Biologia plantarum 2013, 57:547-554 | DOI: 10.1007/s10535-013-0314-4

Viral resistance mediated by shRNA depends on the sequence similarity and mismatched sites between the target sequence and siRNA

L. Zhang1, X. Xie1, Y. Song1, F. Jiang1, C. Zhu1,*, F. Wen1
1 State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, Shandong, P.R. China

Viral resistance can be effectively induced in transgenic plants through their silencing machinery. Thus, we designed nine short hairpin RNAs (shRNA) constructs to target nuclear inclusion protein b (NIb), helper component proteinase (HC-Pro), cylindrical inclusion protein (CI) and viral protein genome linked (VPg) genes of Potato virus Y (PVYN) and Tobacco etch virus (TEV-SD1). The shRNAs were completely complementary to the genes of PVYN, and contained 1-3 nt mismatches to the genes of TEV-SD1. To study the specificity of gene silencing in shRNA-mediated viral resistance, the constructs were introduced into tobacco plants. The results of viral resistance assay revealed that these nine kinds of transgenic tobacco plants can effectively induce viral resistance against both PVYN and TEV-SD1, and the shRNA construct targeting the NIb gene showed higher silencing efficiency. Northern blot and short interfering RNA (siRNA) analyses demonstrated that the viral resistance can be attributed to the degradation of the target RNA through the RNA silencing system. Correlation analysis of siRNA sequence characteristics with its activity suggested that the secondary structure stability of the antisense strand did not influence siRNA activity; 1 to 3 nt 5' end of the sense strand caused a significant effect on siRNA activity where the first base such as U was favourable for silencing; the base mismatch between the siRNA and the target gene may be more tolerated in the 5' end.

Keywords: Nicotiana benthamiana; Potato virus Y; RNA silencing; Tobacco etch virus
Subjects: viral resistence; plant-pathogen interaction; RNA silencing; shRNA; siRNA; transgenic plants; Nothern blot; tobacco; Potato virus Y; Tobacco etch virus
Species: Nicotiana benthamiana

Received: June 6, 2012; Accepted: November 7, 2012; Published: September 1, 2013Show citation

ACS AIP APA ASA Harvard Chicago IEEE ISO690 MLA NLM Turabian Vancouver
Zhang, L., Xie, X., Song, Y., Jiang, F., Zhu, C., & Wen, F. (2013). Viral resistance mediated by shRNA depends on the sequence similarity and mismatched sites between the target sequence and siRNA. Biologia plantarum, 57(3), 547-554. doi: 10.1007/s10535-013-0314-4.
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)

    • Open full article

    References

    1. Ambros, V.: The functions of animal microRNAs. - Nature 431: 350-355, 2004. Go to original source...
    2. Bucher, E., Lohuis, D., Van Poppel, P.M., Geerts-Dimitriadou, C., Goldbach, R., Prins, M.: Multiple virus resistance at a high frequency using a single transgene construct. - J. gen. Virol. 87: 3697-3701, 2006. Go to original source...
    3. Chan, C.Y., Carmack, C.S., Long, D.D., Maliyekkel, A., Shao, Y., Roninson, I.B., Ding, Y.: A structural interpretation of the effect of GC-content on efficiency of RNA interference. - BMC Bioinformatics 10 (Suppl 1): S33, 2009. Go to original source...
    4. Cogoni, C., Romano, N., Macino, G.: Suppression of gene expression by homologous transgenes. - Antonie Van Leeuwenhoek 65: 205-209, 1994. Go to original source...
    5. Donald, B.R., Martin, J.: Automated NMR assignment and protein structure determination using sparse dipolar coupling constraints. - Progr. nucl. magnetic Resonance Spectrosc. 55: 101-127, 2009. Go to original source...
    6. Duan, C.G., Wang, C.H., Fang, R.X., Guo, H.S.: Artificial microRNAs highly accessible to targets confer efficient virus resistance in plants. - J. Virol. 82: 11084-11095, 2008. Go to original source...
    7. Fahim, M., Ayala-Navarrete, L., Millar, A.A., Larkin, P..J.: Hairpin RNA derived from viral NIa gene confer immunity wheat streak mosaic virus infection in transgenic wheat plants. - Plant Biotechnol. J. 8: 821-834, 2010. Go to original source...
    8. Fitchen, J.H., Beachy, R.N.: Genetically engineered protection against viruses in transgenic plants. - Annu. Rev. Microbiol. 47: 739-763, 1993. Go to original source...
    9. Gil, J., Esteban, M.: Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. - Apoptosis 5: 107-114, 2000. Go to original source...
    10. Guo, X.Q., Liu, S.E., Zhu, C,X., Song, Y.Z., Meng, X.B., Zheng, C.C., Wen, F.J.: RNA mediated viral resistance against potato virus Y (PVY) in transgenic tobacco plants. - Acta phytopathol. sin. 31: 349-356, 2011.
    11. Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E., Prydz, H.: Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. - Nucl. Acids Res. 30: 1757-1766, 2002. Go to original source...
    12. Jackson, A.L., Bartz, S.R., Schelter, J., Kobayashi, S.V., Burchard, J., Mao, M., Li, B., Cavet, G., Linsley, P.S.: Expression profiling reveals off-target gene regulation by RNAi. - Nat. Biotechnol. 21: 635-637, 2003. Go to original source...
    13. Jiang, F., Wu, B., Zhang, C., Song, Y., An, H., Zhu, C., Wen, F.: Special origin of stem sequence influence the resistance of hairpin expressing plants against PVY. - Biol. Plant. 55: 528-535, 2011. Go to original source...
    14. Lindbo J.A, Dougherty W. G.. Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts. - Virology 189: 725-733, 1992. Go to original source...
    15. Lindbo, J.A., Silva-Rosales, L., Proebsting, W.M., Dougherty, W.G.: Induction of a highly specific antiviral state in transgenic plant: Implications for regulation of gene expression and virus resistance. - Plant Cell 5: 1749-1759, 1993. Go to original source...
    16. Lucioli, A., Noris, E., Brunetti, A., Tavazza, R., Ruzza, V., Castillo, A.G., Bejarano, E.R., Accotto, G.P., Tavazza, M.: Tomato yellow leaf curl Sardinia virus Rep-derived resistance to homologous and heterologous Geminivirus occurs by different mechanisms and is overcome if virus-mediated transgene silencing is activated. - J. Virol. 77: 6785-6798, 2003. Go to original source...
    17. Molnár, A., Csorba, T., Lakatos, L., Várallyay, E., Lacomme, C., Burgyán, J.: Plant virus-derived small interference RNAs originate predominantly from highly structured single-stranded viral RNAs. - J. Virol. 79: 7812-7818, 2005. Go to original source...
    18. Patzel, V., Rutz, S., Dietrich, I., Koberle, C., Scheffold, A., Kaufmann, S.H.: Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency. - Natur. Biotechnol. 23: 1440-1444, 2005. Go to original source...
    19. Pusch, O., Boden, D., Silbermann, R., Lee, F., Tucker, L., Ramratnam, B.: Nucleotide sequence homology requirements of HIV-1-specific short hairpin RNA. - Nucl. Acids Res. 31: 6444-6449, 2003. Go to original source...
    20. Qi, X., Ba, F.S., Xie, Z.: Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis. - PLoS One 4: e4971, 2009. Go to original source...
    21. Qu, J., Ye, J., Fang, R.: Artificial microRNA-mediated virus resistance in plants. - J. Virol. 81: 6690-6699, 2007. Go to original source...
    22. Ramesh, S.V., Mishra, A.K., Praveen, S.: Hairpin RNA-mediated strategies for silencing of tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants. - Oligonucleotides 17: 251-257, 2007. Go to original source...
    23. Sijen, T., Steiner, F.A., Thijssen, K.L., Thijssen, K., Plasterk, R.: Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. - Science 12: 244-247, 2007. Go to original source...
    24. Szittya, G., Silhavy, D., Molnar, A., Havelda, Z., Lovas, A., Lakatos, L., Banfalvi Z., Burgyan J.: Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. - EMBO J. 22: 633-640, 2003. Go to original source...
    25. Wesley, S.V., Helliwell, C.A., Smith, N.A., Wang, M.B., Rouse, D.T., Liu, Q., Gooding, P.S., Singh, S.P., Abbott, D., Stoutjesdijk, P.A., Robinson, S.P., Gleave, A.P., Green, A.G., Waterhouse, P.M.: Construct design for efficient, effective and high throughout gene silencing in plants. - Plant J. 27: 581-590, 2001. Go to original source...
    26. Xu, L., Song, Y.Z., Zhu, J.H.: Conserved sequences of replicase gene-mediated resistance to potyvirus through RNA silencing. - J. Plant Biol. 9: 550-559, 2009. Go to original source...
    27. Yan, P.Q., Li, L.J., Kang, H.: Cultivation transgenic tobacco against two viruses depending on RNAi. - China Bioeng. 27: 27-31, 2007.
    28. Yu, J.Y., Wang, T.W., Vojtek, A.B., Parent, J.M., Turner, D.L.: Use of short hairpin RNA expression vectors to study mammalian neural development. - Methods Enzymol. 392: 186-199, 2005. Go to original source...
    29. Zamore, P.D.: Ancient pathways programmed by small RNAs. - Science 296: 1265-1269, 2002. Go to original source...
    30. Zeng, Y., Cullen, B.R.: Sequence requirements for micro RNA processing and function in human cell. - RNA 9: 112-123, 2003.
    31. Zhu, C.X., Song, Y.Z., Yin, G.H.: Induction of RNA-mediated multiple virus resistance to potato Y, tomato mosaic and cucumber mosaic viruses. - J. Phytopathol. 157: 101-107, 2009. Go to original source...

    Return to the content