Identification of Tomato Infecting Viruses That Co-Isolate with Nanovesicles Using a Combined Proteomics and Electron-Microscopic Approach
Ramila Mammadova,
Immacolata Fiume,
Ramesh Bokka,
Veronika Kralj-Iglič,
Darja Božič,
Matic Kisovec,
Marjetka Podobnik,
Apolonija Bedina Zavec,
Matej Hočevar,
Gabriella Gellén,
Gitta Schlosser,
Gabriella Pocsfalvi
Affiliations
Ramila Mammadova
Extracellular Vesicles and Mass Spectrometry Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy, 80131 Naples, Italy
Immacolata Fiume
Extracellular Vesicles and Mass Spectrometry Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy, 80131 Naples, Italy
Ramesh Bokka
Extracellular Vesicles and Mass Spectrometry Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy, 80131 Naples, Italy
Veronika Kralj-Iglič
Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, SI-1000 Ljubljana, Slovenia
Darja Božič
Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, SI-1000 Ljubljana, Slovenia
Matic Kisovec
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, SI-1000 Ljubljana, Slovenia
Marjetka Podobnik
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, SI-1000 Ljubljana, Slovenia
Apolonija Bedina Zavec
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, SI-1000 Ljubljana, Slovenia
Matej Hočevar
Institute of Metals and Technology, SI-1000 Ljubljana, Slovenia
Gabriella Gellén
MTA-ELTE Lendület Ion Mobility Mass Spectrometry Research Group, ELTE Eötvös Loránd University, Institute of Chemistry, H-1117 Budapest, Hungary
Gitta Schlosser
MTA-ELTE Lendület Ion Mobility Mass Spectrometry Research Group, ELTE Eötvös Loránd University, Institute of Chemistry, H-1117 Budapest, Hungary
Gabriella Pocsfalvi
Extracellular Vesicles and Mass Spectrometry Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy, 80131 Naples, Italy
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.
