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

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 Xuorescently 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.