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

## TL;DR

This paper provides a detailed protocol for isolating viable protoplasts from a fungus farmed by leafcutter ants.

## Contribution

The study introduces a novel protocol for protoplast isolation from Leucoagaricus gongylophorus, a symbiotic fungus.

## Key findings

- A protocol for culturing and enzymatic digestion of Leucoagaricus gongylophorus mycelia is described.
- Viability and wall removal of protoplasts are assessed using microscopy and fluorescent staining.
- The method enables purification and stabilization of fungal protoplasts for further study.

## Abstract

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

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.