1：Experimental Design and Method Selection:

The TriFC system combines the bimolecular fluorescence complementation (BiFC) system with the MS2 system for RNA imaging. Target RNA is tagged with 6xMS2 repeats, and MCP and RNA-binding protein are fused with YFP fragments. Upon RNA-protein interaction, YFP fragments complement to emit fluorescence, visualized by confocal microscopy.

2：Sample Selection and Data Sources:

Uses Nicotiana benthamiana plants (2–4 weeks old, 6–10 leaves stage). Specific constructs include pENTR-ELENA1 (lncRNA), pENTR-Med19a (RNA-binding protein), and pENTR-MCP.

3：List of Experimental Equipment and Materials:

Includes destination vectors (pBA3130, pBA3132, pBA3134, pBA3136, pBA-6xMS2-DC, pBA-DC-6xMS2), entry clones, LR Clonase II, E. coli DH5α, Agrobacterium GV3101, antibiotics (spectinomycin, kanamycin), LB media, acetosyringone, confocal laser scanning microscope (ZEISS LSM 780), and ZEN image analyzer.

4：Experimental Procedures and Operational Workflow:

Vector construction via LR reaction and transformation into E. coli and Agrobacterium. Agrobacterium cultures are prepared, mixed, and infiltrated into tobacco leaves using a syringe. Plants are incubated for 2–3 days, and YFP signals are observed with confocal microscopy at 514 nm excitation and 520–550 nm emission detection.

5：Data Analysis Methods:

YFP fluorescence signals are analyzed using ZEN image analyzer to confirm RNA-protein interactions.