Isolation of extracellular vesicles from pine (Pinus sp.) leaves and roots

Abstract

Abstract Plants release extracellular vesicles (EVs) for various reasons, such as unconventional protein secretion, RNA transport, and defence against pathogens. However, very little is known about plant EV biosynthesis and diverse and bioactive luminal contents, including how they alter recipient cell biology and function. To study EVs, researchers often isolate them from apoplastic wash or juice fluids using various methods. A method is needed to isolate EVs from non-model plants with tougher leaves such as species in the Pinus genus. Because of their spindly and sharp shape, pines have strong acicular-shaped leaves that are commonly called needles. Unlike all other taproots, pine seedlings grow into a primary root first, which gives rise to lateral roots later. There are also various dimensions and shapes of pine needles, but they are relatively strong compared to the leaves of other plants such as Arabidopsis thaliana, where most EV studies have been concentrated. Extracting EVs from a juice fluid is highly effective for plants with tougher leaves because it ensures that the cells have been broken down and the vesicles are present. Given these reasons, we describe our method for the isolation and purification of EVs from needles and roots of different pine species. A detailed protocol is provided that can guide the collection of juice fluid from pine needles and roots in a clean and efficient manner. A number of other plant species with similar morphologies can greatly benefit from the use of this protocol. Basic protocol 1: Isolation of extracellular vesicles from juice fluid of pine leaves and roots Basic protocol 2: Isolation of pine extracellular vesicles from juice fluid using size exclusion chromatography