Skip to main content
SpringerLink
Log in

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts

  • Research Article
  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m5C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m5C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Adams RLP: DNA methylation. The effect of minor bases on DNA-protein interactions. Biochem J 265: 309–320 (1990).

    PubMed  Google Scholar 

  2. Amasino RM, Powell ALT, Gordon MP: Changes in T-DNA methylation and expression are associated with phenotypic variation and plant regeneration in crown gall tumor lines. Mol Gen Genet 197: 437–446 (1984).

    PubMed  Google Scholar 

  3. Ausubel FM, Brent R, Kingston RE, Moore DD Siedman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. John Wiley, New York (1987).

    Google Scholar 

  4. Banks JA, Masson P, Federoff N: Molecular mechanisms in the developmental regulation of the maizeSuppressor-mutator transposable element. Genes Devel 2: 1367–1380 (1988).

    Google Scholar 

  5. Bianchi MW, Viotti A: DNA methylation and tissue-specific transcription of the storage protein genes of maize. Plant Mol Biol 11: 203–214 (1988).

    Google Scholar 

  6. Brough CL, Hayes RJ, Morgan AJ, Coutts RHA, Buck KW: Effects of mutagenesis in vitro on the ability of cloned tomato golden mosaic virus DNA to infectNicotiana benthamiana plants. J Gen Virol 69: 503–514 (1988).

    Google Scholar 

  7. Cedar H, Razin A: DNA methylation and development. Biochim Biophys Acta 1049: 1–8 (1990).

    PubMed  Google Scholar 

  8. Ehrlich M, Wang RY-H: 5-methylcytosine in eukaryotic DNA. Science 212: 1350–1357 (1981).

    PubMed  Google Scholar 

  9. Elmer JS, Sunter G, Gardiner WE, Brand L, Browning CK, Bisaro DM, Rogers SG:Agrobacterium-mediated inoculation of plants with tomato golden mosaic virus DNAs. Plant Mol Biol 10: 225–234 (1988).

    Google Scholar 

  10. Flavell RB, O'Dell M, Thompson WF: Regulation of cytosine methylation in ribosomal DNA and nucleolus organizer expression in wheat. J Mol Biol 204: 523–534 (1988).

    Article  PubMed  Google Scholar 

  11. Gardiner WE, Sunter G, Brand L, Elmer JS, Rogers SG, Bisaro DM: Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development. EMBO J 7: 899–904 (1988).

    Google Scholar 

  12. Gruenbaum Y, Naveh-Many T, Cedar H, Razin A: Sequence specificity of methylation in higher plant DNA. Nature 292: 860–862 (1981).

    PubMed  Google Scholar 

  13. Hamilton WDO, Bisaro DM, Buck KW: Identification of novel DNA forms in tomato golden mosaic virus infected tissue. Evidence for a two component viral genome. Nucl Acids Res 10: 4901–4912 (1982).

    PubMed  Google Scholar 

  14. Hamilton WDO, Bisaro DM, Coutts RHA, Buck KW: Demonstration of the bipartite nature of the genome of a single-stranded DNA plant virus by infection with the cloned DNA components. Nucl Acids Res 11: 7387–7396 (1983).

    PubMed  Google Scholar 

  15. Hamilton WDO, Stein VE, Coutts RHA, Buck KW: Complete nucleotide sequence of the infectious cloned DNA components of tomato golden mosaic virus: potential coding regions and regulatory sequences. EMBO J 3: 2197–2205 (1984).

    Google Scholar 

  16. Hepburn AG, Clarke LE, Pearson L, White J: The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor. J Mol Appl Genet 2: 315–329 (1983).

    PubMed  Google Scholar 

  17. Hershkovitz M, Gruenbaum Y, Zakai N, Loyter A: Gene transfer in plant protoplasts. Inhibition of gene activity by cytosine methylation and expression of single-stranded DNA constructs. FEBS Lett 253: 167–172 (1989).

    Article  Google Scholar 

  18. John MC, Amasino RM: Extensive changes in DNA methylation patterns accompany activation of a silentipt gene inAgrobacterium tumefaciens-transformed plant cells. Mol Cell Biol 9: 4298–4303 (1989).

    PubMed  Google Scholar 

  19. Klaas M, John MC, Crowell DN, Amasino RM: Rapid induction of genomic demethylation and T-DNA gene expression in plant cells by 5-azacytosine derivatives. Plant Mol Biol 12: 413–423 (1989).

    Article  Google Scholar 

  20. Mettler IJ: A simple and rapid method for minipreparation of DNA from tissue cultured plant cells. Plant Mol Biol Rep 5: 346–349 (1987).

    Google Scholar 

  21. Michalowsky LA, Jones PA: DNA methylation and differentiation. Envir Health Persp 80: 189–197 (1989).

    Google Scholar 

  22. Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F: Hybrid genes in the analysis of tranformation conditions I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol Biol 8: 363–373 (1987).

    Google Scholar 

  23. Nelson M, McClelland M: The effect of site-specific methylation on restriction-modification methyltransferases and restriction endonucleases. Nucl Acids Res 17: r389-r415 (1989).

    Google Scholar 

  24. Potrykus I, Shillito R: Protoplasts: isolation, culture, plant regeneration. In: Weissbach H, Weissbach A (eds) Methods in Enzymology, vol 118, pp. 549–579. Academic Press, New York (1986).

    Google Scholar 

  25. Raleigh EA, Murray NE, Revel H, Blumenthal RM, Westaway D, Reith AD, Rigby PWJ, Elhai J, Hanahan D: McrA and McrB restriction phenotypes of someE. coli strains and implications for gene cloning. Nucl Acids Res 16: 1563–1575 (1988).

    PubMed  Google Scholar 

  26. Razin A, Riggs AD: DNA methylation and gene function. Science 210: 604–610 (1980).

    PubMed  Google Scholar 

  27. Revington GN, Sunter G, Bisaro DM: DNA sequences essential for replication of the B genome component of tomato golden mosaic virus. Plant Cell 1: 985–992 (1989).

    Article  PubMed  Google Scholar 

  28. Rogers SG, Bisaro DM, Horsch RB, Fraley RT, Hoffmann NL, Brand L, Elmer JS, Lloyd AM: Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants. Cell 45: 593–600 (1986).

    Article  PubMed  Google Scholar 

  29. Rushing AE, Sunter G, Gardiner WE, Dute RR, Bisaro DM: Ultrastructural aspects of tomato golden mosaic virus infection in tobacco. Phytopathology 77: 1231–1236 (1987).

    Google Scholar 

  30. Shapiro HS: Distribution of purines and pyrimidines in deoxyribonucleic acids. In: CRC Handbook of Biochemistry and Molecular Biology. CRC Press, Boca Raton, FL (1976).

    Google Scholar 

  31. Shenoy S, Ehrlich ME: Isolation ofin vitro synthesized covalently closed circular double-stranded DNA by selective denaturation and filtration. Prep Biochem 14: 485–497 (1985).

    Google Scholar 

  32. Stein R, Gruenbaum Y, Pollack Y, Razin A, Cedar H: Clonal inheritance of the pattern of DNA methylation in mouse cells. Proc Natl Acad Sci USA 79: 61–65 (1982).

    PubMed  Google Scholar 

  33. Stenger DC, Revington GN, Stevenson MC, Bisaro DM: Replicational release of geminivirus genomes from tandemly repeated copies: Evidence for rolling circle replication of a plant viral DNA. Proc Natl Acad Sci USA 88: 8029–8033.

  34. Subramanian KN: Effect ofin vitro methylation at CpG sites on gene expression in a genome functioning autonomously in a vertebrate host. Nucl Acids Res 10: 3475–3486 (1982).

    PubMed  Google Scholar 

  35. Sunter G, Gardiner WE, Rushing AE, Rogers SG, Bisaro DM: Independent encapsidation of tomato golden mosaic virus A component DNA in transgenic plants. Plant Mol Biol 8: 477–484 (1987).

    Google Scholar 

  36. Sunter G, Hartitz MD, Hormuzdi SG, Brough CL, Bisaro DM: Genetic analysis of tomato golden mosaic virus. ORF AL2 is required for coat protein accumulation while ORF AL3 is necessary for efficient DNA replication. Virology 179: 69–77 (1990).

    Article  PubMed  Google Scholar 

  37. Toth M, Muller U, Doerfler W: Establishment ofde novo methylation patterns: Transcription factor binding and deoxycytidine methylation at CpG and non-CpG sequences in an integrated adenovirus promotor. J Mol Biol 214: 673–683 (1990).

    Article  PubMed  Google Scholar 

  38. Vanyushin BF: Replicative DNA methylation in animals and higher plants. Curr Top Microbiol Immunol 108: 99–114 (1984).

    PubMed  Google Scholar 

  39. Vieira J, Messing J: Production of single-stranded DNA. In: Wu R, Grossman L (eds) Methods in Enzymology, vol. 153, pp. 3–11. Academic Press, New York (1987).

    Google Scholar 

  40. Wagner H, Simon D, Werner E, Gelderblom H, Darai C, Flugel RM: Methylation pattern of fish lymphocystis disease virus DNA. J Virol 53: 1005–1007 (1985).

    PubMed  Google Scholar 

  41. Wagner I, Capesius I: Determination of 5-methylcytosine from plant DNA by high-performance liquid chromatography. Biochim Biophys Acta 654: 52–56 (1981).

    PubMed  Google Scholar 

  42. Wang RY-H, Shenoy S, Ehrlich M: DNA methylation inhibits the transfecting activity of replicative form ΦX174 DNA. J Virol 49: 674–679 (1984).

    PubMed  Google Scholar 

  43. Weber H, Graessmann A: Biological activity of hemimethylated and single-stranded DNA after direct gene transfer into tobacco protoplasts. FEBS Lett 253: 163–166 (1989).

    Article  Google Scholar 

  44. Weber H, Zeichmann C, Graessmann A:In vitro DNA methylation inhibits gene expression in transgenic tobacco. EMBO J 9: 4409–4415 (1990).

    PubMed  Google Scholar 

  45. Willis DB, Essani K, Goorha R, Thompson JP, Granoff A: Transcription of a methylated DNA virus. In: Clawson GA, Willis DB, Weissbach A, Jones PA, (eds) Nucleic Acid Methylation, vol. 128, pp. 139–151. Alan R. Liss, New York (1990).

    Google Scholar 

Download references

Author information

Author notes

  1. Clare L. Brough

    Present address: Department of Biological Sciences, University of Durham, DH1 3LE, Durham, UK

Authors and Affiliations

  1. Biotechnology Center and Department of Molecular Genetics, The Ohio State University, 43210, Columbus, OH, USA

    Clare L. Brough, William E. Gardiner & David M. Bisaro

  2. Department of Biochemistry, Tulane University Medical Center, 70112, New Orleans, LA, USA

    Nilufar M. Inamdar, Xian-Yang Zhang & Melanie Ehrlich

  3. Department of Biological Sciences, Mississippi State University, P.O. Drawer GY, 39762, Starkville, MS, USA

    William E. Gardiner

Authors
  1. Clare L. Brough
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. William E. Gardiner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nilufar M. Inamdar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xian-Yang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Melanie Ehrlich
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David M. Bisaro
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brough, C.L., Gardiner, W.E., Inamdar, N.M. et al. DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts. Plant Mol Biol 18, 703–712 (1992). https://doi.org/10.1007/BF00020012

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00020012

Key words

Search

Navigation