Abstract
Green fluorescent protein (GFP)-tagged viruses are basic research tools widely applied in studies concerning molecular determinants of disease during virus infection. Here, we described a new generation of genetically stable infectious clones of tomato torrado virus isolate Kra (ToTVpJL-Kra) that could infect Nicotiana benthamiana and Solanum lycopersicum. Importantly, a modified variant of the viral RNA2—with inserted sGFP (forming, together with virus RNA1, into ToTVpJL-KraGFP)—was engineered as well. RNA2 of ToTVpJL-KraGFP was modified by introducing an additional open reading frame (ORF) of sGFP flanked with an amino acid-coding sequence corresponding to the putative virus protease recognition site. Our further analysis revealed that sGFP-tagged ToTV-Kra was successfully passaged by mechanical inoculation and spread systemically in plants. Therefore, the clone might be applied in studying the in vivo cellular, tissue, and organ-level localization of ToTV during infection. By performing whole-plant imaging, followed by fluorescence and confocal microscopy, the presence of the ToTVpJL-KraGFP-derived fluorescence signal was confirmed in infected plants. All this information was verified by sGFP-specific immunoprecipitation and western blot analysis. The molecular biology of the torradovirus-plant interaction is still poorly characterized; therefore, the results obtained here opened up new possibilities for further research. The application of sGFP-tagged virus infectious clones and their development method can be used for analyzing plant-virus interactions in a wide context of plant pathology.
Highlights
In vivo monitoring of virus movement, as well as its subcellular localization in an infected host plant, can answer many important questions at the forefront of modern molecular plant virology.Currently, it is possible to characterize virus localization in tissues using a wide range of monitoring approaches
It was demonstrated that the mobilized virus, named hereafter ToTVpJL -Kra, was infectious to N. benthamiana and S. lycopersicum
This was confirmed by disease symptoms manifested on agroinfiltrated plants: yellowing and Tomato torrado virus (ToTV)-specific spoon-like malformations of systemically infected leaves in N. benthamiana and leaf mottling followed by severe necrosis developing near veins of systemically infected leaves in S. lycopersicum (Figure 1A, middle panel)
Summary
In vivo monitoring of virus movement, as well as its subcellular localization in an infected host plant, can answer many important questions at the forefront of modern molecular plant virology. It is possible to characterize virus localization in tissues using a wide range of monitoring approaches (reviewed in [1]). Green fluorescent protein (GFP) seems to be the gold standard in studies focusing on expressing foreign genes from virus genomes [2]. For this purpose, a wide range of virus-based expression systems were developed and applied either in plants [3,4,5] or animal systems [6]. Other plant species were previously described as hosts for ToTV [7], including
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