Developing a mobile RNA delivery system for grafting trait improvement

Systemic RNA movement offers a route for non-transgenic trait improvement by grafting, but progress has been limited by the lack of reliable tools to identify mobile RNA elements. Here, we report a fluorescence-aptamer-based live-imaging platform for screening mobile RNAs and mapping mobility elements. In a transient screening assay, live imaging of 100 RNA-seq-predicted candidates detects three reproducibly mobile mRNAs, CAT3, CK1, and GAI, under our assay conditions, with GAI mRNA exhibiting the highest bidirectional mobility. Truncation analysis defines two independent 30-nt cis-elements, GME1 in the coding region and GME2 in the 3'UTR, with GME2 mediating a higher transport rate and mobility rate. A tandem 2×GME2 cassette in the 3'UTR functions as a high-capacity RNA delivery module and, in our transient assay, outperforms the tRNA-like sequence motif (TLS; tRNA^Met) construct tested here. Using 2×GME2, we deliver three otherwise non-mobile mRNAs across graft junctions and detect associated molecular or phenotypic effects in recipient tissues. Together, these findings establish a live-imaging-guided pipeline for mobile RNA validation and identify 2×GME2 as a non-viral RNA delivery element for graft-mediated trait modification.