Isolation of extracellular vesicles and characterization of their role as shuttle of biologically active small RNAs in intercellular communication

Extracellular vesicles (EVs) are nanoscale particles delimited by a lipid bilayer membrane, secreted by most eukaryotic cells into the extracellular space. Several evidences support the crucial role played by EVs in cell-to-cell communication as carriers of functional biological molecules, such as small non-coding RNAs (sRNAs). In plants, as part of the infection process, a communication mechanism called bi-directional cross-kingdom RNA interference occurs, based on sRNA trafficking via EVs between fungal pathogens and host plants. Therefore, EVs have emerged as nanovectors of genetic information, and the study of their composition and biological properties in different plant species is a field of research in rapid expansion. The aim of this research is the optimization of a protocol for the isolation of EVs from apoplastic washing fluid collected from Nicotiana benthamiana leaves. The final goal is the characterization of the small RNA composition of EVs by comparing samples isolated from wild-type plants with those from plants expressing an antiviral construct against the Grapevine Fan Leaf Virus and the Grapevine Leaf Roll-associated Virus. Differential centrifugation was employed to isolate EVs, and the sedimented material was physically characterized by TEM and DLS. We obtained EVs with an average diameter of 100-150 nm. After treatment with RNAse to remove RNA potentially present outside the vesicles, we proceed with EVs lysis and isolation of the sRNAs. RNA sequencing will be performed to characterize sRNAs present in the EVs samples. The results will have both heuristic relevance, increasing knowledge beyond state of the art concerning the movement of the silencing signal in plants, and applied interest, for exogenous application, using EVs as shuttles of sRNAs for improving crop protection and silencing of target genes.