Multiple cellular compartments engagement in Nicotiana benthamiana-peanut stunt virus-satRNA interactions revealed by systems biology approach

Abstract Key message PSV infection changed the abundance of host plant’s transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and cytosol, affecting photosynthesis, translation, transcription, and splicing. Abstract Virus infection is a process resulting in numerous molecular, cellular, and physiological changes, a wide range of which can be analyzed due to development of many high-throughput techniques. Plant RNA viruses are known to replicate in the cytoplasm; however, the roles of chloroplasts and other cellular structures in the viral replication cycle and in plant antiviral defense have been recently emphasized. Therefore, the aim of this study was to analyze the small RNAs, transcripts, proteins, and phosphoproteins affected during peanut stunt virus strain P (PSV-P)–Nicotiana benthamiana interactions with or without satellite RNA (satRNA) in the context of their cellular localization or functional connections with particular cellular compartments to elucidate the compartments most affected during pathogenesis at the early stages of infection. Moreover, the processes associated with particular cell compartments were determined. The ‘omic’ results were subjected to comparative data analyses. Transcriptomic and small RNA (sRNA)–seq data were obtained to provide new insights into PSV-P–satRNA–plant interactions, whereas previously obtained proteomic and phosphoproteomic data were used to broaden the analysis to terms associated with cellular compartments affected by virus infection. Based on the collected results, infection with PSV-P contributed to changes in the abundance of transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and the cytosol, and the most affected processes were photosynthesis, translation, transcription, and mRNA splicing. Furthermore, sRNA-seq and phosphoproteomic analyses indicated that kinase regulation resulted in decreases in phosphorylation levels. The kinases were associated with the membrane, cytoplasm, and nucleus components. </jats:sec

PCR-RFLP method to distinguish Frankliniella occidentalis, Frankliniella intonsa, Frankliniella pallida and Frankliniella tenuicornis

Thrips from the genus Frankliniella (Thysanoptera, Thripidae) are phytophagous on crops and wild plants. Some of them cause slight economic damage, however, others including F. occidentalis and F. intonsa are responsible for considerable losses in crop production. Moreover, they constitute a double threat for host plants by not only feeding on them but also vectoring viruses, some of which are on the quarantined list of the European Plant Protection Organization. The rapid detection and differentiation between more and less harmful Frankliniella species is, therefore, important in order to combat the pests at the time of their appearance. In this study, we have undertaken to develop a method of detecting F. occidentalis, F. intonsa, F. pallida, and F. tenuicornis. The protocol is based on PCR amplification of ITS1 rDNA fragments of these insects using universal primers pair giving products of slightly distinct length for studied insects. Restriction enzymes digestion which is easy to interpret, allows for visible differentiation of all these Frankliniella species. The method was shown to be species-specific and sensitive. Even single specimens in either the larvae or adult stage could be distinguished

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 (ToTVpJL-Kra) that could infect Nicotiana benthamiana and Solanum lycopersicum. Importantly, a modified variant of the viral RNA2&mdash;with inserted sGFP (forming, together with virus RNA1, into ToTVpJL-KraGFP)&mdash;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