Plant Molecular Biology

, Volume 68, Issue 1–2, pp 61–79 | Cite as

P19-dependent and P19-independent reversion of F1-V gene silencing in tomato

  • M. Lucrecia Alvarez
  • Heidi L. Pinyerd
  • Emel Topal
  • Guy A. Cardineau


As a part of a project to develop a plant-made plague vaccine, we expressed the Yersinia pestis F1-V antigen fusion protein in tomato. We discovered that in some of these plants the expression of the f1-v gene was undetectable in leaves and fruit by ELISA, even though they had multiple copies of f1-v according to Southern-blot analysis. A likely explanation of these results is the phenomenon of RNA silencing, a group of RNA-based processes that produces sequence-specific inhibition of gene expression and may result in transgene silencing in plants. Here we report the reversion of the f1-v gene silencing in transgenic tomato plants through two different mechanisms. In the P19-dependent Reversion or Type I, the viral suppressor of gene silencing, P19, induces the reversion of gene silencing. In the P19-independent Reversion or Type II, the f1-v gene expression is restored after the substantial loss of gene copies as a consequence of transgene segregation in the progeny. The transient and stable expression of the p19 gene driven by a constitutive promoter as well as an ethanol inducible promoter induced a P19-dependent reversion of f1-v gene silencing. In particular, the second generation plant 3D1.6 had the highest P19 protein levels and correlated with the highest F1-V protein accumulation, almost a three-fold increase of F1-V protein levels in fruit than that previously reported for the non-silenced F1-V elite tomato lines. These results confirm the potential exploitation of P19 to substantially increase the expression of value-added proteins in plants.


P19 PTGS RNA silencing Tomato Transgenic plants Viral suppressor 



Post-transcriptional gene silencing


RNA-dependent RNA polymerase.


RNA-induced silencing complex


Transcriptional gene silencing

TBSV-P19 or P19

19 kDa viral suppressor protein of TBSV


Tomato bushy stunt virus


Total soluble protein


Double-stranded RNA


Micro RNA


Small RNA


Short interfering RNA


Single-stranded RNA


Trans-acting short interfering RNA


Virus-induced gene silencing



We are very grateful to Dr. Herman Scholthof for providing the pTBSV-100 and the primary antibody against TBSV-P19, and to Dr. Hugh Mason for plasmid pAlc:p19. We want to thank Angela Rojas and Mike Ewing for technical assistance with tissue culture and to Jason Crisantes for maintenance of the tomato plants at the greenhouse facilities. The authors are also very grateful with Paul Arnold for his help with the editing of the paper. This project was partially supported by the US Department of Defense grant DAMD17–02-2-0015.


  1. Alvarez ML, Guelman S, Halford NG, Lustig S, Reggiardo MI, Ryabushkina N, Shewry P, Stein J, Vallejos RH (2000) Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits. Theor Appl Genet 100:319–327CrossRefGoogle Scholar
  2. Alvarez ML, Pinyerd HL, Crisantes JD, Rigano MM, Pinkhasov J, Walmsley AM, Mason HS, Cardineau GA (2006) Plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice. Vaccine 24:2477–2490PubMedCrossRefGoogle Scholar
  3. Baulcombe DC (2004) RNA silencing in plants. Nature 431:356–363PubMedCrossRefGoogle Scholar
  4. Becker D, Kemper E, Schell J, Materson R (1992) New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol Biol 20:1195–1197PubMedCrossRefGoogle Scholar
  5. Beclin C, Boutet S, Waterhouse P, Vaucheret H (2002) A branched pathway for transgene-induced RNA silencing in plants. Curr Biol 12:684–688PubMedCrossRefGoogle Scholar
  6. Bernstein E, Caudy A, Hammond S, Hannon G (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366PubMedCrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248PubMedCrossRefGoogle Scholar
  8. Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends in Genet 22:268–280CrossRefGoogle Scholar
  9. Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja V, Carrington JC (2004) Viral RNA silencing suppressors inhibit the miRNA pathway at an intermediate step. Genes Dev 18:1179–1186PubMedCrossRefGoogle Scholar
  10. Curtiss R, Cardineau GA (1990) Oral immunization by transgenic plants. World patent application WO 90/02484, 22 March 1990Google Scholar
  11. De Buck S, Jacobs A, Van Montagu M, 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 20:295–304PubMedCrossRefGoogle Scholar
  12. De Buck S, Van Montagu M, Depicker A (2001) Transgene silencing of invertedly repeated transgenes is released upon deletion of one of the transgenes involved. Plant Mol Biol 46:433–445PubMedCrossRefGoogle Scholar
  13. De Wilde C, Van Houdt H, De Buck S, Angenon G, De Jaeger G, Depicker A (2000) Plants as bioreactors for protein production: avoiding the problem of Transgene silencing. Plant Mol Biol 43:347–359PubMedCrossRefGoogle Scholar
  14. Deveaux Y, Peaucelle A, Roberts GR, Coen E, Simon R, Mizukami Y, Traas J, Murray JA, Doonan JH, Laufs P (2003) The ethanol switch: a tool for tissue-specific gene induction during plant development. Plant J 36:918–930PubMedCrossRefGoogle Scholar
  15. Dunoyer P, Lecellier CH, Parizotto EA, Himber Ch, Voinnet O (2004) Probing the MicroRNA and small interfering RNA pathway with virus-encoded suppressors of RNA silencing. The Plant Cell 16:1235–1250PubMedCrossRefGoogle Scholar
  16. Elmayan T, Vaucheret H (1996) Expression of single copies of a strongly expressed 35S transgene can be silenced post-transcriptionally. Plant J 9:787–797CrossRefGoogle Scholar
  17. Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296PubMedCrossRefGoogle Scholar
  18. Haq T, Mason H, Clements J, Arntzen Ch (1995) Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268:714–716PubMedCrossRefGoogle Scholar
  19. Hearne P, Knorr D, Hillman B, Morris T (1990) The complete genome structure and synthesis of infectious RNA from clones of Tomato Bushy Stunt Virus. Virology 177:141–151PubMedCrossRefGoogle Scholar
  20. Heath D, Anderson G, Mauro M, Welkos S, Andrews G, Adamovicz J (1998) Protection against experimental bubonic and pneumonic plague by recombinant capsular F1-V antigen fusion protein vaccine. Vaccine 16:1131–1137PubMedCrossRefGoogle Scholar
  21. Hoekema A, Hirsch PR, Hooykas PJJ, Schilpperoort RA (1983) A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefasiens Ti plasmid. Nature 303:179–180CrossRefGoogle Scholar
  22. Hood E, Gelvin S, Melchers S, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218CrossRefGoogle Scholar
  23. Jorgensen RA, Cluster PD, English J, Que Q, Napoli CA (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 31:957–973PubMedCrossRefGoogle Scholar
  24. Kapila J, De Rycke R, Van montagu M, Angenon G (1997) An Agrobaterium-mediated transient gene expressión system for intact leaves. Plant Sci 122:101–108CrossRefGoogle Scholar
  25. Lakatos L, Szittya G, Silhavy D, Burgyan J (2004) Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses. EMBO J 23:876–884PubMedCrossRefGoogle Scholar
  26. Lakatos L, Csorba T, Pantaleo V, Chapman EJ, Carrington JC, Liu Y, Dolja V, Fernandez Calvino L, Lopez-moya J, Burguyan J (2006) Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. EMBO J 25:2768–2780PubMedCrossRefGoogle Scholar
  27. Li F, Ding S (2006) Virus counter-defense: diverse strategies for evading the RNA-silencing immunity. Annu Rev Microbiol 60:503–531PubMedCrossRefGoogle Scholar
  28. Lippman Z, Martienssen R (2004) The role of RNA interference in heterochromatic silencing. Nature 431:364–370PubMedCrossRefGoogle Scholar
  29. Mallory AC, Parks G, Endres MW, Baulcombe D, Bowman LH, Pruss GJ, Vance VB (2002) The amplicon-plus system for high-level expression of transgenes in plants. Nat Biotechnol 20:622–625PubMedCrossRefGoogle Scholar
  30. Marathe R, Smith TH, Anandalakshmi R, Bowman LH, Fagard M, Mourrain P, Vaucheret H, Vance VB (2000) Plant viral suppressors of post-transcriptional silencing do not suppress transcriptional silencing. Plant J 22:51–59PubMedCrossRefGoogle Scholar
  31. Matzke MA, Priming M, Trnovsky J, Matzke AJ (1989) Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J 8:643–649PubMedGoogle Scholar
  32. Meins F, Si-Ammour A, Blevins T (2005) RNA silencing systems and their relevance to plant development. Annu Rev Cell Dev Biol 21:297–318PubMedCrossRefGoogle Scholar
  33. Moissiard G, Voinnet O (2004) Viral suppression of RNA silencing in plants. Mol Plant Pathol 5:71–82CrossRefGoogle Scholar
  34. Mor TS, Mason HS, Kirk DD, Arntzen CA, Cardineau GA (2004) Plants as a production and delivery vehicles for orally delivered subunit vaccines. In: Levine MM, Woodrow GC, Kaper JB, Cobon GS (eds) New generation vaccines, 3rd edn. Marcel Dekker, New York, pp 305–311Google Scholar
  35. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous gene in trans. Plant Cell 2:279–289PubMedCrossRefGoogle Scholar
  36. Oksman-Caldentey KM, Barz W (eds) (2002) Plant biotechnology and transgenic plants. Marcel Dekker, New YorkGoogle Scholar
  37. Omarov R, Sparks K, Smith L, Zindovic J, Scholthof HB (2006) Biological relevance of a stable biochemical interaction between the tombusvirus-encoded P19 and short interfering RNAs. J Virol 80:3000–3008PubMedCrossRefGoogle Scholar
  38. Palatnik J, Alle E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D (2003) Control of leaf morphogenesis by microRNAs. Nature 425:257–263PubMedCrossRefGoogle Scholar
  39. Papp I, Mette MF, Aufzats W, Daxinger L, Schauer SE, Ray A, Van der Winden J, Matzke M, Matzke AJ (2003) Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors. Plant Physiol 132:1382–1390PubMedCrossRefGoogle Scholar
  40. Qiu W, Park JW, Scholthof HB (2002) Tombosvirus P19-mediated suppression of virus-induced gene silencing is controlled by genetic and dosage features that influence pathogenicity. Mol Plant Microbe Interact 15:269–280PubMedCrossRefGoogle Scholar
  41. Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110:513–520PubMedCrossRefGoogle Scholar
  42. Roth BM, Pruss GJ, Vance VB (2004) Plant viral suppresors of RNA silencing. Virus Res 102:97–108PubMedCrossRefGoogle Scholar
  43. Ru P, Xu L, Ma H, Huang H (2006) Plant fertility defects induced by the enhanced expression of micro-RNA167. Cell Res 16:457–465PubMedCrossRefGoogle Scholar
  44. Sambrook J, Fisher E, Maniatis T (1981) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  45. Scholthof H (2006) The Tombusvirus-encoded P19: from irrelevance to elegance. Nat Rev Microbiol 4:405–411PubMedCrossRefGoogle Scholar
  46. Scholthof H, Morris T, Jackson A (1993) The capsid protein gene of Tomato Bushy Stunt Virus is dispensable for systemic movement and can be replaced for localized expression of foreign genes. Mol Plant Microbe Interact 6:309–322Google Scholar
  47. Siddiqui S, Sarmiento C, Truve E, Lehto H, Lehto K (2008) Phenotypes and functional effects caused by various viral RNA silencing suppressors in transgenic Nicotiana benthamiana and N. tabacum. Mol Plant Microbe Interact 21:178–187PubMedCrossRefGoogle Scholar
  48. Silhavy D, Molnar A, Lucioli A, Sitia G, Homyik C, Tavazza M, Burgyan J (2002) A viral protein suppresses RNA silencing and binds, silencing-generated, 21 to 25-nucleotide double-stranded RNAs. EMBO J 21:3070–3080PubMedCrossRefGoogle Scholar
  49. Sweetman JP, Chu C, Greenland AJ, Sonnewald U, Jepson I (2002) Ethanol vapor is an efficient inducer of the alc gene expression system in model and crop plant species. Plant Physiol 129:943–948PubMedCrossRefGoogle Scholar
  50. Tang W, Newton RJ, Weidner DA (2007) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pines. J Exp Bot 58:545–554PubMedCrossRefGoogle Scholar
  51. Thanavala Y, Huang Z, Mason HS (2006) Plant-derived vaccines: a look back at the highlights and a view to the challenges on the road ahead. Expert Rev Vaccines 5:249–260PubMedCrossRefGoogle Scholar
  52. Thompson C, Movva N, Tizard R, Crameri R, Davies J, Lauwereys M, Botterman J (1987) Characterization of the herbicide resistance gene bar from Streptomyces hygroscopicus. EMBO J 6:2519–2523PubMedGoogle Scholar
  53. Van der Krol AR, Mur LA, Beld M, Mol JNM, Stuitje AR (1990) Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2:291–299PubMedCrossRefGoogle Scholar
  54. Vaucheret H (2005) Micro-RNA dependent Trans-Acting siRNA production. Sci STKE 300:43Google Scholar
  55. Vaucheret H, Beclin Ch, Elmayan T, Feuerbach F, Godon Ch, Morel JB, Mourrain P, Paulaqui JC, Vernhettes S (1998) Transgene-induced gene silencing in plants. Plant J 16:651–619PubMedCrossRefGoogle Scholar
  56. Voinnet O (2001) RNA silencing as a plant immune system against viruses. Trends Genet 17:449–459PubMedCrossRefGoogle Scholar
  57. Voinnet O (2005) Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6:206–220PubMedCrossRefGoogle Scholar
  58. Voinnet O, Pinto YM, Baulcombe DC (1999) Suppression of gene silencing : A general strategy used by diverse DNA and RNA viruses in plants. Proc Natl Acad Sci USA 99:14147–14152CrossRefGoogle Scholar
  59. Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956PubMedCrossRefGoogle Scholar
  60. Walmsley AM, Alvarez ML, Jin Y, Kirk DD, Lee SM, Pinkhasov J, Rigano MM, Arntzen Ch, Mason H (2003) Expression of the B subunit of Escherichia coli heat-labile enterotoxin as a fusion protein in transgenic tomato. Plant cell Rep 21:1020–1026PubMedCrossRefGoogle Scholar
  61. Ye K, Malinina L, Patel DJ (2003) Recognition of small interfering RNA by a viral suppressor of RNA silencing. Nature 426:874–878PubMedCrossRefGoogle Scholar
  62. Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotides intervals. Cell 101:25–33PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • M. Lucrecia Alvarez
    • 1
    • 2
  • Heidi L. Pinyerd
    • 1
    • 2
  • Emel Topal
    • 1
    • 2
  • Guy A. Cardineau
    • 1
    • 2
  1. 1.Center for Infectious Diseases and Vaccinology (CIDV)The Biodesign Institute at Arizona State UniversityTempeUSA
  2. 2.The School of Life SciencesArizona State UniversityTempeUSA

Personalised recommendations