Single-Molecule Fluorescence in situ Hybridization (smFISH)

Approximately 10² to 10³ conidia of the strain RIB40 were inoculated into 100 μL of respective liquid media placed into poly-lysine coated glass bottom dishes (Matsunami) and incubated at 30°C for 12 h in complete medium or for 20 h in minimal medium. Single-molecule fluorescence in situ hybridization (smFISH) analysis was performed essentially according to the manufacturer’s instructions with some modifications based on previous literature (LGC Biosearch Technologies; König et al., 2009; Tutucci et al., 2018). Cultured cells were fixed with 100 μL of 7.4% formaldehyde in PBS (0.8% NaCl, 0.2% KCl, 0.12% Na₂HPO₄, 0.2% KH₂PO₄) at room temperature (RT) for 1 h. After undergoing 2 rinses with 100 μL of fixation buffer (1.2 M sorbitol, 0.1 M K₂HPO₄, pH 7.5), the cultures were permeabilized with 100 μL of 70% ethanol at −20°C overnight. The cells were then dissolved in 100 μL of hybridization buffer containing 10% deionized formamide with or without the addition of 1 μL of 12.5 μM of the probe (final concentration of 125 nM) overnight. The cells were then protected from the light for the procedures that follow. After replacement of the hybridization buffer with 100 μL of buffer A (LGC Biosearch Technologies), containing 10% of deionized formamide, the cells were incubated with 100 μL of buffer A at 30°C for 30 min. To stain the nuclei, the cells were incubated with 100 μL of buffer A containing 5 ng/mL DAPI in DMSO (Molecular Probes) at 30°C for 30 min. Buffer A was then replaced with 100 μL of buffer B (LGC Biosearch Technologies) and incubated at RT for 3 min. The cells were then mounted in 50 μL of Vectashield Antifade Mounting Medium (Vector Laboratories) to minimize fluorescence bleaching and subsequently observed under fluorescence microscope.

The smFISH probes (LGC Biosearch Technologies) were used according to the manufacturer’s instructions. The smFISH probe for amyB consisted of mixtures of 18–22 nt from 47 regions in 1,497 b, each region was linked to fluorescein amidite (FAM; excitation/emission, 495/520 nm). The smFISH probe for actA consisted of mixtures of 18 to 22 nt from 45 regions in 1,128 b, each region was linked to CAL Fluor Red 610 (excitation/emission, 590/610 nm). The probe sequence data are summarized in Supplementary Figures S1, S2.