Technical advances in extracellular vesicle isolation from fungi and oomycetes: Insights from plant-pathogenic species

Research on fungal extracellular vesicles (EVs) has gained a lot of attention due to their role in plant-microbe interaction and intercellular and cross-kingdom communication. However, the isolation and characterization of EVs from plant pathogenic fungi and oomycetes still face challenges. We provide a comprehensive overview of the most recent methods for EV isolation, such as density gradient, ultracentrifugation size exclusion chromatography and differential ultracentrifugation. Quality control measures, such as dynamic light scattering, nanoparticle tracking analysis and transmission electron microscopy to ensure purity and integrity, are discussed. EVs from various organisms display heterogenicity in size and cargo. To ensure reproducibility and cross-study comparisons, we highlight the importance of standardized protocols for EV isolation and characterization. Identification of pan-fungal and pan-oomycetal EV marker proteins are needed to improve our knowledge of their function in plant-pathogen interactions. This work provides a methodological framework for the comparative analysis of EVs from fungi and oomycetes based on approaches from plant pathogens and highlights their potential relevance as targets or tools in the development of innovative plant protection strategies.