Analysis Of The Resistance Mediated By AmiRNA Silencing In Transgenic Tobacco Plants

Recently, the application of artificial microRNA (amiRNA) strategy has supplied a new way for generating virus-resistant transgenic plant. MicroRNAs (miRNAs) processed from nuclear-encoded transcripts control the expression of target transcripts by directing cleavage or translational inhibition. amiRNAs that exploit this endogenous gene silencing mechanism can be designed to target any gene of interest and provide a highly specific approach for effective post-transcriptional gene silencing (PTGS) in plants. Thus, this strategy can be used to obtain virus-resistant transgenic plant in theory. Here, using Arabidopsis thaliana miR159a, miR167b, and miR171a precursors as backbones, we designed two types of amiRNAs targeting sequences encoding the silencing suppressor HC-Pro of Potato virus Y (PVY) and p25 of Potato virus X (PVX), respectively. Then, we investigated the virus-resistance of the T1 generation, and the main results are as follows:1) Three kinds of transgenic tobacco plants harboring different modified backbones which target to PVY or PVX were obtained. Northern blot assays revealed that all of them can generate amiRNAs, but with different efficiency. The plant with the pre-miR159a backbone was the most effective in generating amiRNAs.2) Through virus-challenging experiment, it was showed that all three kinds of transgenic plants could resist to their virus to some extent. ELISA assays and quantitative real-time RT-PCR have showed that the different resistance levels of the transgenic plants were correlated with the expression levels of amiRNAs.3) 5′RLM-RACE assay has showed that amiRNAs could mediated a precise cleavage of their target viral RNAs. Northern blot was carried out in the transgenic plants and the results indicated that the expression level of amiRNA was inversely correlated to the virus RNA accumulation.4) Inoculated the transgenic plants with different virus isolations of the one kind of virus. It was revealed that transgenic plants could present a wide range of resistance against their target viruses. However, the resistance was strongly influenced by the complementarity of the target sequence.5) Two different amiRNAs could be generated from transgenic plants with a dimeric amiRNA precursor, and these amiRNAs mediated double resistance against both PVX and PVY.6) The virus-challenging experiment of the T2 generation transgenic plants with single virus resistance verified that the resistance mediated by amiRNAs is hereditable, and when inoculated with mixed viruses, no synergistic effect was observed.