Biomedical Sciences, School of Health, Leeds Beckett University, Leeds, UK.
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Crete, Greece.
Curr Protoc. 2024 Nov;4(11):e70026. doi: 10.1002/cpz1.70026.
Extracellular vesicles (EVs) are small membranous vesicles secreted by cells into their surrounding extracellular environment. Similar to mammalian EVs, plant EVs have emerged as essential mediators of intercellular communication in plants that facilitate the transfer of biological material between cells. They also play essential roles in diverse physiological processes including stress responses, developmental regulation, and defense mechanisms against pathogens. In addition, plant EVs have demonstrated promising health benefits as well as potential therapeutic effects in mammalian health. Despite the plethora of potential applications using plant EVs, their isolation and characterization remains challenging. In contrast to mammalian EVs, which benefit from more standardized isolation protocols, methods for isolating plant EVs can vary depending on the starting material used, resulting in diverse levels of purity and composition. Additionally, the field suffers from the lack of plant EV markers. Nevertheless, three main EV subclasses have been described from leaf apoplasts: tetraspanin 8 positive (TET8), penetration-1-positive (PEN1), and EXPO vesicles derived from exocyst-positive organelles (EXPO). Here, we present an optimized protocol for the isolation and enrichment of small EVs (sEVs; <200 nm) from the apoplastic fluid from Nicotiana benthamiana leaves by ultracentrifugation. We analyze the preparation through transmitted electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. We believe this method will establish a basic protocol for the isolation of EVs from N. benthamiana leaves, and we discuss technical considerations to be evaluated by each researcher working towards improving their plant sEV preparations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation and enrichment of small extracellular vesicles (sEVs) from the apoplastic fluid of Nicotiana benthamiana leaves.
细胞外囊泡（EVs）是细胞分泌到其周围细胞外环境中的小膜囊泡。与哺乳动物 EVs 类似，植物 EVs 已成为植物细胞间通讯的重要介质，促进细胞间生物物质的转移。它们还在多种生理过程中发挥重要作用，包括应激反应、发育调控和抵御病原体的防御机制。此外，植物 EVs 已被证明对哺乳动物健康具有良好的健康益处和潜在的治疗效果。尽管使用植物 EVs 具有广泛的潜在应用，但它们的分离和表征仍然具有挑战性。与受益于更标准化分离方案的哺乳动物 EVs 不同，植物 EVs 的分离方法因起始材料的不同而有所不同，导致纯度和组成水平的差异。此外，该领域还缺乏植物 EV 标志物。尽管如此，已经从叶片质外体描述了三种主要的 EV 亚类：四跨膜蛋白 8 阳性（TET8）、穿透素 1 阳性（PEN1）和来源于外泌体阳性细胞器的 EXPO 囊泡（EXPO）。在这里，我们提出了一种通过超速离心从拟南芥叶片质外体液中分离和富集小细胞外囊泡（sEVs；<200nm）的优化方案。我们通过透射电子显微镜（TEM）、纳米颗粒跟踪分析（NTA）和蛋白质印迹分析对制备物进行分析。我们相信这种方法将为从拟南芥叶片中分离 EVs 建立一个基本方案，并且我们讨论了每个研究人员在改进其植物 sEV 制备时需要评估的技术考虑因素。© 2024 作者。Wiley Periodicals LLC 出版的《当代协议》。基础方案：从拟南芥叶片质外体液中分离和富集小细胞外囊泡（sEVs）。
