Protocol for generating protoplasts from the leafcutter ant symbiotic fungus Leucoagaricus gongylophorus

We present a protocol to isolate viable protoplasted cells of the basidiomycete fungal mutualist (Leucoagaricus gongylophorus) obligately farmed by leafcutter ants. We describe steps for culturing actively growing mycelia, enzymatic digestion of fungal cell walls under osmotic stabilization, and purification of protoplasts (plasma membrane enclosing cytoplasm and organelles). We then detail procedures for assessing viability and wall removal using microscopy and fluorescent staining.