Growing pains: addressing the pitfalls of plant extracellular vesicle research

Abstract

Summary Extracellular vesicles (EVs) are small, membrane‐enclosed compartments that mediate the intercellular transport of proteins and small RNAs. In plants, EVs are thought to play a prominent role in immune responses and are being championed as the long‐sought‐after mechanism for host‐induced gene silencing. However, parallel research on mammalian EVs is raising concerns about potential pitfalls faced by all EV researchers that will need to be addressed in order to convincingly establish that EVs are the primary mediators of small RNA transfer between organisms. Here we discuss these pitfalls in the context of plant EV research, with a focus on experimental approaches required to distinguish bona fide EV cargo from merely co‐purifying contaminants.

A call for Rigor and standardization in plant extracellular vesicle research

“…This discrepancy and regular focus on an intermediate pelleting fraction seems to be specific to the plant field, since mammalian EV separations have overwhelmingly relied on 10-20 K and 100 K fractions. We refer the reader as well to a summary of good practices for plant EV separation and characterization by Rutter & Innes (Rutter & Innes, 2020). The authors emphasize the importance of distinguishing bona fide EV cargo from merely co-purifying contaminants and point out that plant EV content may vary according to the separation procedure used.…”

“…The functions of EVs in planta open novel opportunities for practical applications. Emerging ideas include the modulation of the plant immune system (Rybak & Robatzek, 2019) or their direct involvement in delivering defense proteins, antimicrobial compounds and sRNA cargos which can be transferred to fungi, although these results are debated (Rutter & Innes, 2020). Nevertheless, possibilities for EV content and release manipulation suggest the exciting avenue of using engineered EVs as agrochemicals for crop protection.…”

Host‐induced gene silencing – mechanisms and applications

“…Although the number of EV‐contained siRNAs was low, we have no information on the minimum concentration of siRNAs inside an EV that is required to induce HIGS. At the same time, EVs emerged as novel players in plant–pathogen interaction, serving as shuttles for proteins and RNAs that interfere with defence responses (Rutter & Innes, 2020). However, multiple questions have arisen that need to be addressed to unravel the molecular mechanisms of EV uptake, loading and release, as well as the role of RNA‐binding proteins (AGOs, DRBs, and others) in stabilizing dsRNAs and siRNAs during HIGS (Fig.…”

Host-induced gene silencing involves Arabidopsis ESCRT-III pathway for the transfer of dsRNA-derived siRNA

“…Isolation of plant EVs from apoplastic washes commonly results in copurification of extravesicular contaminants which can influence the interpretation of data (Rutter and Innes 2020). To overcome this limitation and to distinguish between siRNA from outside or inside EVs, we performed several EV treatments: protease and RNase to digest extracellular RNAs, proteins as well as ribonucleoprotein complexes.…”

“…The many pitfalls in plant EV research were recently reviewed by Rutter and Innes (2020) exemplifying the requirement of EV markers, EV reference proteins/RNAs to ensure comparability, reliability, and reproducibility among different EV preparations. Thus, the central question is how robust are results from one EV preparation and sequencing event to another?…”