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.
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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
References
Aravin A, Tuschl T (2005) Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett 579:5830–5840. doi:10.1016/j.febslet.2005.08.009
Arzola L, Chen J, Rattanaporn K, Maclean JM, McDonald KA (2011) Transient co-expression of post-transcriptional gene silencing suppressors for Increased in planta expression of a recombinant anthrax receptor fusion protein. Int J Mol Sci 12:4975–4990. doi:10.3390/ijms12084975
Baulcombe DC, Molnár A (2004) Crystal structure of p19—a universal suppressor of RNA silencing. Trends Biochem Sci 29:279–281. doi:10.1016/j.tibs.2004.04.007
Boivin EB, Lepage E, Matton DP, De Crescenzo G, Jolicoeur M (2010) Transient expression of antibodies in suspension plant cell suspension cultures is enhanced when co-transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing. Biotechnol Progr 26:1534–1543. doi:10.1002/btpr.485
Bourque JE (1995) Antisense strategies for genetic manipulations in plants. Plant Sci 105:125–149. doi:10.1016/0168-9452(94)04051-6
Brown PH, Ho TD (1987) Biochemical properties and hormonal regulation of barley nuclease. Eur J Biochem 168:357–364. doi:10.1111/j.1432-1033.1987.tb13427.x
Dean JD, Goodwin PH, Hsiang T (2002) Comparison of relative RT-PCR and northern blot analyses to measure expression of beta-1,3-glucanase in Nicotiana benthamiana infected with Colltotrichum destructivum. Plant Mol Biol Rep 20:347–356. doi:10.1007/BF02772122
Fang EF, Ng TB (2011) Ribonucleases of different origins with a wide spectrum of medicinal applications. BBA-Rev Cancer 1815:65–74. doi:10.1016/j.bbcan.2010.09.001
Farage-Barhom S, Burd S, Sonego L, Mett A, Belausov E, Gidoni D, Lers A (2011) Localization of the Arabidopsis senescence- and cell death-associated BFN1 nuclease: from the ER to fragmented nuclei. Mol Plant 4:1062–1073. doi:10.1093/mp/ssr045
Garabagi F, Gilbert E, Loos A, McLean MD, Hall JC (2012) Utility of the P19 suppressor of gene-silencing protein for production of therapeutic antibodies in Nicotiana benthamiana expression hosts. Plant Biotechnol J 10:1118–1128. doi:10.1111/j.1467-7652.2012.00742.x
German MA, Luo S, Schroth G, Meyers BC, Green PJ (2009) Construction of parallel analysis of RNA ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome. Nat Protoc 4:356–362. doi:10.1038/nprot.2009.8
Green PJ (1994) The ribonucleases of higher plants. Annu Rev Plant Phys Plant Mol Biol 45:421–445. doi:10.1146/annurev.pp.45.060194.002225
Hellens R, Mullineaux P, Klee H (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446–451. doi:10.1016/S1360-1385(00)01740-4
Ito J, Fukuda H (2002) ZEN1 is a key enzyme in the degradation of nuclear DNA during programmed cell death of tracheary elements. Plant Cell 14:3201–3211. doi:10.1105/tpc.006411
Jin H, Zhu JK (2010) A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes. Gene Dev 24:853–856. doi:10.1101/gad.1927310
Johansen W, Wilson RC (2008) Viral suppressor proteins show varying abilities and effectiveness to suppress transgene-induced post-transcriptional gene silencing of endogenous Chalcone synthase in transgenic Arabidopsis. Plant Cell Rep 27:911–921. doi:10.1007/s00299-008-0508-y
Kovaľ T, Lipovová P, Podzimek T, Matoušek J, Dušková J, Skálová T, Štěpánková A, Hašek J, Dohnálek J (2013) Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis. Acta Crystallogr D 69:213–226. doi:10.1107/S0907444912043697
Kozomara A, Griffiths-Jones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42:D68–D73. doi:10.1093/nar/gkt1181
Krüger J, Rehmsmeier M (2006) RNAhybrid: microRNA target prediction easy, fast and flexible. Nucleic Acids Res 34:W451–W454. doi:10.1093/nar/gkl243
Matoušek J, Matoušek J (2010) Plant ribonuclease and nucleases as antiproliferative agens targets human tumors growing in mice. Recent Pat DNA Gene Seq 4:29–39. doi:10.2174/187221510790410813
Matoušek J, Vrba L, Škopek J, Orctová L, Pešina K, Heyerick A, Baulcombe D, De Keukeleire D (2006) Sequence analysis of a “true” chalcone synthase (chs_H1) oligofamily from hop (Humulus lupulus L.) and PAP1 activation of chs_H1 in heterologous systems. J Agr Food Chem 54:7606–7615. doi:10.1021/jf061785g
Matoušek J, Kozlová P, Orctová L, Schmitz A, Pešina K, Bannach O, Diermann N, Steger G, Riesner D (2007) Accumulation of viroid-specific small RNAs and increase of nucleolytic activities linked to viroid-caused pathogenesis. Biol Chem 388:1–13. doi:10.1515/BC.2007.001
Matoušek J, Orctová L, Škopek J, Pešina K, Steger G (2008) Elimination of hop latent viroid upon developmental activation of pollen nucleases. Biol Chem 389:905–918. doi:10.1515/BC.2008.096
Matoušek J, Podzimek T, Poučková P, Stehlík J, Škvor J, Souček J, Matoušek J (2009) Antitumor effects and cytotoxicity of recombinant plant nucleases. Oncol Res 18:163–171. doi:10.3727/096504009790217425
Matoušek J, Podzimek T, Poučková P, Stehlík J, Škvor J, Lipovová P, Matoušek J (2010) Antitumor activity of apoptotic nuclease TBN1 from L. esculentum. Neoplasma 57:339–348. doi:10.4149/neo_2010_04_339
Matoušek J, Kocábek T, Patzak J, Füssy Z, Procházková J, Heyerick A (2012) Combinatorial analysis of lupulin gland transcription factors from R2R3Myb, bHLH and WDR families indicates a complex regulation of chs_H1 genes essential for prenylflavonoid biosynthesis in hop (Humulus Lupulus L.) BMC Plant Biol 12:27. doi:10.1186/1471-2229-12-27
Matoušek J, Piernikarczyk RJJ, Týcová A, Duraisamy GS, Kocábek T, Steger G (2015) Expression of SANT/HTH Myb mRNA, a plant morphogenesis-regulating transcription factor, changes due to viroid infection. J Plant Physiol 183:85–94. doi:10.1016/j.jplph.2015.06.001
Muramoto Y, Watanabe A, Nakamura T, Takabe T (1999) Enhanced expression of a nuclease gene in leaves of barley plants under salt stress. Gene 234:315–321. doi:10.1016/S0378-1119(99)00193-6
Panavas T, Pikula A, Reid PD, Rubinstein B, Walker EL (1999) Identification of senescence-associated genes from daylily petals. Plant Mol Biol 40:237–248. doi:10.1023/A:1006146230602
Pérez-Amador MA, Abler M, Jay de Rocher E, Thompson DM, van Hoof A, LeBrasseur ND, Lers A, Green PJ (2000) Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis. Plant Physiol 122:169–179. doi:10.1104/pp.122.1.169
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45. doi:10.1093/nar/29.9.e45
Podzimek T, Matoušek J, Lipovová P, Poučková P, Spiwok V, Šantrůček J (2011) Biochemical properties of three plant nucleases with anticancer potential. Plant Sci 180:343–351. doi:10.1016/j.plantsci.2010.10.006
Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66. doi:10.1016/S0304-3940(02)01423-4
Schumacher J, Meyer N, Riesner D, Weidemann HL (1986) Diagnostic procedure for detection of viroids and viruses with circular RNAs by “return”-gel electrophoresis. J Phytopathol 115:332–343. doi:10.1111/j.1439-0434.1986.tb04346.x
Schwach F, Vaistij FE, Jones L, Baulcombe DC (2005) An RNA-dependent RNA polymerase prevents meristem invasion by potato virus X and is required for the activity but not the production of a systemic silencing signal. Plant Physiol 138:1842–1852. doi:10.1104/pp.105.063537
Siddiqui SA, Sarmiento C, Truve E, Lehto H, Lehto K (2008) Phenotypes and functional effects caused by various viral RNA silencing suppressors in transgenic Nicotiana benthamina and N. tabacum. Mol Plant-Microbe Interact 21:178–187. doi:10.1094/MPMI-21-2-0178
Thomas CL, Lech V, Lederer C, Maule AJ (2003) Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306:33–41. doi:10.1016/S0042-6822(02)00018-1
Vargason JM, Szittya G, Burgyán J, Hall TMT (2003) Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115:799–811. doi:10.1016/S0092-8674(03)00984-X
Veluthambi K, Gupta AK, Sharma A (2003) The current status of plant transformation technologies. Curr Sci India 84:368–380
Voinnet O (2005) Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6:206–220. doi:10.1038/nrg1555
Voinnet O, Pinto YM, Baulcombe DC (1999) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. P Natl Acad Sci USA 96:14147–14152. doi:10.1073/pnas.96.24.14147
Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based in suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956. doi:10.1046/j.1365-313X.2003.01676.x
Vrba L, Matoušek J (2005) Expression of modified 7SL RNA gene in transgenic Solanum tuberosum plants. Biol Plantarum 49:371–380. doi:10.1007/s10535-005-0010-0
Willmann MR, Endres MW, Cook RT, Gregory BD (2011) The functions of RNA-dependent RNA polymerases in Arabidopsis. Arabidopsis Book 9:e0146. doi:10.1199/tab.0146
Wilson CM (1982) Plant nucleases: biochemistry and development of multiple molecular forms. Isozymes-Curr T Biol 6:33–54
Xia Z, Zhu Z, Zhu J, Zhou R (2009) Recognition mechanism of siRNA by viral p19 suppressor of RNA silencing: a molecular dynamics study. Biophys J 96:1761–1769. doi:10.1016/j.bpj.2008.11.047
Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:0642–0652. doi:10.1371/journal.pbio.0020104
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.
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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.
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Týcová, A., Piernikarczyk, R.J.J., Kugler, M. et al. A 5′P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells. Plant Cell Tiss Organ Cult 127, 347–358 (2016). https://doi.org/10.1007/s11240-016-1054-x
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DOI: https://doi.org/10.1007/s11240-016-1054-x