Abstract
In plants, RNA interference (RNAi)-induced gene silencing can spread from the initiation site to nearby cells. The silencing signal moves from cell-to-cell through plasmodesmata and, over long distances, through the phloem. In this study, we employed a nuclear-localized GFP fusion protein to visualize the pattern of gene silencing induced by three different transgenes expressing double-stranded RNA (dsRNA) in Arabidopsis root tips. In all cases, we found that dsRNA-induced silencing did not spread from the silencing initiation site to adjacent cells. In the first set of experiments, in a transgenic background expressing nuclear-localized GFP within a contiguous cell layer that included endodermis, cortex/endodermis (joint) initial (CEI) cells and the quiescent center (QC) cells, expression of the marker gene was silenced specifically in the QC cells without affecting gene expression in the adjacent CEI and endodermal cells. The next two sets of experiments examined the knockdown of two endogenous genes. We observed that silencing was completely restricted to the QC and endodermal cells within which the dsRNA transgenes were expressed. Overall, these results accentuate one important aspect of RNAi-induced gene silencing, that it can be cell autonomous, and demonstrated the feasibility of selective gene knockdown within specific cell types.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Birnbaum K, Shasha DE, Wang JY, Jung JW, Lambert GM, Galbraith DW, Benfey PN (2003) A gene expression map of the Arabidopsis root. Science 302(5652):1956–1960. doi:10.1126/science.1090022
Brosnan CA, Voinnet O (2011) Cell-to-cell and long-distance siRNA movement in plants: mechanisms and biological implications. Curr Opin Plant Biol. doi:10.1016/j.pbi.2011.07.011
Byzova M, Verduyn C, De Brouwer D, De Block M (2004) Transforming petals into sepaloid organs in Arabidopsis and oilseed rape: implementation of the hairpin RNA-mediated gene silencing technology in an organ-specific manner. Planta 218(3):379–387. doi:10.1007/s00425-003-1117-1
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743
Di Laurenzio L, Wysocka-Diller J, Malamy JE, Pysh L, Helariutta Y, Freshour G, Hahn MG, Feldmann KA, Benfey PN (1996) The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86(3):423–433
Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K, Scheres B (1993) Cellular-organization of the Arabidopsis-thaliana root. Development 119(1):71–84
Dunoyer P, Schott G, Himber C, Meyer D, Takeda A, Carrington JC, Voinnet O (2010) Small RNA duplexes function as mobile silencing signals between plant cells. Science 328(5980):912–916. doi:10.1126/science.1185880
Finnegan EJ, Peacock WJ, Dennis ES (1996) Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc Natl Acad Sci USA 93(16):8449–8454
Haecker A, Gross-Hardt R, Geiges B, Sarkar A, Breuninger H, Herrmann M, Laux T (2004) Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana. Development 131(3):657–668. doi:10.1242/dev.00963
Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286(5441):950–952
Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404(6775):293–296. doi:10.1038/35005107
Heidstra R, Welch D, Scheres B (2004) Mosaic analyses using marked activation and deletion clones dissect Arabidopsis SCARECROW action in asymmetric cell division. Gene Dev 18(16):1964–1969. doi:10.1101/Gad.305504
Helliwell CA, Waterhouse PM (2005) Constructs and methods for hairpin RNA-mediated gene silencing in plants. Method Enzymol 392:24–35
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(17):4523–4533
Islam S, Miyazaki T, Tanno F, Itoh K (2005) Dissection of gene function by RNA silencing. Plant Biotechnol 22:4
Limpens E, Ramos J, Franken C, Raz V, Compaan B, Franssen H, Bisseling T, Geurts R (2004) RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula. J Exp Bot 55(399):983–992. doi:10.1093/Jxb/Erh122
Malamy JE, Benfey PN (1997) Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124(1):33–44
McGinnis K, Chandler V, Cone K, Kaeppler H, Kaeppler S, Kerschen A, Pikaard C, Richards E, Sidorenko L, Smith T, Springer N, Wulan T (2005) Transgene-induced RNA interference as a tool for plant functional genomics. Method Enzymol 392:1–24
Morel JB, Mourrain P, Beclin C, Vaucheret H (2000) DNA methylation and chromatin structure affect transcriptional and post-transcriptional transgene silencing in Arabidopsis. Curr Biol 10(24):1591–1594
Palauqui JC, Elmayan T, Pollien JM, Vaucheret H (1997) Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J 16(15):4738–4745
Ronemus MJ, Galbiati M, Ticknor C, Chen JC, Dellaporta SL (1996) Demethylation-induced developmental pleiotropy in Arabidopsis. Science 273(5275):654–657
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn, Cold Spring Harbor Laboratory Press
Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD, Rathore KS (2006) Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc Natl Acad Sci USA 103(48):18054–18059. doi:10.1073/pnas.0605389103
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(2):177–187
Waterhouse PM, Helliwell CA (2003) Exploring plant genomes by RNA-induced gene silencing. Nat Rev Genet 4(1):29–38. doi:10.1038/Nrg982
Wysocka-Diller JW, Helariutta Y, Fukaki H, Malamy JE, Benfey PN (2000) Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot. Development 127(3):595–603
Yoo BC, Kragler F, Varkonyi-Gasic E, Haywood V, Archer-Evans S, Lee YM, Lough TJ, Lucas WJ (2004) A systemic small RNA signaling system in plants. Plant Cell 16(8):1979–2000. doi:10.1105/tpc.104.023614
Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101(1):25–33
Zhang C, Gong FC, Lambert GM, Galbraith DW (2005) Cell type-specific characterization of nuclear DNA contents within complex tissues and organs. Plant Methods 1(1):7. doi:10.1186/1746-4811-1-7
Zhang C, Barthelson RA, Lambert GM, Galbraith DW (2008) Global characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiol 147(1):30–40. doi:10.1104/pp.107.115246
Acknowledgments
The authors thank Georgina Lambert and Rangasamy Elumalai for critical discussions of the results and for many helpful suggestions. This work was part of the Ph.D. training program of C.Q.Z., and was made possible by support from the Galbraith Laboratory, the School of Plant Sciences, and by grant DBI 0211857 to D.W.G. from the National Science Foundation.
Author information
Authors and Affiliations
Corresponding author
Additional information
GenBank accession numbers: DQ231581, DQ231580.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhang, C., Galbraith, D.W. RNA interference-mediated gene knockdown within specific cell types. Plant Mol Biol 80, 169–176 (2012). https://doi.org/10.1007/s11103-012-9937-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11103-012-9937-7