(JIA-2016-0023) RNAi-mediated transgenic rice resistance to Rice stripe virus
LI Li1*, GUO Cheng2*, WANG Biao1, ZHOU Tong3, LEI Yang1, DAI Yu-hua1, HE Wen1, LIANG Chun1, 2, 4, WANG Xi-feng1
1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China 2 Department of Biology, Miami University, Oxford, OH 45056, USA 3 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China 4 Department of Computer Science and Software Engineering, Miami University, Oxford, OH 45056, USA
AbstractRice stripe virus (RSV) often causes severe rice yield loss in temperate regions of East Asia. Although the correlation of small interfering RNAs (siRNAs) with transgenic virus resistance of plants using RNA interference (RNAi) is known for decades, no systematical research have been done on the profiing of siRNAs from a genomic scale. Our research is aiming to systematically study the RNAi impact in RSV-resistant transgenic rice, which was generated by introducing an inverted repeat construct that targets RSV nucleocapsid protein (NCP) gene. In this paper, three independent RSV-resistant transgenic rice lines were generated, their stable integration of the T-DNA fragment and the expression of siRNAs were confimed by Southern blotting and Northern blotting analyses, and the majority of siRNAs were in lengths of 21, 22, and 24 nucleotides (nt), which have validated a connection between the presence of the RSV NCP homologous siRNAs and the RSV resistance in those transgenic rice lines. In one of these transgenic lines (T4-B1), the T-DNA fragment was found to have been inserted at chromosome 1 of the rice genome, substituting the rice genome fragment from 32 158 773 to 32 158 787 nt. Bioinformatics analysis of small RNA-Seq data on the T4-B1 line also confimed the large population of NCP-derived siRNAs in transgenic plants, and the RSV-infected library (T4-B1-V) possessed more siRNAs than its mock inoculated libraries (T4-B1-VF), these results indicating the inverted repeat construct and RSV could introduce abundance of siRNAs in transgenic rice. Moreover, a varied expression level of specifi siRNAs was found among different segments of the NCP gene template, about 47% of NCP-derived siRNAs reads aligned with the fragment from 594 to 832 nt (239 nt in length) in NCP gene (969 nt in length) in the T4-B1-V, indicating a potential usage of hotspot regions for RNAi silencing in future research. In conclusion, as the fist study to address the siRNA profie in RSV-resistant transgenic plant using next generation sequencing (NGS) technique, we confimed that the massive abundance of siRNA derived from the inverted repeat of NCP is the major reason for RSV-resistance.
Financial support was provided by the National Key Basic Research of China (2012CB114004), the Special Fund for Agro-Scientifi Research in the Public Interest, China (201303021) and the National R&D Project of Transgenic Crops of China (2012ZX08009001).