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Designing Effective amiRNA and Multimeric amiRNA Against Plant Viruses

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siRNA Design

Part of the book series: Methods in Molecular Biology ((MIMB,volume 942))

Abstract

RNA-mediated virus resistance is increasingly becoming a method of choice for antiviral defense in plants when effective natural resistance is unavailable. In this chapter we discuss the design principles of artificial micro RNA (amiRNA), in which a natural miRNA precursor gene is modified to target a different species of RNA, in particular viral RNA. In addition, we explore the advantages and effectiveness of multiple amiRNAs within one polycistronic amiRNA precursor against a virus, as illustrated with Wheat streak mosaic virus, WSMV. The judicious selection of amiRNAs, which are sequences of short length as compared to other related methodologies of RNA interference, greatly assists in avoiding unintended off-targets in the host plant. The viral sequences targeted can be genomic or replicative and should be derived from conserved regions of the published WSMV genome. In short, using published folding and miRNA selection rules and algorithms, candidate miRNA sequences are selected from conserved regions between a number of WSMV genomes, and are BLASTed against wheat TIGR ESTs. Five miRNAs are selected that are least likely to interfere with the expression of transcripts from the wheat host. Then, the natural miRNA in each of the five arms of the polycistronic rice miR395 is replaced in silico with the chosen artificial miRNAs. This artificial precursor is transformed into wheat behind a ubiquitin promoter, and its integration into transformed wheat plants is confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of this methodology using an amiRNA precursor that we have termed Fanguard. The processing of amiRNAs in transgenic leaves is verified through splinted ligation assay, and the functionality of the transgene in preventing viral replication is verified by virus bioassay. Resistance is confirmed using mechanical virus inoculation over two subsequent generations. This example demonstrates the potential of polycistronic amiRNA to achieve stable immunity to economically important viruses.

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References

  1. Abel PP et al (1986) Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232(4751):738–743

    Article  CAS  Google Scholar 

  2. Bendahmane M et al (2007) Coat protein-mediated resistance to TMV infection of Nicotiana tabacum involves multiple modes of interference by coat protein. Virology 366(1):107–116

    Article  CAS  Google Scholar 

  3. Register JC 3rd, Beachy RN (1988) Resistance to TMV in transgenic plants results from interference with an early event in infection. Virology 166(2):524–32

    Article  CAS  Google Scholar 

  4. Nejidat A, Beachy RN (1990) Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses. Mol Plant Microbe Interact 3(4):247–251

    Article  CAS  Google Scholar 

  5. Lindbo JA, Dougherty WG (2005) Plant pathology and RNAi: a brief history. Annu Rev Phytopathol 43:191–204

    Article  CAS  Google Scholar 

  6. Lomonossoff GP (1995) Pathogen-derived resistance to plant viruses. Annu Rev Phytopathol 33:323–343

    Article  CAS  Google Scholar 

  7. Baulcombe DC (1996) Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8(10):1833–1844

    CAS  Google Scholar 

  8. Beachy RN (1997) Mechanisms and applications of pathogen-derived resistance in transgenic plants. Curr Opin Biotechnol 8(2):215–220

    Article  CAS  Google Scholar 

  9. Bucher E et al (2006) Multiple virus resistance at a high frequency using a single transgene construct. J Gen Virol 87(Pt 12):3697–3701

    Article  CAS  Google Scholar 

  10. Goldbach R, Bucher E, Prins M (2003) Resistance mechanisms to plant viruses: an overview. Virus Res 92(2):207–212

    Article  CAS  Google Scholar 

  11. Nejidat A, Beachy RN (1989) Decreased levels of TMV coat protein in transgenic tobacco plants at elevated temperatures reduce resistance to TMV infection. Virology 173(2):531–538

    Article  CAS  Google Scholar 

  12. Waterhouse PM, Graham HW, Wang MB (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 95(23):13959–13964

    Article  CAS  Google Scholar 

  13. Kalantidis K et al (2002) The occurrence of CMV-specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Mol Plant Microbe Interact 15(8):826–833

    Article  CAS  Google Scholar 

  14. Di Nicola-Negri E et al (2005) Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus. Transgenic Res 14(6):989–994

    Article  Google Scholar 

  15. Tougou M et al (2006) Development of resistant transgenic soybeans with inverted repeat-coat protein genes of soybean dwarf virus. Plant Cell Rep 25(11):1213–1218

    Article  CAS  Google Scholar 

  16. Fuentes A et al (2006) Intron-hairpin RNA derived from replication associated protein C1 gene confers immunity to tomato yellow leaf curl virus infection in transgenic tomato plants. Transgenic Res 15(3):291–304

    Article  CAS  Google Scholar 

  17. Fahim M et al (2010) Hairpin RNA derived from viral NIa gene confers immunity to wheat streak mosaic virus infection in transgenic wheat plants. Plant Biotechnol J 8(7):821–834

    Article  CAS  Google Scholar 

  18. Jackson AL et al (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21(6):635–637

    Article  CAS  Google Scholar 

  19. Jackson AL, Linsley PS (2004) Noise amidst the silence: off-target effects of siRNAs? Trends Genet 20(11):521–524

    Article  CAS  Google Scholar 

  20. Jackson AL et al (2006) Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA 12(7):1179–1187

    Article  CAS  Google Scholar 

  21. Jackson AL et al (2006) Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12(7):1197–1205

    Article  CAS  Google Scholar 

  22. Fuchs M, Gonsalves D (2007) Safety of virus-resistant transgenic plants two decades after their introduction: lessons from realistic field risk assessment studies. Annu Rev Phytopathol 45:173–202

    Article  CAS  Google Scholar 

  23. Schwab R et al (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18(5):1121–1133

    Article  CAS  Google Scholar 

  24. Schnippenkoetter WH et al (2001) Forced recombination between distinct strains of Maize streak virus. J Gen Virol 82:3081–3090

    CAS  Google Scholar 

  25. Brodersen P et al (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320(5880):1185–1190

    Article  CAS  Google Scholar 

  26. Meister G et al (2004) Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10(3):544–550

    Article  CAS  Google Scholar 

  27. Israsena N et al (2009) Inhibition of rabies virus replication by multiple artificial microRNAs. Antiviral Res 84(1):76–83

    Article  CAS  Google Scholar 

  28. Khraiwesh B et al (2008) Specific gene silencing by artificial microRNAs in Physcomitrella patens: an alternative to targeted gene knockouts. Plant Physiol 148(2):684–693

    Article  CAS  Google Scholar 

  29. Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53(4):674–690

    Article  CAS  Google Scholar 

  30. Niu QW et al (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24(11):1420–1428

    Article  CAS  Google Scholar 

  31. Duan CG et al (2008) Artificial MicroRNAs highly accessible to targets confer efficient virus resistance in plants. J Virol 82(22):11084–11095

    Article  CAS  Google Scholar 

  32. Ai T et al (2011) Highly efficient virus resistance mediated by artificial microRNAs that target the suppressor of PVX and PVY in plants. Plant Biol 13(2):304–316

    Article  CAS  Google Scholar 

  33. Fahim M et al (2012) Resistance to wheat streak mosaic virus generated by expression of an artificial polycistronic microRNA in wheat. Plant Biotechnol J 10(2):150–163

    Article  CAS  Google Scholar 

  34. Kung YJ et al (2012) Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense single-stranded RNA plant virus. Mol Plant Pathol 13(3):303–317

    Article  CAS  Google Scholar 

  35. Qu J, Ye J, Fang R (2007) Artificial microRNA-mediated virus resistance in plants. J Virol 81(12):6690–6699

    Article  CAS  Google Scholar 

  36. Zhang X et al (2011) Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner. Transgenic Res 20(3):569–581

    Article  CAS  Google Scholar 

  37. Sivamani E et al (2000) Resistance to wheat streak mosaic virus in transgenic wheat expressing the viral replicase (NIb) gene. Mol Breed 6(5):469–477

    Article  CAS  Google Scholar 

  38. Sivamani E et al (2002) Resistance to wheat streak mosaic virus in transgenic wheat engineered with the viral coat protein gene. Transgenic Res 11(1):31–41

    Article  CAS  Google Scholar 

  39. Jiang F et al (2011) The choice of target site is crucial in artificial miRNA-mediated virus resistance in transgenic Nicotiana tabacum. Physiol Mol Plant Pathol 76(1):2–8

    Article  CAS  Google Scholar 

  40. Jiang F et al (2011) Special origin of stem sequence influence the resistance of hairpin expressing plants against PVY. Biol Plantarum 55(3):528–535

    Article  CAS  Google Scholar 

  41. Anderson E et al (2008) Identifying siRNA-induced off-targets by microarray analysis. Methods Mol Biol 442:45–63

    Article  CAS  Google Scholar 

  42. Bartz SR et al (2002) Off-target activity of siRNA oligos in mammalian cells. Mol Biol Cell 13:409a–410a

    Google Scholar 

  43. Jackson AL, Linsley PS (2010) Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov 9(1):57–67

    Article  CAS  Google Scholar 

  44. Petri S et al (2011) Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects. RNA 17(4):737–749

    Article  CAS  Google Scholar 

  45. Smith NA, Eamens AL, Wang MB (2011) Viral small interfering RNAs target host genes to mediate disease symptoms in plants. PLoS Pathog 7(5):e1002022

    Article  CAS  Google Scholar 

  46. Shimura H et al (2011) A viral satellite RNA induces yellow symptoms on tobacco by targeting a gene involved in chlorophyll biosynthesis using the RNA silencing machinery. PLoS Pathog 7(5):e1002021

    Article  CAS  Google Scholar 

  47. Fortier E, Belote JM (2000) Temperature-dependent gene silencing by an expressed inverted repeat in Drosophila. Genesis 26(4):240–244

    Article  CAS  Google Scholar 

  48. Szittya G et al (2003) Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. EMBO J 22(3):633–640

    Article  CAS  Google Scholar 

  49. Kameda T et al (2004) A hypothermic-temperature-sensitive gene silencing by the mammalian RNAi. Biochem Biophys Res Commun 315(3):599–602

    Article  CAS  Google Scholar 

  50. Chen J et al (2004) Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of MicroRNA in host gene expression. Plant Cell 16(5):1302–1313

    Article  CAS  Google Scholar 

  51. Schubert S et al (2005) Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions. J Mol Biol 348(4):883–893

    Article  CAS  Google Scholar 

  52. Praveen S, Mishra AK, Antony G (2006) Viral suppression in transgenic plants expressing chimeric transgene from tomato leaf curl virus and cucumber mosaic virus. Plant Cell Tissue Organ Cult 84(1):47–53

    Article  CAS  Google Scholar 

  53. Antony G, Mishra AK, Praveen S (2005) A single chimeric transgene derived from two distinct viruses for multiple virus resistance. J Plant Biochem Biotechnol 14(2):101–105

    Article  CAS  Google Scholar 

  54. Lafforgue G et al (2011) Tempo and mode of plant RNA virus escape from RNA interference-mediated resistance. J Virol 85(19):9686–9695

    Article  CAS  Google Scholar 

  55. Rodrigo G et al (2011) Optimal viral strategies for bypassing RNA silencing. J R Soc Interface 8(55):257–268

    Article  CAS  Google Scholar 

  56. Liu YP et al (2008) Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron. Nucleic Acids Res 36(9):2811–2824

    Article  CAS  Google Scholar 

  57. Westerhout EM et al (2005) HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res 33(2):796–804

    Article  CAS  Google Scholar 

  58. Simon-Mateo C, Garcia JA (2006) MicroRNA-guided processing impairs Plum pox virus replication, but the virus readily evolves to escape this silencing mechanism. J Virol 80(5):2429–2436

    Article  CAS  Google Scholar 

  59. Lin SS et al (2009) Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-mediated silencing. Plos Pathogens 5(2):e1000312

    Article  Google Scholar 

  60. ter Brake O et al (2006) Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol Ther 14(6):883–892

    Article  Google Scholar 

  61. Guddeti S et al (2005) Molecular evolution of the rice miR395 gene family. Cell Res 15:631–638

    Article  CAS  Google Scholar 

  62. Overhoff M et al (2005) Local RNA target structure influences siRNA efficacy: a systematic global analysis. J Mol Biol 348(4):871–881

    Article  CAS  Google Scholar 

  63. Fahim M et al (2011) Resistance to wheat streak mosaic virus – a survey of resources and development of molecular markers. Plant Pathol 61(3):425-440

    Google Scholar 

  64. Kawashima CG et al (2009) Sulphur starvation induces the expression of microRNA-395 and one of its target genes but in different cell types. Plant J 57(2):313–321

    Article  CAS  Google Scholar 

  65. Chiou TJ (2007) The role of microRNAs in sensing nutrient stress. Plant Cell Environ 30(3):323–332

    Article  CAS  Google Scholar 

  66. Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant MicroRNAs and their targets, including a stress-induced miRNA. Mol Cell 14(6):787–799

    Article  CAS  Google Scholar 

  67. Wang M-B, Abbott DC, Waterhouse PM (2000) A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol Plant Pathol 1(6):347–356

    Article  CAS  Google Scholar 

  68. Pellegrineschi A et al (2002) Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants. Genome 45(2):421–430

    Article  CAS  Google Scholar 

  69. Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3(6):1077–1084

    Article  CAS  Google Scholar 

  70. Maroney PA et al (2008) Direct detection of small RNAs using splinted ligation. Nat Protoc 3(2):279–287

    Article  CAS  Google Scholar 

  71. Maroney PA et al (2007) A rapid, quantitative assay for direct detection of microRNAs and other small RNAs using splinted ligation. RNA 13(6):930–936

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Lab of Plant Developmental Molecular Genetics, School of Life Science and Biotechnology, Korea University, Seoul, South Korea

    Muhammad Fahim

  2. CSIRO Plant Industry, Canberra, ACT, Australia

    Philip J. Larkin

Authors
  1. Muhammad Fahim
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  2. Philip J. Larkin
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Corresponding author

Correspondence to Philip J. Larkin .

Editor information

Editors and Affiliations

  1. , Lineberger Comprehensive Cancer Center, University of North Carolina, West Drive 450, Chapel Hill, 27599, North Carolina, USA

    Debra J. Taxman

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Fahim, M., Larkin, P.J. (2013). Designing Effective amiRNA and Multimeric amiRNA Against Plant Viruses. In: Taxman, D. (eds) siRNA Design. Methods in Molecular Biology, vol 942. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-119-6_19

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  • DOI: https://doi.org/10.1007/978-1-62703-119-6_19

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-118-9

  • Online ISBN: 978-1-62703-119-6

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