Isolation, Characterization, and Proteomic Analysis of Crude and Purified Extracellular Vesicles Extracted from f. sp. .

Extracellular vesicles (EVs) produced by f. sp. () play vital roles in plant-pathogen interactions; however, the isolation of purified TR4-EVs and their pathogenicity and proteomic profiles are not well studied. This study aims to isolate and characterize purified TR4-EVs and compare their pathogenic effects and protein profiles with crude TR4-EVs. TR4-EVs were isolated using ultracentrifugation and purified by iodixanol gradient centrifugation. After characterization and evaluation of the pathogenicity effects on banana leaves, LC-MS/MS was performed to conduct the proteomics assay. Results indicated that Fraction 2 EVs exhibited clearer spherical structures (TEM), excessive abundance (1.70 × 10 particles/mL), greater intensity (400 a.u), mean size (154.5 nm), moderate protein content (333.16 ng/µL), and protein profile (25-77 kDa), which were superior to Fractions 1, 3, and crude EVs. Crude EVs displayed significant background interference with EV structures (TEM), highest abundance (2.11 × 10 particles/mL), lower intensity (7.0 a.u), higher protein content (528.33 ng/µL), and higher molecular weight proteins (55-70 kDa) compared to gradient EVs. A non-significant biocontrol effect of Foc-EVs on the growth of TR4 spores was observed. Pathogenicity assays revealed that crude EVs caused the largest (2.805 cm), while Fraction 2 (1.386 cm) and Fraction 3 (1.255 cm) resulted in moderate lesions on banana leaves. Proteomic analysis identified 807 unique proteins in Fraction 2, enriched in pathways related to EV trafficking and signaling. In comparison, crude EVs contained 179 unique non-EV proteins related to metabolism and secondary metabolites, indicating that non-EV proteins of crude EVs also influence the pathogenicity observed in banana leaves. This study emphasizes the importance of EV purification, with Fraction 2 being a critical focus for future research on EV pathogenicity.