RNA-triggered fluorescence controlled by RNA switches for real-time RNA expression tracking in living plants

Real-time visualization of RNA dynamics across spatial scales in living plants is desirable yet remains challenging, hindered by the absence of imaging tools that simultaneously offer high sensitivity and deployability in whole living plants. Here, we engineered a modular, RNA-triggered fluorescence (RTF) reporter system de novo for spatiotemporal imaging of RNA from cellular to whole-plant scales in vivo. This system integrates three functional modules: a target-specific allosteric RNA switch, a degradable adapter for background suppression, and a fluorescent reporter. All components are stably expressed from a single vector and assemble in vivo, enabling uniform deployment throughout entire plants. The modified RTF (RTFst) achieves single-molecule sensitivity (signal-to-noise ratio >60) for dynamic tracking of developmentally regulated, tissue-specific, circadian, and stress-responsive mRNAs. Crucially, it enables the first direct, real-time observation of graft-transmissible mRNA trafficking across tissue scales and cross-kingdom transfer of the aphid-secreted long non-coding RNA Ya1 to plant cells. RTF establishes a versatile platform for studying gene regulation, signaling, and transcript mobility in plants, with broad applicability in synthetic biology and crop improvement.