Abstract
Genetically engineered resistance to protect plants against virus infections can be based on protein- and RNA-mediated defense mechanisms. RNA silencing that leads to high-level virus resistance is triggered by virus-specific double-stranded (ds)RNA. The most efficient means to produce such dsRNA in transgenic plants is the introduction and expression of hairpin (hp) RNA constructs. Successful induction of the RNA silencing pathway is witnessed by the accumulation of virus-specific small interfering (si)RNAs that guide destruction of complementary viral RNA. Here, we describe strategies and methods for the efficient generation of hpRNA constructs and for the extraction and detection of siRNAs.
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References
Waterhouse PM, Graham MW, Wang M-B (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 95:13959–13964
Smith NA, Singh SP, Wang M-B, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320
Fusaro AF, Matthew L, Smith NA, Curtin SJ, Dedic-Hagan J, Ellacott GA et al (2006) RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway. EMBO Rep 7:1168–1175
Mitter N, Sulistyowati E, Graham MW, Dietzgen RG (2001) Suppression of gene silencing: a threat to virus-resistant transgenic plants? Trends Plant Sci 6:246–247
Dietzgen RG, Mitter N (2006) Transgenic gene silencing strategies for virus control. Australas Plant Pathol 35:605–618
Wesley SV, Helliwell CA, Smith N, Wang MB, Rouse DT, Liu Q et al (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27: 581–590
Chen Y-K, Lohuis D, Goldbach R, Prins M (2004) High frequency induction of RNA-mediated resistance against Cucumber mosaic virus using inverted repeat constructs. Mol Breed 14:215–226
Jan F-J, Fagoaga C, Pang S-Z, Gonsalves D (2000) A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing. J Gen Virol 81:2103–2109
Bonfim K, Faria JC, Nogueira EOPL, Mendes EA, Aragão FJL (2007) RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol Plant Microbe Interact 20:717–726
Vanderschuren H, Alder A, Zhang P, Gruissem W (2009) Dose-dependent RNAi-mediated geminivirus resistance in the tropical root crop cassava. Plant Mol Biol 70:265–272
Roy G, Sudarshana MR, Ullman DE, Ding S-W, Dandekar AM, Falk BW (2006) Chimeric cDNA sequences from Citrus tristeza virus confer RNA silencing-mediated resistance in transgenic Nicotiana benthamiana plants. Phytopathology 96:819–827
Tougou M, Furutani N, Yamagishi N, Shizukawa Y, Takahata Y, Hidaka S (2006) Development of resistant transgenic soybeans with inverted-repeat coat protein genes of soybean dwarf virus. Plant Cell Rep 25: 1213–1218
Mitter N, Sulistyowati E, Dietzgen RG (2003) Cucumber mosaic virus infection transiently breaks dsRNA-induced transgenic immunity to Potato virusY in tobacco. Mol Plant Microbe Interact 16:936–944
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: 347–356
Gleave AP (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20:1203–1207
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Chapman EJ, Prokhnevsky A, Gopinath K, Dolja VV, Carrington JC (2004) Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18:1179–1186
Lózsa R, Csorba T, Lakatos L, Burgyán J (2008) Inhibition of 3′ modification of small RNAs in virus-infected plants require spatial and temporal co-expression of small RNAs and viral silencing-suppressor proteins. Nucleic Acids Res 36:4099–4107
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Mitter, N., Dietzgen, R.G. (2012). Use of Hairpin RNA Constructs for Engineering Plant Virus Resistance. In: Watson, J., Wang, MB. (eds) Antiviral Resistance in Plants. Methods in Molecular Biology, vol 894. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-882-5_13
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DOI: https://doi.org/10.1007/978-1-61779-882-5_13
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