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. • Steps for enzymatic digestion of Leucoagaricus gongylophorus cell walls • Instructions for purification and stabilization of isolated fungal protoplasts • Guidance on microscopy validation and quantitative protoplast assessment Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.