Plant Molecular Biology

, Volume 43, Issue 1, pp 67–82 | Cite as

High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation

  • Ming-Bo Wang
  • Peter M. Waterhouse


Two transgenic callus lines of rice, stably expressing a β-glucuronidase (GUS) gene, were supertransformed with a set of constructs designed to silence the resident GUS gene. An inverted-repeat (i/r) GUS construct, designed to produce mRNA with self-complementarity, was much more effective than simple sense and antisense constructs at inducing silencing. Supertransforming rice calluses with a direct-repeat (d/r) construct, although not as effective as those with the i/r construct, was also substantially more effective in silencing the resident GUS gene than the simple sense and antisense constructs. DNA hybridisation analyses revealed that every callus line supertransformed with either simple sense or antisense constructs, and subsequently showing GUS silencing, had the silence-inducing transgenes integrated into the plant genome in inverted-repeat configurations. The silenced lines containing i/r and d/r constructs did not necessarily have inverted-repeat T-DNA insertions. There was significant methylation of the GUS sequences in most of the silenced lines but not in the unsilenced lines. However, demethylation treatment of silenced lines with 5-azacytidine did not reverse the post-transcriptional gene silencing (PTGS) of GUS. Whereas the levels of RNA specific to the resident GUS gene were uniformly low in the silenced lines, RNA specific to the inducer transgenes accumulated to a substantial level, and the majority of the i/r RNA was unpolyadenylated. Altogether, these results suggest that both sense- and antisense-mediated gene suppression share a similar molecular basis, that unpolyadenylated RNA plays an important role in PTGS, and that methylation is not essential for PTGS.

antisense direct repeat dsRNA gene silencing GUS inverted repeat 


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  1. Baulcombe, D.C. and English, J.J. 1996. Ectopic pairing of homologous DNA and post-transcriptional gene silencing in transgenic plants. Curr. Opin. Biotechnol. 7: 173–180.Google Scholar
  2. Bolllmann, J., Carpenter, R. and Coen, E.S. 1991. Allelic interactions at the nivea locus of Antirrhinum. Plant Cell 3: 1327–1336.Google Scholar
  3. Carter, M.S., Li, S. and Wilkinson, M.F. 1996. A splicing-dependent regulatory mechanism that detects translation signals. EMBO J. 15: 5965–5975.Google Scholar
  4. Christensen, A.H. and Quail, P.H. 1996. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5: 213–218.Google Scholar
  5. Cock, J.M., Swarup, R. and Dumas, C. 1997. Natural antisense transcripts of the S locus receptor kinase gene and related sequences in Brassica oleracea. Mol. Gen. Genet. 255: 514–524.Google Scholar
  6. Cogoni, C. and Macino, G. 1999. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399: 166–169.Google Scholar
  7. Cornelissen, M. 1989. Nuclear and cytoplasmic sites for anti-sense control. Nucl. Acids Res. 17: 7203–7209.Google Scholar
  8. Covey, S.N., Al-Kaff, N.S., Lángara, A. and Turner, D.S. 1997. Plants combat infection by gene silencing. Nature 385: 781–782.Google Scholar
  9. de Vries, S., Hoge, H. and Bisseling, T. 1988. Isolation of total and polysomal RNA from plant tissues. In: S.B. Gelvin, R.A. Schilperoort and D.P.S. Verma (Eds.), Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht, Netherlands, pp. B6/1–B6/13.Google Scholar
  10. Ditta, G., Stanfield, S., Corbin, D. and Helinski, D.R. 1980. Broad host-range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77: 7347–7351.Google Scholar
  11. Dougherty, W.G. and Parks, T.D. 1995. Transgenes and gene suppression: telling us something new? Curr. Opin. Cell Biol. 7: 399–405.Google Scholar
  12. Draper, J. and Scott, R. 1988. The isolation of plant nucleic acids. In: J. Draper, R. Scott, P. Armitage and R. Walden (Eds.), Plant Genetic Transformation and Gene Expression: A Laboratory Manual, Alden Press, Oxford, pp. 199–236.Google Scholar
  13. English, J.J., Mueller, E. and Baulcombe, D.C. 1996. Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell 8: 179–188.Google Scholar
  14. Gough, K.H. and Shukla, D.D. 1993. Nucleotide sequence of Johnsongrass mosaic potyvirus genomic RNA. Intervirology 36: 181–192.Google Scholar
  15. Hamilton, A.J., Brown, S., Han, Y., Ishizuka, M., Lowe, A., Alpuche Solis, A.G. and Grierson, D. 1998. A transgene with repeated DNA causes high frequency, post-transcriptional suppression of ACC-oxidase gene expression in tomato. Plant J. 15: 737–746.Google Scholar
  16. Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901–3907.Google Scholar
  17. Jones, A.L., Thomas, C.L. and Maule, A.J. 1998. De novo methylation and co-suppression induced by a cytoplasmically replicating RNA virus. EMBO J. 17: 6385–6393.Google Scholar
  18. Jorgensen, R.A., Cluster, P.D., English, J., Que, Q. and Napoli, C.A. 1996. Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and singlecopy vs. complex T-DNA sequences. Plant Mol. Biol. 31: 957–973.Google Scholar
  19. Koch, K.S. and Leffert, H.L. 1998. Giant hairpins formed by CUG repeats in myotonic messenger RNAs might sterically block RNA export through nuclear pores. J. Theor. Biol. 192: 505–514.Google Scholar
  20. Kumar, M. and Carmichael, G.G. 1997. Nuclear antisense RNA induces extensive adenosine modifications and nuclear retention of target transcripts. Proc. Natl. Acad. Sci. USA 94: 3542–3547.Google Scholar
  21. Kumar, M. and Carmichael, G.G. 1998. Antisense RNA: function and fate of duplex RNA in cells of higher eukaryotes. Microbiol. Mol. Biol. Rev. 62: 1415–1434.Google Scholar
  22. Lee, K.Y., Baden, C., Howie, W.J., Bedbrook, J. and Dunsmuir, P. 1997. Post-transcriptional gene silencing of ACC synthase in tomato results from cytoplasmic RNA degradation. Plant J. 12: 1127–1137.Google Scholar
  23. Liu, Z., Batt, D.B. and Carmichael, G.G. 1994. Targeted nuclear antisense RNA mimics natural antisense-induced degradation of polyoma virus early RNA. Proc. Natl. Acad. Sci. USA 91: 4258–4262.Google Scholar
  24. Luff, B., Pawlowski, L. and Bender, J. 1999. An inverted repeat triggers cytosine methylation of identical sequences in Arabidopsis. Mol. Cell 3: 505–511.Google Scholar
  25. Mette, M.F., van der Winden, J., Matzke, M.A. and Matzke, A.J.M. 1999. Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans. EMBO J. 18: 241–248.Google Scholar
  26. Metzlaff, M., O'Dell, M., Cluster, P.D. and Flavell, R.B. 1997. RNA-mediated RNA degradation and chalcone synthase A silencing in Petunia. Cell 88: 845–854.Google Scholar
  27. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.Google Scholar
  28. Que, Q., Wang, H.Y., English, J.J. and Jorgensen, R.A. 1997. The frequency and degree of cosuppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence. Plant Cell 9: 1357–1368.Google Scholar
  29. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  30. Selker, E.U. 1999. Gene silencing: repeats that count. Cell 97: 157–160.Google Scholar
  31. Sijen, T., Wellink, J., Hiriart, J.B. and van Kammen, A. 1996. RNA-mediated virus resistance: role of repeated transgenes and delineation of targeted regions. Plant Cell 8: 2277–2294.Google Scholar
  32. Stam, M., de Bruin, R., Kenter, S., van der Hoorn, R.A.L., van Blokland, R., Mol, J.N.M. and Kooter, J.M. 1997a. Posttranscriptional silencing of chalcone synthase in Petunia by inverted transgene repeats. Plant J. 12: 63–82.Google Scholar
  33. Stam, M., Mol, J.N.M. and Kooter, J.M. 1997b. The silence of genes in transgenic plants. Ann. Bot. 79: 3–12.Google Scholar
  34. Stam, M., Viterbo, A., Mol, J.N.M. and Kooter, J.M. 1998. Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants. Mol. Cell. Biol. 18: 6165–6177.Google Scholar
  35. van Eldik, G.J., Litiè re, K., Jacobs, J.J.M.R., Van Montagu, M. and Cornelissen, M. 1998. Silencing of β-1,3-glucanase genes in tobacco correlates with an increased abundance of RNA degradation intermediates. Nucl. Acids Res. 26: 5176–5181.Google Scholar
  36. van Hoof, A. and Green, P.J. 1996. Premature nonsense codons decrease the stability of phytohemagglutinin mRNA in a positiondependent manner. Plant J. 10: 415–424.Google Scholar
  37. Van Houdt, H., Ingelbrecht, I., Van Montagu, M. and Depicker, A. 1997. Post-transcriptional silencing of a neomycin phosphotransferase II transgene correlates with the accumulation of unproductive RNAs and with increased cytosine methylation of 3' flanking regions. Plant J. 12: 379–392.Google Scholar
  38. Vaucheret, H., Béclin, C., Elmayan, T., Feuerbach, F., Godon, C., Morel, J.B., Mourrain, P., Palauqui, I.C. and Vernhettes, S. 1998. Transgene-induced gene silencing in plants. Plant J. 16: 651–659.Google Scholar
  39. Voinnet, O., Vain, P., Angell, S. and Baulcombe, D.C. 1998. Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA. Cell 85: 177–187.Google Scholar
  40. Wagner, E.G.H. and Simons, R.S. 1994. Antisense RNA control in bacteria, phages, and plasmids. Annu. Rev. Microbiol. 48: 713–742.Google Scholar
  41. Wang, M.B., Upadhyaya, N.M., Brettell, R.I.S. and Waterhouse, P.M. 1997. Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. J. Genet. Breed. 51: 325–334.Google Scholar
  42. Wang, M.B., Li, Z.Y., Matthews, P.R., Upadhyaya, N.M. and Waterhouse, P.M. 1998. Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants. Acta Hort. 461: 401–407.Google Scholar
  43. Wassenegger, M. and Pélissier, T. 1998. A model for RNA-mediated gene silencing in higher plants. Plant Mol. Biol. 37: 349–362.Google Scholar
  44. Wassenegger, M., Heimes, S., Riedel, L. and Sänger, H.L. 1994. RNA-directed de novo methylation of genomic sequences in plants. Cell 76: 567–576.Google Scholar
  45. Waterhouse, P.M., Graham, M.W. and Wang, M.B. 1998. Virus 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.Google Scholar
  46. Waterhouse, P.M., Smith, N.A. and Wang, M.B. 1999. Virus resistance and gene silencing: killing the messenger. Trends Plant Sci. 4: 452–457.Google Scholar
  47. Zheng, Z., Hayashimoto, A., Li, Z. and Murai, N. 1991. Hygromycin resistance gene cassettes for vector construction and selection of transformed rice protoplasts. Plant Physiol. 97: 832–835.Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Ming-Bo Wang
    • 1
  • Peter M. Waterhouse
    • 1
  1. 1.CSIRO Plant IndustryCanberraAustralia

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