Isolation, Characterization, and Proteomic Analysis of Crude and Purified Extracellular Vesicles Extracted from <i>Fusarium oxysporum</i> f. sp. <i>cubense</i>
Mudassar Ahmad,
Yushan Liu,
Shiyi Huang,
Yile Huo,
Ganjun Yi,
Chongfei Liu,
Wajeeha Jamil,
Xiaofang Yang,
Wei Zhang,
Yuqing Li,
Dandan Xiang,
Huang Huoqing,
Siwen Liu,
Wei Wang,
Chunyu Li
Affiliations
Mudassar Ahmad
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Yushan Liu
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Shiyi Huang
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Yile Huo
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Ganjun Yi
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Chongfei Liu
Department of Pomology, College of Horticulture, China Agricultural University, Beijing 100193, China
Wajeeha Jamil
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Xiaofang Yang
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Wei Zhang
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Yuqing Li
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Dandan Xiang
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Huang Huoqing
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Siwen Liu
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Wei Wang
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Chunyu Li
Key Laboratory of South Subtropical Fruit Biology and Genetic Research Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Extracellular vesicles (EVs) produced by Fusarium oxysporum f. sp. cubense (Foc) play vital roles in plant–pathogen interactions; however, the isolation of purified Foc TR4-EVs and their pathogenicity and proteomic profiles are not well studied. This study aims to isolate and characterize purified Foc TR4-EVs and compare their pathogenic effects and protein profiles with crude TR4-EVs. Foc 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 × 109 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 × 109 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 cm2), while Fraction 2 (1.386 cm2) and Fraction 3 (1.255 cm2) 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 Foc EV pathogenicity.
