Reappraisal of Spinach-based RNA Visualization in Plants

Abstract

RNAs can be imaged in living cells using molecular beacons, RNA-binding labeled proteins and RNA aptamer-based approaches. However, Spinach RNA-mimicking GFP (RMG) has not been successfully used to monitor cellular RNAs in plants. In this study, we re-evaluated Spinach-based RNA visualization in different plants via transient, transgenic, and virus-based expression strategies. We found that like bacterial, yeast and human cellular tRNAs, plant tRNAs such as tRNALys (K) can protect and/or stabilize the spinach RNA aptamer interaction with the fluorophore DFHBI enabling detectable levels of green fluorescence to be emitted. The tRNALys-spinach-tRNALys (KSK), once delivered into "chloroplast-free" onion epidermal cells can emit strong green fluorescence in the presence of DFHBI. Transgenic or virus-based expression of monomer KSK, in either stably transformed or virus-infected Nicotinana benthamiana plants, failed to show RMG fluorescence. However, incorporating tandem repeats of KSK into recombinant viral RNAs, enabled qualitative and quantitative detection, both in vitro and ex vivo (ex planta), of KSK-specific green fluorescence, though RMG was less obvious in vivo (in planta). These findings demonstrate Spinach-based RNA visualization has the potential for ex vivo and in vivo monitoring RNAs in plant cells. One sentence summarySpinach-based RMG technology was reevaluated to have potential for ex vivo and in vivo monitoring RNAs in plant cells.