Molecular Aspects Of Rice Stripe Virus

Rice stripe virus (RSV) is one of the most economically important rice pathogens in China. It has broken out many times in different provinces, causing huge rice yield losses. To get a better insight into pathogenesis and epidemics of RSV, the population genetic diversity of RSV and the mechanism of RSV counterdefense againt RNA silencing were studied.1Sequencing of six RSV isolates and Moleculary evolutionary genetic analysis of RSV isolates from East AsiaThe genomes of six RSV isolates from Yunnan, Zhejiag, Jiangsu and Liaoning were sequenced. The genome sequences of RSV isolates from China, Japan and South Korea were down load from NCBI (http://www.ncbi.nlm.nih.gov/) GenBank, and Moleculary evolutionary genetic analysis of all the isolates was done. It indicated that RSV isolates from East Asia had a low level of genetic diversity, and all the genes of RSV were under negative selection. But RSV isolates from Yunnan were more diverse than the other areas of East Asia, and a clade of phylogenetic tree were composited exclusively by RSV isolate from Yunan.2Analysis of genetic diversity and molecular evolution of RSV quasispecies through High-throughput SequencingWe analyzed genetic diversity of RSV quasispecies in Oryza sativa (a natural host of RSV) and compared it with the RSV quasispecies obtained from a RSV infected Nicotiana benthamiana (an experimental host of RSV) using the high-throughput sequencing technology. Results of our study showed that the RSV quasispecies from infected N. benthamiana is more diveres than that obtained from rice. Our results also suggest that three RSV genes, RdRp, NSvc2and CP were under the positive selection. Here we also demonstrated that the high-throughput sequencing technology is a powerful technology that can be used to investigate population genetic diversity across the RSV quasispecies. 3. Characterization of small interfering RNAs derived from RSV infected Oryza sativa and Nicotiana benthamianaWe profiled small interfering RNAs (sRNA) derived from RSV infected Oryza sativa and Nicotiana benthamiana using Solexa-based deep sequencing. RSV derived sRNA accumulated preferentially20-22nt species in both hosts, but the polarity distribution was different. In infected Oryza sativa, vRNA derived sRNA were much more than vcRNA derived sRNA; while in infected Nicotiana benthamiana vRNA and vcRNA derived sRNA were closed to1:1ratio. Furthermore, the polarity distributions among different size of sRNA were not conserved. Briefly, there are more23and24nt sRNAs derived from vRNA than from vcRNA in both hosts.4. Genetic engineering for RSV resistance in Rice Plant Mediated by dsRNAIn order to effectively transcribe viral dsRNA, a partial sequence of RSV RdRP was inserted into pCAMBIA1031plasmid downstream of35S promotor in inverted repeat manner. Rice plants expressing dsRNA were generated by Agrobacterium tumefaciens mediated transformation. The transgenic plants were tested for their resistance against RSV infection.6out of12lines and two generations of their offspring were conformed resistant to RSV infection.5Screening for host factor interacting with RSV NS3Rice cellular factors interacting with RSV NS3protein were screened by yeast two-hybridization. NS3was clone to pGBK-T7vector as bait, and transformed into yeast strain Y2HGold, and subsequencingly mate with the yeast strain Y187containing rice cDNA library. One of the screened interacting-positive protein was OsSO, and its interaction with NS3was further confirmed by bimolecular fluorescence complementation analysis. The fluorescence showed that these two proteins interacted both in nuclear and cytoplasm, and they accumulated in unkown cell bodies.OsSO is a sulfite oxidase, usually reacts in response of abiotic stress. The mechanism of their interaction should be explored in following works.