Reactivation of rolB transgene expression in Vitis amurensis Rupr. cells upon retransformation with 2b gene from Cucumovirus isolate NK

  • Alexey P. TyuninEmail author
  • Nikolay N. Nityagovsky
  • Yuri A. Karetin
  • Konstantin V. Kiselev
Original Research Paper



Studies concerning 2b protein from Cucumovirus showed 2b to effectively repress functioning of the plant silencing complex, current study aimed whether retransformation with 2b gene able to restore silenced transgene expression in plant cells.


A rolB-transgenic cell culture of Vitis amurensis Rupr. that was continuously subcultured during more than 10 years and exhibited decreased transcription of the rolB transgene was retransformed with the 2b gene of Cucumovirus-NK. Three cell lines retransformed with 2b showed a significant up-regulation of rolB expression accompanied with enhancements in their stilbenes content level in more than 2,7-fold compared to parental rolB-transgenic cell line. The mentioned increase in the level of stilbenes content was due to activation of certain stilbene synthase genes expression responsible for stilbenes biosynthesis in V. amurensis cells. Restoration of rolB expression upon 2b-retransformation led to increase in the expression levels of VaSTS2-VaSTS5 and VaSTS7 isoforms.


2b from CMV-NK can reactivate a silenced transgene expression, even after 10 years of subcultivation, nevertheless, optimization of the methods concerning 2b introduction in plant genomes is necessary to avoid undesirable silencing effects.


2b protein Cucumovirus Stilbenes Stilbene synthase t-Resveratrol Transgene silencing 



Authors express their gratitude to Dr. Nesmelov I.B. for providing cDNA of CMV-NK and to Dr. Dubrovina A.S. for vise recommendation on article preparation. This work was supported by The Russian Foundation for Basic Research, Grant Number: 17-04-01381.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10529_2018_2642_MOESM1_ESM.doc (34 kb)
Supplementary material 1 (DOC 33 kb)


  1. Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, Takada Y (2004) Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res 24:2783–2840Google Scholar
  2. Aleynova OA, Dubrovina AS, Manyakhin AY, Karetin YA, Kiselev KV (2015) Regulation of resveratrol production in Vitis amurensis cell cultures by calcium-dependent protein kinases. Appl Biochem Biotechnol 175:1460–1476CrossRefGoogle Scholar
  3. Bekesiova I, Nap JP, Mlynarova L (1999) Isolation of high quality DNA and RNA from leaves of the carnivorous plant Drosera rotundifolia. Plant Mol Biol Rep 17:269–277CrossRefGoogle Scholar
  4. Butaye KMJ, Cammue BPA, Delaure SL, De Bolle MFC (2005) Approaches to minimize variation of transgene expression in plants. Mol Breed 16:79–91CrossRefGoogle Scholar
  5. Diveki Z, Salanki K, Balazs E (2004) The necrotic pathotype of the cucumber mosaic virus (CMV) ns strain is solely determined by amino acid 461 of the 1a protein. Mol Plant Microbe Interact 17:837–845CrossRefGoogle Scholar
  6. Du Z, Chen F, Zhao Z, Liao Q, Palukaitis P, Chen J (2008) The 2b protein and the C-terminus of the 2a protein of Cucumber mosaic virus subgroup I strains both play a role in viral accumulation and induction of symptoms. Virology 380:363–370CrossRefGoogle Scholar
  7. Dubrovina AS, Kiselev KV (2012) Effect of long-term cultivation on resveratrol accumulation in a high-producing cell culture of Vitis amurensis. Acta Physiol Plant 34:1101–1106CrossRefGoogle Scholar
  8. Gelvin SB, Kim SI (2007) Effect of chromatin upon Agrobacterium T-DNA integration and transgene expression. Biochim Biophys Acta-Gene Struct Exp 1769:410–421CrossRefGoogle Scholar
  9. Goto K, Kobori T, Kosaka Y, Natsuaki T, Masuta C (2007) Characterization of silencing suppressor 2b of Cucumber mosaic virus based on examination of its small RNA-binding abilities. Plant Cell Physiol 48:1050–1060CrossRefGoogle Scholar
  10. Hamera S, Yan Y, Song X, Chaudhary SU, Murtaza I, Su L, Tariq M, Chen X, Fang R (2016) Expression of Cucumber mosaic virus suppressor 2b alters FWA methylation and its siRNA accumulation in Arabidopsis thaliana. Biol Open 5:1727–1734CrossRefGoogle Scholar
  11. Hou WN, Duan CG, Fang RX, Zhou XY, Guo HS (2011) Satellite RNA reduces expression of the 2b suppressor protein resulting in the attenuation of symptoms caused by Cucumber mosaic virus infection. Mol Plant Pathol 12:595–605CrossRefGoogle Scholar
  12. Inaba JI, Kim BM, Shimura H, Masuta C (2011) Virus-induced necrosis is aconsequence of direct protein-protein interaction between a viral RNA-silencing suppressor and a host catalase. Plant Physiol 156:2026–2036CrossRefGoogle Scholar
  13. Kiselev KV (2011) Perspectives for production and application of resveratrol. Appl Microbiol Biotechnol 90:417–425CrossRefGoogle Scholar
  14. Kiselev KV, Dubrovina AS, Veselova MV, Bulgakov VP, Fedoreyev SA, Zhuravlev YN (2007) The rolB gene-induced overproduction of resveratrol in Vitis amurensis transformed cells. J Biotechnol 128:681–692CrossRefGoogle Scholar
  15. Kiselev KV, Dubrovina AS, Bulgakov VP (2009) Phenylalanine ammonialyase and stilbene synthase gene expression in rolB transgenic cell cultures of Vitis amurensis. Appl Microbiol Biotechnol 82:647–655CrossRefGoogle Scholar
  16. Kiselev KV, Tyunin AP, Karetin YA (2013) Influence of 5-azacytidine and salicylic acid on demethylase gene expression in cell cultures of Vitis amurensis Rupr. Acta Physiol Plant 35:1843–1851CrossRefGoogle Scholar
  17. Koizumi M, Shimotori Y, Saeki Y, Hirai S, Oka SI, Kodama H (2017) Effects of the 2b protein of Cucumber mosaic virus subgroup IB strain IA on different transgene-induced RNA silencing pathways. Plant Mol Biol Report 35:265–272CrossRefGoogle Scholar
  18. Langcake P, Pryce RJ (1977) A new class of phytoalexins from grapevines. Experientia 33:151–152CrossRefGoogle Scholar
  19. Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11:204-220Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408Google Scholar
  20. Murota K, Shimura H, Takeshita M, Chikara M (2017) Interaction between Cucumber mosaic virus 2b protein and plant catalase induces a specific necrosis in association with proteasome activity. Plant Cell Rep 36:37–47CrossRefGoogle Scholar
  21. Palukaitis P, Garcia-Arenal F (2003) Cucumoviruses. Adv Virus Res 62:241–323CrossRefGoogle Scholar
  22. Pumplin N, Voinnet O (2013) RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nat Rev Microbiol 11:745–760CrossRefGoogle Scholar
  23. Roossinck MJ (2002) Evolutionary history of Cucumber mosaic virus deduced by phylogenetic analyses. J Virol 76:3382–3387CrossRefGoogle Scholar
  24. Shankar S, Singh G, Srivastava RK (2007) Chemoprevention by resveratrol: molecular mechanisms and therapeutic potential. Front Biosci 12:4839–4854CrossRefGoogle Scholar
  25. Shumakova OA, Manyakhin AY, Kiselev KV (2011) Resveratrol content and expression of phenylalanine ammonia-lyase and stilbene synthase genes in cell cultures of Vitis amurensis treated with coumaric acid. Appl Biochem Biotechnol 165:1427–1436CrossRefGoogle Scholar
  26. Siddiqui SA, Valkonen JPT, Rajamaki ML, Lehto K (2011) The 2b silencing suppressor of a mild strain of Cucumber mosaic virus alone is sufficient for synergistic interaction with Tobacco mosaic virus and induction of severe leaf malformation in 2b-transgenic tobacco plants. Mol Plant Microbe Interact 24:685–693CrossRefGoogle Scholar
  27. Tyunin AP, Kiselev KV, Zhuravlev YN (2012) Effects of 5-azacytidine induced DNA demethylation on methyltransferase gene expression and resveratrol production in cell cultures of Vitis amurensis. Plant Cell Tissue Organ Cult 111:91–100CrossRefGoogle Scholar
  28. Tzfira T, Tian GW, Lacroix B, Vyas S, Li JX, Leitner-Dagan Y, Krichevsky A, Taylor T, Vainstein A, Citovsky V (2005) PSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants. Plant Mol Biol 57:503–516CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Laboratory of Biotechnology, Federal Scientific Center of East Asia Terrestrial BiodiversityFar Eastern Branch of Russian Academy of SciencesVladivostokRussia
  2. 2.Department of Biochemistry and BiotechnologyFar Eastern Federal UniversityVladivostokRussia
  3. 3.Department of Cell Biology and Genetics, The School of Natural SciencesFar Eastern Federal UniversityVladivostokRussia
  4. 4.Laboratory of Embryology, National Scientific Center of Marine BiologyFar Eastern Branch of the Russian Academy of SciencesVladivostokRussia

Personalised recommendations