Regulation of extracellular vesicles for protein secretion in Aspergillus nidulans

Fungi were among the first eukaryotes to transition from aquatic to terrestrial life, developing multicellular hyphae, polar growth, and expanded secretomes for nutrient processing, defense, and symbiosis. We present a reliable method for purifying and characterizing extracellular vesicles (EVs) from <i>Aspergillus nidulans</i> and demonstrate that the induction of xylanase C is associated with increased EV release and EV-associated enzymatic activity. Using a mCherry reporter replacing xylanase C, we generalized this effect, showing that reporter induction increases EV production and reporter loading into EVs. This phenomenon primarily depends on the signal peptide (SP), suggesting that the induction of endoplasmic reticulum (ER)- trafficked proteins has a pronounced effect on EV production and cargo loading. We speculate that EV biogenesis may originate at the ER, where ER-translated proteins could be selectively loaded into vesicles and subsequently trafficked directly to the plasma membrane or through multivesicular bodies (MVBs). EV secretion is minimal in the first 24-48 hours but increases later in growth, coinciding with biofilm formation. This timing allows <i>A. nidulans</i> to modify the secretome, adapting it to new nutrient sources.