A 5′P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells
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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.
KeywordsPlant nuclease I mRNA quantification siRNA Nicotiana benthamiana Nicotiana tabacum Post-transcriptional gene silencing
Post-transcriptional gene silencing
Tomato bifunctional nuclease 1
Nicotiana benthamiana nuclease 1
RNA-dependent RNA polymerase
Real-time quantitative reverse-transcription polymerase-chain-reaction
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.
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.
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