Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3–9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana
Gi-Ho Lee A , Seong-Han Sohn B , Eun-Young Park A and Young-Doo Park A C
+ Author Affiliations
- Author Affiliations
A Department of Horticultural Biotechnology, Kyung Hee University, Seocheon-dong, Giheung-gu, Yongin-si, Gyeonggi-do, 446-701, Korea.
B National Academy of Agricultural Science, Rural Development Administration, Suwon-si, Gyeonggi-do, 441-100, Korea.
C Corresponding author. Email: ydpark@khu.ac.kr
Functional Plant Biology 39(9) 764-773 https://doi.org/10.1071/FP12051
Submitted: 16 February 2012 Accepted: 21 July 2012 Published: 20 August 2012
Abstract
The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host’s defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3–9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.
Additional keywords: molecular genetics, transcription, transgenic plants.
References
Ahmed I, Sarazin A, Bowler C, Colot V, Quesneville H (2011) Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis. Nucleic Acids Research 39, 6919–6931.| Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtFehsbfE&md5=adb253aa27df9ffe24409509bd376a02CAS |
Brookes G, Barfoot P (2009) ‘GM crops: global socioeconomic and environmental impact 1996–2007.’ (PG Economics Ltd: Dochester, UK)
Cao X, Aufsatz W, Zilberman D, Mette MF, Huang MS, Matzke M, Jacobsen SE (2003) Role of the DRM and CMT3 methyltransferases in RNA directed DNA methylation. Current Biology 13, 2212–2217.
| Role of the DRM and CMT3 methyltransferases in RNA directed DNA methylation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXhtVSjsrnM&md5=cb719e1098270edc491132cb7030cb01CAS |
Chen ZJ, Pikaard CS (1997) Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance. Genes & Development 11, 2124–2136.
| Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXls1Gqu78%3D&md5=ec069896a4c50b897ac33d574ad37182CAS |
Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, Pradhan S, Nelson SF, Pellegrini M, Jacobsen SE (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219.
| Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXjt1GnurY%3D&md5=171472bc3f9e953937a7ecc9e3f4bb61CAS |
Daxinger L, Hunter B, Sheikh M, Jauvion V, Gasciolli V, Vaucheret H, Matzke M, Furner I (2008) Unexpected silencing effects from T-DNA tags in Arabidopsis. Trends in Plant Science 13, 4–6.
| Unexpected silencing effects from T-DNA tags in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXlsFaisw%3D%3D&md5=27dec730e3f98c4395f26e9c79b2d61eCAS |
De Buck S, Peck I, De Wilde C, Marjanac G, Nolf J, De Paepe A, Depicker A (2007) Generation of single-copy T-DNA transformants in Arabidopsis by the CRE/loxP recombination-mediated resolution system. Plant Physiology 145, 1171–1182.
| Generation of single-copy T-DNA transformants in Arabidopsis by the CRE/loxP recombination-mediated resolution system.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhsVCntLrI&md5=4c58a3fc45e9c979d960dd3b66c0af99CAS |
Dietz-Pfeilstetter A (2010) Stability of transgene expression as a challenge for genetic engineering. Plant Science 179, 164–167.
| Stability of transgene expression as a challenge for genetic engineering.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXosVyqu7g%3D&md5=f1b99617d78722400724a6e2be8c8305CAS |
Ding L, Li S, Gao J, Wang Y, Yang G, He G (2009) Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat. Molecular Biology Reports 36, 29–36.
| Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtl2mu7vJ&md5=64e336cf7f3b0c37ca1262f9795ed4aeCAS |
Eamens A, Wang MB, Smith NA, Waterhouse PM (2008) RNA silencing in plants: yesterday, today, and tomorrow. Plant Physiology 147, 456–468.
| RNA silencing in plants: yesterday, today, and tomorrow.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXnsVyhsb8%3D&md5=8b75ae2027803d293877d15babeaf30eCAS |
Fan J, Liu X, Xu SX, Xu Q, Guo WW (2011) T-DNA direct repeat and 35S promoter methylation affect transgene expression but do not cause silencing in transgenic sweet orange. Plant Cell, Tissue and Organ Culture 107, 225–232.
| T-DNA direct repeat and 35S promoter methylation affect transgene expression but do not cause silencing in transgenic sweet orange.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXht12qs7%2FK&md5=99c31005f771d30f710029dd330a0878CAS |
Hetzl J, Foerster AM, Raidl G, Mittelsten Scheid O (2007) CyMATE: a new tool for methylation analysis of plant genomic DNA after bisulphite sequencing. The Plant Journal 51, 526–536.
| CyMATE: a new tool for methylation analysis of plant genomic DNA after bisulphite sequencing.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXpsVKju7c%3D&md5=9ae40612cd7747f904c88c89408fcc79CAS |
Hsieh TF, Ibarra CA, Silva P, Zemach A, Eshed-Williams L, Fischer RL, Zilberman D (2009) Genome-wide demethylation of Arabidopsis endosperm. Science 324, 1451–1454.
| Genome-wide demethylation of Arabidopsis endosperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXntVSiu7g%3D&md5=835f8cfc459b64477d87f4776268815aCAS |
International Service for the Acquisition of Agric-biotech Applications (2011) ISAAA Briefs No. 43 – preview: global status of commercialized biotech/GM crops: 2011. Available at http://www.isaaa.org [Verified 16 February 2012]
Jaenisch R, Bird A (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nature Genetics 33, 245–254.
| Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXhsV2kt7s%3D&md5=a0b210806e987efc7586f25cd56619a7CAS |
Johnson LM, Law JA, Khattar A, Henderson IR, Jacobsen SE (2008) SRA-domain proteins required for DRM2-mediated de novo DNA methylation. PLOS Genetics 4, e1000280
| SRA-domain proteins required for DRM2-mediated de novo DNA methylation.Crossref | GoogleScholarGoogle Scholar |
Liu C, Lu F, Cui X, Cao X (2010) Histone methylation in higher plants. Annual Review of Plant Biology 61, 395–420.
| Histone methylation in higher plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXnslSjsLk%3D&md5=5abe475600b59c01e3116fec94eadcc7CAS |
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T))method. Methods 25, 402–408.
| Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T))method.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XhtFelt7s%3D&md5=86e5fd6e2b10f00206b12a43a671f6c8CAS |
Matzke MA, Mette MF, Aufsatz W, Jakowitsch J, Matzke AJ (1999) Host defenses to parasitic sequences and the evolution of epigenetic control mechanisms. Genetica 107, 271–287.
| Host defenses to parasitic sequences and the evolution of epigenetic control mechanisms.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXmtVChs74%3D&md5=f0412e0035f1d3843cd99e7a43f331d2CAS |
Matzke M, Kanno T, Huettel B, Daxinger L, Matzke AJ (2007) Targets of RNA-directed DNA methylation. Current Opinion in Plant Biology 10, 512–519.
| Targets of RNA-directed DNA methylation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtFekurjL&md5=9ff17a41153b67e4320be37d8991b644CAS |
McCouch SR, Kochert G, Yu ZH, Wang ZY, Khush GS, Coffman WR, Tanksley SD (1988) Molecular mapping of rice chromosomes. Theoretical and Applied Genetics 76, 815–829.
| Molecular mapping of rice chromosomes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1MXhsVyqu7w%3D&md5=4ab25fa4d53a6a0dfb27e348a3a411c1CAS |
McDonald JF, Matzke MA, Matzke AJ (2005) Host defenses to transposable elements and the evolution of genomic imprinting. Cytogenetic and Genome Research 110, 242–249.
| Host defenses to transposable elements and the evolution of genomic imprinting.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2Mvisleltg%3D%3D&md5=386bd6706f4c3063d4d8307e53bd77cbCAS |
Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJ (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO Journal 19, 5194–5201.
| Transcriptional silencing and promoter methylation triggered by double-stranded RNA.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXnvF2qtLk%3D&md5=35b7d2029d1477014a2bb919417b3e8aCAS |
Meyer P, Linn F, Heidmann I, Meyer H, Niedenhof I, Saedler H (1992) Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype. Molecular Genetics and Genomics 231, 345–352.
| Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK38XhvVyrtr4%3D&md5=411f6dfc6d67a2dc95ed2c066f9d1538CAS |
Mishiba K, Nishihara M, Nakatsuka T, Abe Y, Hirano H, Yokoi T, Kikuchi A, Yamamura S (2005) Consistent transcriptional silencing of 35S-driven transgenes in gentian. The Plant Journal 44, 541–556.
| Consistent transcriptional silencing of 35S-driven transgenes in gentian.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXht1Grtr7O&md5=73f32779adbd8d4c82306f08799d0af6CAS |
Park YD, Papp I, Moscone EA, Iglesias VA, Vaucheret H, Matzke AJ, Matzke MA (1996) Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity. The Plant Journal 9, 183–194.
| Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28XitVyjur0%3D&md5=d12dfd5508b6968fc70e55ec8cc4a610CAS |
Roa-Rodrigues C (2003) ‘Promoters used to regulate gene expression.’ (CAMBIA Intellectual Property: Canberra)
Rojas CA, Hemerly AS, Ferreira PCG (2010) Genetically modified crops for biomass increase genes and strategies. GM Crops 1, 137–142.
| Genetically modified crops for biomass increase genes and strategies.Crossref | GoogleScholarGoogle Scholar |
Sambrook J, Russell DW (2001) ‘Molecular cloning: a laboratory manual.’ (3rd edn) (Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY)
Sohn SH, Choi MS, Kim KH, Lomonossoff G (2011) The epigenetic phenotypes in transgenic Nicotiana benthamiana for CaMV 35S-GFP are mediated by spontaneous transgene silencing. Plant Biotechnology Reports 5, 273–281.
| The epigenetic phenotypes in transgenic Nicotiana benthamiana for CaMV 35S-GFP are mediated by spontaneous transgene silencing.Crossref | GoogleScholarGoogle Scholar |
Stam M, Mol JNM, Kooter JM (1997) The silence of genes in transgenic plants. Annals of Botany 79, 3–12.
| The silence of genes in transgenic plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXhtFGjt7w%3D&md5=64b4d8fe8cd3d05882c250a789be7ccbCAS |
Tang W, Newton RJ, Weidner DA (2007) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. Journal of Experimental Botany 58, 545–554.
| Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhvFWlsLc%3D&md5=fa2db010c56dfb11497479cd82edc239CAS |
Tian L, Chen ZJ (2001) Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development. Proceedings of the National Academy of Sciences of the United States of America 98, 200–205.
| Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXkslOjtQ%3D%3D&md5=6409413be2100e5984c33733fe73d6d5CAS |
Ülker B, Allen GC, Thompson WF, Spiker S, Weissinger AK (1999) A tobacco MAR increases transgene expression and protects against gene silencing in the progeny of transgenic tobacco plants. The Plant Journal 18, 253–263.
Vaucheret H, Fagard M (2001) Transcriptional gene silencing in plants: targets, inducers and regulators. Trends in Plant Science 17, 29–35.
Widman N, Jacobsen SE, Pellegrini M (2009) Determining the conservation of DNA methylation in Arabidopsis. Epigenetics 4, 119–124.
| Determining the conservation of DNA methylation in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXptlWms7o%3D&md5=0eb2a6e36da3add9a327c60704f87851CAS |
Yao Q, Cong L, Chang JL, Li KX, Yang GX, He GY (2006) Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment. Journal of Experimental Botany 57, 3737–3746.
| Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtlCnsLnE&md5=bf3fcdb26518e8c5bae48ff8e85000a3CAS |
