Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants
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 (ToTV<sub>pJL&l...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-10-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/12/10/1195 |
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| author | Przemysław Wieczorek Marta Budziszewska Patryk Frąckowiak Aleksandra Obrępalska-Stęplowska |
| author_facet | Przemysław Wieczorek Marta Budziszewska Patryk Frąckowiak Aleksandra Obrępalska-Stęplowska |
| author_sort | Przemysław Wieczorek |
| collection | DOAJ |
| description | 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 (ToTV<sub>pJL</sub>-Kra) that could infect <i>Nicotiana benthamiana</i> and <i>Solanum lycopersicum</i>. Importantly, a modified variant of the viral RNA2—with inserted sGFP (forming, together with virus RNA1, into ToTV<sub>pJL</sub>-Kra<sub>GFP</sub>)—was engineered as well. RNA2 of ToTV<sub>pJL</sub>-Kra<sub>GFP</sub> 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 ToTV<sub>pJL</sub>-Kra<sub>GFP</sub>-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. |
| first_indexed | 2024-03-10T15:28:53Z |
| format | Article |
| id | doaj.art-b1289c5a0cee4c66b93ef3278fbc4b61 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T15:28:53Z |
| publishDate | 2020-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-b1289c5a0cee4c66b93ef3278fbc4b612023-11-20T17:52:08ZengMDPI AGViruses1999-49152020-10-011210119510.3390/v12101195Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in PlantsPrzemysław Wieczorek0Marta Budziszewska1Patryk Frąckowiak2Aleksandra Obrępalska-Stęplowska3Department of Molecular Biology and Biotechnology, Institute of Plant Protection—National Research Institute, Władysława Węgorka 20St, 60-318 Poznań, PolandDepartment of Molecular Biology and Biotechnology, Institute of Plant Protection—National Research Institute, Władysława Węgorka 20St, 60-318 Poznań, PolandDepartment of Molecular Biology and Biotechnology, Institute of Plant Protection—National Research Institute, Władysława Węgorka 20St, 60-318 Poznań, PolandDepartment of Molecular Biology and Biotechnology, Institute of Plant Protection—National Research Institute, Władysława Węgorka 20St, 60-318 Poznań, PolandGreen 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 (ToTV<sub>pJL</sub>-Kra) that could infect <i>Nicotiana benthamiana</i> and <i>Solanum lycopersicum</i>. Importantly, a modified variant of the viral RNA2—with inserted sGFP (forming, together with virus RNA1, into ToTV<sub>pJL</sub>-Kra<sub>GFP</sub>)—was engineered as well. RNA2 of ToTV<sub>pJL</sub>-Kra<sub>GFP</sub> 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 ToTV<sub>pJL</sub>-Kra<sub>GFP</sub>-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.https://www.mdpi.com/1999-4915/12/10/1195tomato torrado virussGFPplant pathologyinfectious cloneplant-virus interaction |
| spellingShingle | Przemysław Wieczorek Marta Budziszewska Patryk Frąckowiak Aleksandra Obrępalska-Stęplowska Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants Viruses tomato torrado virus sGFP plant pathology infectious clone plant-virus interaction |
| title | Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants |
| title_full | Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants |
| title_fullStr | Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants |
| title_full_unstemmed | Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants |
| title_short | Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants |
| title_sort | development of a new tomato torrado virus based vector tagged with gfp for monitoring virus movement in plants |
| topic | tomato torrado virus sGFP plant pathology infectious clone plant-virus interaction |
| url | https://www.mdpi.com/1999-4915/12/10/1195 |
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