Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2016, Vol. 42 ›› Issue (11): 1647-1655.doi: 10.3724/SP.J.1006.2016.01647


RNAi-mediated SMV-P3 Silencing Increases Soybean Resistance to Soybean Mosaic Virus

YANG Xiang-Dong,NIU Lu,ZHANG Wei,YANG Jing,DU Qian,XING Guo-Jie,GUO Dong-Quan,LI Qi-Yun,DONG Ying-Shan   

  1. Jilin Academy of Agricultural Sciences, Agricultural Biotechnology Institute / Jilin Provincial Key laboratory of Agricultural Biotechnology, Changchun 130033, China
  • Received:2016-02-16 Revised:2016-06-20 Online:2016-11-12 Published:2016-07-04
  • Contact: Dong Yingshan, E-mail: ysdong@cjaas.com, Tel: +86-431-87063008 E-mail:xdyang020918@126.com
  • Supported by:

    This study was supported by the China National Novel Transgenic Organisms Breeding Project (2016ZX08004-004) and the Science & Technology Development Project of Jilin Province in China(20150204011NY)。


Soybean mosaic virus (SMV) is one of the most important diseases in major soybean production areas and has severe effects on soybean production and seed quality in China. Breeding disease-resistant varieties is the most economical and effective strategy to prevent and control SMV. In this study, RNAi fragments of the gene encoding P3 protein, which is involved in SMV mobility and affecting host range, were introduced into soybean by plant-mediated RNA interference (RNAi) techniques to explore the influence of RNAi-mediated SMV-P3 silencing on soybean SMV resistance. Southern blot analysis revealed that exogenous RNAi fragments were integrated into the soybean genome at low copy numbers (1–4). T1–T3 generation transgenic soybeans were sprayed with herbicide and inserted fragments were examined using PCR. The results indicated that T-DNA insertion fragments could be stably inherited between generations of transgenic soybean. Inoculation of T2 and T3 generation transgenic soybeans with SMV suggested that transgenic soybeans exhibited significantly higher resistance to the prevailing SMV strain, SC-3, in major soybean production areas than the non-transgenic control varieties Williams 82 and SN9. The disease index was reduced by 4.37%-18.51%. Further, the resistance could be stably inherited. In conclusion, RNAi-mediated SMV-P3 silencing can significantly increase the SMV resistance of transgenic soybeans.

Key words: Soybean, Soybean mosaic virus, SMV-P3, RNAi-mediated gene silencing

[1]Ross J P. Effect of soybean mosaic on component yields from blends of mosaic resistant and susceptible soybeans. Crop Sci, 1983, 23: 343–346 [2]Yang Y Q, Zheng G J, Han L, Wang D G, Yang X F, Yuan Y, Huang S H, Zhi H J. Genetic analysis and mapping of genes for resistance to multiple strains of Soybean mosaic virus in a single resistant soybean accession PI 96983. Theor Appl Genet, 2013, 126: 1783–1791 [3]Gao L, Ding X N, Li K, Liao W L, Zhong Y K, Ren R, Liu Z T, Karthikeyan A, Zhi H J. Characterization of Soybean mosaic virus resistance derived from inverted repeat SMV HC Pro genes in multiple soybean cultivars. Theor Appl Genet, 2015, 128: 1489–1505 [4]智海剑, 盖钧镒. 大豆花叶病毒及抗性遗传的研究进展. 大豆科学, 2006, 25: 174–180 Zhi H J, Gai J Y. Advances in the studies on soybean mosaic virus. Soybean Sci, 2006, 25: 174–180 [5]Yang Y Q, Lin J, Zheng G J, Zhang M C, Zhi H J. Recombinant soybean mosaic virus is prevalent in Chinese soybean fields. Arch Virol, 2014, 159: 1793–1796 [6]李延华, 吕文清. 大豆花叶病毒的显症率与大豆品种及病毒株系的关系研究. 植物保护学报, 1991, 18: 334–334 Li Y H, Lü W Q. Study on the relationship between the rate of SMV symptom appearance and soybean cultivars and SMV strains. Acta Phytophyl Sin, 1991, 18: 334–334 [7]Koo J M, Choi B K, Ahn H J, Yum H J, Choi C W. First report of an Rsv resistance-breaking isolate of Soybean mosaic virus in Korea. Plant Pathol, 2005, 54 : 573 [8]Choi B K, Koo J M, Ahn H J, Yum H J, Choi C W, Ryu K H, Chen P, Tolin S A. Emergence of Rsv-resistance breaking soybean mosaic virus isolates from Korean soybean cultivars. Virus Res, 2005, 112: 42–51 [9]Gagarinova A G, Babu M, Poysa V, Hill J H, Wang A. Identification and molecular characterization of two naturally occurring Soybean mosaic virus isolates that are closely related but differ in their ability to overcome Rsv4 resistance. Virus Res, 2008, 138: 50–56 [10]KusabaM. RNA interference in crop plant. Curr Opin Biotechno, 2004, 15: 139–143 [11]Ding S W. RNA-based antiviral immunity. Nat Rev Immunol, 2010, 10: 632–641 [12]侯静, 刘青青, 徐明良. 植物抗病毒侵染的分子机制. 作物学报, 2012, 38: 761–772 Hou J, Liu Q Q, Xu M L. Molecular mechanism of plant defense against virus attack. Acta Agron Sin, 2012, 38: 761–772 [13]Bucher E, Lohuis D, van Poppel P M J A, Geerts-Dimitriadou C, Goldbach R, Prins M. Multiple virus resistance at a high frequency using a single transgene construct. J General Virol, 2006, 87: 3697–3701 [14]Kreuze J F, Klein I S, Lazaro M U, Chuquiyuri W J C, Morgan G L, Mejía P G C, Ghislain M, Valkonen J P T. RNA silencing-mediated resistance to a crinivirus (Closteroviridae) in cultivated sweetpotato (Ipomoea batatas L.) and development of sweetpotato virus disease following co-infection with a potyvirus. Mol Plant Pathol, 2008, 9: 589–598 [15]Zhu C X, Song Y Z, Yin G H, Wen F J. Induction of RNA-mediated multiple virus resistance to Potato virus Y, Tobacco mosaic virus, and Cucumber mosaic virus. J Phytopathol, 2009, 157: 101–107 [16]Wang X Y, Eggenberger A L, Nutter F W J, Hill J H. Pathogen derived transgenic resistance to soybean mosaic virus in soybean. Mol Breed, 2001, 8: 119–127 [17]Furutani N, Hidaka S, Kosaka Y, Shizukawa Y, Kanematsu S. Coat protein gene-mediated resistance to soybean mosaic virus in transgenic soybean. Breed Sci, 2006, 56: 119–124 [18]Zhang X C, Sato S, Ye X H, Dorrance A E, Morris T J, Clemente T E, Qu F. Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene. Phytopathology, 2011, 101: 1264–1269 [19]Kim H J, Kim M J, Pak J H, Jung H W, Choi H K, Lee Y H, Baek I Y, Ko J M, Jeong S C, Pack I S, Ryu K H, Chung Y S. Characterization of SMV resistance of soybean produced by genetic transformation of SMV-CP gene in RNAi. Plant Biotechnol Rep, 2013, 7: 425–433 [20]Chen L Y, Cheng X F, Cai J Y, Zhan L L, Wu X X, Liu Q, Wu X Y. Multiple virus resistance using artificial trans-acting siRNAs. J Virol Methods, 2016, 228: 16–20 [21]Hill J H, Benner H I. Properties of Soybean mosaic virus ribonucleic acid. Phytopathology, 1980, 70: 236–239 [22]Adams M J, Antoniw J F, Beaudoin F. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Mol Plant Pathol, 2005, 6: 471–487 [23]Jermer C E, Wang X, Tomimura K, Ohshima K, Ponz F, Walsh J A. The dual role of the Potyvirus P3 Protein of turnip mosaic virus as asymptom and avirulence determinant in Brassica. Mol Plant-Microbe Interact, 2003, 16: 777–784 [24]SuehiroN, Natsuaki T, Watanabe T, Okuda S. An important determinant of the ability of turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein. J General Virol, 2004, 85 : 2087–2098 [25]Wen R H, Saghai Maroof M A, Hajimorad M R. Amino acid changes in P3, and not the overlapping pipo-encoded protein, determine virulence of Soybean mosaic virus on functionally immune Rsv1-genotype soybean. Mol Plant Pathol, 2011, 12: 799–807 [26]Wen R H, Khatabi B, Ashfield T, Saghai Maroof M A, Hajimorad M R. The HC-Pro and P3 cistrons of an avirulent soybean mosaic virus are recognized by different resistance genes at the complex Rsv1 locus. Mol Plant-Microbe Interact, 2013, 26: 203–215 [27]Hajimorad M R, Eggenberger A L, Hill J H. Strain-specific P3 of Soybean mosaic virus elicits Rsv1-mediated extreme resistance, but absence of P3 elicitor function alone is insufficient for virulence on Rsv1-genotype soybean. Virology, 2006, 345: 156–166 [28]Chowda-Reddy R V, Sun H Y, Chen H Y, Poysa V, Ling H, Gijzen M and Wang A M. Mutations in the P3 Protein of soybean mosaic virus G2 isolates determine virulence on Rsv4-genotype soybean. Mol Plant-Microbe Interact, 2011, 24: 37–43 [29]Wang Y, Khatabi B and Hajimorad M R. Amino acid substitution in P3 of soybean mosaic virus to convert avirulence to virulence on Rsv4-genotype soybean is influenced by the genetic composition of P3. Mol Plant Pathol, 2015, 16: 301–307 [30]Wen R H, Hajimorad M R. Mutational analysis of the putative pipo of soybean mosaic virus suggests disruption of PIPO protein impedes movement. Virology, 2010, 400: 1–7 [31]Zhang L, Yang X D, Zhang Y Y, Yang J, Qi G X, Guo D Q, Xing G J, Yao Y, Xu W J, Li H Y, Li Q Y, Dong Y S. Changes in oleic acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing. Intl J Genom, 2014: 104–116 [32]Edwards K, Johnstone C, and Thompson C, A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl Acids Res, 1991, 19: 1349 [33]Tel-Zur N, Abbo S, Myslabodski D, Mizrahi Y. Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Plant Mol Biol Rep, 1999, 17: 249–254 [34]智海剑, 盖钧镒, 何小红. 大豆对SMV数量(程度)抗性的综合分级方法研究. 大豆科学, 2005, 24(2): 5–11 Zhi H J, Gai J Y, He X H. Study on methods of classification of quantitative resistance to soybean mosaic virus in soybean. Soybean Sci, 2005, 24(2): 5–11 [35]Tougou M, Furutani N, Yamagishi N, Shizukawa Y, Takahata Y, Hidaka S. Development of resistant transgenic soybeans with inverted repeat-coat protein genes of soybean dwarf virus. Plant Cell Rep, 2006, 25: 1213–1218 [36]Meyer P. Transcriptional transgene silencing and chromatin components. Plant Mol Biol, 2000, 43: 221–234 [37]杨清华. 我国大豆花叶病毒的株系分化、P3基因序列特征以及大豆对强毒株系抗病基因的标记定位. 南京农业大学博士学位论文, 江苏南京, 2009. p 80 Yang Q H. Strain differentiation and P3 sequence characteristics of soybean mosaic virus in China and gene mapping of resistance to a virulent strain in soybean. PhD Dissertation of Nanjing Agricultural University, Nanjing, China, 2009. p 80

[1] CHEN Ling-Ling, LI Zhan, LIU Ting-Xuan, GU Yong-Zhe, SONG Jian, WANG Jun, QIU Li-Juan. Genome wide association analysis of petiole angle based on 783 soybean resources (Glycine max L.) [J]. Acta Agronomica Sinica, 2022, 48(6): 1333-1345.
[2] YANG Huan, ZHOU Ying, CHEN Ping, DU Qing, ZHENG Ben-Chuan, PU Tian, WEN Jing, YANG Wen-Yu, YONG Tai-Wen. Effects of nutrient uptake and utilization on yield of maize-legume strip intercropping system [J]. Acta Agronomica Sinica, 2022, 48(6): 1476-1487.
[3] YU Chun-Miao, ZHANG Yong, WANG Hao-Rang, YANG Xing-Yong, DONG Quan-Zhong, XUE Hong, ZHANG Ming-Ming, LI Wei-Wei, WANG Lei, HU Kai-Feng, GU Yong-Zhe, QIU Li-Juan. Construction of a high density genetic map between cultivated and semi-wild soybeans and identification of QTLs for plant height [J]. Acta Agronomica Sinica, 2022, 48(5): 1091-1102.
[4] LI A-Li, FENG Ya-Nan, LI Ping, ZHANG Dong-Sheng, ZONG Yu-Zheng, LIN Wen, HAO Xing-Yu. Transcriptome analysis of leaves responses to elevated CO2 concentration, drought and interaction conditions in soybean [Glycine max (Linn.) Merr.] [J]. Acta Agronomica Sinica, 2022, 48(5): 1103-1118.
[5] PENG Xi-Hong, CHEN Ping, DU Qing, YANG Xue-Li, REN Jun-Bo, ZHENG Ben-Chuan, LUO Kai, XIE Chen, LEI Lu, YONG Tai-Wen, YANG Wen-Yu. Effects of reduced nitrogen application on soil aeration and root nodule growth of relay strip intercropping soybean [J]. Acta Agronomica Sinica, 2022, 48(5): 1199-1209.
[6] WANG Hao-Rang, ZHANG Yong, YU Chun-Miao, DONG Quan-Zhong, LI Wei-Wei, HU Kai-Feng, ZHANG Ming-Ming, XUE Hong, YANG Meng-Ping, SONG Ji-Ling, WANG Lei, YANG Xing-Yong, QIU Li-Juan. Fine mapping of yellow-green leaf gene (ygl2) in soybean (Glycine max L.) [J]. Acta Agronomica Sinica, 2022, 48(4): 791-800.
[7] LI Rui-Dong, YIN Yang-Yang, SONG Wen-Wen, WU Ting-Ting, SUN Shi, HAN Tian-Fu, XU Cai-Long, WU Cun-Xiang, HU Shui-Xiu. Effects of close planting densities on assimilate accumulation and yield of soybean with different plant branching types [J]. Acta Agronomica Sinica, 2022, 48(4): 942-951.
[8] DU Hao, CHENG Yu-Han, LI Tai, HOU Zhi-Hong, LI Yong-Li, NAN Hai-Yang, DONG Li-Dong, LIU Bao-Hui, CHENG Qun. Improving seed number per pod of soybean by molecular breeding based on Ln locus [J]. Acta Agronomica Sinica, 2022, 48(3): 565-571.
[9] ZHOU Yue, ZHAO Zhi-Hua, ZHANG Hong-Ning, KONG You-Bin. Cloning and functional analysis of the promoter of purple acid phosphatase gene GmPAP14 in soybean [J]. Acta Agronomica Sinica, 2022, 48(3): 590-596.
[10] WANG Juan, ZHANG Yan-Wei, JIAO Zhu-Jin, LIU Pan-Pan, CHANG Wei. Identification of QTLs and candidate genes for 100-seed weight trait using PyBSASeq algorithm in soybean [J]. Acta Agronomica Sinica, 2022, 48(3): 635-643.
[11] ZHANG Guo-Wei, LI Kai, LI Si-Jia, WANG Xiao-Jing, YANG Chang-Qin, LIU Rui-Xian. Effects of sink-limiting treatments on leaf carbon metabolism in soybean [J]. Acta Agronomica Sinica, 2022, 48(2): 529-537.
[12] SONG Li-Jun, NIE Xiao-Yu, HE Lei-Lei, KUAI Jie, YANG Hua, GUO An-Guo, HUANG Jun-Sheng, FU Ting-Dong, WANG Bo, ZHOU Guang-Sheng. Screening and comprehensive evaluation of shade tolerance of forage soybean varieties [J]. Acta Agronomica Sinica, 2021, 47(9): 1741-1752.
[13] CAO Liang, DU Xin, YU Gao-Bo, JIN Xi-Jun, ZHANG Ming-Cong, REN Chun-Yuan, WANG Meng-Xue, ZHANG Yu-Xian. Regulation of carbon and nitrogen metabolism in leaf of soybean cultivar Suinong 26 at seed-filling stage under drought stress by exogenous melatonin [J]. Acta Agronomica Sinica, 2021, 47(9): 1779-1790.
[14] ZHANG Ming-Cong, HE Song-Yu, QIN Bin, WANG Meng-Xue, JIN Xi-Jun, REN Chun-Yuan, WU Yao-Kun, ZHANG Yu-Xian. Effects of exogenous melatonin on morphology, photosynthetic physiology, and yield of spring soybean variety Suinong 26 under drought stress [J]. Acta Agronomica Sinica, 2021, 47(9): 1791-1805.
[15] YU Tao-Bing, SHI Qi-Han, NIAN-Hai , LIAN Teng-Xiang. Effects of waterlogging on rhizosphere microorganisms communities of different soybean varieties [J]. Acta Agronomica Sinica, 2021, 47(9): 1690-1702.
Full text



No Suggested Reading articles found!