org/10.1099/vir.0.043869-0
/content/journal/jgv/10.1099/vir.0.043869-0
/content/journal/jgv/10.1099/vir.0.043869-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

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