Skip to main content
The Plant Cell logoLink to The Plant Cell
. 1996 Dec;8(12):2277–2294. doi: 10.1105/tpc.8.12.2277

RNA-Mediated Virus Resistance: Role of Repeated Transgenes and Delineation of Targeted Regions.

T Sijen 1, J Wellink 1, J B Hiriart 1, A Van Kammen 1
PMCID: PMC161352  PMID: 12239378

Abstract

Resistance to cowpea mosaic virus (CPMV) in transgenic Nicotiana benthamiana plants is RNA mediated. In resistant CPMV movement protein (MP) gene-transformed lines, transgene steady state mRNA levels were low, whereas nuclear transcription rates were high, implying that a post-transcriptional gene-silencing mechanism is at the base of the resistance. The silencing mechanism can also affect potato virus X (PVX) RNAs when they contain CPMV MP gene sequences. In particular, sequences situated in the 3[prime] part of the transcribed region of the MP transgene direct elimination of recombinant PVX genomes. Remarkably, successive portions of this 3[prime] part, which can be as small as 60 nucleotides, all tag PVX genomes for degradation. These observations suggest that the entire 3[prime] part of the MP transgene mRNA is the initial target of the silencing mechanism. The arrangement of transgenes in the plant genome plays an important role in establishing resistance because the frequency of resistant lines increased from 20 to 60% when transformed with a transgene containing a direct repeat of MP sequences rather than a single MP transgene. Interestingly, we detected strong methylation in all of the plants containing directly repeated MP sequences. In sensitive lines, only the promoter region was found to be heavily methylated, whereas in resistant lines, only the transcribed region was strongly methylated.

Full Text

The Full Text of this article is available as a PDF (3.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bochardt A., Hodal L., Palmgren G., Mattsson O., Okkels F. T. DNA methylation is involved in maintenance of an unusual expression pattern of an introduced gene. Plant Physiol. 1992 Jun;99(2):409–414. doi: 10.1104/pp.99.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chapman S., Kavanagh T., Baulcombe D. Potato virus X as a vector for gene expression in plants. Plant J. 1992 Jul;2(4):549–557. doi: 10.1046/j.1365-313x.1992.t01-24-00999.x. [DOI] [PubMed] [Google Scholar]
  3. Dorer D. R., Henikoff S. Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila. Cell. 1994 Jul 1;77(7):993–1002. doi: 10.1016/0092-8674(94)90439-1. [DOI] [PubMed] [Google Scholar]
  4. Dougherty W. G., Lindbo J. A., Smith H. A., Parks T. D., Swaney S., Proebsting W. M. RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation. Mol Plant Microbe Interact. 1994 Sep-Oct;7(5):544–552. [PubMed] [Google Scholar]
  5. Dougherty W. G., Parks T. D. Transgenes and gene suppression: telling us something new? Curr Opin Cell Biol. 1995 Jun;7(3):399–405. doi: 10.1016/0955-0674(95)80096-4. [DOI] [PubMed] [Google Scholar]
  6. English J. J., Mueller E., Baulcombe D. C. Suppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes. Plant Cell. 1996 Feb;8(2):179–188. doi: 10.1105/tpc.8.2.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hart C. M., Fischer B., Neuhaus J. M., Meins F., Jr Regulated inactivation of homologous gene expression in transgenic Nicotiana sylvestris plants containing a defense-related tobacco chitinase gene. Mol Gen Genet. 1992 Nov;235(2-3):179–188. doi: 10.1007/BF00279359. [DOI] [PubMed] [Google Scholar]
  8. Hellwald K. H., Palukaitis P. Viral RNA as a potential target for two independent mechanisms of replicase-mediated resistance against cucumber mosaic virus. Cell. 1995 Dec 15;83(6):937–946. doi: 10.1016/0092-8674(95)90209-0. [DOI] [PubMed] [Google Scholar]
  9. Hobbs S. L., Kpodar P., DeLong C. M. The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants. Plant Mol Biol. 1990 Dec;15(6):851–864. doi: 10.1007/BF00039425. [DOI] [PubMed] [Google Scholar]
  10. Hohn T., Corsten S., Rieke S., Müller M., Rothnie H. Methylation of coding region alone inhibits gene expression in plant protoplasts. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8334–8339. doi: 10.1073/pnas.93.16.8334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jorgensen R. A. Cosuppression, flower color patterns, and metastable gene expression States. Science. 1995 May 5;268(5211):686–691. doi: 10.1126/science.268.5211.686. [DOI] [PubMed] [Google Scholar]
  12. Judelson H. S., Whittaker S. L. Inactivation of transgenes in Phytophthora infestans is not associated with their deletion, methylation, or mutation. Curr Genet. 1995 Nov;28(6):571–579. doi: 10.1007/BF00518171. [DOI] [PubMed] [Google Scholar]
  13. Keshet I., Lieman-Hurwitz J., Cedar H. DNA methylation affects the formation of active chromatin. Cell. 1986 Feb 28;44(4):535–543. doi: 10.1016/0092-8674(86)90263-1. [DOI] [PubMed] [Google Scholar]
  14. Lee R. C., Feinbaum R. L., Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993 Dec 3;75(5):843–854. doi: 10.1016/0092-8674(93)90529-y. [DOI] [PubMed] [Google Scholar]
  15. Lewin B. Chromatin and gene expression: constant questions, but changing answers. Cell. 1994 Nov 4;79(3):397–406. doi: 10.1016/0092-8674(94)90249-6. [DOI] [PubMed] [Google Scholar]
  16. Lindbo J. A., Dougherty W. G. Pathogen-derived resistance to a potyvirus: immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence. Mol Plant Microbe Interact. 1992 Mar-Apr;5(2):144–153. doi: 10.1094/mpmi-5-144. [DOI] [PubMed] [Google Scholar]
  17. Lindbo J. A., Silva-Rosales L., Proebsting W. M., Dougherty W. G. Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance. Plant Cell. 1993 Dec;5(12):1749–1759. doi: 10.1105/tpc.5.12.1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Matzke M. A., Primig M., Trnovsky J., Matzke A. J. Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J. 1989 Mar;8(3):643–649. doi: 10.1002/j.1460-2075.1989.tb03421.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meyer P., Saedler H. HOMOLOGY-DEPENDENT GENE SILENCING IN PLANTS. Annu Rev Plant Physiol Plant Mol Biol. 1996 Jun;47(NaN):23–48. doi: 10.1146/annurev.arplant.47.1.23. [DOI] [PubMed] [Google Scholar]
  20. Palmgren G., Mattson O., Okkels F. T. Treatment of Agrobacterium or leaf disks with 5-azacytidine increases transgene expression in tobacco. Plant Mol Biol. 1993 Feb;21(3):429–435. doi: 10.1007/BF00028801. [DOI] [PubMed] [Google Scholar]
  21. Prins M., Resende R. de O., Anker C., van Schepen A., de Haan P., Goldbach R. Engineered RNA-mediated resistance to tomato spotted wilt virus is sequence specific. Mol Plant Microbe Interact. 1996 Jul;9(5):416–418. doi: 10.1094/mpmi-9-0416. [DOI] [PubMed] [Google Scholar]
  22. Rivier D. H., Pillus L. Silencing speaks up. Cell. 1994 Mar 25;76(6):963–966. doi: 10.1016/0092-8674(94)90373-5. [DOI] [PubMed] [Google Scholar]
  23. Schiebel W., Haas B., Marinković S., Klanner A., Sänger H. L. RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties. J Biol Chem. 1993 Jun 5;268(16):11858–11867. [PubMed] [Google Scholar]
  24. Smith H. A., Swaney S. L., Parks T. D., Wernsman E. A., Dougherty W. G. Transgenic plant virus resistance mediated by untranslatable sense RNAs: expression, regulation, and fate of nonessential RNAs. Plant Cell. 1994 Oct;6(10):1441–1453. doi: 10.1105/tpc.6.10.1441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Swaney S., Powers H., Goodwin J., Rosales L. S., Dougherty W. G. RNA-mediated resistance with nonstructural genes from the tobacco etch virus genome. Mol Plant Microbe Interact. 1995 Nov-Dec;8(6):1004–1011. doi: 10.1094/mpmi-8-1004. [DOI] [PubMed] [Google Scholar]
  26. Wassenegger M., Heimes S., Riedel L., Sänger H. L. RNA-directed de novo methylation of genomic sequences in plants. Cell. 1994 Feb 11;76(3):567–576. doi: 10.1016/0092-8674(94)90119-8. [DOI] [PubMed] [Google Scholar]
  27. Weber H., Ziechmann C., Graessmann A. In vitro DNA methylation inhibits gene expression in transgenic tobacco. EMBO J. 1990 Dec;9(13):4409–4415. doi: 10.1002/j.1460-2075.1990.tb07891.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wellink J., Verver J., Van Lent J., Van Kammen A. Capsid proteins of cowpea mosaic virus transiently expressed in protoplasts form virus-like particles. Virology. 1996 Oct 1;224(1):352–355. doi: 10.1006/viro.1996.0541. [DOI] [PubMed] [Google Scholar]
  29. Wellink J., van Lent J. W., Verver J., Sijen T., Goldbach R. W., van Kammen A. The cowpea mosaic virus M RNA-encoded 48-kilodalton protein is responsible for induction of tubular structures in protoplasts. J Virol. 1993 Jun;67(6):3660–3664. doi: 10.1128/jvi.67.6.3660-3664.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wilson T. M. Strategies to protect crop plants against viruses: pathogen-derived resistance blossoms. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3134–3141. doi: 10.1073/pnas.90.8.3134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zhang D. L., Ehrlich K. C., Supakar P. C., Ehrlich M. A plant DNA-binding protein that recognizes 5-methylcytosine residues. Mol Cell Biol. 1989 Mar;9(3):1351–1356. doi: 10.1128/mcb.9.3.1351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. ten Lohuis M., Müller A., Heidmann I., Niedenhof I., Meyer P. A repetitive DNA fragment carrying a hot spot for de novo DNA methylation enhances expression variegation in tobacco and petunia. Plant J. 1995 Dec;8(6):919–932. doi: 10.1046/j.1365-313x.1995.8060919.x. [DOI] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES