Current Genetics

, 52:259 | Cite as

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

  • Christine Rech
  • Ines Engh
  • Ulrich Kück
Technical Note


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.


Sordaria 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).

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)


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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

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

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