Trimolecular Fluorescence Complementation (TriFC) Assay for Visualization of RNA-Protein Interaction in Plants

  • Jun Sung Seo
  • Nam-Hai ChuaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1933)


RNA-protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA-protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with the MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) to tag lncRNA. Target RNA is tagged with 6xMS2, and MCP and RNA-binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-protein interaction in vivo is observed by confocal microscopy.

Key words

Long noncoding RNA RNA-protein interaction TriFC Tobacco transient expression In vivo visualization 



We thank Dr. Ulrich Z. Hammes for the Gateway 6xMS2 tagging vectors. This work was supported by Singapore NRF RSSS Grant (NRF-RSSS-002).


  1. 1.
    St Laurent G, Wahlestedt C, Kapranov P (2015) The Landscape of long noncoding RNA classification. Trends Genet 31(5):239–251CrossRefGoogle Scholar
  2. 2.
    Christensen NM, Oparka KJ, Tilsner J (2010) Advances in imaging RNA in plants. Trends Plant Sci 15(4):196–203CrossRefGoogle Scholar
  3. 3.
    Schonberger J, Hammes UZ, Dresselhaus T (2012) In vivo visualization of RNA in plants cells using the λN22 system and a GATEWAY-compatible vector series for candidate RNAs. Plant J 71(1):173–181CrossRefGoogle Scholar
  4. 4.
    Valegård K, Murray JB, Stockley PG, Stonehouse NJ, Liljas L (1994) Crystal structure of an RNA bacteriophage coat protein-operator complex. Nature 371(6498):623–626CrossRefGoogle Scholar
  5. 5.
    LeCuyer KA, Behlen LS, Uhlenbeck OC (1996) Mutagenesis of a stacking contact in the MS2 coat protein-RNA complex. EMBO J 15(24):6847–6853CrossRefGoogle Scholar
  6. 6.
    Campalans A, Kondorosi A, Crespi M (2004) Enod40, a short open reading frame-containing mRNA, induces cytoplasmic localization of a nuclear RNA binding protein in Medicago truncatula. Plant Cell 16(4):1047–1059CrossRefGoogle Scholar
  7. 7.
    Fujioka Y, Utsumi M, Ohba Y, Watanabe Y (2007) Location of a possible miRNA processing site in SmD3/SmB nuclear bodies in Arabidopsis. Plant Cell Physiol 48(9):1243–1253CrossRefGoogle Scholar
  8. 8.
    Golding I, Cox EC (2004) RNA dynamics in live Escherichia coli cells. PNAS 101(31):11310–11315CrossRefGoogle Scholar
  9. 9.
    Seo JS, Sun HX, Park BS, Huang CH, Yeh SD, Jung C, Chua NH (2017) ELF18-INDUCED LONG-NONCODING RNA associates with mediator to enhance expression of innate immune response genes in Arabidopsis. Plant Cell 29(5):1024–1038CrossRefGoogle Scholar
  10. 10.
    Höfgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16(20):9877CrossRefGoogle Scholar
  11. 11.
    Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Plant Molecular BiologyRockefeller UniversityNew YorkUSA
  2. 2.TEMASEK Life Sciences LaboratorySingaporeSingapore

Personalised recommendations