Plant Molecular Biology

, Volume 58, Issue 4, pp 575–583 | Cite as

Evidence implying only unprimed RdRP activity during transitive gene silencing in plants



RNA silencing is a sequence-specific RNA degradation mechanism found in most eukaryotes, where small cleavage products (siRNAs) of double stranded RNA (dsRNA) mediate silencing of genes with sequence identity to the dsRNA inducer. In several systems, silencing has been found to spread from the dsRNA inducer sequence into upstream or downstream regions of the target RNA, a phenomenon termed transitive silencing. In nematodes, silencing spreads only in the 3′–5′ direction along the target mRNA by siRNAs serving as primers for cRNA synthesis by RNA-dependent RNA polymerase. In plants, transitive silencing is seen in both directions suggesting that at least some cRNA synthesis occurs by un-primed initiation at the 3′ end of mRNAs. Replicating plant viruses trigger an RNA silencing defence response that degrades the viral RNA, thus tempering the virus infection. Likewise, fragments of plant genes inserted into a virus will become targets for degradation, leading to virus-induced gene silencing (VIGS) of the homologous plant mRNAs. We have analyzed the spreading of gene silencing in VIGS experiments using a transgene and two endogenous genes as targets. In Nicotiana benthamiana plants expressing a β-glucuronidase (GUS) transgene, a Potato virus X vector carrying a 5′ fragment of the GUS gene induced silencing which spread to downstream regions of the transgene mRNA including the 3′-untranslated region. Conversely, silencing induced by a 3′ fragment spread only for a limited distance in the 3′–5′ direction. Silencing induced by a central GUS gene fragment spread only into downstream regions. Similar analyses using the endogenous plant genes, magnesium chelatase subunit I (ChlI) and an RNase L inhibitor homologue (RLIh), revealed no spreading along target sequences. This implies that transitive silencing in plants occurs by un-primed cRNA synthesis from the 3′ end of targeted (transgene) transcripts, and not by siRNA-primed cRNA synthesis.


RNA silencing siRNAs transitive gene silencing virus-induced gene silencing (VIGS) 



magnesium chelatase subunit I


double-stranded RNA


enhanced green fluorescent protein


green fluorescent protein




Potato virus X


RNA dependent RNA polymerase


RNA-induced silencing complex


RNase L inhibitor homologue


salicylic acid


small interfering RNA


untranslated region


virus-induced gene silencing


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  1. Alder, M.N., Dames, S., Gaudet, J., Mango, S.E. 2003Gene silencing in Caenorhabditis elegans by transitive RNA interferenceRNA92532Google Scholar
  2. Baulcombe, D. 2004RNA silencing in plantsNature431356363Google Scholar
  3. Braunstein, T.H., Moury, B., Johannessen, M., Albrechtsen, M. 2002Specific degradation of 3′ regions of GUS mRNA in posttranscriptionally silenced tobacco lines may be related to 5′–3′ spreading of silencingRNA8103444Google Scholar
  4. Burch-Smith, T.M., Anderson, J.C., Martin, G.B., Dinesh-Kumar, S.P. 2004Applications and advantages of virus-induced gene silencing for gene function studies in plantsPlant J.39734746Google Scholar
  5. Cogoni, C., Macino, G. 1999Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymeraseNature399166169Google Scholar
  6. Dalmay, T., Hamilton, A., Rudd, S., Angell, S., Baulcombe, D.C. 2000An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not be a virusCell101543553Google Scholar
  7. Gazzani, S., Lawrenson, T., Woodward, C., Headon, D., Sablowski, R. 2004A link between mRNA turnover and RNA interference in ArabidopsisScience30610461048Google Scholar
  8. Giordano, E., Rendina, R., Peluso, I., Furia, M. 2002RNAi triggered by symmetrically transcribed transgenes in Drosophila melanogasterGenetics160637648Google Scholar
  9. Himber, C., Dunoyer, P., Moissiard, G., Ritzenthaler, C., Voinnet, O. 2003Transitivity-dependent and –independent cell-to-cell movement of RNA silencingEMBO J.2245234533Google Scholar
  10. Hutvágner, G., Mlynárová, L., Nap, J.-P. 2000Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobaccoRNA614451454Google Scholar
  11. Jones, L., Hamilton, A.J., Voinnet, O., Thomas, C.L., Maule, A.J., Baulcombe, D.C. 1999RNA-DNA interactions and DNA methylation in post-transcriptional gene silencingPlant Cell1122912301Google Scholar
  12. Kjemtrup, S., Sampson, K.S., Peele, C.G., Nguyen, L.V., Conkling, M.A., Thompson, W.F., Robertson, D. 1998Gene silencing from plant DNA carried by a geminivirusPlant J.1491100Google Scholar
  13. Klahre, U., Crété, P., Leuenberger, S.A., Iglesias, V.A., Meins, F.,Jr. 2002High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plantsProc. Natl. Acad. Sci. USA991198111986Google Scholar
  14. Lipardi, C., Wei, Q., Paterson, B.M. 2001RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAsCell107297307Google Scholar
  15. Makeyev, E.V., Bamford, D.H. 2002Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modesMol. Cell1014171427Google Scholar
  16. Martens, H., Novotny, J., Oberstrass, J., Steck, T.L., Postlethwait, P., Nellen, W. 2002RNAi in Dictyostelium: the role of RNA-directed RNA polymerases and double-stranded RNaseMol. Biol. Cell13445453Google Scholar
  17. Mourrain, P., Béclin, C., Elmayan, T., Feuerbach, F., Godon, C., Morel, J.-B., Jouette, D., Lacombe, A.-M., Nikic, S., Picault, N., Rémoué, K., Sanial, M., Vo, T.-A., Vaucheret, H. 2000Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistanceCell101533542Google Scholar
  18. Nicolás, F.E., Torres-Martínez, S., Ruiz-Vázquez, R.M. 2003Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenesEMBO J.2239833991Google Scholar
  19. Papaefthimiou, I., Hamilton, A.J., Denti, M.A., Baulcombe, D.C., Tsagris, M., Tabler, M. 2001Replicating potato spindle tuber viroid RNA is accompanied by short RNA fragments that are characteristic of post-transcriptional gene silencingNucleic Acids Res2923952400Google Scholar
  20. Petersen, B.O., Jørgensen, B., Albrechtsen, M. 2004Isolation and RNA silencing of homologues of the RNase L inhibitor in Nicotiana speciesPlant Science16712831289Google Scholar
  21. Roignant, J.-Y., Carré, C., Mugat, B., Szymczak, D., Lepesant, J.-A., Antoniewski, C. 2003Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in DrosophilaRNA9299308Google Scholar
  22. Ruiz, M.T., Voinnet, O., Baulcombe, D.C. 1998Initiation and maintenance of virus-induced gene silencingPlant Cell10937946Google Scholar
  23. Sanders, M., Maddelein, W., Depicker, A., Montagu, M., Cornelissen, M., Jacobs, J. 2002An active role for endogenous beta-1,3-glucanase genes in transgene-mediated co-suppression in tobaccoEMBO J.2158245832Google Scholar
  24. Schiebel, W., Haas, B., Marinkovic, S., Klanner, A., Sanger, H.L. 1993aRNA-directed RNA polymerase from tomato leavesI. Purification and physical properties. J. Biol. Chem.2681185111857Google Scholar
  25. Schiebel, W., Haas, B., Marinkovic, S., Klanner, A., Sanger, H.L. 1993bRNA-directed RNA polymerase from tomato leaves. I. Catalytic in vitro propertiesJ. Biol. Chem.2681185811867Google Scholar
  26. Schwarz, D.S., Hutvágner, G., Haley, B., Zamore, P.D. 2002Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathwaysMol. Cell10537548Google Scholar
  27. Sijen, T., Fleenor, J., Simmer, F., Thijssen, K.L., Parrish, S., Timmons, L., Pasterk, R.H.A., Fire, A. 2001On the role of RNA amplification in dsRNA-triggered gene silencingCell107465476Google Scholar
  28. Smardon, A., Spoerke, J.M., Stacey, S.C., Klein, M.E., Mackin, N., Maine, E.M. 2000EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans.Curr. Biol.10169178Google Scholar
  29. Smith, N.A., Singh, S.P., Wang, M.-B., Stoutjesdijk, P.A., Green, A.G., Waterhouse, P.M. 2000Total silencing by intron-spliced hairpin RNAsNature407319320Google Scholar
  30. Stein, P., Svoboda, P., Anger, M., Schultz, R.M. 2003RNAi: mammalian oocytes do it without RNA-dependent RNA polymeraseRNA9187192Google Scholar
  31. Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Fire, A., Mello, C.C. 1999The rde-1 gene, RNA interference, and transposon silencing in C.elegansCell99123132Google Scholar
  32. Tang, G., Reinhart, B.J., Bartel, D.P., Zamore, P.D. 2003A biochemical framework for RNA silencing in plantsGenes & Devel.174963Google Scholar
  33. Tijsterman, M., Ketting, R.F., Plasterk, R.H.A. 2002The genetics of RNA silencingAnnu. Rev. Genet.36489519Google Scholar
  34. Vaistij, F.E., Jones, L., Baulcombe, D.C. 2002Spreading of␣RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative␣RNA-dependent RNA polymerasePlant Cell14857867Google Scholar
  35. Voinnet, O. 2001RNA silencing as a plant immune system against virusesTrends. Genet.17449459Google Scholar
  36. Voinnet, O., Vain, P., Angell, S., Baulcombe, D.C. 1998Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNACell95177187Google Scholar
  37. Waterhouse, P.M., Wang, M.-B., Lough, T. 2001Gene silencing as an adaptive defence against virusesNature411834842Google Scholar
  38. Yamada, K., Lim, J., Dale, J.M., Chen, H., Shinn, P., Palm, C.J., et al.,  2003Empirical analysis of transcriptional activity in the Arabidopsis genomeScience302842846Google Scholar
  39. Yang, S.-J., Carter, S.A., Cole, A.B., Cheng, N.-H., Nelson, R.S. 2004A natural variant of a host RNA-dependent RNA polymerase is associated with increased susceptibility to viruses by Nicotiana benthamianaProc. Natl. Acad. Sci. USA10162976302Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  1. 1.Biotechnology GroupDanish Institute of Agricultural SciencesFrederiksberg CDenmark

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