ABSTRACT
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.
Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Gene Silencing , Pore Forming Cytotoxic Proteins/genetics , Solanum lycopersicum/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Blotting, Southern , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Ethanol/pharmacology , Fruit/genetics , Fruit/growth & development , Gene Expression/drug effects , Gene Fusion , Solanum lycopersicum/growth & development , Plant Leaves/genetics , Plant Leaves/growth & development , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Pore Forming Cytotoxic Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transformation, GeneticABSTRACT
Yersinia pestis, the causative agent of plague, is an extremely virulent bacterium but there are no approved vaccines for protection against it. Our goal was to produce a vaccine that would address: ease of delivery, mucosal efficacy, safety, rapid scalability, and cost. We developed a novel production and delivery system for a plague vaccine of a Y. pestis F1-V antigen fusion protein expressed in tomato. Immunogenicity of the F1-V transgenic tomatoes was confirmed in mice that were primed subcutaneously with bacterially-produced F1-V and boosted orally with transgenic tomato fruit. Expression of the plague antigens in fruit allowed producing an oral vaccine candidate without protein purification and with minimal processing technology.
