Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 Apr 25;16(8):3127–3140. doi: 10.1093/nar/16.8.3127

Selective recovery of foreign gene transcripts as virus-like particles in TMV-infected transgenic tobaccos.

D E Sleat 1, D R Gallie 1, J W Watts 1, C M Deom 1, P C Turner 1, R N Beachy 1, T M Wilson 1
PMCID: PMC336483  PMID: 2453837

Abstract

A short origin-of-assembly sequence (OAS) located in the 30kDa movement protein gene, about 1.0kb from the 3'-end of the common strain of tobacco mosaic virus (TMV) RNA, nucleates encapsidation of the 6395-nucleotide-long genome by TMV coat protein in vitro, and presumably also in vivo. Single-stranded RNAs containing a foreign reporter gene sequence and the TMV OAS at their 5' - and 3' -ends, respectively, can be synthesized in vitro from recombinant SP6-transcription plasmids and will assemble spontaneously in vitro to form TMV-like 'pseudovirus' particles. In this paper, we show that foreign gene transcripts derived from the nuclear DNA of plants transformed by Agrobacterium tumefaciens, and which contain the TMV OAS, can be assembled into stable 'pseudovirus' particles in vivo during a systemic infection by TMV (helper). This is the first report of structural complementation between a heritable function bestowed on a transgenic plant and an infecting virus. As a route to protect, accumulate and recover a specific mRNA in vivo, in transgenic plant cells, this novel approach may find wider applications in developmental plant molecular biology.

Full text

PDF
3127

Images in this article

Selected References

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

  1. A simple and general method for transferring genes into plants. Science. 1985 Mar 8;227(4691):1229–1231. doi: 10.1126/science.227.4691.1229. [DOI] [PubMed] [Google Scholar]
  2. Abel P. P., Nelson R. S., De B., Hoffmann N., Rogers S. G., Fraley R. T., Beachy R. N. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science. 1986 May 9;232(4751):738–743. doi: 10.1126/science.3457472. [DOI] [PubMed] [Google Scholar]
  3. Bevan M. W., Mason S. E., Goelet P. Expression of tobacco mosaic virus coat protein by a cauliflower mosaic virus promoter in plants transformed by Agrobacterium. EMBO J. 1985 Aug;4(8):1921–1926. doi: 10.1002/j.1460-2075.1985.tb03871.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bruening G., Beachy R. N., Scalla R., Zaitlin M. In vitro and in vivo translation of the ribonucleic acids of a cowpea strain of tobacco mosaic virus. Virology. 1976 Jun;71(2):498–517. doi: 10.1016/0042-6822(76)90377-9. [DOI] [PubMed] [Google Scholar]
  5. Butler P. J. The current picture of the structure and assembly of tobacco mosaic virus. J Gen Virol. 1984 Feb;65(Pt 2):253–279. doi: 10.1099/0022-1317-65-2-253. [DOI] [PubMed] [Google Scholar]
  6. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  7. Deom C. M., Oliver M. J., Beachy R. N. The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement. Science. 1987 Jul 24;237(4813):389–394. doi: 10.1126/science.237.4813.389. [DOI] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. Gallie D. R., Plaskitt K. A., Wilson T. M. The effect of multiple dispersed copies of the origin-of-assembly sequence from TMV RNA on the morphology of pseudovirus particles assembled in vitro. Virology. 1987 Jun;158(2):473–476. doi: 10.1016/0042-6822(87)90225-x. [DOI] [PubMed] [Google Scholar]
  10. Gallie D. R., Sleat D. E., Watts J. W., Turner P. C., Wilson T. M. In vivo uncoating and efficient expression of foreign mRNAs packaged in TMV-like particles. Science. 1987 May 29;236(4805):1122–1124. doi: 10.1126/science.3472350. [DOI] [PubMed] [Google Scholar]
  11. Gallie D. R., Sleat D. E., Watts J. W., Turner P. C., Wilson T. M. The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucleic Acids Res. 1987 Apr 24;15(8):3257–3273. doi: 10.1093/nar/15.8.3257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goelet P., Lomonossoff G. P., Butler P. J., Akam M. E., Gait M. J., Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5818–5822. doi: 10.1073/pnas.79.19.5818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Leberman R. The isolation of plant viruses by means of "simple" coacervates. Virology. 1966 Nov;30(3):341–347. doi: 10.1016/0042-6822(66)90112-7. [DOI] [PubMed] [Google Scholar]
  17. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  19. Rochon D., Kelly R., Siegel A. Encapsidation of 18 S rRNA by tobacco mosaic virus coat protein. Virology. 1986 Apr 15;150(1):140–148. doi: 10.1016/0042-6822(86)90273-4. [DOI] [PubMed] [Google Scholar]
  20. Rochon D., Siegel A. Chloroplast DNA transcripts are encapsidated by tobacco mosaic virus coat protein. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1719–1723. doi: 10.1073/pnas.81.6.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Siegel A. Pseudovirions of tobacco mosaic virus. Virology. 1971 Oct;46(1):50–59. doi: 10.1016/0042-6822(71)90005-5. [DOI] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Turner D. R., Butler P. J. Essential features of the assembly origin of tobacco mosaic virus RNA as studied by directed mutagenesis. Nucleic Acids Res. 1986 Dec 9;14(23):9229–9242. doi: 10.1093/nar/14.23.9229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zimmern D., Butler P. J. The isolation of tobacco mosaic virus RNA fragments containing the origin for viral assembly. Cell. 1977 Jul;11(3):455–462. doi: 10.1016/0092-8674(77)90064-2. [DOI] [PubMed] [Google Scholar]
  27. Zimmern D., Hunter T. Point mutation in the 30-K open reading frame of TMV implicated in temperature-sensitive assembly and local lesion spreading of mutant Ni 2519. EMBO J. 1983;2(11):1893–1900. doi: 10.1002/j.1460-2075.1983.tb01676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zimmern D., Wilson T. M. Location of the origin for viral reassembly on tobacco mosaic virus RNA and its relation to stable fragment. FEBS Lett. 1976 Dec 1;71(2):294–298. doi: 10.1016/0014-5793(76)80954-4. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES