Slide 8 well microscopy chambers

Approximately 1,000 conidiospores of each strain were inoculated in Ibidi® µ-Slide 8-well microscopy chambers containing 400 µL liquid MM_PABA and were grown vegetatively for 20 h at 37 °C for confocal microscopy. To analyze the subcellular localization of GFP-tagged proteins, the Zeiss AxioObserver Z.1 inverted confocal microscope connected with a Plan-Apochromat 100×/1.4 oil objective and a QuantEM:512SC camera were used. The SlideBook version 6.0 software was used with default settings to record and process the pictures. The GFP-fused proteins were detected at around 510 nm. The autofluorescence of each strain was corrected against the background signal of the wild-type strain. The visualization of fungal nuclei occurred either by DAPI (4´,6-diamidin-2-phenylindol) staining or by the production of RFP-Histone 2A. DAPI was added in a final concentration of 1 µg/mL to the fungal cultures, which were subsequently incubated for additional 30 min at room temperature (RT) in the darkness prior to microscopy. DAPI signals were detected at around 460 nm. In case of strains producing RFP-Histone 2A, the RFP was visualized by illumination at around 588 nm. 100 or 1,000 millisecond illumination was used to visualize the DIC (differential interference contrast), GFP, RFP, and DAPI signals.