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

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.

Extracellular vesicles isolated from dsRNA-sprayed barley plants exhibit no growth inhibition or gene silencing in Fusarium graminearum

“…In our recent studies, we observed that state-of-the-art EV purification from apoplastic fluids leads to impure EV isolates containing additional co-purified apoplastic substances [47]. This finding aligns with recent debates discussing the pitfalls of current plant EV research methods and the need for standardization, with different contamination risks reported for different plant EV separation and characterization methods [33,40,46]. To avoid such pitfalls that may lead to false conclusions, we performed a stringent digestive treatment of EV isolates to degrade extravesicular proteins and RNAs before in vitro treatment of F. graminearum with plant EVs.…”

“…thaliana in vitro. The impurity of plant EV isolates raised concerns about the reliability of findings and their interpretation [33,46]; we thus performed rigorous digestive treatments of EV isolates before F. graminearum in vitro testing. Encouraged by our previous finding that drop inoculation of barley EVs on F. graminearum cultures grown on solid agar plates caused an increase in purple pigmentation, indicative of the stress-induced premature formation of fruiting bodies [48], we expected to observe similar effects in liquid cultures.…”

Protein Biocargo and Anti-Inflammatory Effect of Tomato Fruit-Derived Nanovesicles Separated by Density Gradient Ultracentrifugation and Loaded with Curcumin

“…In recent years, plant-derived nanovesicles (PDNVs) have emerged due to their perspective towards biomedical and nanotechnological applications. They have been isolated from many homogenized plants and fruit juices, including tomato [ 1 , 2 ], citrus species [ 3 , 4 , 5 ], strawberry [ 6 , 7 ] and ginseng [ 8 , 9 ], just to mention some. In this paper, we use PDNVs as an umbrella term to refer to all kind of nano-sized particles, nanolipids, exosomes, nanovectors, etc.…”

“…Recently we have reported the isolation and characterization of NVs from tomato ( Solanum lycopersicum L.) fruit [ 1 , 2 ]. Tomatoes are healthy, versatile and an essential fruit of the Mediterranean diet with associated health benefits, including improved antioxidant defense and reduced risk of inflammatory diseases.…”

Interaction between Microalgae P. tricornutum and Bacteria Thalassospira sp. for Removal of Bisphenols from Conditioned Media

“…Samples of SCPs were prepared using Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA). Quantifoil ®® R 2/2, 200 mesh holey carbon grids (Quantifoil Micro Tools GmbH, Großlöbichau, Germany) were glow-discharged for 60 s at 20 mA and positive polarity in the air (GloQube ®® Plus, Quorum, Laughton, UK) [ 55 ]. Conditions were set at 4 °C, 100% relative humidity, blot time: 5 s, and blot force: 4.…”