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

Plant Mol Biol. 2008 Sep;68(1-2):61-79. doi: 10.1007/s11103-008-9352-2. Epub 2008 Jun 5.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antigens, Bacterial / genetics*
  • Antigens, Bacterial / metabolism
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Blotting, Southern
  • Blotting, Western
  • Enzyme-Linked Immunosorbent Assay
  • Ethanol / pharmacology
  • Fruit / genetics
  • Fruit / growth & development
  • Gene Expression / drug effects
  • Gene Fusion
  • Gene Silencing*
  • Plant Leaves / genetics
  • Plant Leaves / growth & development
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / growth & development
  • Pore Forming Cytotoxic Proteins / genetics*
  • Pore Forming Cytotoxic Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Solanum lycopersicum / genetics*
  • Solanum lycopersicum / growth & development
  • Transformation, Genetic

Substances

  • Antigens, Bacterial
  • Bacterial Proteins
  • LcrV protein, Yersinia
  • Pore Forming Cytotoxic Proteins
  • caf1 protein, Yersinia pestis
  • Ethanol