Co-immunoprecipitation-Based Isolation of Double-Stranded RNA-Associated Protein Complexes in Nicotiana benthamiana

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.

Keywords: Double-stranded RNA; GFP antibody; Nicotiana benthamiana; Protein-complex immunoprecipitation; Turnip mosaic virus; dsRNA binding protein B2.