Subcellular Localization and Apoptosis Assay for Fon

To analyze septation and subcellular localization, fresh conidia and mycelia of the Fon strains were washed with sterile ddH₂O₂ and co-stained with CFW or Hoechst 33342, respectively [9 (link)]. For apoptotic cell death analysis, the germ tubes of the Fon strains were treated with/without apoptosis-inducing compound farnesol (FOH; Shanghai Aladdin Bio-Chem, Shanghai, China) at 25 or 50 μM for 4 h at 26 °C. Necrotic cells and nuclei were co-stained with propidium iodide (PI; Shanghai Yeasen Biotech, Shanghai, China) and Hoechst 33342 (Shanghai Yeasen Biotech, China), respectively [35 (link),36 (link)]. To examine autophagy, the Fon strains were cultivated in liquid CM for 12 h, and the collected mycelia were rinsed with sterile ddH₂O₂. After washing, the mycelia were transferred into nitrogen-starved MM medium (MM-N) with/without 4 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO, USA) for 1 h. The mycelia were stained with the fluorescent dye monodansylcadaverine (MDC; Sigma-Aldrich, St. Louis, MO, USA) for 30 min in the dark, followed by ddH₂O₂ washing [37 (link),38 (link)]. All aforementioned samples were observed under a Zeiss LSM 780 Meta confocal microscope (Gottingen, Niedersachsen, Germany) with the appropriate excitation and emission filters for corresponding dyes and signals.