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Construction of Mismatched Inverted Repeat (IR) Silencing Vectors for Maximizing IR Stability and Effective Gene Silencing in Plants

  • M. E. Chrissie Rey
  • Johan Harmse
  • Sarah H. Taylor
  • Patrick Arbuthnot
  • Marc S. Weinberg
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1287)

Abstract

Inverted repeat (IR) RNA silencing vectors containing homologous fragments of target endogenous plant genes, or pathogen genes, are the most widely used vectors to either study the function of genes involved in biotic stress or silence pathogens to induce plant resistance, respectively. RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of IR constructs during cloning and plant transformation. We have therefore developed a robust method for the production of long IR vector constructs by introducing base pair mismatches in the form of cytosine to thymine mutations on the sense arm by exposure to sodium bisulfite prior to assembly of the IR.

Key words

Inverted repeat RNA silencing Plants Mismatches Sodium bisulfite 

Notes

Acknowledgements

We would like to thank the South African National Research Foundation, Casquip Starch Manufacturing Pty Ltd. (Jim Casey), and The Technical Innovation Agency for financial contributions to this project.

References

  1. 1.
    Pumplin N, Voinnet O (2013) RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nat Rev Microbiol 11:745–760CrossRefPubMedGoogle Scholar
  2. 2.
    Wang MB, Masuta C, Smith NA, Shimura H (2012) RNA silencing and plant viral diseases. Mol Plant Microb Interact 25(10):1275–1285CrossRefGoogle Scholar
  3. 3.
    Ruiz-Ferrer V, Voinnet O (2009) Roles of plant small RNAs in biotic stress responses. Annu Rev Plant Biol 60:485–510CrossRefPubMedGoogle Scholar
  4. 4.
    Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends Genet 22:268–280CrossRefPubMedGoogle Scholar
  5. 5.
    Pooggin MM (2013) How can plant viruses evade siRNA-directed DNA methylation and silencing? Int J Mol Sci 14:15233–15259CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Vanderschuren H, Stupak M, Futterer J et al (2007) Engineering resistance to geminiviruses—review and perspectives. Plant Biotechnol J 5:207–220CrossRefPubMedGoogle Scholar
  7. 7.
    Aregger M, Borah BK, Seguin J et al (2012) Primary and secondary siRNAs in geminivirus-induced gene silencing. PLoS Pathog 8:e1002941CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Llave C (2010) Virus-derived small interfering RNAs at the core of plant-virus interactions. Trends Plant Sci 15:701–707CrossRefPubMedGoogle Scholar
  9. 9.
    Pantaleo V (2011) Plant RNA silencing in viral defence. Adv Exp Med Biol 722:39–58CrossRefPubMedGoogle Scholar
  10. 10.
    Rajeswaran R, Pooggin MM (2012) Role of virus-derived small RNAs in plant antiviral defence: insights from DNA viruses. In: Sunkar R (ed) MicroRNAs in plant development and stress response. Springer-Verlag, Berlin, pp 261–289CrossRefGoogle Scholar
  11. 11.
    Hohn T, Vazquez F (2011) RNA silencing pathways of plants: Silencing and its suppression by plant DNA viruses. Biochim Biophys Acta 1809:588–600CrossRefPubMedGoogle Scholar
  12. 12.
    Zvereva AS, Pooggin MM (2012) Silencing and innate immunity in plant defense against viral and non-viral pathogens. Viruses 4:2578–2597CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Jackson AL, Linsley PS (2004) Noise amidst the silence: off-target effects of siRNAs? Trends Genet 20:521–524CrossRefPubMedGoogle Scholar
  14. 14.
    Senthil-Kumar M, Mysore KS (2011) Caveat of RNAi in plants: the off-target effect. In: Kodama H, Komamine A (eds) RNAi and plant gene function analysis, methods in molecular biology 744. Human, New York, pp 13–25CrossRefGoogle Scholar
  15. 15.
    Chuang CF, Meyerowitz EM (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci U S A 97:4985–4990CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Gebow D, Miselis N, Liber HL (2000) Homologous and non-homologous recombination resulting in deletion: effects of p53 status, micro-homology, and repetitive DNA length and orientation. Mol Cell Biol 20:4028–4035CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Brunier D, Michel B, Ehrlich SD (1988) Copy choice illegitimate DNA recombination. Cell 52:883–892CrossRefPubMedGoogle Scholar
  18. 18.
    Duckett DR, Murchie AIH, Diekmann S et al (1988) The structure of the Holliday junction and its resolution. Cell 55:79–89CrossRefPubMedGoogle Scholar
  19. 19.
    Connelly JC, Kirkham LA, Leach DRF (1998) The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA. Proc Natl Acad Sci U S A 95:7969–7974CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Leach DR (1994) Long DNA palindromes, cruciform structures, genetic instability and secondary structure repair. Bioessays 16:893–900CrossRefPubMedGoogle Scholar
  21. 21.
    Sharples GJ, Chan SN, Mahdi AA et al (1994) Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions. EMBO J 13:6133–6142PubMedCentralPubMedGoogle Scholar
  22. 22.
    Taylor SH, Harmse J, Arbuthnot P et al (2012) Construction of effective inverted repeat silencing constructs using sodium bisulfite treatment coupled with strand-specific PCR. Biotechniques 52(4):254–262CrossRefPubMedGoogle Scholar
  23. 23.
    Gleave AP (1992) A versatile binary vector system with a T-DNA organizational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20:1203–1207CrossRefPubMedGoogle Scholar
  24. 24.
    Holsters M, de Waele D, Depicker A et al (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187CrossRefPubMedGoogle Scholar
  25. 25.
    Zuker M (2003) MFOLD web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • M. E. Chrissie Rey
    • 1
  • Johan Harmse
    • 1
  • Sarah H. Taylor
    • 1
  • Patrick Arbuthnot
    • 1
  • Marc S. Weinberg
    • 1
  1. 1.School of Molecular and Cell BiologyUniversity of the WitwatersrandJohannesburgSouth Africa

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