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Journal of Virology, November 2008, p. 11084-11095, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.01377-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Artificial MicroRNAs Highly Accessible to Targets Confer Efficient Virus Resistance in Plants{triangledown}

Cheng-Guo Duan,1,2 Chun-Han Wang,1,{dagger} Rong-Xiang Fang,1 and Hui-Shan Guo1*

State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China,1 Graduate University of Chinese Academy of Sciences, Beijing 100049, China2

Received 1 July 2008/ Accepted 25 August 2008

Short-hairpin RNAs based on microRNA (miRNA) precursors to express the artificial miRNAs (amiRNAs) can specifically induce gene silencing and confer virus resistance in plants. The efficacy of RNA silencing depends not only on the nature of amiRNAs but also on the local structures of the target mRNAs. However, the lack of tools to accurately and reliably predict secondary structures within long RNAs makes it very hard to predict the secondary structures of a viral genome RNA in the natural infection conditions in vivo. In this study, we used an experimental approach to dissect how the endogenous silencing machinery acts on the 3' untranslated region (UTR) of the Cucumber mosaic virus (CMV) genome. Transiently expressed 3'UTR RNAs were degraded by site-specific cleavage. By comparing the natural cleavage hotspots within the 3'UTR of the CMV-infected wild-type Arabidopsis to those of the triple dcl2/3/4 mutant, we acquired true small RNA programmed RNA-induced silencing complex (siRISC)-mediated cleavage sites to design valid amiRNAs. We showed that the tRNA-like structure within the 3'UTR impeded target site access and restricted amiRNA-RISC-mediated cleavage of the target viral RNA. Moreover, target recognition in the less-structured area also influenced siRISC catalysis, thereby conferring different degrees of resistance to CMV infection. Transgenic plants expressing the designed amiRNAs that target the putative RISC accessible target sites conferred high resistance to the CMV challenge from both CMV subgroup strains. Our work suggests that the experimental approach is credible for studying the course of RISC target recognition to engineer effective gene silencing and virus resistance in plants by amiRNAs.

* Corresponding author. Mailing address: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Phone: 86 (010) 64847989. Fax: 86 (010) 64847989. E-mail: guohs{at}im.ac.cn

{triangledown} Published ahead of print on 3 September 2008.

{dagger} Present address: Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

Journal of Virology, November 2008, p. 11084-11095, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.01377-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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