Ahmad Mudassar, Liu Yushan, Huang Shiyi, Huo Yile, Yi Ganjun, Liu Chongfei, Jamil Wajeeha, Yang Xiaofang, Zhang Wei, Li Yuqing, Xiang Dandan, Huoqing Huang, Liu Siwen, Wang Wei, Li Chunyu
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.
Department of Pomology, College of Horticulture, China Agricultural University, Beijing 100193, China.
Plants (Basel). 2024 Dec 18;13(24):3534. doi: 10.3390/plants13243534.
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.
由尖孢镰刀菌（Fusarium oxysporum f. sp.）产生的细胞外囊泡（EVs）在植物与病原体的相互作用中起着至关重要的作用；然而，纯化的香蕉枯萎病菌4号生理小种（TR4）-EVs的分离及其致病性和蛋白质组学特征尚未得到充分研究。本研究旨在分离和鉴定纯化的TR4-EVs，并将其致病作用和蛋白质谱与粗制TR4-EVs进行比较。采用超速离心法分离TR4-EVs，并通过碘克沙醇梯度离心法进行纯化。在对香蕉叶片进行致病性影响的表征和评估后，进行液相色谱-串联质谱（LC-MS/MS）分析以进行蛋白质组学检测。结果表明，第2组分的EVs呈现出更清晰的球形结构（透射电子显微镜观察）、数量过多（1.70×10个颗粒/毫升）、强度更大（400任意单位）、平均大小（154.5纳米）、蛋白质含量适中（333.16纳克/微升）以及蛋白质谱范围为（25-77千道尔顿），这些均优于第1、3组分以及粗制EVs。与梯度EVs相比，粗制EVs在EV结构（透射电子显微镜观察）上显示出明显的背景干扰、数量最多（2.11×10个颗粒/毫升）、强度较低（7.0任意单位）、蛋白质含量较高（528.33纳克/微升）以及含有分子量较高的蛋白质（55-70千道尔顿）。未观察到尖孢镰刀菌-EVs对TR4孢子生长有显著的生物防治效果。致病性检测表明，粗制EVs导致的病斑最大（2.805厘米），而第2组分（1.386厘米）和第3组分（1.255厘米）在香蕉叶片上导致中等程度的病斑。蛋白质组学分析在第2组分中鉴定出807种独特蛋白质，这些蛋白质富集于与EV运输和信号传导相关的途径。相比之下，粗制EVs含有179种与代谢和次生代谢产物相关的独特非EV蛋白质，这表明粗制EVs中的非EV蛋白质也影响在香蕉叶片中观察到的致病性。本研究强调了EV纯化的重要性，第2组分是未来关于EV致病性研究的关键重点。
