In Vivo RNA Visualization in Plants Using MS2 Tagging

Abstract

Intracellular trafficking and asymmetric localization of RNA molecules within cells are a prevalent process across phyla involved in developmental control and signaling and thus in the determination of cell fate. In addition to intracellular localization, plants support the trafficking of RNA molecules also between cells through plasmodesmata (PD), which has important roles in the cell-to-cell and systemic communication during plant growth and development. Viruses have developed strategies to exploit the underlying plant RNA transport mechanisms for the cell-to-cell and systemic dissemination of infection. In vivo RNA visualization methods have revolutionized the study of RNA dynamics in living cells. However, their application in plants is still in its infancy. To gain insights into the RNA transport mechanisms in plants, we study the localization and transport of Tobacco mosaic virus RNA using MS2 tagging. This technique involves the tagging of the RNA of interest with repeats of an RNA stem–loop (SL) that is derived from the origin of assembly of the bacteriophage MS2 and recruits the MS2 coat protein (MCP). Thus, expression of MCP fused to a fluorescent marker allows the specific visualization of the SL-carrying RNA. Here we describe a detailed protocol for Agrobacterium tumefaciens-mediated transient expression and in vivo visualization of MS2-tagged mRNAs in Nicotiana benthamiana leaves.