P19-dependent and P19-independent reversion of F1-V gene silencing in tomato
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.
KeywordsP19 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
Short interfering 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.
- Curtiss R, Cardineau GA (1990) Oral immunization by transgenic plants. World patent application WO 90/02484, 22 March 1990Google Scholar
- 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
- Oksman-Caldentey KM, Barz W (eds) (2002) Plant biotechnology and transgenic plants. Marcel Dekker, New YorkGoogle Scholar
- Sambrook J, Fisher E, Maniatis T (1981) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
- 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
- Vaucheret H (2005) Micro-RNA dependent Trans-Acting siRNA production. Sci STKE 300:43Google Scholar