Effective methods for isolation and purification of extracellular vesicles from plants,Journal of Integrative Plant Biology

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Effective methods for isolation and purification of extracellular vesicles from plants
Journal of Integrative Plant Biology ( IF 9.3 ) Pub Date : 2021-10-20 , DOI: 10.1111/jipb.13181
Yifan Huang _{1,

2} , Shumei Wang ₃ , Qiang Cai _{1,

2} , Hailing Jin ₃

Affiliation

Plant extracellular vesicles (EVs) play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes, most notably in plant defense responses. However, the isolation of pure, intact EVs from plants remains challenging. A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid (AWF). Here, we compare published plant EV isolation methods, and provide our recommended method for the isolation and purification of plant EVs. This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves, followed by EV isolation via differential centrifugation. To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations, density gradient ultracentrifugation and immunoaffinity capture are then utilized. We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications.

中文翻译：

从植物中分离纯化细胞外囊泡的有效方法

植物细胞外囊泡 (EV) 在分子从宿主到相互作用的微生物的跨界运输中起着关键作用，尤其是在植物防御反应中。然而，从植物中分离出纯净、完整的 EV 仍然具有挑战性。已使用多种方法从质外体洗涤液 (AWF) 中分离植物 EV。在这里，我们比较了已发表的植物 EV 分离方法，并提供了我们推荐的植物 EV 分离和纯化方法。此方法包括从拟南芥中收集干净 AWF 的详细协议叶，然后通过差速离心分离 EV。为了进一步从异质囊泡群中分离和纯化 EV 的特定亚类，然后使用密度梯度超速离心和免疫亲和捕获。我们发现，当有合适的特定 EV 生物标志物及其相应抗体可用时，免疫亲和捕获是分离特定 EV 亚类的最精确方法。总体而言，本研究为所需下游应用的 EV 隔离方法的选择和优化提供了指导。

更新日期：2021-12-28

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