Identification of Tomato Infecting Viruses That Co-Isolate with Nanovesicles Using a Combined Proteomics and Electron-Microscopic Approach.

Ramila Mammadova,Matej Hočevar,Gabriella Gellén,Matic Kisovec,Marjetka Podobnik,Ramesh Bokka,Immacolata Fiume,Gabriella Pocsfalvi,Veronika Kralj-Iglič,Apolonija Bedina Zavec,Darja Božič,Gitta Schlosser

doi:10.3390/nano11081922

Abstract

Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato (Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC–MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry–based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.

Highlights

This article is an open access articleCell-derived submicron particles isolated from plant resources [1,2,3,4,5,6,7] are gaining attention both as complex biomaterials with health-promoting effects [8,9] and as delivery vectors for exogenous substances [10]
An NV sample isolated from plant material typically contains a very heterogeneous and dynamic mixture of intra and extracellular vesicles, as well as vesicles that formed in the isolation process
We have shown that viruses in the fruit of tomatoes can co-purify with bulk membrane-bound nanovesicles in the dUC or size-exclusion chromatography (SEC)-based isolation protocols

Summary

Introduction

This article is an open access articleCell-derived submicron particles isolated from plant resources [1,2,3,4,5,6,7] are gaining attention both as complex biomaterials with health-promoting effects [8,9] and as delivery vectors for exogenous substances [10]. Nanomaterials 2021, 11, 1922 dried plant material [5] Several of these isolates were shown to have anticancer [3,17,18], anti-inflammatory [7,11,15] or anti-senescence [19] activities in vitro or in vivo. Native NVs from ginger root have been shown to be efficient in the treatment of alcohol-induced liver damage in vivo [11] Both broccoli- and grapefruit-derived nanoparticles prevent dextran sulfate sodium–induced colitis in murine models [15,23]. NVs isolated from wheat grass juice exert activity in the wound-healing process based on in vitro testing [16] and ginseng-derived vesicles exhibited anti-senescence effects on human skin cells [19]. The administration of ginger-exosome-like NVs loaded with exogenous therapeutic RNA efficiently inhibited tumor growth in a mice model [10]

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