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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 127, Issue 2, pp 347–358 | Cite as

A 5′P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells

  • Anna Týcová
  • Rajen J. J. Piernikarczyk
  • Michael Kugler
  • Petra Lipovová
  • Tomáš Podzimek
  • Gerhard Steger
  • Jaroslav MatoušekEmail author
Original Article

Abstract

Tomato bifunctional nuclease 1 (TBN1) is a polyfunctional protein with anticancerogenic activity originally isolated as an overexpressed protein from viroid-infected tomato. Its molecular farming in plant cells could be a non-expensive source for its biotechnology preparation. So we analysed TBN1 expression in Agrobacterium-infiltrated leaf sectors of Nicotiana benthamiana and in transformed suspension culture of tobacco BY-2 cells. During its transient expression, TBN1 mRNA was strongly degraded within a hot spot localized in the 3′ region. This early degradation process was inhibited by PTGS suppressors p19 and p38 resulting in increased TBN1 mRNA and protein yield. In parallel to degradation of TBN1 mRNA, high mRNA levels of two RNA-dependent RNA polymerases were detected in infiltrated leaf sectors, as well as in the transformed tobacco suspension culture BY-2, where low expression of the nuclease was stably maintained. Higher TBN1 mRNA and nuclease activity levels were found during its molecular farming in RDR6-deficient N. benthamiana plants. By fluorescent microscopy of infiltrated and transformed plant cells, the nuclease-GFP fusion protein was shown to be organized in filament-like structures.

Keywords

Plant nuclease I mRNA quantification siRNA Nicotiana benthamiana Nicotiana tabacum Post-transcriptional gene silencing 

Abbreviations

PTGS

Post-transcriptional gene silencing

TBN1

Tomato bifunctional nuclease 1

NBBN1

Nicotiana benthamiana nuclease 1

RDR

RNA-dependent RNA polymerase

qRT-PCR

Real-time quantitative reverse-transcription polymerase-chain-reaction

Notes

Acknowledgments

Authors acknowledge Ing. Lidmila Orctová, Ing. Olga Horáková, Helena Matoušková, and Dr. G. S. Duraisamy (BC CAS, v. v. i., IPMB) for their help and excellent technical assistance. Authors would like to acknowledge Dr. A. K. Mishra (BC CAS, v. v. i., IPMB) for his help and reading of the manuscript. The project was supported by the Alexander von Humboldt Foundation, Research Group Linkage Programme, by the cooperative project FP7-REGPOT-2012-2013-1 MODBIOLIN No. 316304, and by the institutional support RVO:60077344 of the Biology Centre of the CAS, v. v. i., IPMB. The support by Grantová agentura Jihočeské univerzity v Českých Budějovicích Grant No. 143/2013/P is also acknowledged.

Author contributions

J. M., P. L. planned the experiments; A. T., R. J. J. P., M. K., P. L., J. M. performed the experiments and analysed the data. A. T., R. J. J. P., P. L., T. P., G. S. and J. M. wrote the paper.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

Supplementary material 1 (AVI 14382 KB)

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Supplementary material 2 (AVI 1782 KB)
11240_2016_1054_MOESM3_ESM.pdf (950 kb)
Supplementary material 3 (PDF 949 KB)
11240_2016_1054_MOESM4_ESM.pdf (14 kb)
Supplementary material 4 (PDF 14 KB)

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Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Anna Týcová
    • 1
    • 2
  • Rajen J. J. Piernikarczyk
    • 3
  • Michael Kugler
    • 4
  • Petra Lipovová
    • 4
  • Tomáš Podzimek
    • 4
  • Gerhard Steger
    • 3
  • Jaroslav Matoušek
    • 1
    Email author
  1. 1.Biology Centre of the CASv. v. i., Institute of Plant Molecular BiologyČeské BudějoviceCzech Republic
  2. 2.Faculty of ScienceUniversity of South Bohemia in České BudějoviceČeské BudějoviceCzech Republic
  3. 3.Institute of Physical BiologyHeinrich-Heine-Universität DüsseldorfDüsseldorfGermany
  4. 4.University of Chemistry and Technology PraguePragueCzech Republic

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