Extracellular Vesicle Isolation from Botrytis cinerea Culture and B. cinerea-Infected Arabidopsis Plants

Extracellular vesicles (EVs) are universally conserved and essential players in the communication between cells across nearly all organisms of life. Botrytis cinerea produces and secretes EVs that are critical in its virulence on plants. Numerous studies in various fungal species reveal that EVs are important across different pathosystems to facilitate fungal infection. For characterization of the cargo and role of EVs in fungal growth and communication, it is necessary to carefully and precisely purify fungal EVs from fungal culture or the plant interaction interface. Here, we present a method for the isolation of Botrytis cinerea EVs from culture and the infection site of B. cinerea on Arabidopsis thaliana. Fungal EVs isolated using this method are free from cellular contamination from fungal or plant cells, remain viable in functional assays, and provide high-quality RNA and protein.