Current Genetics

, 52:259 | Cite as

Detection of hyphal fusion in filamentous fungi using differently fluorescence-labeled histones

Technical Note

Abstract

Cell fusion occurs regularly during the vegetative and sexual phases of the life cycle in filamentous fungi. Here, we present a simple and efficient method that can detect even rare hyphal fusion events. Using the homothallic ascomycete Sordaria macrospora as an experimental system, we developed a histone-assisted merged fluorescence (HAMF) assay for the investigation of hyphal fusion between vegetative mycelia. For this purpose, two reporter vectors were constructed encoding the histone proteins HH2B or HH2A fused at their C terminus either with the cyan or yellow fluorescent protein, respectively. The chimeric proteins generate fluorescently labeled nuclei and thus enable the distinction between different strains in a mycelial mixture. For example, hyphae with nuclei that show both cyan as well as yellow fluorescence indicate the formation of a heterokaryon as a result of hyphal fusion. To test the applicability of our HAMF assay, we used two S. macrospora developmental mutants that are supposed to have reduced hyphal fusion rates. The simple and efficient HAMF assay described here could detect even rare fusion events and should be applicable to a broad range of diverse fungal species including those lacking male or female reproductive structures or asexual spores.

Keywords

Sordaria macrospora Cell fusion Histone-assisted merged fluorescence assay Sterile mutant 

Supplementary material

294_2007_158_MOESM1_ESM.pdf (182 kb)
Physical maps of pCH2B, pYH2A, pCHN3 and pYHN3. Details of constructions are given in the main text. Abbreviations: bla, ampicillin resistance gene; ecfp, gene for enhanced cyan fluorescent protein (ECFP); eyfp, gene for enhanced yellow fluorescent protein (EYFP); hh2A, histone H2A gene; hh2B, histone H2B gene; hph, hygromycin B phosphotransferase gene; Pgpd, A. nidulans gpd promoter; pUCori, E. coli replication origin; TtrpC, A. nidulans trpC terminator (PDF 182 kb)
294_2007_158_MOESM2_ESM.pdf (14 kb)
Fusion sequence between histone hh2b-ecfp (a) and hh2a-eyfp (b). Coding sequences and deduced amino acid residues are marked in black letters, intron sequences are given in grey letters, heterologous sequences adapted from N. crassa sequence are shown in italics. Transcriptional start codons and stop codons are highlighted in bold letters. Underlined are NcoI restriction sites that were used for fusion of the open reading frames encoding the histone and the fluorescent protein (PDF 14 kb)

References

  1. Baxevanis AD, Arents G, Moudrianakis EN, Landsman D (1995) A variety of DNA-binding and multimeric proteins contain the histone fold motif. Nucleic Acids Res 23:2685–2691PubMedCrossRefGoogle Scholar
  2. Bistis GN (1981) Chemotropic interactions between trichogynes and conidia of opposite mating-type in Neurospora crassa. Mycologia 73:959–975CrossRefGoogle Scholar
  3. Engh I, Würtz C, Witzel-Schlömp K, Zhang HY, Hoff B, Nowrousian M, Rottensteiner H, Kück U (2007) The WW domain protein PRO40 is required for fungal fertility and associates with Woronin bodies. Eukaryot Cell 6:831–843PubMedCrossRefGoogle Scholar
  4. Esser K (1982) Cryptogams—cyanobacteria, algae, fungi, lichens. Cambridge University Press, LondonGoogle Scholar
  5. Esser K, Straub J (1958) Genetische Untersuchungen an Sordaria macrospora Auersw.: Kompensation und Induktion bei genbedingten Entwicklungsdefekten. Z Vererbungsle 89:729–746CrossRefGoogle Scholar
  6. Fleißner A, Sarkar S, Jacobson DJ, Roca MG, Read ND, Glass NL (2005) The so locus is required for vegetative cell fusion and postfertilization events in Neurospora crassa. Eukaryot Cell 4:920–930PubMedCrossRefGoogle Scholar
  7. Freitag M, Hickey PC, Raju NB, Selker EU, Read ND (2004) GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa. Fungal Genet Biol 41:897–910PubMedCrossRefGoogle Scholar
  8. Glass NL, Fleißner A (2006) Re-wiring the network: understanding the mechanism and fuction of anstomosis in filamentous ascomycete fungi. In: Kües U, Fischer R (eds) The Mycota I. Springer, Berlin, pp 123–140Google Scholar
  9. Glass NL, Jacobson DJ, Shiu PK (2000) The genetics of hyphal fusion and vegetative incompatibility in filamentous ascomycete fungi. Annu Rev Genet 34:165–186PubMedCrossRefGoogle Scholar
  10. Gregory P (1984) The fungal mycelium: a historical perspective. Trans Br Mycol Soc 82:1–11CrossRefGoogle Scholar
  11. Hoff B, Kück U (2005) Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of living cells from the filamentous fungus Acremonium chrysogenum. Curr Genet 47:132–138PubMedCrossRefGoogle Scholar
  12. Hou Z, Xue C, Peng Y, Katan T, Kistler HC, Xu JR (2002) A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility, heterokaryon formation, and plant infection. Mol Plant Microbe Interact 15:1119–1127PubMedCrossRefGoogle Scholar
  13. Jerpseth B, Greener A, Short JM, Viola J, Kretz PL (1992) XL1-Blue MRF` E. coli cells: mcrA-, mcrCB-, mcrF-, mmr-, hsdR- derivative of XL1-Blue cells. Strateg Mol Biol 5:81–83Google Scholar
  14. Kück U (2005) A Sordaria macrospora mutant lacking the leu1 gene shows a developmental arrest during fruiting body formation. Mol Genet Genomics 274:307–315PubMedCrossRefGoogle Scholar
  15. Lemke PA, Peng M (1995) Genetic manipulation of fungi by DNA-mediated transformation. In: Kück U (ed) The Mycota II. Springer, Berlin, pp 109–139Google Scholar
  16. Li L, Schmelz M, Kellner EM, Galgiani JN, Orbach MJ (2007) Nuclear labeling of Coccidioides posadasii with green fluorescent protein (GFP). Ann N Y Acad Sci (in press)Google Scholar
  17. Maruyama J, Nakajima H, Kitamoto K (2001) Visualization of nuclei in Aspergillus oryzae with EGFP and analysis of the number of nuclei in each conidium by FACS. Biosci Biotechnol Biochem 65:1504–1510PubMedCrossRefGoogle Scholar
  18. Masloff S, Pöggeler S, Kück U (1999) The pro1(+) gene from Sordaria macrospora encodes a C6 zinc finger transcription factor required for fruiting body development. Genetics 152:191–199PubMedGoogle Scholar
  19. Meeks-Wagner D, Hartwell LH (1986) Normal stoichiometry of histone dimer sets is necessary for high fidelity of mitotic chromosome transmission. Cell 44:43–52PubMedCrossRefGoogle Scholar
  20. Mullaney EJ, Hamer JE, Roberti KA, Yelton MM, Timberlake WE (1985) Primary structure of the trpC gene from Aspergillus nidulans. Mol Gen Genet 199:37–45PubMedCrossRefGoogle Scholar
  21. Nolan S, Cowan AE, Koppel DE, Jin H, Grote E (2006) FUS1 regulates the opening and expansion of fusion pores between mating yeast. Mol Biol Cell 17:2439–2450PubMedCrossRefGoogle Scholar
  22. Nowrousian M, Cebula P (2005) The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora. BMC Microbiol 5:64PubMedCrossRefGoogle Scholar
  23. Nowrousian M, Masloff S, Pöggeler S, Kück U (1999) Cell differentiation during sexual development of the fungus Sordaria macrospora requires ATP citrate lyase activity. Mol Cell Biol 19:450–460PubMedGoogle Scholar
  24. Nowrousian M, Würtz C, Pöggeler S, Kück U (2004) Comparative sequence analysis of Sordaria macrospora and Neurospora crassa as a means to improve genome annotation. Fungal Genet Biol 41:285–292PubMedCrossRefGoogle Scholar
  25. Pandey A, Roca MG, Read ND, Glass NL (2004) Role of a mitogen-activated protein kinase pathway during conidial germination and hyphal fusion in Neurospora crassa. Eukaryot Cell 3:348–358PubMedCrossRefGoogle Scholar
  26. Pöggeler S, Kück U (2004) A WD40 repeat protein regulates fungal cell differentiation and can be replaced functionally by the mammalian homologue striatin. Eukaryot Cell 3:232–240PubMedCrossRefGoogle Scholar
  27. Pöggeler S, Masloff S, Hoff B, Mayrhofer S, Kück U (2003) Versatile EGFP reporter plasmids for cellular localization of recombinant gene products in filamentous fungi. Curr Genet 43:54–61PubMedGoogle Scholar
  28. Pontecorvo G (1956) The parasexual cycle in fungi. Annu Rev Microbiol 10:393–400PubMedCrossRefGoogle Scholar
  29. Punt PJ, Dingemanse MA, Kuyvenhoven A, Soede RD, Pouwels PH, van den Hondel CA (1990) Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase. Gene 93:101–109PubMedCrossRefGoogle Scholar
  30. Ramon A, Muro-Pastor MI, Scazzocchio C, Gonzalez R (2000) Deletion of the unique gene encoding a typical histone H1 has no apparent phenotype in Aspergillus nidulans. Mol Microbiol 35:223–233PubMedCrossRefGoogle Scholar
  31. Read ND (1994) Cellular nature and multicellular morphogenesis of higher fungi. In: Ingram D, Hudson A (eds) Shape and form in plants and fungi. Academic, London, pp 254–271Google Scholar
  32. Roca MG, Arlt J, Jeffree CE, Read ND (2005) Cell biology of conidial anastomosis tubes in Neurospora crassa. Eukaryot Cell 4:911–919PubMedCrossRefGoogle Scholar
  33. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  34. Walz M, Kück U (1995) Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis. Curr Genet 29:88–95PubMedCrossRefGoogle Scholar
  35. Wei H, Requena N, Fischer R (2003) The MAPKK kinase SteC regulates conidiophore morphology and is essential for heterokaryon formation and sexual development in the homothallic fungus Aspergillus nidulans. Mol Microbiol 47:1577–1588PubMedCrossRefGoogle Scholar
  36. Xiang Q, Rasmussen C, Glass NL (2002) The ham-2 locus, encoding a putative transmembrane protein, is required for hyphal fusion in Neurospora crassa. Genetics 160:169–180PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Lehrstuhl für Allgemeine und Molekulare BotanikRuhr-Universität BochumBochumGermany

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