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.
Alvarez
M Lucrecia
ML
Center for Infectious Diseases and Vaccinology, The Biodesign Institute at Arizona State University, Tempe, AZ 85287-5401, USA. lucrecia.alvarez@asu.edu
Pinyerd
Heidi L
HL
Topal
Emel
E
Cardineau
Guy A
GA
D000942:Antigens, Bacterial / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015139:Blotting, Southern; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D000431:Ethanol / Q000494:pharmacology; D005638:Fruit / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression / Q000187:drug effects; D050939:Gene Fusion; D020868:Gene Silencing; D018551:Solanum lycopersicum / Q000235:genetics* / Q000254:growth & development; D018515:Plant Leaves / Q000235:genetics / Q000254:growth & development; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C043908:LcrV protein, Yersinia; D052899:Pore Forming Cytotoxic Proteins; C086725:caf1 protein, Yersinia pestis; D000431:Ethanol