Plant Biotechnology Reports

The Korean Society of Plant Biotechnology (한국식물생명공학회)
권호 표지
DOI QR코드

DOI QR Code

The epigenetic phenotypes in transgenic Nicotiana benthamiana for CaMV 35S-GFP are mediated by spontaneous transgene silencing

  • Received : 2011.04.17
  • Accepted : 2011.05.03
  • Published : 2011.07.31
Download PDF
⟨ Previous Next ⟩

Abstract

Diverse epigenetic phenotypes are frequently found during research on transgenic plants. To understand the factors underlying such diversity, hundreds of independent 35S-GFP transgenic N. benthamiana plants were analyzed. The diverse GFP-expression phenotypes of the transgenic plants were classified into three major types based on the GFP expression patterns and their response to 35S-GFP agroinfiltration: steady-green, silenced and non-uniform phenotype. The non-uniform phenotype was further sub-divided into five minor phenotypes: variegated, red-dropped, on-silencing, partitioned and misty, according to the distribution of GFP expression on the leaves. Many of transgenic plants continuously generated diverse phenotypes over several generations despite the transgene identity. Such epigenetic GFP phenotyping was found to be the result of spontaneous transgene silencing mediated by either or both of post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). This finding was verified by the detection of 21- and 24-nt small interfering RNA (siRNA) molecules, and DNA methylation in the transgenic plants that showed repeated epigenetic variation. Agroinfiltration demonstrated that irregular distribution of GFP on a leaf was the result of erratic transgene silencing, and the technique also proved to be a rapid and effective method for selecting fully silenced plants within 3 days. Furthermore, two novel phenotypes described are potential materials for in-depth investigations into the genes and mechanisms responsible for spontaneous transgene silencing.

Keywords

References

  1. Bender J (2004) DNA methylation and epigenetics. Annu Rev Plant Biol 55:41-68 https://doi.org/10.1146/annurev.arplant.55.031903.141641
  2. Brigneti G, Voinnet O, Li WX, Ji LH, Ding SW, Baulcombe DC (1998) Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J 17: 6739-6746 https://doi.org/10.1093/emboj/17.22.6739
  3. Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends Genet 22:268-280 https://doi.org/10.1016/j.tig.2006.03.003
  4. Bucher E, Sijen T, de Haan P, Goldbach R, Prins M (2003) Negativestrand tospoviruses and tenuiviruses carry a gene for a suppressor of gene silencing at analogous genomic positions. J Virol 77:1329-1336 https://doi.org/10.1128/JVI.77.2.1329-1336.2003
  5. Cakir C, To¨r M (2010) Factors influencing barley stripe mosaic virusmediated gene silencing in wheat. Physiol Mol Plant Pathol 74:246-253 https://doi.org/10.1016/j.pmpp.2010.04.001
  6. Cheung P, Lau P (2005) Epigenetic regulation by histone methylation and histone variants. Mol Endocrinol 19:563-573 https://doi.org/10.1210/me.2004-0496
  7. Choi MS, Yoon I-S, Rhee Y, Choi SK, Lim S-H, Won S-Y, Lee Y-H, Choi H-S, Lee S-C, Kim K-H, Lomonossoff G, Sohn S-H (2008) The effect of cucumber mosaic virus 2b protein to transient expression and transgene silencing mediated by agro-infiltration. Plant Pathol J 24:296-304 https://doi.org/10.5423/PPJ.2008.24.3.296
  8. Dunoyer P, Himber C, Voinnet O (2005) DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal. Nature Genet 37:1356-1360 https://doi.org/10.1038/ng1675
  9. Eamens A, Wang M-B, Smith NA, Waterhouse PM (2008) RNA silencing in plants: yesterday, today, and tomorrow. Plant Physiol 147:456-468 https://doi.org/10.1104/pp.108.117275
  10. Fagard M, Boutet S, Morel JB, Bellini C, Vaucheret H (2000) AGO1, QDE-2, and RDE-1 are related proteins required for posttranscriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proc Natl Acad Sci USA 97:1650-11654
  11. Fojtova M, van Houdt H, Depicker A, Kovarik A (2003) Epigenetic switch from posttranscriptional to transcriptional silencing is correlated with promoter hypermethylation. Plant Physiol 133:1240-1250 https://doi.org/10.1104/pp.103.023796
  12. Frizzi A, Huang S (2010) Tapping RNA silencing pathways for plant biotechnology. Plant Biotech J 8:655-677 https://doi.org/10.1111/j.1467-7652.2010.00505.x
  13. Goto K, Kanazawa A, Kusaba M, Masuta C (2003) A simple and rapid method to detect plant siRNAs using nonradioactive probes. Plant Mol Biol Rep 21:51-58 https://doi.org/10.1007/BF02773396
  14. Goto K, Kobori T, Kosaka Y, Natsuaki T, Masuta C (2007) Characterization of silencing suppressor 2b of cucumber mosaic virus based on examination of its small RNA-binding abilities. Plant Cell Physiol 48:1050-1060 https://doi.org/10.1093/pcp/pcm074
  15. Harper BK, Stewart CN (2000) Patterns of green fluorescent protein expression in transgenic plants. Plant Mol Biol Rep 18:141a-141i https://doi.org/10.1007/BF02824023
  16. Haseloff J, Siemering KR, Prasher DC (1997) Removal of cryptic intron and sub-cellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94:2122-2127 https://doi.org/10.1073/pnas.94.6.2122
  17. Herr AJ, Molnar A, Jones A, Baulcombe DC (2006) Defective RNA processing enhances RNA silencing and influences flowering of Arabidopsis. Proc Natl Acad Sci USA 103:14994-15001 https://doi.org/10.1073/pnas.0606536103
  18. Jones L, Hamilton AJ, Voinnet O, Thomas CL, Maule AJ, Baulcombe DC (1999) RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing. Plant Cell 11:2291-2301 https://doi.org/10.1105/tpc.11.12.2291
  19. Kalantidis K, Schumacher HT, Alexiadis T, Helm M (2008) RNA silencing movement in plants. Biol Cell 100:13-26 https://doi.org/10.1042/BC20070079
  20. Kanazawa A (2008) RNA silencing manifested as visibly altered phenotypes in plants. Plant Biotech 25:423-435 https://doi.org/10.5511/plantbiotechnology.25.423
  21. Kanazawa A, O'Dell M, Hellens RP (2007) Epigenetic inactivation of chalcone synthase-A transgene transcription in petunia leads to a reversion of the post-transcriptional gene silencing phenotype. Plant Cell Physiol 48:638-647 https://doi.org/10.1093/pcp/pcm028
  22. Li HW, Lucy AP, Guo H-S, Li WX, Ji LH, Wong SM, Ding SW (1999) Strong host resistance targeted against a viral suppressor of the plant gene silencing defense mechanism. EMBO J 18:2683-2691 https://doi.org/10.1093/emboj/18.10.2683
  23. Lucy AP, Guo HS, Li WX (2002) Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus. EMBO J 19:1672-1680
  24. Luo Z, Chen Z (2007) Improperly terminated, unpolyadenylated mRNA of sense transgenes is targeted by RDR6-mediated RNA silencing in Arabidopsis. Plant Cell 19:943-958 https://doi.org/10.1105/tpc.106.045724
  25. Matzke MA, Matzke AJM (1995) How and why do plants inactivate homologous (trans) gene? Plant Physiol 107:679-685 https://doi.org/10.1104/pp.107.3.679
  26. Morel JB, Godon C, Mourrain P, Beclin C, Boutet S, Feuerbach F, Proux F, Vaucheret H (2002) Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in posttranscriptional gene silencing and virus resistance. Plant Cell 14:629-639 https://doi.org/10.1105/tpc.010358
  27. Palauqui JC, Vaucheret H (1998) Transgenes are dispensable for the RNA degradation step of cosuppression. Proc Natl Acad Sci USA 95:9675-9680 https://doi.org/10.1073/pnas.95.16.9675
  28. Robert BH, Joyce F, Nancy H, Jeanne N, Stephen GR, Robert TF (1988) Leaf disc transformation. In: Stanton BG, Robert AS (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp A5/1-9
  29. Ruiz MT, Voinnet O, Baulcombe DC (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10:937-946 https://doi.org/10.1105/tpc.10.6.937
  30. Schubert D, Lechtenberg B, Forsbach A, Gils M, Bahadur S, Schmidt R (2004) Silencing in Arabidopsis T-DNA transformants: the predominant role of gene-specific RNA sensing mechanism versus position effects. Plant Cell 16:2561-2572 https://doi.org/10.1105/tpc.104.024547
  31. Stokes TL, Kunkel BN, Richards EJ (2002) Epigenetic variation in Arabidopsis disease resistance. Genes Dev 16:171-182 https://doi.org/10.1101/gad.952102
  32. Tang W, Newton RJ, Weidner DA (2007) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. J Exp Bot 58:545-554
  33. 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 14:857-867 https://doi.org/10.1105/tpc.010480
  34. Vaucheret H (2005) RNA polymerase IV and transcriptional silencing. Nature Genet 37:659-660 https://doi.org/10.1038/ng0705-659
  35. Voinnet O, Rivas S, Mestre P, Baulcombe DC (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949-956 https://doi.org/10.1046/j.1365-313X.2003.01676.x
  36. Wassenegger M, Krczal G (2006) Nomenclature and functions of RNA-directed RNA polymerases. Trends Plant Sci 11:142-151 https://doi.org/10.1016/j.tplants.2006.01.003
  37. Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:642-652

Cited by

  1. Effect of Rice stripe virus NS3 on Transient Gene Expression and Transgene Co-Silencing vol.27, pp.4, 2011, https://doi.org/10.5423/ppj.2011.27.4.310
  2. Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3 - 9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana vol.39, pp.9, 2012, https://doi.org/10.1071/fp12051
  3. Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants vol.13, pp.None, 2013, https://doi.org/10.1186/1471-2229-13-99
  4. From immunity to susceptibility: virus resistance induced in tomato by a silenced transgene is lost as TGS overcomes PTGS vol.75, pp.6, 2011, https://doi.org/10.1111/tpj.12253
  5. Cell-autonomous-like silencing of GFP-partitioned transgenic Nicotiana benthamiana vol.65, pp.15, 2011, https://doi.org/10.1093/jxb/eru200
  6. 오이모자이크바이러스 2b 유전자 발현 담배의 형태 및 전사체 분석 vol.21, pp.3, 2011, https://doi.org/10.5423/rpd.2015.21.3.186
  7. Elevating seed oil content in a polyploid crop by induced mutations in SEED FATTY ACID REDUCER genes vol.18, pp.11, 2011, https://doi.org/10.1111/pbi.13381
  8. The Capsicum baccatum-Specific Truncated NLR Protein CbCN Enhances the Innate Immunity against Colletotrichum acutatum vol.22, pp.14, 2011, https://doi.org/10.3390/ijms22147672