Plant Molecular Biology

, Volume 63, Issue 6, pp 803–813 | Cite as

Generation of secondary small interfering RNA in cell-autonomous and non-cell autonomous RNA silencing in tobacco

  • Katsuyoshi Shimamura
  • Shin-ichiro Oka
  • Yumi Shimotori
  • Takashi Ohmori
  • Hiroaki Kodama


Small interfering RNA (siRNA) species with 21–25 nucleotides in length guide mRNA cleavage, translational arrest, and heterochromatin formation in RNA interference (RNAi). To delineate the target region of RNAi, a construct harboring a transcriptional fusion between parts of the target mRNA and the β-glucuronidase gene was biolistically delivered into tobacco leaves showing an RNAi phenotype and the assay sequence was transiently expressed. The RNAi effect was monitored by amplification of this chimeric transcript. By using this assay method, we addressed the transitive RNA silencing of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase gene (NtFAD3). In the NtFAD3 RNAi plants, the target region of RNAi was restricted in the inducer region corresponding to a stem sequence of the hairpin double-stranded RNA, indicating that endogenous NtFAD3 mRNA was not a template for an RNA-dependent RNA polymerase. The secondary NtFAD3 siRNAs were produced in the crossbred plants between the NtFAD3 overexpressed plant and the NtFAD3 RNAi plant. Similarly, the secondary siRNAs were generated in the systemically silenced scion. Although these secondary siRNAs originated preferentially from the 3′ region downstream of the inducer region, the secondary siRNAs produced in the silenced scion (non-cell autonomous secondary siRNAs) resulted in the strong degradation of the target mRNA, but the secondary siRNAs in the crossbred plants (cell-autonomous secondary siRNAs) showed limited RNA degradation activity. These results showed that this in vivo assay for determination of RNAi efficiency is a useful tool to delineate RNAi mechanisms.


Fatty acid desaturase RdRP RNA silencing Secondary siRNA Systemic gene silencing Tobacco 



Cauliflower mosaic virus


Complimentary RNA


Double-stranded RNA


Inverted repeat-post-transcriptional gene silencing


Post-transcriptional gene silencing


RNA-dependent RNA polymerase


RNA-induced silencing complex


RNA interference


Small interfering RNA


Sense-post-transcriptional gene silencing


Virus-induced gene silencing


Wild type


  1. Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK (2003) RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev 67:657–685PubMedCrossRefGoogle Scholar
  2. Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing. Curr Opin Plant Biol 2:109–113PubMedCrossRefGoogle Scholar
  3. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366PubMedCrossRefGoogle Scholar
  4. Bezanilla M, Pan A, Quatrano RS (2003) RNA interference in the moss Physcomitrella patens. Plant Physiol 133:470–474PubMedCrossRefGoogle Scholar
  5. Braunstein TH, Moury B, Johannessen M, Albrechtsen M (2002) Specific degradation of 3′ regions of GUS mRNA in posttranscriptionally silenced tobacco lines may be related to 5′-3′ spreading of silencing. RNA 8:1034–1044PubMedCrossRefGoogle Scholar
  6. Crété P, Leuenberger S, Iglesias VA, Suarez V, Schöb H, Holtorf H, van Eeden S, Meins F Jr (2001) Graft transmission of induced and spontaneous post-transcriptional silencing of chitinase genes. Plant J 28:493–501PubMedCrossRefGoogle Scholar
  7. Davuluri GR, van Tuinen A, Mustilli AC, Manfredonia A, Newman R, Burgess D, Brummell DA, King SR, Palys J, Uhlig J, Pennings HMJ, Bowler C (2004) Manipulation of DET1 expression in tomato results in photomorphogenic phenotypes caused by post-transcriptional gene silencing. Plant J 40:344–354PubMedCrossRefGoogle Scholar
  8. Ebhardt HA, Thi EP, Wang M, Unrau PJ (2005) Extensive 3′ modification of plant small RNAs is modulated by helper component-proteinase expression. Proc Natl Acad Sci USA 102:13398–13403PubMedCrossRefGoogle Scholar
  9. Hamada T, Kodama H, Nishimura M, Iba K (1994) Cloning of a cDNA encoding tobacco ω-3 fatty acid desaturase. Gene 147:293–294PubMedCrossRefGoogle Scholar
  10. Hamada T, Kodama H, Takeshita K, Utsumi H, Iba K (1998) Characterization of transgenic tobacco with an increased α-linolenic acid level. Plant Physiol 118:591–598PubMedCrossRefGoogle Scholar
  11. Hamada T, Kodama H (2006) Phenotype of the transgene in plants: expression and silencing. In: Teixeira da Silva JA (ed) Floriculture, ornamental and plant biotechnology: advances and topical issues, 1st edn. vol. II, Global Science Books, London, UK, pp 98–107Google Scholar
  12. Hamilton A, Voinnet O, Chappell L, Baulcombe D (2002) Two classes of short interfering RNA in RNA silencing. EMBO J 21:4671–4679PubMedCrossRefGoogle Scholar
  13. Hammond SM, Bernstein E, Beach D, Hannon GL (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296PubMedCrossRefGoogle Scholar
  14. Han Y, Grierson D (2002a) Relationship between small antisense RNAs and aberrant RNAs associated with sense transgene mediated gene silencing in tomato. Plant J 29:509–519CrossRefGoogle Scholar
  15. Han Y, Grierson D (2002b) The influence of inverted repeats on the production of small antisense RNAs involved in gene silencing. Mol Genet Genomics 267:629–635CrossRefGoogle Scholar
  16. Himber C, Dunoyer P, Moissiard G, Ritzenthaler C, Voinnet O (2003) Transitivity-dependent and independent cell-to-cell movement of RNA silencing. EMBO J 22:4523–4533PubMedCrossRefGoogle Scholar
  17. Horiguchi G (2004) RNA silencing in plants: a shortcut to functional analysis. Differentiation 72:65–73PubMedCrossRefGoogle Scholar
  18. Hutvágner G, Mlynárová L, Nap J-P (2000) Detailed characterization of the post-transcriptional gene silencing-related small RNA in a GUS gene-silenced tobacco. RNA 6:1445–1454PubMedCrossRefGoogle Scholar
  19. Jorgensen RA, Atkinson RG, Forster RLS, Lucas WJ (1998) An RNA-based information superhighway in plants. Science 279:1486–1487PubMedCrossRefGoogle Scholar
  20. Kalantidis K (2004) Grafting the way to the systemic silencing signal in plants. PLoS Biol 2:e224Google Scholar
  21. Klahre U, Crété P, Leuenberger SA, Iglesias VA, Meins F Jr (2002) High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants. Proc Natl Acad Sci USA 99:11981–11986PubMedCrossRefGoogle Scholar
  22. Kodama H, Ito M, Ohnishi N, Suzuka I, Komamine A (1991) Molecular cloning of the gene for plant proliferating-cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells. Eur J Biochem 197:495–503PubMedCrossRefGoogle Scholar
  23. Kodama H, Hamada T, Horiguchi G, Nishimura M, Iba K (1994) Genetic enhancement of cold tolerance by expression of a gene for chloroplast ω-3 fatty acid desaturase in transgenic tobacco. Plant Physiol 105:601–605PubMedGoogle Scholar
  24. Kościańska E, Kalantidis K, Wypijewski K, Sadowski J, Tabler M (2005) Analysis of RNA silencing in agroinfiltrated leaves of Nocotiana benthamiana and Nicotiana tabacum. Plant Mol Biol 59:647–661PubMedCrossRefGoogle Scholar
  25. Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14:1605–1619PubMedCrossRefGoogle Scholar
  26. Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431:343–349PubMedCrossRefGoogle Scholar
  27. Miki D, Itoh R, Shimamoto K (2005) RNA silencing of single and multiple members in a gene family of rice. Plant Physiol 138:1903–1913PubMedCrossRefGoogle Scholar
  28. Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohashi Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49–59PubMedGoogle Scholar
  29. Mitsui M, Murohashi Y, Asano Y, Masada M, Kodama H (2003) Transient assay for in vivo splicing in tobacco leaf cells by particle bombardment. Plant Mol Biol Rep 21:21–30Google Scholar
  30. Motamedi MR, Verdel A, Colmenares SU, Gerber SA, Gygi SP, Moazed D (2004) Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119:789–802PubMedCrossRefGoogle Scholar
  31. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2:279–289PubMedCrossRefGoogle Scholar
  32. Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expression in tobacco of a β-glucuronidase reporter gene containing an intron with the coding sequence. Plant Cell Physiol 31:805–813Google Scholar
  33. Palauqui J-C, Elmayan T, Pollien J-M, Vaucheret H (1997) Systemic acquired silencing: transgene-specific post-transcriptional silencing is trasmitted by grafting from silenced stocks to non-silenced scions. EMBO J 16:4738–4745PubMedCrossRefGoogle Scholar
  34. Peterson BO, Albrechtsen M (2005) Evidence implying only unprimed RdRP activity during transitive gene silencing in plants. Plant Mol Biol 58:575–583CrossRefGoogle Scholar
  35. Qi Y, Hannon GJ (2005) Uncovering RNAi mechanisms in plants: biochemistry enters the foray. FEBS Lett 579:5899–5903PubMedCrossRefGoogle Scholar
  36. Sanders M, Maddelein W, Depicker A, Montagu MV, Cornelissen M, Jacobs J (2002) An active role for endogenous β-1,3-glucanase genes in transgene-mediated co-suppression in tobacco. EMBO J 21:5824–5832PubMedCrossRefGoogle Scholar
  37. Schiebel W, Pélissier T, Riedel L, Thalmeir S, Schiebel R, Kempe D, Lottspeich F, Sänger HL, Wassenegger M (1998) Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato. Plant Cell 10:2087–2101PubMedCrossRefGoogle Scholar
  38. Schwach F, Vaisitij FE, Jones L, Baulcombe DC (2005) An RNA-dependent RNA polymerase prevents meristem invasion by potato virus X and is required for the activity but not the production of a systemic silencing signal. Plant Physiol 138:1842–1852PubMedCrossRefGoogle Scholar
  39. Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RHA, Fire A (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465–476PubMedCrossRefGoogle Scholar
  40. Sugiyama T, Cam H, Verdel A, Moazed D, Grewal SIS (2005) RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterchromatin assembly to siRNA production. Proc Natl Acad Sci USA 102:152–157PubMedCrossRefGoogle Scholar
  41. Tang G, Reinhart BJ, Bartel DP, Zamore PD (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17:49–63PubMedCrossRefGoogle Scholar
  42. Tomita R, Hamada T, Horiguchi G, Iba K, Kodama H (2004) Transgene overexpression with cognate small interfering RNA in tobacco. FEBS Lett 573:117–120PubMedCrossRefGoogle Scholar
  43. Vaistij FE, Jones L, Baulcombe DC (2002) Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14:857–867PubMedCrossRefGoogle Scholar
  44. van der Krol AR, Mur LA, Beld M, Mol JNM, Stuitje AR (1990) Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of a gene expression. Plant Cell 2:291–299PubMedCrossRefGoogle Scholar
  45. Van Houdt H, Bleys A, Depicker A (2003) RNA target sequences promote spreading of RNA silencing. Plant Physiol 131:245–253PubMedCrossRefGoogle Scholar
  46. Voinnet O (2005) Non-cell autonomous RNA silencing. FEBS Lett 579:5858–5871PubMedCrossRefGoogle Scholar
  47. Voinnet O, Vain P, Angell S, Baulcombe DC (1998) Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA. Cell 95:177–187PubMedCrossRefGoogle Scholar
  48. Wang M-B, Rezaian A, Watson JM, Waterhouse PM, Metzlaff M (2006) Understanding and exploiting RNA silencing-mediated antiviral defense in plants. In: Teixeira da Silva JA (ed) Floriculture, ornamental and plant biotechnology: advances and topical issues. 1st edn. vol. III, Global Science Books, London, UK, pp 509–522Google Scholar
  49. Waterhouse PM, Smith NA, Wang M-B (1999) Virus resistance and gene silencing: killing the messenger. Trends Plant Sci 4:452–457PubMedCrossRefGoogle Scholar
  50. Waterhouse PM, Helliwell CA (2003) Exploring plant genomes by RNA-induced gene silencing. Nature Rev Genet 4:29–38CrossRefGoogle Scholar
  51. Watson JM, Fusaro AF, Wang M, Waterhouse PM (2005) RNA silencing platforms in plants. FEBS Lett 579:5982–5987PubMedCrossRefGoogle Scholar
  52. Xie Q, Guo H (2006) Systemic antiviral silencing in plants. Virus Res 118:1–6PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Katsuyoshi Shimamura
    • 1
  • Shin-ichiro Oka
    • 1
  • Yumi Shimotori
    • 1
  • Takashi Ohmori
    • 2
  • Hiroaki Kodama
    • 2
  1. 1.Graduate School of Science and TechnologyChiba UniversityChiba Japan
  2. 2.Department of Bioproduction Science, Faculty of HorticultureChiba UniversityChiba Japan

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