Abstract
Transgene transcripts were recently shown to accumulate at higher levels in roots, relative to leaves, of silenced-transgenic Nicotiana benthamiana plants and to be inversely related with the accumulation of small interfering RNAs (siRNAs), suggesting that RNA silencing is less active in roots than in leaves (Andika et al., 2005. Mol. Plant–Microbe Interact. 18: 194). Here we show that the lower transgene RNA silencing activity in roots was associated with lower transgene methylation levels at non-symmetrical CpNpN context but not at symmetrical CpG or CpNpG context in three sets of transformant plants with different exogenous genes. In contrast, such a difference between roots and leaves was not observed for the Tnt1 retrotransposon: no Tnt1 transcript was detected in roots or in leaves of N. benthamiana, while equal levels of Tnt1-derived siRNA accumulation and Tnt1 methylation were found. From our data and previously reported information, we suggest that roots have less of an activity that acts at the step of generation of siRNAs.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- BNYVV:
-
Beet necrotic yellow vein virus
- C:
-
cytosine
- DIG:
-
digoxigenin
- dsRNA:
-
double-stranded RNA
- GFP:
-
green fluorescent protein
- LTR:
-
long terminal repeat
- Nos:
-
nopaline synthase
- ORF:
-
open reading frame
- PTGS:
-
post-transcriptional gene silencing
- RdDM:
-
RNA-directed DNA methylation
- RdRP:
-
RNA-dependent RNA polymerase
- RNAi:
-
RNA interfering
- RTD:
-
readthrough domain
- siRNA:
-
small interfering RNA
- VIGS:
-
virus-induced gene silencing
References
I.B. Andika H. Kondo T. Tamada (2005) ArticleTitleEvidence that RNA silencing-mediated resistance to Beet necrotic yellow vein virus is less effective in roots than in leaves Mol. Plant-Microbe Interact. 18 194–204 Occurrence Handle1:CAS:528:DC%2BD2MXhs1eltb4%3D Occurrence Handle15782633
W. Aufsatz M.F. Mette J. Winden Particlevan der A.J. Matzke M. Matzke (2002) ArticleTitleRNA-directed DNA methylation in Arabidopsis Proc. Natl. Acad. Sci. USA 99 16499–16506 Occurrence Handle10.1073/pnas.162371499 Occurrence Handle1:CAS:528:DC%2BD38Xps12htL4%3D Occurrence Handle12169664
B.L. Bass (2000) ArticleTitleDouble-stranded RNA as a template for gene silencing Cell 101 235–238 Occurrence Handle10.1016/S0092-8674(02)71133-1 Occurrence Handle1:CAS:528:DC%2BD3cXjtFSrt7g%3D Occurrence Handle10847677
J. Bender (2001) ArticleTitleA vicious cycle: RNA silencing and DNA methylation in plants Cell 106 129–132 Occurrence Handle10.1016/S0092-8674(01)00441-X Occurrence Handle1:CAS:528:DC%2BD3MXlvVCls7k%3D Occurrence Handle11511339
E. Bernstein A.A. Caudy S.M. Hammond G.J. Hannon (2001) ArticleTitleRole for a bidentate ribonuclease in the initiation step of RNA interference Nature 409 363–366 Occurrence Handle10.1038/35053110 Occurrence Handle1:CAS:528:DC%2BD3MXms12ksA%3D%3D Occurrence Handle11201747
M.E. Byrne C.A. Kidner R.A. Martienssen (2003) ArticleTitlePlant stem cells: divergent pathways and common themes in shoots and roots Curr. Opin. Genet. Dev. 13 551–557 Occurrence Handle10.1016/j.gde.2003.08.008 Occurrence Handle1:CAS:528:DC%2BD3sXnvVOlsr0%3D Occurrence Handle14550423
X. Cao S.E. Jacobsen (2002) ArticleTitleRole of the Arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing Curr. Biol. 12 1138–1144 Occurrence Handle10.1016/S0960-9822(02)00925-9 Occurrence Handle1:CAS:528:DC%2BD38XlsVyjsbo%3D Occurrence Handle12121623
S.N. Covey N.S. Al-Kaff A. Langara D.S. Turner (1997) ArticleTitlePlants combat infection by gene silencing Nature 385 781–782 Occurrence Handle10.1038/385781a0 Occurrence Handle1:CAS:528:DyaK2sXhsFWqsbc%3D
T. Dalmay A. Hamilton S. Rudd S. Angell D.C. Baulcombe (2000) ArticleTitleAn RNA-dependent RNA polymerase gene in Arabidopsis is required for post-transcriptional gene silencing mediated by a transgene but not by a virus Cell 101 543–553 Occurrence Handle10.1016/S0092-8674(00)80864-8 Occurrence Handle1:CAS:528:DC%2BD3cXjvFWntLY%3D Occurrence Handle10850496
C. Wilde ParticleDe N. Podevin P. Windels A. Depicker (2001) ArticleTitleSilencing of antibody genes in plants with single-copy transgene inserts as a result of gene dosage effects Mol. Genet. Genom. 265 647–653
J.J. English G.F. Davenport T. Elmayan H. Vaucheret D.C. Baulcombe (1997) ArticleTitleRequirement of sense transcription for homology-dependent virus and trans-inactivation Plant J. 12 597–603 Occurrence Handle1:CAS:528:DyaK2sXntVCktb0%3D
M. Fagard S. Boutet J.B. Morel C. Bellini H. Vaucheret (2000) ArticleTitleAGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals Proc. Natl. Acad. Sci. USA 97 11650–11654 Occurrence Handle10.1073/pnas.200217597 Occurrence Handle1:CAS:528:DC%2BD3cXnsF2qtr0%3D Occurrence Handle11016954
M.A. Grandbastien A. Spielmann M. Caboche (1989) ArticleTitleTnt1, a mobile retroviral-like transposable element of tobacco isolated by plant cell genetics Nature 337 376–380 Occurrence Handle10.1038/337376a0 Occurrence Handle1:CAS:528:DyaL1MXhs1OisbY%3D Occurrence Handle2536143
A. Grishok A.E. Pasquinelli D. Conte N. Li S. Parrish I. Ha D.L. Baillie A. Fire G. Ruvkun C.C. Mello (2001) ArticleTitleGenes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing Cell 106 23–34 Occurrence Handle10.1016/S0092-8674(01)00431-7 Occurrence Handle1:CAS:528:DC%2BD3MXlsVCqtrw%3D Occurrence Handle11461699
A.J. Hamilton D.C. Baulcombe (1999) ArticleTitleA species of small antisense RNA in post-transcriptional gene silencing in plants Science 286 950–952 Occurrence Handle10.1126/science.286.5441.950 Occurrence Handle1:CAS:528:DyaK1MXntFaktbY%3D Occurrence Handle10542148
A. Hamilton O. Voinnet L. Chappell D.C. Baulcombe (2002) ArticleTitleTwo classes of short interfering RNA in RNA silencing EMBO J. 21 4671–4679 Occurrence Handle10.1093/emboj/cdf464 Occurrence Handle1:CAS:528:DC%2BD38Xms1KmsrY%3D Occurrence Handle12198169
S.M. Hammond E. Bernstein D. Beach G.J. Hannon (2000) ArticleTitleAn RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells Nature 404 293–296 Occurrence Handle1:CAS:528:DC%2BD3cXit1agtrw%3D Occurrence Handle10749213
G.J. Hannon (2002) ArticleTitleRNA interference Nature 418 244–251 Occurrence Handle10.1038/418244a Occurrence Handle1:CAS:528:DC%2BD38XltFGmu7Y%3D Occurrence Handle12110901
A.J. Herr M.B. Jensen T. Dalmay D.C. Baulcombe (2005) ArticleTitleRNA polymerase IV directs silencing of endogenous DNA Science 308 118–120 Occurrence Handle10.1126/science.1106910 Occurrence Handle1:CAS:528:DC%2BD2MXivVaqsrw%3D Occurrence Handle15692015
R.F. Ketting T.H. Haverkamp H.G. Luenen Particlevan R.H Plasterk (1999) ArticleTitlemut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD Cell 99 133–141 Occurrence Handle10.1016/S0092-8674(00)81645-1 Occurrence Handle1:CAS:528:DyaK1MXmvFCls7c%3D Occurrence Handle10535732
R.F. Ketting S.E. Fischer E. Bernstein T. Sijen G.J. Hannon R.H. Plasterk (2001) ArticleTitleDicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans Genes Dev. 15 2654–2659 Occurrence Handle10.1101/gad.927801 Occurrence Handle1:CAS:528:DC%2BD3MXnvVWksLY%3D Occurrence Handle11641272
H. Kumagai H. Kouchi (2003) ArticleTitleGene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules Mol. Plant–Microbe Interact. 16 663–668 Occurrence Handle1:CAS:528:DC%2BD3sXlvF2itrc%3D Occurrence Handle12906110
E. Limpens J. Ramos C. Franken V. Raz B. Compaan H. Franssen T. Bisseling R. Geurts (2004) ArticleTitleRNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula J. Exp. Bot. 55 983–992 Occurrence Handle10.1093/jxb/erh122 Occurrence Handle1:CAS:528:DC%2BD2cXjs1Wrur0%3D Occurrence Handle15073217
J.A. Lindbo L. Silva-Rosales W.M. Proebsting W.G. Dougherty (1993) ArticleTitleInduction of highly specific antiviral state in transgenic plants: implication of regulation of gene expression and virus resistance Plant Cell 5 1749–1759 Occurrence Handle10.1105/tpc.5.12.1749 Occurrence Handle1:CAS:528:DyaK2cXisVSgurg%3D Occurrence Handle12271055
O. Mathieu J. Bender (2004) ArticleTitleRNA-directed DNA methylation J. Cell Sci. 117 4881–4888 Occurrence Handle10.1242/jcs.01479 Occurrence Handle1:CAS:528:DC%2BD2cXhtVSns7rN Occurrence Handle15456843
A. Miura S. Yonebayashi K. Watanabe T. Toyama H. Shimada T. Kakutani (2001) ArticleTitleMobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis Nature 411 212–214 Occurrence Handle10.1038/35075612 Occurrence Handle1:CAS:528:DC%2BD3MXjslGlurg%3D Occurrence Handle11346800
J.B. Morel P. Mourrain C. Beclin H. Vaucheret (2000) ArticleTitleDNA methylation and chromatin structure affect transcriptional and post-transcriptional transgene silencing in Arabidopsis Curr. Biol. 10 1591–1594 Occurrence Handle10.1016/S0960-9822(00)00862-9 Occurrence Handle1:CAS:528:DC%2BD3cXovFKgur8%3D Occurrence Handle11137011
P. Mourrain C. Béclin T. Elmayan F. Feuerbach C. Godon J.B. Morel D. Jouette A.M. Lacombe S. Nikic N. Picault K. Remoue M. Sanial T.A. Vo H. Vaucheret (2000) ArticleTitleArabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance Cell 101 533–542 Occurrence Handle10.1016/S0092-8674(00)80863-6 Occurrence Handle1:CAS:528:DC%2BD3cXjvFWntLk%3D Occurrence Handle10850495
C. Napoli C. Lemieux R. Jorgensen (1990) ArticleTitleIntroduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans Plant Cell 2 279–289 Occurrence Handle10.1105/tpc.2.4.279 Occurrence Handle1:CAS:528:DyaK3cXkvFalu7c%3D Occurrence Handle12354959
A. Nykänen B. Haley P.D. Zamore (2001) ArticleTitleATP requirements and small interfering RNA structure in the RNA interference pathway Cell 107 309–321 Occurrence Handle11701122
J.C. Palauqui T. Elmayan J.M. Pollien H. Vaucheret (1997) ArticleTitleSystemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions EMBO J. 16 4738–4745 Occurrence Handle10.1093/emboj/16.15.4738 Occurrence Handle1:CAS:528:DyaK2sXlsVClsbk%3D Occurrence Handle9303318
R. Paulin G.W. Grigg M.W. Davey A.A. Piper (1998) ArticleTitleUrea improves efficiency of bisulphite-mediated sequencing of 5′-methylcytosine in genomic DNA Nucl. Acids Res. 26 5009–5010 Occurrence Handle1:CAS:528:DyaK1cXnsVKgur0%3D Occurrence Handle9776768
F. Ratcliff B.D. Harrison D.C. Baulcombe (1997) ArticleTitleA similarity between viral defense and gene silencing in plants Science 276 1558–1560 Occurrence Handle10.1126/science.276.5318.1558 Occurrence Handle1:CAS:528:DyaK2sXjslGktr8%3D
M.T. Ruiz O. Voinnet D.C. Baulcombe (1998) ArticleTitleInitiation and maintenance of virus-induced gene silencing Plant Cell 10 937–946 Occurrence Handle10.1105/tpc.10.6.937 Occurrence Handle1:CAS:528:DyaK1cXktl2hu7s%3D Occurrence Handle9634582
G. Tang B.J. Reinhart D.P. Bartel P.D. Zamore (2003) ArticleTitleA biochemical framework for RNA silencing in plants Genes Dev. 17 49–63 Occurrence Handle10.1101/gad.1048103 Occurrence Handle1:CAS:528:DC%2BD3sXktlKrug%3D%3D Occurrence Handle12514099
R. Tomita T. Hamada G. Horiguchi K. Iba H. Kodama (2004) ArticleTitleTransgene overexpression with cognate small interfering RNA in tobacco FEBS Lett. 573 117–120 Occurrence Handle10.1016/j.febslet.2004.07.063 Occurrence Handle1:CAS:528:DC%2BD2cXmvFyrtrs%3D Occurrence Handle15327985
Topping, J.F. 1998. Tobacco transformation. In: G.D. Foster (Ed.), Methods in Molecular Biology, Vol. 20: Plant Virology. Humana Press, Totowa, NJ, pp. 365–372
V. Vance H. Vaucheret (2001) ArticleTitleRNA silencing in plants – defense and counterdefense Science 292 2277–2280 Occurrence Handle10.1126/science.1061334 Occurrence Handle1:CAS:528:DC%2BD3MXks1yrtr8%3D Occurrence Handle11423650
O. Voinnet D.C. Baulcombe (1997) ArticleTitleSystemic signalling in gene silencing Nature 389 553 Occurrence Handle10.1038/39215 Occurrence Handle1:CAS:528:DyaK2sXmslWgtr8%3D Occurrence Handle9335491
O. Voinnet P. Vain S. Angell D.C. Baulcombe (1998) ArticleTitleSystemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA Cell 95 177–187 Occurrence Handle10.1016/S0092-8674(00)81749-3 Occurrence Handle1:CAS:528:DyaK1cXntVSmu7o%3D Occurrence Handle9790525
P.M. Waterhouse M.W. Graham M.B. Wang (1998) ArticleTitleVirus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA Proc. Natl. Acad. Sci. USA 95 13959–13964 Occurrence Handle10.1073/pnas.95.23.13959 Occurrence Handle1:CAS:528:DyaK1cXnsVGhurk%3D Occurrence Handle9811908
D. Wu-Scharf B. Jeong C. Zhang H. Cerutti (2000) ArticleTitleTransgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase Science 290 1159–1162 Occurrence Handle10.1126/science.290.5494.1159 Occurrence Handle1:CAS:528:DC%2BD3cXotVChsbg%3D Occurrence Handle11073454
Z. Xie L.K. Johansen A.M. Gustafson K.D. Kasschau A.D. Lellis D. Zilberman S.E. Jacobsen J.C. Carrington (2004) ArticleTitleRelated genetic and functional diversification of small RNA pathways in plants PLoS Biol. 2 1–11 Occurrence Handle10.1371/journal.pbio.0020104
Y. Yoshioka S. Matsumoto S. Kojima K. Ohshima N. Okada Y. Machida (1993) ArticleTitleMolecular characterization of a short interspersed repetitive element from tobacco that exhibits sequence homology to specific tRNAs Proc. Natl. Acad. Sci. USA 90 6562–6566 Occurrence Handle1:CAS:528:DyaK3sXlsFeitb4%3D Occurrence Handle8341669
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Andika, I.B., Kondo, H., Rahim, M.D. et al. Lower Levels of Transgene Silencing in Roots is Associated with Reduced DNA Methylation Levels at Non-Symmetrical Sites but not at Symmetrical Sites. Plant Mol Biol 60, 423–435 (2006). https://doi.org/10.1007/s11103-005-4429-7
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11103-005-4429-7