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RNAi-Mediated, Stable Resistance to Triticum mosaic virus in Wheat

Jessica L. Shoup Rupp, Luisa F. Cruz, Harold N. Trick, John P. Fellers
Crop science 2016 v.56 no.4 pp. 1602-1610
RNA interference, Triticum aestivum, Triticum mosaic virus, biolistics, coat proteins, crop production, crossing, cultivars, disease resistance, gene expression, genetic transformation, glufosinate, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, transgenes, transgenic plants, viral load, viruses, winter wheat
Triticum mosaic virus (TriMV) was discovered in 2006 and affects wheat (Triticum spp.) production systems in the Great Plains of the United States. Genetic resistance is available in a few commercial varieties; however, it is not effective above 20°C. RNA interference (RNAi) was evaluated as an alternative strategy to generate resistance to TriMV. An RNAi pANDA-mini-based hairpin expression vector was created from 272 bp of the TriMV coat protein (CP) sequence. Immature embryos of the wheat cultivar ‘Bobwhite’ were co-transformed by biolistic particle delivery system with the pANDA::TriMVCP vector and pAHC20, containing the bar gene for glufosinate selection. T₀ plants were analyzed by PCR for the presence of the RNAi TriMV CP fragment. Transgenic T₁ lines were tested for transgene expression via reverse transcription PCR Virus resistance was evaluated by mechanical inoculation with TriMV and indirect enzyme-linked immunosorbant assay (ELISA). Several lines were identified that were resistant, having little or no viral RNA. Resistant lines underwent single plant selection to the T₆ generation. Real-time PCR and ELISA showed that T₆ lines had a high level of resistance when challenged with the virus. To test if the transgene would behave similarly in an adapted variety, homozygous transgenic T₃ lines were crossed with the virus-susceptible winter wheat, ‘Overley.’ Real-time PCR results indicate decreased viral titer by as much as 20-fold in the T₆ transgenic lines, the F₁ crosses, and the BC₁F₁ lines when compared with control plants, providing evidence that this RNAi-silencing construct can provide stable resistance to TriMV and has great potential benefits to both breeders and producers.