水稻矮缩病毒Pns6和P8蛋白在病毒侵染水稻原生质体后的表达
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摘要
【目的】为明确水稻矮缩病毒(Rice dwarf virus, RDV) Pns6和P8蛋白在病毒侵染水稻原生质体后的表达动态,【方法】利用PEG介导的病毒侵染原生质体体系,通过免疫荧光和电镜技术分析Pns6、P8蛋白以及病毒粒体在水稻原生质体中的定位;同时,通过Western blot和实时荧光定量PCR分析Pns6和P8蛋白及其RNA在水稻原生质体中的积累量。【结果】病毒接种水稻原生质体48h后,Pns6蛋白在细胞质中可以形成类似于病毒原质(viroplasm)的点状内含体,P8蛋白也大量的表达。同时,病毒粒体在水稻原生质体中也形成内含体状的结构。病毒接种水稻原生质体12 h后,均可检测到Pns6和P8蛋白的表达,并且在36 h后达到最高值。病毒接种水稻原生质体后,Pns6 RNA在24 h时表达量达到最高,P8 RNA在36 h时表达量达到最高。【结论】RDV侵染水稻原生质体后,Pns6和P8蛋白均有表达,并且病毒也可能通过形成病毒原质来完成病毒在寄主细胞的复制和装配。
【Objective】In order to clarify expression dynamics of Pns6 and P8 proteins of Rice dwarf virus after virus infecting rice protoplasts, 【Method】subcellular localization of Pns6 and P8 proteins as well as viral particles in rice protoplasts were conducted with a PEG mediated virus infecting protoplast system by immunofluorescence and electron microscope. Meanwhile, accumulation of Pns6 and P8 proteins and their RNAs in rice protoplasts were measured by Western blot and real-time fluorescence quantitative PCR. 【Result】Pns6 protein could form viroplasm-like punctate inclusion bodies in cytoplasm 48 h after virus inoculation, and P8 protein was also expressed in large quantities.Meanwhile, viral particles could form inclusion body-like structure in rice protoplasts. Expression of Pns6 and P8 proteins was both detected at 12 h after virus inoculation, and reached the maximum level after 36 h. At the same time,the expression level of Pns6 RNA peaked at 24 h and that of P8 RNA at 36 h after virus inoculation. 【Conclusion】Pns6 and P8 proteins were both expressed after virus infection, and Rice dwarf virus may also formed viroplasm-like inclusion bodies to complete virus replication and assembly in host cells.
【Objective】In order to clarify expression dynamics of Pns6 and P8 proteins of Rice dwarf virus after virus infecting rice protoplasts, 【Method】subcellular localization of Pns6 and P8 proteins as well as viral particles in rice protoplasts were conducted with a PEG mediated virus infecting protoplast system by immunofluorescence and electron microscope. Meanwhile, accumulation of Pns6 and P8 proteins and their RNAs in rice protoplasts were measured by Western blot and real-time fluorescence quantitative PCR. 【Result】Pns6 protein could form viroplasm-like punctate inclusion bodies in cytoplasm 48 h after virus inoculation, and P8 protein was also expressed in large quantities.Meanwhile, viral particles could form inclusion body-like structure in rice protoplasts. Expression of Pns6 and P8 proteins was both detected at 12 h after virus inoculation, and reached the maximum level after 36 h. At the same time,the expression level of Pns6 RNA peaked at 24 h and that of P8 RNA at 36 h after virus inoculation. 【Conclusion】Pns6 and P8 proteins were both expressed after virus infection, and Rice dwarf virus may also formed viroplasm-like inclusion bodies to complete virus replication and assembly in host cells.
引文
[1]Omura T,Yan J.Role of outer capsid proteins in transmission of Phytoreovirus by insect vectors.Adv Virus Res,1999,54(1):15-43.
[2]谢联辉,林奇英,魏太云,吴祖建.水稻病毒.北京:科学出版社,2016:158-182.Xie L H,Lin Q Y,Wei T Y,Wu Z J.Rice Viruses.Beijing:Science Press,2016:158-182.(in Chinese)
[3]谢联辉,林奇英.我国水稻病毒病研究的进展.中国农业科学,1984,17(6):58-65.Xie L H,Lin Q Y.Advances in the studies of rice virus disease in China.Sci Agric Sin,1984,17(6):58-65.(in Chinese with English abstract)
[4]Suzuki N,Tanimura M,Watanabe Y,Kusano T,Kitagawa Y,Suda N,Kudo H,Uyeda I,Shikata E.Molecular analysis of rice dwarf phytoreovirus segment S1:Interviral homology of the putative RNA-dependent RNApolymerase between plant-and animal-infecting reoviruses.Virology,1992,190(1):240-247.
[5]Omura T,Yan J,Zhong B,Wada M,Zhu Y,Tomaru M,Maruyama W,Kikuchi A,Watanabe Y,Kimura I,Hibino H.The P2 protein of rice dwarf phytoreovirus is required for adsorption of the virus to cells of the insect vector.JVirol,1998,72(11):9370-9373.
[6]Zhou F,Pu Y,Wei T,Liu H,Deng W,Wei C,Ding B,Omura T,Li Y.The P2 capsid protein of the nonenveloped rice dwarf phytoreovirus induces membrane fusion in insect host cells.Proc Natl Acad Sci,2007,104(49):19547-19552.
[7]Zhu S,Gao F,Cao X,Chen M,Ye G,Wei C,Li Y.The rice dwarf virus P2 protein interacts with ent-kaurene oxidases in vivo,leading to reduced biosynthesis of gibberellins and rice dwarf symptoms.Plant Physiol,2005,139(4):1935-1945.
[8]Jin L,Qin Q,Wang Y,Pu Y,Liu L,Wen X,Ji S,Wu J,Wei C,Ding B,Li Y.Rice dwarf virus P2 protein hijacks auxin signaling by directly targeting the rice Os IAA10protein,enhancing viral infection and disease development.PLo S Pathog,2016,12(9):e1005847.
[9]Hagiwara K,Higashi T,Miyazaki N,Naitow H,Cheng RH,Nakagawa A,Mizuno H,Tsukihara T,Omura T.The amino-terminal region of major capsid protein P3 is essential for self-assembly of single-shelled core-like particles of Rice dwarf virus.J Virol,2004,78(6):3145-3148.
[10]Wei T,Kikuchi A,Suzuki N,Shimizu T,Hagiwara K,Chen H,Omura T.Pns4 of rice dwarf virus is a phosphoprotein,is localized around the viroplasm matrix,and forms minitubules.Arch Virol,2006,151(9):1701-1712.
[11]Chen Q,Zhang L,Chen H,Xie L,Wei T.Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector.Virol J,2015,12:211.
[12]Suzuki N,Kusano T,Matsuura Y,Omura T.Novel NTPbinding property of rice dwarf phytoreovirus minor core protein P5.Virology,1996,19(2):471-474.
[13]Ji X,Qian D,Wei C,Ye G,Zhang Z,Wu Z,Xie L,Li Y.Movement protein Pns6 of rice dwarf phytoreovirus has both ATPase and RNA binding activities.PLo S One,2011,6(9):e24986.
[14]Li Y,Bao Y M,Wei C H,Kang Z S,Zhong Y W,Mao P,Wu G,Chen Z L,Schiemann J,Nelson R S.Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function.J Virol,2004,78:5382-5389.
[15]Chen Q,Chen H,Jia D,Mao Q,Xei L,Wei T.Nonstructural protein Pns12 of rice dwarf virus is a principal regulator for viral replication and infection in its insect vector.Virus Res,2015,210:54-61.
[16]Wei T,Shimizu T,Hagiwara K,Kikuchi A,Moriyasu Y,Suzuki N,Chen H,Omura T.Pns12 protein of Rice dwarf virus is essential for formation of viroplasms and nucleation of viral-assembly complexes.J Gen Virol,2006,87(2):429-438.
[17]Ueda S,Uyeda I.The rice dwarf phytoreovirus structural protein P7 possesses non-specific nucleotide acids binding activity in vitro.Mol Plant Pathol,1997,http://www.bspp.org.uk/mppol/1997/0123Ueda/.
[18]Zhong B,Shen Y,Omura T.RNA-binding domain of the key structural protein P7 for the Rice dwarf virus particle assembly.Acta Bioch Bioph Sin,2005,37(1):55-60.
[19]Miyazaki N,Hagiwara K,Naitow H,Higashi T,Cheng RH,Tsukihara T,Nakagawa A,Omura T.Transcapsidation and the conserved interactions of two major structural proteins of a pair of phytoreoviruses confirm the mechanism of assembly of the outer capsid layer.J Mol Biol,2005,345:229237.
[20]Zhou F,Wu G,Deng W,Pu Y,Wei C,Li Y.Interaction of rice dwarf virus outer capsid P8 protein with rice glycolate oxidase mediates relocalization of P8.Febs Lett,2007,581(1):34-40.
[21]Zhong B,Kikuchi A,Moriyasu Y,Higashi T,Hagiwara K,Omura T.A minor outer capsid protein,P9,of Rice dwarf virus.Arch Virol,2003,148(11):2275-2280.
[22]尹哲,吉栩,吴云峰.水稻矮缩病毒外壳蛋白P9具有体内转录激活活性.中国农业科技导报,2007,9(3):61-65.Yin Z,Ji X,Wu Y F.Minor outer capsid protein P9 of Rice dwarf virus shows transcriptional activation in vivo.J Agric Sci Technol,2007,9(3):61-65.(in Chinese with English abstract)
[23]Cao X,Zhou P,Zhang X,Zhu S,Zhong X,Xiao Q,Ding B,Li Y.Identification of an RNA silencing suppressor from a plant double-stranded RNA virus.J Virol,2005,79(20):13018.
[24]Wei T,Kikuchi A,Moriyasu Y,Suzuki N,Shimizu T,Hagiwara K,Chen H,Takahashi M,Ichiki-Uehara T,Omura T.The spread of Rice dwarf virus among cells of its insect vector exploits virus-induced tubular structures.J Virol,2006,80(17):8593-8602.
[25]Wei T,Shimizu T,Omura T.Endomembranes and myosin mediate assembly into tubules of Pns10 of rice dwarf virus and intercellular spreading of the virus in cultured insect vector cells.Virology,2008,372(2):349-356.
[26]Xu H,Li Y,Mao Z,Li Y,Wu Z,Qu L,An C,Ming X,Schiemann J,Casper R,Chen Z.Rice dwarf phytoreovirus segment S11 encodes a nucleic acidbinding protein.Virology,1998,40(2):267-272.
[27]吴建国.水稻矮缩病毒非结构蛋白Pns11的核仁定位及其与核仁蛋白Fibrillarin的互作.福州:福建农林大学,2010.Wu J.Rice dwarf virus non-structural protein Pns11localized in nucleolus and interacted with the major nucleolar protein Fibrillarin.Fuzhou:Fujian Agriculture and Forestry University,2010.(in Chinese with English abstract)
[28]Zhao S,Hong W,Wu J,Wang Y,Ji S,Zhu S,Wei C,Zhang J,Li Y.A viral protein promotes host SAMS1activity and ethylene production for the benefit of virus infection.Elife,2017,6:e27529.
[29]张洁,陈晓敏,吴锦鸿,朱重庆,丁新伦,吴祖建.水稻齿叶矮缩病毒Pns10蛋白在水稻原生质体内的表达.中国水稻科学,2017,31(3):232-237.Zhang J,Chen X M,Wu J H,Zhu C Q,Ding X L,Wu Z J.Expression of Pns10 protein of rice ragged stunt virus in rice protoplasts.Chin J Rice Sci,2017,31(3):232-237.(in Chinese with English abstract)
[30]张洁,陈晓敏,宛柏杰,吴锦鸿,林文武,吴祖建.水稻齿叶矮缩病毒非结构蛋白Pns7在水稻原生质体内的表达动态.植物病理学报,2017,47(1):61-67.Zhang J,Chen X M,Wan B J,Wu J H,Lin W W,Wu ZJ.Expression dynamics of Rice ragged stunt virus nonstructural protein Pns7 in rice protoplast.Acta Phytopathol Sin,2017,47(1):61-67.(in Chinese with English abstract)
[31]段炼,钱君,郭小雨,朱英.一种快速高效的水稻原生质体制备和转化方法的建立.植物生理学报,2014,50(3):351-357.Duan L,Qian J,Guo X Y,Zhu Y.A rapid and efficient method for isolation and transformation of rice protoplast.Plant Physiol J,2014,50(3):351-357.(in Chinese with English abstract)
[32]Kikkert M,van Poelwijk F,Storms M,Kassies W,Bloksma H,van Lent J,Kormelink R,Goldbach R.Aprotoplast system for studying tomato spotted wilt virus infection.J Gen Virol,1997,78(7):1755-1763.
[33]贾东升.SRBSDV和RRSV在介体飞虱体内的侵染机理.福州:福建农林大学,2013.Jia D S.Infection mechanisms of SRBSDV and RRSV in vector planthoppers.Fuzhou:Fujian Agriculture and Forestry University,2013.(in Chinese with English abstract)
[34]郑胜兰.SRBSDV非结构蛋白P6在病毒侵染白背飞虱过程中的功能分析.福州:福建农林大学,2014.Zheng S L.The functional analysis of P6 encoded by SRBSDV during virus infecting the white backed planthopper.Fuzhou:Fujian Agriculture and Forestry University,2013.(in Chinese with English abstract)
[35]陈晓敏,宛柏杰,吴锦鸿,林文武,吴祖建,张洁.水稻齿叶矮缩病毒P8蛋白的抗体制备及其在水稻原生质体内的表达动态.中国科技论文,2016,11(6):670-674.Chen X M,Wan B J,Wu J H,Lin W W,Wu Z J,Zhang J.Preparation of polyclonal antibody against P8 protein of rice ragged stunt virus and its expression dynamic trends in rice protoplast.China Sciencepaper,2016,11(6):670-674.(in Chinese with English abstract)
[2]谢联辉,林奇英,魏太云,吴祖建.水稻病毒.北京:科学出版社,2016:158-182.Xie L H,Lin Q Y,Wei T Y,Wu Z J.Rice Viruses.Beijing:Science Press,2016:158-182.(in Chinese)
[3]谢联辉,林奇英.我国水稻病毒病研究的进展.中国农业科学,1984,17(6):58-65.Xie L H,Lin Q Y.Advances in the studies of rice virus disease in China.Sci Agric Sin,1984,17(6):58-65.(in Chinese with English abstract)
[4]Suzuki N,Tanimura M,Watanabe Y,Kusano T,Kitagawa Y,Suda N,Kudo H,Uyeda I,Shikata E.Molecular analysis of rice dwarf phytoreovirus segment S1:Interviral homology of the putative RNA-dependent RNApolymerase between plant-and animal-infecting reoviruses.Virology,1992,190(1):240-247.
[5]Omura T,Yan J,Zhong B,Wada M,Zhu Y,Tomaru M,Maruyama W,Kikuchi A,Watanabe Y,Kimura I,Hibino H.The P2 protein of rice dwarf phytoreovirus is required for adsorption of the virus to cells of the insect vector.JVirol,1998,72(11):9370-9373.
[6]Zhou F,Pu Y,Wei T,Liu H,Deng W,Wei C,Ding B,Omura T,Li Y.The P2 capsid protein of the nonenveloped rice dwarf phytoreovirus induces membrane fusion in insect host cells.Proc Natl Acad Sci,2007,104(49):19547-19552.
[7]Zhu S,Gao F,Cao X,Chen M,Ye G,Wei C,Li Y.The rice dwarf virus P2 protein interacts with ent-kaurene oxidases in vivo,leading to reduced biosynthesis of gibberellins and rice dwarf symptoms.Plant Physiol,2005,139(4):1935-1945.
[8]Jin L,Qin Q,Wang Y,Pu Y,Liu L,Wen X,Ji S,Wu J,Wei C,Ding B,Li Y.Rice dwarf virus P2 protein hijacks auxin signaling by directly targeting the rice Os IAA10protein,enhancing viral infection and disease development.PLo S Pathog,2016,12(9):e1005847.
[9]Hagiwara K,Higashi T,Miyazaki N,Naitow H,Cheng RH,Nakagawa A,Mizuno H,Tsukihara T,Omura T.The amino-terminal region of major capsid protein P3 is essential for self-assembly of single-shelled core-like particles of Rice dwarf virus.J Virol,2004,78(6):3145-3148.
[10]Wei T,Kikuchi A,Suzuki N,Shimizu T,Hagiwara K,Chen H,Omura T.Pns4 of rice dwarf virus is a phosphoprotein,is localized around the viroplasm matrix,and forms minitubules.Arch Virol,2006,151(9):1701-1712.
[11]Chen Q,Zhang L,Chen H,Xie L,Wei T.Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector.Virol J,2015,12:211.
[12]Suzuki N,Kusano T,Matsuura Y,Omura T.Novel NTPbinding property of rice dwarf phytoreovirus minor core protein P5.Virology,1996,19(2):471-474.
[13]Ji X,Qian D,Wei C,Ye G,Zhang Z,Wu Z,Xie L,Li Y.Movement protein Pns6 of rice dwarf phytoreovirus has both ATPase and RNA binding activities.PLo S One,2011,6(9):e24986.
[14]Li Y,Bao Y M,Wei C H,Kang Z S,Zhong Y W,Mao P,Wu G,Chen Z L,Schiemann J,Nelson R S.Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function.J Virol,2004,78:5382-5389.
[15]Chen Q,Chen H,Jia D,Mao Q,Xei L,Wei T.Nonstructural protein Pns12 of rice dwarf virus is a principal regulator for viral replication and infection in its insect vector.Virus Res,2015,210:54-61.
[16]Wei T,Shimizu T,Hagiwara K,Kikuchi A,Moriyasu Y,Suzuki N,Chen H,Omura T.Pns12 protein of Rice dwarf virus is essential for formation of viroplasms and nucleation of viral-assembly complexes.J Gen Virol,2006,87(2):429-438.
[17]Ueda S,Uyeda I.The rice dwarf phytoreovirus structural protein P7 possesses non-specific nucleotide acids binding activity in vitro.Mol Plant Pathol,1997,http://www.bspp.org.uk/mppol/1997/0123Ueda/.
[18]Zhong B,Shen Y,Omura T.RNA-binding domain of the key structural protein P7 for the Rice dwarf virus particle assembly.Acta Bioch Bioph Sin,2005,37(1):55-60.
[19]Miyazaki N,Hagiwara K,Naitow H,Higashi T,Cheng RH,Tsukihara T,Nakagawa A,Omura T.Transcapsidation and the conserved interactions of two major structural proteins of a pair of phytoreoviruses confirm the mechanism of assembly of the outer capsid layer.J Mol Biol,2005,345:229237.
[20]Zhou F,Wu G,Deng W,Pu Y,Wei C,Li Y.Interaction of rice dwarf virus outer capsid P8 protein with rice glycolate oxidase mediates relocalization of P8.Febs Lett,2007,581(1):34-40.
[21]Zhong B,Kikuchi A,Moriyasu Y,Higashi T,Hagiwara K,Omura T.A minor outer capsid protein,P9,of Rice dwarf virus.Arch Virol,2003,148(11):2275-2280.
[22]尹哲,吉栩,吴云峰.水稻矮缩病毒外壳蛋白P9具有体内转录激活活性.中国农业科技导报,2007,9(3):61-65.Yin Z,Ji X,Wu Y F.Minor outer capsid protein P9 of Rice dwarf virus shows transcriptional activation in vivo.J Agric Sci Technol,2007,9(3):61-65.(in Chinese with English abstract)
[23]Cao X,Zhou P,Zhang X,Zhu S,Zhong X,Xiao Q,Ding B,Li Y.Identification of an RNA silencing suppressor from a plant double-stranded RNA virus.J Virol,2005,79(20):13018.
[24]Wei T,Kikuchi A,Moriyasu Y,Suzuki N,Shimizu T,Hagiwara K,Chen H,Takahashi M,Ichiki-Uehara T,Omura T.The spread of Rice dwarf virus among cells of its insect vector exploits virus-induced tubular structures.J Virol,2006,80(17):8593-8602.
[25]Wei T,Shimizu T,Omura T.Endomembranes and myosin mediate assembly into tubules of Pns10 of rice dwarf virus and intercellular spreading of the virus in cultured insect vector cells.Virology,2008,372(2):349-356.
[26]Xu H,Li Y,Mao Z,Li Y,Wu Z,Qu L,An C,Ming X,Schiemann J,Casper R,Chen Z.Rice dwarf phytoreovirus segment S11 encodes a nucleic acidbinding protein.Virology,1998,40(2):267-272.
[27]吴建国.水稻矮缩病毒非结构蛋白Pns11的核仁定位及其与核仁蛋白Fibrillarin的互作.福州:福建农林大学,2010.Wu J.Rice dwarf virus non-structural protein Pns11localized in nucleolus and interacted with the major nucleolar protein Fibrillarin.Fuzhou:Fujian Agriculture and Forestry University,2010.(in Chinese with English abstract)
[28]Zhao S,Hong W,Wu J,Wang Y,Ji S,Zhu S,Wei C,Zhang J,Li Y.A viral protein promotes host SAMS1activity and ethylene production for the benefit of virus infection.Elife,2017,6:e27529.
[29]张洁,陈晓敏,吴锦鸿,朱重庆,丁新伦,吴祖建.水稻齿叶矮缩病毒Pns10蛋白在水稻原生质体内的表达.中国水稻科学,2017,31(3):232-237.Zhang J,Chen X M,Wu J H,Zhu C Q,Ding X L,Wu Z J.Expression of Pns10 protein of rice ragged stunt virus in rice protoplasts.Chin J Rice Sci,2017,31(3):232-237.(in Chinese with English abstract)
[30]张洁,陈晓敏,宛柏杰,吴锦鸿,林文武,吴祖建.水稻齿叶矮缩病毒非结构蛋白Pns7在水稻原生质体内的表达动态.植物病理学报,2017,47(1):61-67.Zhang J,Chen X M,Wan B J,Wu J H,Lin W W,Wu ZJ.Expression dynamics of Rice ragged stunt virus nonstructural protein Pns7 in rice protoplast.Acta Phytopathol Sin,2017,47(1):61-67.(in Chinese with English abstract)
[31]段炼,钱君,郭小雨,朱英.一种快速高效的水稻原生质体制备和转化方法的建立.植物生理学报,2014,50(3):351-357.Duan L,Qian J,Guo X Y,Zhu Y.A rapid and efficient method for isolation and transformation of rice protoplast.Plant Physiol J,2014,50(3):351-357.(in Chinese with English abstract)
[32]Kikkert M,van Poelwijk F,Storms M,Kassies W,Bloksma H,van Lent J,Kormelink R,Goldbach R.Aprotoplast system for studying tomato spotted wilt virus infection.J Gen Virol,1997,78(7):1755-1763.
[33]贾东升.SRBSDV和RRSV在介体飞虱体内的侵染机理.福州:福建农林大学,2013.Jia D S.Infection mechanisms of SRBSDV and RRSV in vector planthoppers.Fuzhou:Fujian Agriculture and Forestry University,2013.(in Chinese with English abstract)
[34]郑胜兰.SRBSDV非结构蛋白P6在病毒侵染白背飞虱过程中的功能分析.福州:福建农林大学,2014.Zheng S L.The functional analysis of P6 encoded by SRBSDV during virus infecting the white backed planthopper.Fuzhou:Fujian Agriculture and Forestry University,2013.(in Chinese with English abstract)
[35]陈晓敏,宛柏杰,吴锦鸿,林文武,吴祖建,张洁.水稻齿叶矮缩病毒P8蛋白的抗体制备及其在水稻原生质体内的表达动态.中国科技论文,2016,11(6):670-674.Chen X M,Wan B J,Wu J H,Lin W W,Wu Z J,Zhang J.Preparation of polyclonal antibody against P8 protein of rice ragged stunt virus and its expression dynamic trends in rice protoplast.China Sciencepaper,2016,11(6):670-674.(in Chinese with English abstract)