Advertisement

Plant Molecular Biology

, Volume 46, Issue 4, pp 433–445 | Cite as

Transgene silencing of invertedly repeated transgenes is released upon deletion of one of the transgenes involved

  • Sylvie De Buck
  • Marc Van Montagu
  • Ann Depicker
Article

Abstract

To analyse experimentally the correlation between transgene silencing and the presence of an inverted repeat in transgenic Arabidopsis thaliana plants, expression of the β-glucuronidase (gus) gene was studied when present as a convergently transcribed inverted repeat or as a single copy in otherwise isogenic lines. In transformants containing two invertedly repeated gus genes separated by a 732 bp palindromic sequence, gus expression was low, as exemplified by the expression levels in the parental line KH15. The parental KH15 locus could induce efficiently in trans silencing of gus copies at allelic and non-allelic positions. In transformants containing two invertedly repeated gus genes separated by a 826 bp non-repetitive spacer region, gus expression was high or intermediate, especially in hemizygous state and at late developmental stages, as demonstrated in detail for line KHsb67. Removal of one of the gus copies by Cre recombinase resulted in all cases in constitutively high gus expression in hemizygous as well as in homozygous state. The derived deletion lines could no longer induce in trans silencing of homologous gus copies. The results show that convergent transcription of transgenes in an inverted repeat is an important parameter to trigger their silencing and that co-transformation of two T-DNAs with identical transgenes can be used to obtain inverted repeats and targeted co-suppression of the homologous endogenes. Moreover, the data suggest that the spacer region in between the inverted genes plays a role in the efficiency of initiating and maintaining silencing.

co-transformation Cre/lox inverted repeats transgene silencing 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Angell, S.M. and Baulcombe, D.C. 1999. Potato virus X amplicon-mediated silencing of nuclear genes. Plant J. 20: 357–362.Google Scholar
  2. Assaad, F.F., Lee Tucker, K. and Signer, E.R. 1993. Epigenetic repeat-induced gene silencing (RIGS) in Arabidopsis. Plant Mol. Biol. 22: 1067–1085.Google Scholar
  3. Bass, B.L. 2000. Double-stranded RNA as a template for gene silencing. Cell 101: 235–238.Google Scholar
  4. Baulcombe, D.C. 2000. Unwinding RNA silencing. Science 290:1108–1109.Google Scholar
  5. 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
  6. Bollmann, J., Carpenter, R. and Coen, E.S. 1991. Allelic interactions at the nivea locus of Anterrhinum. Plant Cell 3: 1327–1336.Google Scholar
  7. Bradford, M.N. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.Google Scholar
  8. Breyne, P., De Loose, M., Dedonder, A., Van Montagu, M. and De-picker A. 1993. Quantitative kinetic analysis of βglucuronidase activities using a computer-directed microtiterplate reader. Plant Mol. Biol. Rep. 11: 21–31.Google Scholar
  9. Chuang, C.-F. and Meyerowitz, E.N. 2000. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 97: 4985–4990.Google Scholar
  10. Cluster, P.D., O'Dell, M., Metzlaff, M. and Flavell, R.B. 1996. Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression. Plant Mol. Biol. 32: 1197–1203.Google Scholar
  11. Covey, S.N. 2000. Silencing genes silencing genes. Trends Plant Sci 5: 405–406.Google Scholar
  12. Dale, E.O. and Ow, D.W. 1990. Intra-and intermolecular site-specific recombination in plant cell mediated by bacteriophage P1 recombinase. Gene 91: 79–85.Google Scholar
  13. De Buck, S. and Depicker, A. 2001. Disruption of their palindromic arrangement leads to selective loss of DNA methylation in inversely repeated gus transgenes in Arabidopsis. Mol. Gen. Genom., in press.Google Scholar
  14. De Buck, S., Jacobs, A., Van Montagu, M. and Depicker, A. 1998. Agrobacterium tumefaciens transformation and cotransformation frequencies of Arabidopsis thaliana root explants and tobacco protoplasts. Mol. Plant-Microbe Interact. 11: 449–457.Google Scholar
  15. De Buck, S., Jacobs, A., Van Montagu, M. and Depicker, A. 1999.The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration. Plant J. 20: 295–304.Google Scholar
  16. De Buck, S., De Wilde, C., Van Montagu, M. and Depicker, A. 2000. Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root explants. Mol. Plant-Microbe Interact. 13: 658–665.Google Scholar
  17. de Carvalho, F., Gheysen, G., Kushnir, S., Van Montagu, M., Inzé, D. and Castresana, C. 1992. Suppression of β-1,3-glucanase transgene expression in homozygous plants. EMBO J. 11: 2595–2602.Google Scholar
  18. De Neve, M., De Buck, S., Jacobs, A., Van Montagu, M. and Depicker, A. 1997. T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from ligation of separate T-DNAs. Plant J. 11: 15–29.Google Scholar
  19. De Neve, M., De Buck, S., De Wilde, C., Van Houdt, H., Strobbe, I., Jacobs, A., Van Montagu, M. and Depicker, A. 1999. Gene silencing results in instability of antibody production in transgenic plants. Mol. Genet. Genom. 260: 582–592.Google Scholar
  20. De Wilde, Podevin, N., Windels, P. and Depicker, A. 2001. Silencing of antibody genes in plants with single-copy transgene inserts as a result of gene dosage effects. Mol. Genet. Genom., in press.Google Scholar
  21. Deblaere, R., Reynaerts, A., Höfte, H., Hernalsteens, J.-P., Lee-mans, J. and Van Montagu, M. 1987. Vectors for cloning in plant cells. In: R. Wu, and L. Grossman (Eds.) Recombinant DNA Part D (Methods in Enzymology Vol. 153), Academic Press, New York, pp. 277–292.Google Scholar
  22. Depicker, A. and Van Montagu, M. 1997. Post-transcriptional gene silencing in plants. Curr. Opin. Cell Biol. 9: 373–382.Google Scholar
  23. Depicker, A., Ingelbrecht, I., Van Houdt, H., De Loose, M. and Van Montagu, M. 1996. Post-transcriptional reporter trans-gene silencing in transgenic tobacco. In: D. Grierson, C.W. Lycett and G.A. Tucker (Eds.) Mechanisms and Applications of Gene Silencing, Nottingham University Press, Nottingham, UK, pp. 71–84.Google Scholar
  24. Dougherty, W.G. and Parks, D.T. 1995. Transgenes and gene sup-pression: telling us something new? Curr. Opin. Cell Biol. 7: 399–405.Google Scholar
  25. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811.Google Scholar
  26. Garrick, U., Fiering, S., Martin, D.I.K. and Whitelaw, E. 1998. Repeat-induced gene silencing in mammals. Nature Genet. 18: 56–59.Google Scholar
  27. Hamilton, A.J. and Baulcombe, D.C. 1999. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286: 950–952.Google Scholar
  28. Hamilton, A.J., Brown, S., Yuanhai, H., Ishizuka, M., Lowe, A., Alpuche Solis, A.-G. and Grierson, U. 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
  29. Hammond, S.N., Bernstein, E., Beach, D. and Hannon, G.J. 2000.An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404: 293–295.Google Scholar
  30. Hobbs, S.L.A., Warkentin, T.D. and DeLong, C.M.O. 1993. Transgene copy number can be positively or negatively associated with transgene expression. Plant. Mol. Biol. 21: 17–26.Google Scholar
  31. Hutvágner, G., Mlynárová, L. and Nap, J.-P. 2000. Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco. RNA 6: 1445–1454.Google Scholar
  32. Jorgensen, RA., Cluster, P.D., English, J. Que, A.Q. and Napoli, C.A. 1996. Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol. Biol. 31: 957–973.Google Scholar
  33. Kooter, J.M., Matzke, M.A. and Meyer P. 1999. Listening to the silent genes: transgene silencing, gene regulation and pathogen control. Trends Plant Sci. 4: 340–347.Google Scholar
  34. Levin, J.Z., de Framond, A.J., Tuttle, A., Bauer, M.W. and Heifetz, P.B. 2000. Methods of double-stranded RNA-mediated gene inactivation in Arabidopsis and their use to define an essential gene in methionine biosynthesis. Plant Mol. Biol. 44: 759–775.Google Scholar
  35. Llave, C., Kasschau, K.D. and Carrington, J.C. 2000. Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc. Natl. Acad. Sci. USA 97: 13401–13406.Google Scholar
  36. 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
  37. Matzke, A.J.M., Neuhuber, F., Park, Y.-D., Ambros, P.F. and Matzke, M.A. 1994. Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes. Mol. Gen. Genet. 244: 219–229.Google Scholar
  38. Matzke, M.A., Mette, M.F. and Matzke, A.J.M. 2000. Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol. Biol. 43: 401–415.Google Scholar
  39. Mette, M.F., Aufsatz, W., van der Winden, J., Matzke, M.A. and Matzke, A.J.M. 2000. Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J. 19: 5194–5201.Google Scholar
  40. Meyer, P. 1998. Stabilities and instabilities in transgene expression.In: K. Lindsey (Ed.) Transgenic Plant Research, Harwood Academic Publishers, Amsterdam, pp. 263–275.Google Scholar
  41. Muskens, M.W.M., Vissers, A.P.A., Mol, J.N.M. and Kooter, J.M. 2000. Role of inverted DNA repeats in transcriptional and post-transcriptional gene silencing. Plant Mol. Biol. 43: 243–260.Google Scholar
  42. Ngô, H., Tschudi, C., Gull, K. and Ullu, E. 1998. Double-stranded RNA induces RNA degradation in Trypanosoma brucei.Proc. Natl. Acad. Sci. USA 95: 14687–14692.Google Scholar
  43. Odell, J.T., Nagy, F. and Chua, N.-H. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812.Google Scholar
  44. Peach, C. and Velten, J. 1991. Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol. Biol. 17: 49–60.Google Scholar
  45. Sijen, T. and Kooter, J.M. 2000. Post-transcriptional gene-silencing: RNAs on the attack or on the defense? BioEssays 22: 520–531.Google Scholar
  46. 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
  47. Smith, N.A., Singh, S.P., Wang, M.-B., Stoutjesdijk, P.A., Green, A.G. and Waterhouse, P.M. 2000. Total silencing by intron-spliced hairpin RNAs. Nature 407: 319–320.Google Scholar
  48. Stam, M., de Bruin, R., Kenter, S., van der Hoorn, R.A.L., van Blok-land, R., Mol, J.N.M. and Kooter, J.M. 1997. Post-transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats. Plant J. 12: 63–82.Google Scholar
  49. 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 posttran-scriptional silencing of homologous host genes in plants. Mol. Cell. Biol. 18: 6165–6177.Google Scholar
  50. Stam, M., de Bruin, R., van Blokland, R., van der Hoorn, R.A.L., Mol, J.N.M. and Kooter, J.M. 2000. Distinct features of post-transcriptional gene silencing by antisense transgenes in single copy and inverted T-DNA repeat loci. Plant J. 21: 27–42.Google Scholar
  51. Todd, J.J. and Vodkin, L.O. 1996. Duplications that suppress and deletions that restore expression from a chalcone synthase multigene family. Plant Cell 8: 687–699.Google Scholar
  52. Van Houdt, H., Kovaŕík, A., Van Montagu, M. and Depicker, A. 2000. Cross-talk between posttranscriptionally silenced neomycin phosphotransferase II transgenes. FEBS Lett. 467: 41–46.Google Scholar
  53. Vaucheret, H. 1993. Identification of general silencer for 19S and 35S promoters in a transgenic tobacco plant: 90 bp of homology in the promoter sequence are sufficient for trans-inactivation. C.R. Acad. Sci. Paris 316: 1471–1483.Google Scholar
  54. Vaucheret, H., Béclin, C., Elmayan, T., Feuerbach, F., Godon, C., Morel, J.-B., Mourrain, P., Palauqui, J.-C. and Vernhettes, S. 1998. Transgene-induced gene silencing in plants. Plant J. 16:651–659.Google Scholar
  55. Wang, M.-B. and Waterhouse, P.M. 2000. High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation. Plant Mol. Biol. 43: 67–82.Google Scholar
  56. Wassenegger, M. 2000. RNA-directed DNA methylation. Plant Mol. Biol. 43: 203–220.Google Scholar
  57. Waterhouse, F.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
  58. 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
  59. Wianny, F. and Zernicka-Goetz, M. 2000. Specific interference with gene function by double-stranded RNA in early mouse development. Nature Cell Biol. 2: 70–75.Google Scholar
  60. Wolffe, A.P. 1997. Transcription control: repressed repeats express themselves. Curr. Biol. 7: R796–R798.Google Scholar
  61. Yang, D., Lu, H. and Erickson, J.W. 2000. Evidence that processed small dsRNAs may mediate sequence-specific mRNA degra-dation during RNAi in Drosophila embryos. Curr. Biol. 10: 1191–1200.Google Scholar
  62. Zamore, P.D., Tuschl, T., Sharp. P.A. and Bartel, D.P. 2000. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25–33.Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Sylvie De Buck
    • 1
  • Marc Van Montagu
    • 1
  • Ann Depicker
    • 1
  1. 1.Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB)Universiteit GentGentBelgium

Personalised recommendations