Skip to main content
SpringerLink
Log in
Book cover

Plasmodesmata pp 329–341Cite as

In Vivo RNA Labeling Using MS2

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1217))

Abstract

The trafficking and asymmetric distribution of cytoplasmic RNA is a fundamental process during development and signaling across phyla. Plants support the intercellular trafficking of RNA molecules such as gene transcripts, small RNAs, and viral RNA genomes by targeting these RNA molecules to plasmodesmata (PD). Intercellular transport of RNA molecules through PD has fundamental implications in the cell-to-cell and systemic signaling during plant development and in the systemic spread of viral disease. Recent advances in time-lapse microscopy allow researchers to approach dynamic biological processes at the molecular level in living cells and tissues. These advances include the ability to label RNA molecules in vivo and thus to monitor their distribution and trafficking. In a broadly used RNA labeling approach, the MS2 method, the RNA of interest is tagged with a specific stem-loop (SL) RNA sequence derived from the origin of assembly region of the bacteriophage MS2 genome that binds to the bacteriophage coat protein (CP) and which, if fused to a fluorescent protein, allows the visualization of the tagged RNA by fluorescence microscopy. Here we describe a protocol for the in vivo visualization of transiently expressed SL-tagged RNA and discuss key aspects to study RNA localization and trafficking to and through plasmodesmata in Nicotiana benthamiana plants.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. St Johnston D (2005) Moving messages: the intracellular localization of mRNAs. Nat Rev Mol Cell Biol 6:363–375

    Article  PubMed  CAS  Google Scholar 

  2. Holt CE, Bullock SL (2009) Subcellular mRNA localization in animal cells and why it matters. Science 326:1212–1216

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. Nevo-Dinur K, Nussbaum-Shochat A, Ben-Yehuda S, Amster-Choder O (2011) Translation-independent localization of mRNA in E. coli. Science 331:1081–1084

    Article  PubMed  CAS  Google Scholar 

  4. Martin KC, Ephrussi A (2009) mRNA localization: gene expression in the spatial dimension. Cell 136:719–730

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  5. Lecuyer E, Yoshida H, Parthasarathy N et al (2007) Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131:174–187

    Article  PubMed  CAS  Google Scholar 

  6. Okita TW, Choi SB (2002) mRNA localization in plants: targeting to the cell’s cortical region and beyond. Curr Opin Plant Biol 5:553–559

    Article  PubMed  CAS  Google Scholar 

  7. Chuong SD, Good AG, Taylor GJ et al (2004) Large-scale identification of tubulin-binding proteins provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells. Mol Cell Proteomics 3:970–983

    Article  PubMed  CAS  Google Scholar 

  8. Ruiz-Medrano R, Xoconostle-Cazares B, Lucas WJ (1999) Phloem long-distance transport of CmNACP mRNA: implications for supracellular regulation in plants. Development 126:4405–4419

    PubMed  CAS  Google Scholar 

  9. Kim M, Canio W, Kessler S, Sinha N (2001) Developmental changes due to long-distance movement of a homeobox fusion transcript in tomato. Science 293:287–289

    Article  PubMed  CAS  Google Scholar 

  10. Lucas WJ, Lee JY (2004) Plasmodesmata as a supracellular control network in plants. Nat Rev Mol Cell Biol 5:712–726

    Article  PubMed  CAS  Google Scholar 

  11. Banerjee AK, Chatterjee M, Yu Y et al (2006) Dynamics of a mobile RNA of potato involved in a long-distance signaling pathway. Plant Cell 18:3443–3457

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  12. Lucas WJ, Bouche-Pillon S, Jackson DP et al (1995) Selective trafficking of KNOTTED1 homeodomain protein and its mRNA through plasmodesmata. Science 270:1980–1983

    Article  PubMed  CAS  Google Scholar 

  13. Melnyk CW, Molnar A, Baulcombe DC (2005) Intercellular and systemic movement of RNA silencing signals. EMBO J 30:3553–3563

    Article  Google Scholar 

  14. Peña EJ, Heinlein M (2012) RNA transport during TMV cell-to-cell movement. Front Plant Sci 3:193

    Article  PubMed  PubMed Central  Google Scholar 

  15. Christensen N, Tilsner J, Bell K et al (2009) The 5′ cap of Tobacco mosaic virus (TMV) is required for virion attachment to the actin/endoplasmic reticulum network during early infection. Traffic 10:536–551

    Article  PubMed  CAS  Google Scholar 

  16. Wang Y, Opperman L, Wickens M, Hall TM (2009) Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein. Proc Natl Acad Sci U S A 106:20186–20191

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  17. Tilsner J, Linnik O, Christensen NM et al (2009) Live-cell imaging of viral RNA genomes using a Pumilio-based reporter. Plant J 57:758–770

    Article  PubMed  CAS  Google Scholar 

  18. Bertrand E, Chartrand P, Schaefer M et al (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2:437–445

    Article  PubMed  CAS  Google Scholar 

  19. Schönberger 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 71:173–181

    Article  PubMed  Google Scholar 

  20. Chen J, Nikolaitchik O, Singh J et al (2009) High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis. Proc Natl Acad Sci U S A 106:13535–13540

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Park HY, Buxbaum AR, Singer RH (2010) Single mRNA tracking in live cells. Methods Enzymol 472:387–406

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  22. Hamada S, Ishiyama K, Choi SB et al (2003) The transport of prolamine RNAs to prolamine protein bodies in living rice endosperm cells. Plant Cell 15:2253–2264

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  23. Sambade A, Brandner K, Hofmann C et al (2008) Transport of TMV movement protein particles associated with the targeting of RNA to plasmodesmata. Traffic 9:2073–2088

    Article  PubMed  CAS  Google Scholar 

  24. Boyko V, Hu Q, Seemanpillai M et al (2007) Validation of microtubule-associated Tobacco mosaic virus RNA movement and involvement of microtubule-aligned particle trafficking. Plant J 51:589–603

    Article  PubMed  CAS  Google Scholar 

  25. Niehl A, Peña EJ, Amari K, Heinlein M (2013) Microtubules in viral replication and transport. Plant J 75:290–308

    Article  PubMed  CAS  Google Scholar 

  26. Peña EJ, Heinlein M (2013) Cortical microtubule-associated ER sites: organization centers of cell polarity and communication. Curr Opin Plant Biol 16:764–773

    Article  PubMed  Google Scholar 

  27. Citovsky V, Knorr D, Schuster G, Zambryski P (1990) The P30 movement protein of Tobacco mosaic virus is a single-strand nucleic acid binding protein. Cell 60:637–647

    Article  PubMed  CAS  Google Scholar 

  28. Heinlein M, Epel BL, Padgett HS, Beachy RN (1995) Interaction of tobamovirus movement proteins with the plant cytoskeleton. Science 270:1983–1985

    Article  PubMed  CAS  Google Scholar 

  29. Heinlein M, Padgett HS, Gens JS et al (1998) Changing patterns of localization of the Tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection. Plant Cell 10:1107–1120

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  30. Boyko V, Ferralli J, Ashby J et al (2000) Function of microtubules in intercellular transport of plant virus RNA. Nat Cell Biol 2:826–832

    Article  PubMed  CAS  Google Scholar 

  31. Ashby J, Boutant E, Seemanpillai M et al (2006) Tobacco mosaic virus movement protein functions as a structural microtubule-associated protein. J Virol 80:8329–8344

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  32. Peiro A, Martinez-Gil L, Tamborero S et al (2014) The Tobacco mosaic virus movement protein associates with but does not integrate into biological membranes. J Virol 88:3016–3026

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  33. Fusco D, Accornero N, Lavoie B et al (2003) Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr Biol 13:161–167

    Article  PubMed  CAS  Google Scholar 

  34. Dahm R, Zeitelhofer M, Gotze B et al (2008) Visualizing mRNA localization and local protein translation in neurons. Methods Cell Biol 85:293–327

    Article  PubMed  CAS  Google Scholar 

  35. Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  PubMed  CAS  Google Scholar 

  36. Karimi M, Bleys A, Vanderhaeghen R, Hilson P (2007) Building blocks for plant gene assembly. Plant Physiol 145:1183–1191

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  37. Karimi M, Depicker A, Hilson P (2007) Recombinational cloning with plant gateway vectors. Plant Physiol 145:1144–1154

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  38. Hellens R, Mullineaux P, Klee H (2000) Technical focus: a guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446–451

    Article  PubMed  CAS  Google Scholar 

  39. Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  40. Gillespie T, Boevink P, Haupt S et al (2002) Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of Tobacco mosaic virus. Plant Cell 14:1207–1222

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  41. Ruiz MT, Voinnet O, Baulcombe DC (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10:937–946

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  42. Mathur J (2007) The illuminated plant cell. Trends Plant Sci 12:506–513

    Article  PubMed  CAS  Google Scholar 

  43. Martin K, Kopperud K, Chakrabarty R et al (2009) Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta. Plant J 59:150–162

    Article  PubMed  CAS  Google Scholar 

  44. Buschmann H, Green P, Sambade A et al (2011) Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells. New Phytol 190:258–267

    Article  PubMed  CAS  Google Scholar 

  45. Rogowsky PM, Close TJ, Chimera JA et al (1987) Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. J Bacteriol 169:5101–5112

    PubMed  CAS  PubMed Central  Google Scholar 

  46. 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 33:949–956

    Article  PubMed  CAS  Google Scholar 

  47. Buschmann H, Sambade A, Pesquet E et al (2010) Microtubule dynamics in plant cells. Methods Cell Biol 97:373–400

    Article  PubMed  CAS  Google Scholar 

  48. Collins TJ (2007) ImageJ for microscopy. Biotechniques 43(Suppl 1):S25–S30

    Article  Google Scholar 

  49. Thevenaz P, Ruttimann UE, Unser M (1998) A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7:27–41

    Article  PubMed  CAS  Google Scholar 

  50. Sage D, Neumann FR, Hediger F et al (2005) Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamics. IEEE Trans Image Process 14:1372–1383

    Article  PubMed  Google Scholar 

Download references

Acknowledgement

Work of E.P. was supported by a grant from the Agence National de la Recherche (ANR, grant ANR-08-BLAN-244) to M.H.

Author information

Authors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes (IBMP), Centre National de la Recherche Scientifique (CNRS), 12 rue du Général Zimmer, 67084, Strasbourg, France

    Eduardo Peña & Manfred Heinlein

  2. Botany, Department of Environmental Sciences, University of Basel, Basel, Switzerland

    Manfred Heinlein

  3. Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universidad de Valencia, 46980, Valencia, Spain

    Adrian Sambade

  4. Instituto de Biotecnología y Biología Molecular, CCT - La Plata CONICET, Fac. Cs. Exactas, U.N.L.P., Calles 49 y 115, La Plata, Argentina

    Eduardo Peña

Authors
  1. Eduardo Peña
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Manfred Heinlein
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adrian Sambade
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Manfred Heinlein .

Editor information

Editors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Strasbourg, France

    Manfred Heinlein

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Peña, E., Heinlein, M., Sambade, A. (2015). In Vivo RNA Labeling Using MS2. In: Heinlein, M. (eds) Plasmodesmata. Methods in Molecular Biology, vol 1217. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1523-1_21

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1523-1_21

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1522-4

  • Online ISBN: 978-1-4939-1523-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation