In vivo visualization of RNA localization and transport in plants

MATÍAS AGÜERO; EDUARDO PEÑA; MANFRED HEINLEIN

Berlin

Workshop; Intercellular comunication in development and disease; 2017

EMBO

During transcription and maturation, a nascent plant mRNA is imprinted with cis-localized elements that will not only coordinate translation and decay but also determine anchorage to a specific subcellular localization or a journey to long distance tissues. Ongoing research is focused on the identification and functional characterization of these elements and factors triggering RNA mobility. Tobacco mosaic virus (TMV), our model to study RNA trafficking in plants, depends on its movement protein (MP) to move cell-to-cell. TMV cell-to-cell movement involves the formation of MP particles that move along cortical endoplasmic reticulum membranes that are connected between cells through plasmodesmata (PD) and intimately associated with the cortical microtubule lattice. To address the composition and trafficking mechanism of these particles, we adapted and compared three different RNA-labeling methods, ?MS2?, ?λN? and ?BglG?, to visualize ectopically expressed MP mRNA. These methods rely on the tagging of the RNA of interest with a protein-binding motif (stem loop, SL) and the co-expression of the cognate RNA-binding protein (RBP). As the RBP is itself fluorescently labelled, it allows the in vivo visualization of the tagged RNA. We found that the system based on the Escherichia coli transcriptional anti-terminator BglG is the most suitable for detecting MP mRNA in planta. The method showed to be highly specific as well as sensitive. We found the mRNA exclusively in association with MP, mostly on MP particles and too much lesser extent accumulates at PDs. Moreover, we observed that in addition to the MP, also the MP mRNA can move cell-to-cell through PD. By subcellular fractionation and RNA immunoprecipitation, we isolated these ribonucleoprotein particles to address the protein composition. Association of MP particles to components of the cortical microtubules network and their possible roles on RNA trafficking will be discussed.