Detection of hyphal fusion in filamentous fungi using differently fluorescence-labeled histones
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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.
KeywordsSordaria macrospora Cell fusion Histone-assisted merged fluorescence assay Sterile mutant
We thank Ms. Susanne Schlewinski and Ms. Ingeborg Godehardt for excellent technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 480, A1). I.E. received a stipend from the Studienstiftung des deutschen Volkes (Bonn, Germany).
- Esser K (1982) Cryptogams—cyanobacteria, algae, fungi, lichens. Cambridge University Press, LondonGoogle Scholar
- 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
- 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
- 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
- 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
- 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
- Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar