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RNA Tagging pp 181–194Cite as

Utilizing Potato Virus X to Monitor RNA Movement

Part of the Methods in Molecular Biology book series (MIMB,volume 2166)

Abstract

Mobility assays coupled with RNA profiling have revealed the presence of hundreds of full-length non-cell-autonomous messenger RNAs that move through the whole plant via the phloem cell system. Monitoring the movement of these RNA signals can be difficult and time consuming. Here we describe a simple, virus-based system for surveying RNA movement by replacing specific sequences within the viral RNA genome of potato virus X (PVX) that are critical for movement with other sequences that facilitate movement. PVX is a RNA virus dependent on three small proteins that facilitate cell-to-cell transport and a coat protein (CP) required for long-distance spread of PVX. Deletion of the CP blocks movement, whereas replacing the CP with phloem-mobile RNA sequences reinstates mobility. Two experimental models validating this assay system are discussed. One involves the movement of the flowering locus T RNA that regulates floral induction and the second involves movement of StBEL5, a long-distance RNA signal that regulates tuber formation in potato.

Key words

  • Potato virus X
  • RNA mobility assay
  • FT
  • BEL5

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  • DOI: 10.1007/978-1-0716-0712-1_10
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References

  1. Lucas WJ, Groover A, Lichtenberger R, Furuta K, Yadav SR, Helariutta Y, He XQ, Fukuda H, Kang J, Brady SM, Patrick JW, Sperry J, Yoshida A, López-Millán AF, Grusak MA, Kachroo P (2013) The plant vascular system: evolution, development and functions. J Integr Plant Biol 55:294–388

    CAS  CrossRef  Google Scholar 

  2. Asano T, Masumura T, Kusano H, Kikuchi S, Kurita A, Shimada H, Kadowaki K (2002) Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: Toward comprehensive analysis of the genes expressed in the rice phloem. Plant J 32:401–408

    CAS  CrossRef  Google Scholar 

  3. Vilaine F, Palauqui JC, Amselem J, Kusiak C, Lemoine R, Dinant S (2003) Towards deciphering phloem: a transcriptome analysis of the phloem of Apium graveolens. Plant J 36:67–81

    CAS  CrossRef  Google Scholar 

  4. Omid A, Keilin T, Glass A, Leshkowitz D, Wolf S (2007) Characterization of phloem-sap transcription profile in melon plants. J Exp Bot 58:3645–3656

    CAS  CrossRef  Google Scholar 

  5. Deeken R, Ache P, Kajahn I, Klinkenberg J, Bringmann G, Hedrich R (2008) Identification of Arabidopsis thaliana phloem RNAs provides a search criterion for phloem-based transcripts hidden in complex datasets of microarray experiments. Plant J 55:746–759

    CAS  CrossRef  Google Scholar 

  6. Gaupels F, Buhtz A, Knauer T, Deshmukh S, Waller F, van Bel AJE, Kogel KH, Kehr J (2008) Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap. J Exp Bot 59:3297–3306

    CAS  CrossRef  Google Scholar 

  7. Lough TJ, Lucas WJ (2006) Integrative plant biology: role of phloem long-distance macromolecular trafficking. Annu Rev Plant Biol 57:203–232

    CAS  CrossRef  Google Scholar 

  8. Notaguchi M, Higashiyama T, Suzuki T (2015) Identification of mRNAs that move over long distances using a RNA-Seq analysis of Arabidopsis/Nicotiana benthamiana heterografts. Plant Cell Physiol 56:311–321

    CAS  CrossRef  Google Scholar 

  9. Thieme CJ, Rojas-Triana M, Stecyk E, Schudoma C, Zhang W, Yang L, Miñambres M, Walther D, Schulze WX, Paz-Ares J, Scheible WR, Kragler F (2015) Endogenous Arabidopsis messenger RNAs transported to distant tissues. Nat Plants 1:15025

    CAS  CrossRef  Google Scholar 

  10. Banerjee AK, Chatterjee M, Yu Y, Suh SG, Miller WA, Hannapel DJ (2006) Dynamics of a mobile RNA of potato involved in a long-distance signaling pathway. Plant Cell 18:3443–3457

    CAS  CrossRef  Google Scholar 

  11. Ghate TH, Sharma P, Khondare KR, Hannapel DJ, Banerjee AK (2017) The mobile RNAs, StBEL11 and StBEL29, suppress growth of tubers in potato. Plant Mol Biol 93:563–578

    CAS  CrossRef  Google Scholar 

  12. Mahajan A, Bhogle S, Kang IH, Hannapel DJ, Banerjee AK (2012) The mRNA of a Knotted1-like transcription factor of potato is phloem mobile. Plant Mol Biol 79:595–608

    CAS  CrossRef  Google Scholar 

  13. Haywood V, Yu TS, Huang NC, Lucas WJ (2005) Phloem long-distance trafficking of gibberellic acid-insensitive RNA regulates leaf development. Plant J 42:49–68

    CAS  CrossRef  Google Scholar 

  14. 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

    CAS  CrossRef  Google Scholar 

  15. Notaguchi M, Wolf S, Lucas WJ (2012) Phloem-mobile Aux/IAA transcripts target to the root tip and modify root architecture. J Integr Plant Biol 54:760–772

    CAS  CrossRef  Google Scholar 

  16. Li C, Gu M, Shi N, Zhang H, Yang X, Osman T, Liu Y, Wang H, Vatish M, Jackson S, Hong Y (2011) Mobile FT mRNA contributes to the systemic florigen signalling in floral induction. Sci Rep 1:73

    CrossRef  Google Scholar 

  17. Lu KJ, Huang NC, Liu YS, Lu CA, Yu TS (2012) Long-distance movement of Arabidopsis flowering locus T RNA participates in systemic floral regulation. RNA Biol 9:653–662

    CAS  CrossRef  Google Scholar 

  18. Huang NC, Jane WN, Chen J, Yu TS (2012) Arabidopsis centroradialis homologue acts systemically to inhibit floral initiation in Arabidopsis. Plant J 72:175–184

    CAS  CrossRef  Google Scholar 

  19. Baulcombe D, Gilbert J, Goulden M, Köhm B, Cruz SS (1994) Molecular biology of resistance to potato virus X in potato. Biochem Soc Symp 60:207–218

    CAS  PubMed  Google Scholar 

  20. Lico C, Benvenuto E, Baschieri S (2015) The two-faced potato virus X: from plant pathogen to smart nanoparticle. Front Plant Sci 6:1009

    CrossRef  Google Scholar 

  21. Van Wezel R, Hong Y (2004) Virus survival of RNA silencing without deploying protein-mediated suppression in Nicotiana benthamiana. FEBS Lett 562:65–70

    CrossRef  Google Scholar 

  22. Hong Y, Saunders K, Stanley J (1997) Transactivation of dianthin transgene expression by African cassava mosaic virus AC2. Virology 228:383–387

    CAS  CrossRef  Google Scholar 

  23. Van Wezel R, Dong X, Liu H, Tien P, Stanley J, Hong Y (2002) Mutations of three cysteine residues in Tomato yellow leaf curl virus-China C2 protein causes dysfunction in pathogenesis and posttranscriptional gene silencing suppression. Mol Plant-Microbe Interact 15:203–208

    CrossRef  Google Scholar 

  24. Li C, Zhang K, Zeng X, Jackson S, Zhou Y, Hong Y (2009) A cis element within Flowering Locus T mRNA determines its mobility and facilitates trafficking of heterologous viral RNA. J Virol 83:3540–3548

    CAS  CrossRef  Google Scholar 

  25. Banerjee AK, Lin T, Hannapel DJ (2009) Untranslated regions of a mobile transcript mediate RNA metabolism. Plant Physiol 151:1831–1843

    CAS  CrossRef  Google Scholar 

  26. Cho SK, Sharma P, Butler NM, Kang IH, Shah S, Rao AG, Hannapel DJ (2015) Polypyrimidine tract-binding proteins of potato mediate tuberization through an interaction with StBEL5 RNA. J Exp Bot 66:6835–6847

    CAS  CrossRef  Google Scholar 

  27. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408

    CAS  CrossRef  Google Scholar 

  28. Wirjanata G, Handayuni I, Zaloumis SG, Chalfein F, Prayoga P, Kenangalem E, Poespoprodjo JR, Noviyanti R, Simpson JA, Price RN, Marfurt J (2016) Analysis of ex vivo drug response data of Plasmodium clinical isolates: the pros and cons of different computer programs and online platforms. Malar J 15:137

    CrossRef  Google Scholar 

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Acknowledgments

This work was in part funded by Ministry of Science and Technology of China (National Key R&D Program 2017YFE0110900), Ministry of Agriculture of China (National Transgenic Program 2016ZX08009001-004), the National Natural Science Foundation of China (31872636, 31370180), Zhejiang Provincial Natural Science of Foundation (LY19C020002), Hangzhou Normal University (Sino-EU Plant RNA Signaling S&T Platform Initiative 9995C5021841101), and a NSF Plant Genome Research Program award no. DBI-0820659 to DH.

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Authors and Affiliations

  1. Research Centre for Plant RNA Signaling, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China

    Zhiming Yu, Pengcheng Zhang & Yiguo Hong

  2. Dura-Line Corporation, Clinton, TN, USA

    Sung Ki Cho

  3. Warwick-Hangzhou RNA Signaling Joint Laboratory, School of Life Sciences, University of Warwick, Warwick, UK

    Yiguo Hong

  4. Worcester-Hangzhou Joint Molecular Plant Health Laboratory, Institute of Science and the Environment, University of Worcester, Worcester, UK

    Yiguo Hong

  5. Plant Biology Major, Iowa State University, Ames, IA, USA

    David J. Hannapel

Authors
  1. Zhiming Yu
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  2. Sung Ki Cho
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  3. Pengcheng Zhang
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  4. Yiguo Hong
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  5. David J. Hannapel
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Corresponding authors

Correspondence to Yiguo Hong or David J. Hannapel .

Editor information

Editors and Affiliations

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

    Manfred Heinlein

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Yu, Z., Cho, S.K., Zhang, P., Hong, Y., Hannapel, D.J. (2020). Utilizing Potato Virus X to Monitor RNA Movement. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_10

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  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

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