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Journal of Integrative Agriculture  2016, Vol. 15 Issue (11): 2539-2549    DOI: 10.1016/S2095-3119(16)61369-2
Plant Protection Advanced Online Publication | Current Issue | Archive | Adv Search |
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
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Abstract      Rice 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 has been done on the profiling 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 confirmed 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 confirmed 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 specific 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 first study to address the siRNA profile in RSV-resistant transgenic plant using next generation sequencing (NGS) technique, we confirmed that the massive abundance of siRNA derived from the inverted repeat of NCP is the major reason for RSV-resistance.
Keywords:  Rice stripe virus (RSV)        transgenic rice        deep sequencing        siRNA       resistance  
Received: 08 January 2016   Accepted:
Fund: 

Financial support was provided by the National Key Basic Research of China (2012CB114004), the Special Fund for Agro-Scientific Research in the Public Interest, China (201303021) and the National R&D Project of Transgenic Crops of China (2012ZX08009001).

Corresponding Authors:  WANG Xi-feng, Tel: +86-10-62815928, E-mail: xfwang@ippcaas.cn; LIANG Chun, E-mail: liangc@miamioh.edu   
About author:  LI Li, E-mail: lli@ippcaas.cn; GUO Cheng, E-mail: guoc2@miamioh.edu

Cite this article: 

LI Li, GUO Cheng, WANG Biao, ZHOU Tong, LEI Yang, DAI Yu-hua, HE Wen, LIANG Chun, WANG Xi-feng. 2016. RNAi-mediated transgenic rice resistance to Rice stripe virus. Journal of Integrative Agriculture, 15(11): 2539-2549.

Ares X, Calamante G, Cabral S, Lodge J, Hemenway P, Beachy R N, Mentaberry A. 1998. Transgenic plants expressing potato virus X ORF2 protein (p24) are resistant to tobacco mosaic virus and Ob tobamoviruses. Journal of Virology, 72, 731–738.

Bonfim K, Faria J C, Nogueira E O P L, Mendes E A, Aragão F J L. 2007. RNAi-mediated resistance to bean (Phaseolus vulgaris) in genetically engineered common bean Molecular Plant-Microbe Interactions, 20, 717–726.

Chen Y K, Lohuis D, Goldbach R, Prins M. 2004. High frequency induction of RNA-mediated resistance against Cucumber mosaic virus using inverted repeat constructs. Molecular Breeding, 14, 215–226.

Fuentes A, Ramos P L, Fiallo E, Callard D, Sánchez Y, Peral R, Rodríguez R, Pujol M. 2006. Intron-hairpin RNA derived from replication associated protein C1 gene confers immunity to Tomato Yellow Leaf Curl Virus infection in transgenic tomato plants. Transgenic Research, 15, 291–304.

Guo C, Li L, Wang X, Liang C. 2015. Alterations in siRNA and miRNA expression profiles detected by deep sequencing of transgenic rice with siRNA-mediated viral resistance. PLoS ONE, 10, e0116175.

Hajano J, Wang B, Ren Y, Lu C, Wang X. 2015. Quantification of southern rice black streaked dwarf virus and rice black streaked dwarf virus in the organs of their vector and nonvector insect over time. Virus Research, 208, 146–155.

Hiei Y, Ohta S, Komari T, Kumashiro T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6, 271–282.

Helliwell C, Waterhouse P. 2003. Constructs and methods for high-throughput gene silencing in plants. Methods, 30, 289–295.

Hayano-Saito Y, Saito K, Nakamura S, Kawasaki S, Iwasaki M. 2000. Fine physical mapping of the rice stripe resistance gene locus, Stvb-i. Theoretical Applied Genetics, 101, 59–63.

Huo Y, Liu W, Zhang F, Chen X, Li L, Liu Q, Zhou Y, Wei T, Fang R, Wang X. 2014. Transovarial transmission of a plant virus is mediated by vitellogenin of its insect vector. PLoS Pathogens, 10, e1003949.

Jaiswal P. 2011. Gramene database: A hub for comparative plant genomics. Methods in Molecular Biology, 678, 247–275.

Jinek M, Doudna J A. 2009. A three-dimensional view of the molecular machinery of RNA interference. Nature, 457, 405–412.

Kalantidis K, Psaradakis S, Tabler M, Tsagris M. 2002. The occurrence of CMV-specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Molecular Plant-Microbe Interactions, 15, 826–833.

Liu W, Gray S, Huo Y, Li L, Wei T, Wang X. 2015. Proteomic analysis of interaction between a plant virus and its vector insect reveals new functions of hemipteran cuticular protein. Molecular & Cellular Proteomics, 14, 2229–2242

Ma J, Song Y, Wu B, Jiang M, Li K, Zhu C, Wen F. 2011. Production of transgenic rice new germplasm with strong resistance against two isolations of Rice stripe virus by RNA interference. Transgenic Research, 20, 1367–1377.

Mansoor S, Amin I, Hussain M, Zafar Y, Briddon R W. 2006. Engineering novel traits in plants through RNA interference. Trends in Plant Science, 11, 559–565.

Mar T, Liu W, Wang X. 2014. Proteomic analysis of interaction between P7-1 of Southern rice black-streaked dwarf virus and the insect vector reveals diverse insect proteins involved in successful transmission. Journal of Proteomics, 102, 83–97.

Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, Wu L, Li S, Zhou H, Long C, Chen S, Hannon G J, Qi Y. 2008. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5´ terminal nucleotide. Cell, 133, 116–127.

Nicola-Negri E D, Brunetti A, Tavazza M, Ilardi V. 2005. Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro  genes for efficient and predictable resistance to the virus. Transgenic Research, 14, 989–994.

Park H M, Choi M S, Kwak D Y, Lee B C, Lee J H, Kim M K, Kim Y G, Shin D B, Park S K, Kim Y H. 2012. Suppression of NS3 and MP is important for the stable inheritance of RNAi-mediated rice stripe virus (RSV) resistance obtained by targeting the fully complementary RSV-CP gene. Molecules and Cells, 33, 43–51.

Prins M. 2003. Broad virus resistance in transgenic plants. Trends in Biotechnology, 21, 373–375.

Prins M, Laimer M, Noris E, Schubert J, Wassenegger M, Tepfer M. 2008. Strategies for antiviral resistance in transgenic plants. Molecular Plant Pathology, 9, 73–83.

Rajeswaran R, Aregger M, Zvereva A S, Borah B K, Gubaeva E G, Pooggin M M. 2012. Sequencing of RDR6-dependent double-stranded RNAs reveals novel features of plant siRNA biogenesis. Nucleic Acids Research, 40, 6241–6254.

Sasaya T, Nakazono-Nagaoka E, Saika H, Aoki H, Hiraguri A, Netsu O, Uehara-Ichiki T, Onuki M, Toki S, Saito K, Yatou O. 2014. Transgenic strategies to confer resistance against viruses in rice plants. Frontiers in Microbiology, 4, 409.

Schwach F, Adam G, Heinze C. 2004. Expression of a modified nucleocapsid-protein of Tomato spotted wilt virus (TSWV) confers resistance against TSWV and Groundnut ringspot virus (GRSV) by blocking systemic spread. Molecular Plant Pathology, 5, 309–316.

Shimizu T, Nakazono-Nagaoka E, Uehara-Ichiki T, Sasaya T, Omura T. 2011. Targeting specific genes for RNA interference is crucial to the development of strong resistance to Rice stripe virus. Plant Biotechnology Journal, 9, 503–512.

Shu L, Hu Z. 2012. Characterization and differential expression of microRNAs elicited by sulfur deprivation in Chlamydomonas reinhardtii. BMC Genomics, 13, 108.

Siomi H, Siomi M C. 2009. On the road to reading the RNA-interference code. Nature, 457, 396–404.

Takeda A, Iwasaki S, Watanabe T, Utsumi M, Watanabe Y. 2008. The mechanism selecting the guide strand from small RNA duplexes is different among argonaute proteins. Plant and Cell Physiology, 49, 493–500.

Toriyama S, Takahashi M, Sano Y, Shimizu T, Ishihama A. 1994. Nucleotide sequence of RNA 1, the largest genomic segment of rice stripe virus, the prototype of the Tenuiviruses. Journal of General Virology, 75, 3569–3579.

Truss M, Swat M, Kielbasa S M, Schäfer R, Herzel H, Hagemeier C. 2005. HuSiDa-the human siRNA database: an open-access database for published functional siRNA sequences and technical details of efficient transfer into recipient cells. Nucleic Acids Research, 33, D108–D111.

Tuschl T, Zamore P D, Lehmann R, Bartel D P, Sharp P A. 1999. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Development, 13, 3191–3197.

Wang B, Hajano J, Ren Y, Lu C, Wang X. 2015. iTRAQ-based quantitative proteomics analysis of rice leaves infected by Rice stripe virus reveals several proteins involved in symptom formation. Virology Journal, 12, 99.

Wang Q, Liu Y, He J, Zheng X, Hu J, Liu Y, Dai H, Zhang Y, Wang B, Wu W, Gao H, Zhang Y, Tao X, Deng H, Yuan D, Jiang L, Zhang X, Guo X, Cheng X, Wu C, et al. 2014. STV11 encodes a sulphotransferase and confers durable resistance to rice stripe virus. Nature Communications, 5, 4768–4775.

Washio O, Ezaka A, Sakurai Y. 1967. Studies on the breeding of rice varieties to stripe disease. I Varietial difference in resistance to stripe disease. Japan Journal of Breeding, 17, 91–98.

Wei T Y, Yang J G, Liao F R, Liao F L, Gao F L, Lu L M, Zhang X T, Li F, Wu Z J, Lin Q Y, Xie L H, Lin H X. 2009. Genetic diversity and population structure of rice stripe virus in China. Journal of General Virology, 90, 1025–1034.

Wintermantel W M, Zaitlin M. 2000. Transgene translatability increases effectiveness of replicase-mediated resistance to Cucumber mosaic virus. Journal of General Virology, 81, 587–595.

Wu L, Zhang Q, Zhou H, Ni F, Wu X, Qi Y. 2009. Rice microRNA effector complexes and targets. The Plant Cell, 21, 3421–3435.

Wu T D, Nacu S. 2010. Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics, 26, 873–881.

Yan F, Zhang H, Adams M J, Yang J, Peng J, Antoniw J F, Zhou Y, Chen J. 2010. Characterization of siRNAs derived from rice stripe virus in infected rice plants by deep sequencing. Archives of Virology, 155, 935–940.

Zhang G Y, Liu R R, Xu G, Zhang P, Li Y, Tang K X, Liang G H, Liu Q Q. 2013. Increased α-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis γ-tocopherol methyltransferase. Transgenic Research, 22, 89–99.

Zhang P, Mar T, Liu W, Li L, Wang X. 2013. Simultaneous detection and differentiation of Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) by duplex real time RT-PCR. Virology Journal, 10, 24.

Zhang S, Li L, Wang X, Zhou G. 2007. Transmission of Rice stripe virus acquired from frozen infected leaves by the small brown planthopper (Laodelphax striatellus Fallen). Journal of Virological Methods, 146, 359–362.

Zhang X, Wang X, Zhou G. 2008. A one-step real time RT-PCR assay for quantifying rice stripe virus (RSV) in rice and in the small brown planthopper (Laodelphax striatellus Fallen). Journal of Virological Methods, 151, 181–187.

Zheng W, Ma L, Zhao J, Li Z, Sun F, Lu X. 2013. Comparative transcriptome analysis of two rice varieties in response to Rice stripe virus and small brown planthoppers during early interaction. PLoS ONE, 8, e82126.

Zhou T, Nelson S C, Hu J S, Wang L, Fan Y, Cheng Z, Zhou Y. 2011. Inheritance and mechanism of resistance to rice stripe disease in cv. Zhendao 88, a Chinese rice cultivar. Journal of Phytopathology, 159, 159–164.
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