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

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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.

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Abbreviations

PTGS:

Post-transcriptional gene silencing

RDR:

RNA-dependent RNA polymerase.

RISC:

RNA-induced silencing complex

TGS:

Transcriptional gene silencing

TBSV-P19 or P19:

19 kDa viral suppressor protein of TBSV

TBSV:

Tomato bushy stunt virus

TSP:

Total soluble protein

dsRNA:

Double-stranded RNA

miRNA:

Micro RNA

sRNA:

Small RNA

siRNA:

Short interfering RNA

ssRNA:

Single-stranded RNA

ta-siRNA:

Trans-acting short interfering RNA

VIGS:

Virus-induced gene silencing

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Acknowledgments

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.

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Authors and Affiliations

  1. Center for Infectious Diseases and Vaccinology (CIDV), The Biodesign Institute at Arizona State University, 1001 South McAllister avenue, Tempe, AZ, 85287-5401, USA

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

  2. The School of Life Sciences, Arizona State University, 1001 South McAllister Avenue, Tempe, AZ, 85287-5401, USA

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

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  1. M. Lucrecia Alvarez
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  2. Heidi L. Pinyerd
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Correspondence to M. Lucrecia Alvarez.

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Alvarez, M.L., Pinyerd, H.L., Topal, E. et al. P19-dependent and P19-independent reversion of F1-V gene silencing in tomato. Plant Mol Biol 68, 61–79 (2008). https://doi.org/10.1007/s11103-008-9352-2

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