A binding-activated CRISPR/dCas13X imaging system enables precise RNA tracking in plants.

Abstract

Visualizing the spatiotemporal dynamics of native RNA molecules with single-transcript-level sensitivity in living plant cells remains a formidable challenge due to the high background noise caused by the unique cellular architecture of plants. Here, using transiently transformed Nicotiana benthamiana cells and stable Arabidopsis thaliana lines, we present a high-signal-to-noise RNA imaging platform that couples CRISPR-dCas13X with a binding-associated fluorescence stabilization strategy. We identified a novel plant-specific degron that selectively targets unbound probes for proteasomal degradation while preserving target-associated signal in a manner consistent with binding-dependent protection, enabling high-fidelity detection of endogenous mRNA puncta with minimal perturbation. Leveraging this platform, we characterized dynamics of FT mRNA trafficking in living plant cells, including plasmodesmata-targeting, cell-to-cell trafficking, and long-distance movement. Loss-of-function analysis further identified the established RNA-binding protein GRP7 as a factor that contributes to efficient FT mRNA transport. This work establishes a useful toolkit for investigating plant RNA biology and provides mechanistic insight into the systemic transport of florigenic signals.