Measuring Fungal Membrane Potential

To analyze the fungal membrane potential, protoplasts were first prepared. The conidial suspension of the indicated strains in CY (15 mL, 5 × 10⁷ conidia mL⁻¹) was shaken for 20 h at 25 °C and the germinating mycelia were incubated in enzymatic digestion buffer (0.8 M MgSO₄, 1 % w/v Lysing Enzymes from Trichoderma (L1412, Sigma), 0.1 % w/v Snailase (S8280, Solarbio, Beijing, China)) for 2 h with gentle shaking (100 rpm) in dark. Protoplasts were collected by centrifugation for 5 min (1500× g) and then shifted to pH 5 or pH 8 conditions and continually shaken (100 rpm) for another 1 h. 2 mM bis (1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3), Invitrogen, Carlsbad, CA, USA) was added to the sample and incubated for 10 min at 4 °C in the dark. Fluorescence was examined using a confocal laser scanning microscope with 488 nm excitation and 509 nm emission using a confocal Zeiss 980 laser scanning microscope with Elyra7 (Zeiss, Oberkochen, Germany) [28 (link),29 (link)]. Images and fluorescence measurements of the confocal data were captured with ZEISS ZEN 3.2 (blue edition) software (Zeiss, Oberkochen, Germany) under the same parameters.