Abstract
Extracellular vesicles (EVs) secreted by Fusarium oxysporum play an important role in the process of its infestation of the host, but the in vitro research system for EVs of F. oxysporum (Fo-EVs) has not yet been improved, and the mechanism of its action remains unclear. In this study, particle size distribution, particle concentration, number of particles per unit of protein, number of particles per unit of mycelial biomass, and concentration of contaminated proteins were used as indicators to evaluate the yield and purity of Fo-EVs. The optimal method for Fo-EV preparation and extraction was screened by comparing liquid culture, solid culture, and solid culture with enzymatic cell wall hydrolysis. The optimal system for Fo-EVs separation and purification was screened by a pairwise combination of three primary methods (Ultracentrifugation (UC), Ultrafiltration (UF), and Polyethylene glycol precipitation method (PEG)) and two secondary methods (Size-exclusion chromatography (SEC) and Aqueous two-phase system (ATPS)), respectively. The protein composition was identified via mass spectrometry technology, followed by GO annotation and GO enrichment analysis using whole-genome proteins as the background. Based on these steps, a Fo-EV protein library was constructed to reveal Fo-EV's most active biological functions. The results showed that solid culture combined with the UC-SEC method could effectively enrich Fo-EVs with a typical cup-shaped membrane structure. The obtained Fo-EVs had an average particle size of 253.50 nm, a main peak value of 200.60 nm, a particle concentration of 2.04 × 10(10) particles/mL, and a particle number per unit protein of 1.09 × 10(8) particles/μg, which were significantly superior to those of other combined methods. Through proteomic analysis, 1931 proteins enriched in Fo-EVs were identified, among which 350 contained signal peptides and 375 had transmembrane domains. GO enrichment analysis revealed that these proteins were mainly involved in cell wall synthesis, vesicle transport, and pathogenicity-related metabolic pathways. Additionally, 9 potential fungal EV markers, including Hsp70, Rho GTPase family, and SNARE proteins, were screened. This study constructed an isolation system and a marker database for Fo-EVs, providing a methodological and theoretical basis for in-depth analysis of the biological functions of Fo-EVs.