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.
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References
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
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
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
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
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
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
Lough TJ, Lucas WJ (2006) Integrative plant biology: role of phloem long-distance macromolecular trafficking. Annu Rev Plant Biol 57:203–232
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
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
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
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
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
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
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
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
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
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
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
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
Lico C, Benvenuto E, Baschieri S (2015) The two-faced potato virus X: from plant pathogen to smart nanoparticle. Front Plant Sci 6:1009
Van Wezel R, Hong Y (2004) Virus survival of RNA silencing without deploying protein-mediated suppression in Nicotiana benthamiana. FEBS Lett 562:65–70
Hong Y, Saunders K, Stanley J (1997) Transactivation of dianthin transgene expression by African cassava mosaic virus AC2. Virology 228:383–387
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
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
Banerjee AK, Lin T, Hannapel DJ (2009) Untranslated regions of a mobile transcript mediate RNA metabolism. Plant Physiol 151:1831–1843
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
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
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
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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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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