Abstract
Double-stranded RNA (dsRNA) is associated with most viral infections, and is generated in host cells during viral replication. Viral RNA replication occurs within the viral factories called the viral replication complexes (VRCs). In addition to viral genome, viral-derived dsRNA and replicase, the VRCs composition remains largely unexplored. The dsRNA binding domain of the B2 protein from Flock house virus has been reported to be used for detecting viral-derived long dsRNA in plants efficiently. Nicotiana benthamiana is widely used as a model plant for plant-microbe interactions owing to its susceptibility to diverse plant diseases, especially viral diseases. Here, we describe the use of Nicotiana benthamiana stably expressing GFP-tagged dsRNA binding protein (B2: GFP) to pull down dsRNA and associated host and viral proteins from turnip mosaic virus-infected plants. The obtained protein complexes are compatible with functional assays, Western blotting, and mass spectrometry. This system provides a valuable and robust tool to study VRC proteome in N. benthamiana upon plant viral infections.
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Acknowledgments
We are indebted to Prof. Christophe Ritzenthaler (Université de Strasbourg, France) for the transgenic T2 dsRNA reporter N. benthamiana line B2-GFP. This research was funded by grants from the National Natural Science Foundation of China (3207016) to GW, the China Agriculture Research System of MOF and MARA (CARS-24-C-04) to FY, and the K. C. Wong Magna Fund in Ningbo University.
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Jia, Z., Chen, J., Yan, F., Wu, G. (2024). Co-immunoprecipitation-Based Isolation of Double-Stranded RNA-Associated Protein Complexes in Nicotiana benthamiana. In: Cheng, X., Wu, G. (eds) Double-Stranded RNA. Methods in Molecular Biology, vol 2771. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3702-9_13
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DOI: https://doi.org/10.1007/978-1-0716-3702-9_13
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