Growth conditions shape the proteome and diversity of Neurospora crassa extracellular vesicles

Extracellular vesicles (EVs) are nanosized, lipid bilayer-enclosed particles secreted by all living organisms. While EV research has primarily focused on mammalian systems, fungal EVs are gaining attention for their biological significance. Here, we investigated how growth conditions influence the protein cargo of EVs produced by <i>Neurospora crassa</i>, a non-pathogenic filamentous fungus and well-established model organism. EVs were isolated from cultures grown on glucose for 16 h (G16) and on sucrose for 16 (S16) and 24 h (S24). Dynamic light scattering (DLS) revealed similar size distributions for S16 and S24 EVs (24-165 nm), whereas G16 EVs exhibited a broader range (32-825 nm). Across all conditions, particles <50 nm were detected, potentially corresponding to mitochondrial-derived vesicles (MDVs) or exomeres, EV subtypes described in mammalian systems. Proteomic profiling identified 682 proteins in G16, 668 in S16, and a reduced set of 367 proteins in S24. Regardless of condition, EVs were enriched in proteins related to cell wall remodeling, protein synthesis, and carbohydrate metabolism. A high proportion of intracellular proteins confirms that fungal EVs participate in unconventional secretion. In addition, the detection of proteins involved in vesicle biogenesis and trafficking suggests that EV formation may also involve the classical secretory pathway. These findings demonstrate that EV composition and biogenesis in <i>N. crassa</i> are modulated by growth conditions and highlight the importance of physiological context in fungal EV research. Notably, the data reveal a diversity of EV types, including forms potentially unrelated to exosomes, expanding our understanding of fungal EV complexity.