Fluorescence anisotropy.

The membrane fluidity was indicated by fluorescence anisotropy. For obtaining an abundance of young germlings, 10⁸ conidia were cultured in 100 ml of liquid MM at 30°C at 220 rpm (15 h for the wild-type and cybE complement strains; 20 h for the cybE deletion strain). The subsequent procedures refer to the previous descriptions (57). Briefly, mycelia were collected and mixed with 11% mannitol that contained 0.25% (vol/vol) formaldehyde for fixation (0.5 h). Mycelia were resuspended in 10 ml osmotic medium (1.2 M MgSO₄, 6.8 mM NaH₂PO₄, and 3.2 mM Na₂HPO₄, pH 5.8) containing 20 mg yatalase and 30 mg lysing enzymes for 4 h at 28°C. Then, protoplasts were washed twice with PBS buffer (pH 7.4) containing 0.25% (vol/vol) formaldehyde and incubated for 1 h at 37°C with 5 μM 1,6-diphenyl-1,3,5-hexatriene (DPH) probe. The unlabeled probe in solution was removed by centrifugation at 3 × 10³ × g for 5 min. Then, cells were resuspended in PBS buffer, and the optical density at 600 nm (OD₆₀₀) of the mixture was adjusted to 0.5. Fluorescence anisotropy was measured at 37°C using a circular dichroism spectrometer with emission at 430 nm and excitation at 360 nm. Anisotropy values (r) were calculated as the formula (I_VV − I_VH)/(I_VV + 2I_VH), where I is the corrected fluorescence intensity, and the subscripts H and V indicate the values obtained with horizontal and vertical orientations, respectively, of the emission analyzer and excitation polarizer. Increases in fluorescence anisotropy indicated decreases in the fluidity of the lipid bilayer.