Mysterious travellers: Identification of viral and host defense-related components in tomato fruit-derived nanovesicles

Plant viruses are obligate intracellular parasites, and their replication and translocation throughout the host requires host cell functions. Although viruses are assumed to spend their whole infection cycle exclusively in the symplast of their hosts, evidences are emerging that virus-derived molecules and host defense related components with a putative antiviral activity could accumulate in exportable membrane-enclosed vesicles. In this study, nanovesicles (NVs, approximate diameter 100nm) were isolated from tomato fruit tissues. An efficient density gradient ultracentrifugation (gUC) protocol was used for a first separation of host NVs from viral particles that occurred in apparently healthy looking tomatoes. Cryogenic transmission electron microscopy (cryo-TEM) from gUC identified low-density purified fractions enriched with NVs and high-density fractions where mostly viral particles accumulated. However, proteomics data revealed the presence of different virus derived-products in all gUC fractions. Proteomics and reverse-transcription PCR analysis on NV-extracted RNA confirmed the presence of different viruses such as Tomato brown rugose fruit virus (ToBRFV), Tomato spotted wilt orthotospovirus (TSWV), Potato virus Y (PVY), Pepino mosaic virus (PepMV), Tobacco rattle virus (TRV), and Southern tomato virus (STV). Interestingly, plant stress-related components were identified in NV-associated fractions. Host components included both fragments of stress-related proteins and microRNAs involved in plant responses against viruses (e.g. sly-miR162, sly-miR168). These findings, together with previous similar reports on different plant-virus systems, indicate a role of plant NVs in viral infection mechanisms, and suggest the hypothesis that viral components may travel outside of plant cells by exploiting the extracellular vesicle-based pathways.