Fungal Cell Wall Digestion Protocol

Fixation in formaldehyde was adapted from the protocols described by Bertaux and Wang (Bertaux et al., 2003 (link); Wang et al., 2017 (link)). Fungal cultures grown under varied conditions were fixed in a 4% solution of formaldehyde (w/v, Thermo Fisher Scientific) in PBS overnight at 4°C. Fungal biomass was washed 3× with PBS and then treated with cocktail combinations of cell wall digestive enzymes: 1 mg•ml⁻¹ solution of lysozyme, or 0.5 mg•ml⁻¹ solution of chitinase, and/or 5 mg•ml⁻¹ solution of glucanase (reagents purchased from Sigma-Aldrich) for 1 h at 37°C and washed 3× with PBS. The cultures were then dehydrated with a step-wise series of ethanol treatments: 50%, 75%, 100%, 75%, 50%, and finally replaced with PBS for 3 min between each step.