Extracting Protoplasts from Filamentous Fungi Using Extralyse, An Enzyme Used in the Wine Industry

The ability to extract protoplasts has contributed significantly to the study of fungi and plants. Protoplasts have historically been used to determine chromosome number via pulsed-field electrophoresis and for the functional characterization of genes via protoplast transformation. More recently, protoplasts have been used to extract the high-molecular-weight DNA required for long-read sequencing projects. The availability of efficient protoplast extraction protocols is thus integral to the study and experimental manipulation of model and non-model fungi. One major hurdle to the development of such protocols has been the discontinuation of enzymes and enzyme cocktails used to digest the fungal cell wall. Here, we provide five protoplast extraction protocols for use in various filamentous ascomycete species spanning the genera Ceratocystis, Fusarium, Metarhizium, Ophiostoma, and Sclerotinia. These protocols all use an inexpensive, readily available enzyme cocktail called Extralyse, a commercially available product commonly used in the wine making industry. Using this enzyme cocktail overcomes reliance on the laboratory-grade enzymes that have frequently been discontinued and are often cost prohibitive at the concentrations required. The protocols described here will allow further research, including genome editing, to be conducted in these fungal genera. Importantly, these protocols also provide a starting point for the development of protoplast extraction techniques in other filamentous fungi. This resource can therefore be used to expand the molecular toolkits available for fungi beyond the species described here, including those with relevance in both medical and biotechnological industries. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protoplast extractions from Ceratocystis eucalypticola and Ceratocystis fimbriata Basic Protocol 2: Protoplast extractions from Fusarium circinatum Basic Protocol 3: Protoplast extractions from Metarhizium acridum, Metarhizium brunneum, and Metarhizium guizhouense Basic Protocol 4: Protoplast extractions from Ophiostoma novo-ulmi Basic Protocol 5: Protoplast extractions from Sclerotinia sclerotiorum.