水稻条纹病毒功能蛋白与水稻蛋白的互作研究
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摘要
水稻条纹病毒(Rice stripe virus,RSV)引起的水稻条纹叶枯病是水稻上的一种重要病毒病,该病在我国的分布极为广泛,给人们带来巨大的损失。为了对该病毒的蛋白功能、致病机制及寄主对病毒的抗感病作用机制有深入的了解,我们从分子水平上研究了病毒蛋白及其与寄主蛋白之间的互作关系。本研究在蛋白筛选的基础上,利用酵母双杂交系统对病毒蛋白与寄主蛋白之间的互作做进一步的验证,并针对RSV编码的功能蛋白NS2,对其互作情况进行了研究。
在构建水稻文库(“武育粳3号”和“KT95-418”)的基础上,分别以RSV CP、SP、NSvc4为诱饵蛋白,通过酵母双杂交系统,筛选出一系列与病毒蛋白存在相互作用的水稻蛋白。从筛选结果中挑取了两个比较有意思的克隆CB18和NB17,RT-PCR扩增其基因全长,将全长CB18和NB17构建到酵母双杂交捕获载体pGADT7上,鉴定筛选获得了重组子pGAD-CB18和pGAD-NB17。将pGAD-CB18和pGAD-NB17分别与诱饵质粒pGBK-CP、pGBK-SP、pGBK-NSvc4共转化酵母菌AH109,并涂布不同的营养缺陷型培养基平板,检测CB18和NB17全长基因表达产物与RSV CP、SP和NSvc4的互作情况。结果表明,CB18与CP存在互作现象,但互作不强烈,与NSVc4、SP不具有互作关系;NB17与NSVc4具有互作,而与CP、SP不具有互作关系。
利用荧光定量PCR(Real Time PCR)技术检测CB18、NB17基因mRNA在RSV侵染后的水稻叶片和健康水稻叶片中的表达情况。比较mRNA表达量的差异,从而分析RSV侵染及病毒蛋白与寄主蛋白互作对其mRNA表达情况的影响。结果表明,CB18 mRNA在RSV侵染后的水稻中与在健康水稻中的表达量相比表现为上调;NB17 mRNA在RSV侵染后的水稻中与在健康水稻中的表达量相比表现为下调。
借助原核表达载体PGEX-4T-1,构建了CB18的融合蛋白原核表达重组子PGEX-CB18。转化大肠杆菌(Escherichia coli)BL21(DE3)并诱导表达,SDS-PAGE检测其表达产物与预期结果大小一致,表达的融合蛋白GST-CB18相对分子量约为37KD。进一步制备了CB18的抗血清,Western blot结果显示所制备的抗血清能与诱导表达的融合蛋白发生特异性反应,并进一步利用Western blot检测了在RSV侵染后的水稻和健康水稻中CB18蛋白表达量变化,结果发现,CB18在RSV侵染后的水稻中蛋白表达量比在健康水稻中表达量高。
除上述研究之外,本研究还通过RT-PCR扩增获得了水稻条纹病毒(RSV)功能蛋白基因NS2,将其构建到酵母双杂交捕获载体pGADT7和诱饵载体pGBKT7上,通过筛选鉴定获得了重组子pGAD-NS2、pGBK-NS2。将上述重组子分别单独转化到酵母菌AH109中,转化产物涂布不同营养缺陷型培养基,检测NS2自激活活性。将重组子分别与NSVc4、CP、SP酵母表达载体共转化酵母菌AH109,转化产物涂布不同营养缺陷型培养基,从而检测NS2蛋白自身以及与NSVc4、CP、SP的互作情况。结果表明,RSV NS2蛋白对酵母菌AH109未表现出自激活活性,NS2蛋白具有自身互作,而NS2与NSvc4、CP、SP蛋白之间未检测到互作现象。
Rice stripe disease caused by Rice stirpe virus (RSV) is an important disease to rice. It distributes in a large scale of China and brings huge loss to people. In order to further study the function of some viral proteins, pathogenic mechanism of the virus, and molecular mechanisms of plant resistance and susceptibility to RSV, we researched the relationship between viral proteins and host proteins at molecular level. Based on library screening, the yeast two hybrid system was employed to further confirm the interaction of viral protein and host protein. we also studied the potential interactions of the function protein NS2 with other proteins encoded by RSV.
Using rice libraries (“Wuyujing 3”and“KT95-418”), we previourly identified a number of rice proteins that interact with used RSV CP,SP and NSVc4 respectively in the yeast two hybrid system. In this study, 2 interesting clones namely CB18 and NB17 were selected from the yeast two-hybrid screening results. The full-length gene was amplified by RT-PCR, and were then inserted into yeast two-hybrid prey vector pGADT7, thus constructing the recombinants pGAD-CB18 and pGAD-NB17. After that the recombinants pGAD-CB18 or pGAD-NB17 were co-transformed into yeast cell AH109 with bait plasmids pGBK-CP, pGBK-SP and pGBK-NSvc4 respectively, and the co-transformants were plated on different synthetic dropout nutrient medium. The results showed that CB18 interacted with CP weakly but not with NSvc4 and SP; NB17 could interact with NSvc4 but not CP and SP.
The expressions of CB18 and NB17 mRNA in the healthy and RSV-infected rice leaves were examined by Real-Time PCR. The differences of mRNA expression were compared, supposing that any changes in the mRNA expression could be a result of RSV infection or interactions with RSV encoded protein. The results showed: the expression of CB18 mRNA in the RSV-infected rice leaves was up-regulated compared with that in the health rice leaves; The expression of NB17 mRNA in the RSV-infected rice leaves was down-regulated compared with that in the health rice leaves.
Fusion protein expression vector PGEX-CB18 was constructed by using prokaryotic expression vector PGEX-4T-1. The recombinants pGEX-CB18 were transformed into E.coli BL21(DE3) and induced to express. The expression products of GST-fusion protein were detected by SDS-PAGE which showed that it is in same size as expected. Then antiserum of CB18 was prepared using the expressed protein, Western-blot analysis showed that the antiserums could react with the expression products of the fusion genes specifically. Then the antiserum was used to investigate the protein expression difference in RSV infection rice and healthy rice. The results showed that: the protein expression of CB18 in the RSV infected rice is more than that in the healthy rice.
Additionally, RSV NS2 genes was amplified by RT-PCR and inserted into yeast two-hybrid system bait vector pGBKT7 and prey vector pGADT7 respectively. The recombinants were transformed into yeast cell AH109 respectively, and plated on the different synthetic dropout nutrient medium to detect the self-activity .Then, the recombinants were co-transformed into yeast cell AH109 with yeast expression vector CP, SP and NSvc4 respectively and plated on the different synthetic dropout nutrient medium to detect the interactions between NS2 and CP, SP, and NSVc4. The results showed: NS2 have no self-activity .it could interact with itself, but could not interact with CP, SP, and NSVc4.
在构建水稻文库(“武育粳3号”和“KT95-418”)的基础上,分别以RSV CP、SP、NSvc4为诱饵蛋白,通过酵母双杂交系统,筛选出一系列与病毒蛋白存在相互作用的水稻蛋白。从筛选结果中挑取了两个比较有意思的克隆CB18和NB17,RT-PCR扩增其基因全长,将全长CB18和NB17构建到酵母双杂交捕获载体pGADT7上,鉴定筛选获得了重组子pGAD-CB18和pGAD-NB17。将pGAD-CB18和pGAD-NB17分别与诱饵质粒pGBK-CP、pGBK-SP、pGBK-NSvc4共转化酵母菌AH109,并涂布不同的营养缺陷型培养基平板,检测CB18和NB17全长基因表达产物与RSV CP、SP和NSvc4的互作情况。结果表明,CB18与CP存在互作现象,但互作不强烈,与NSVc4、SP不具有互作关系;NB17与NSVc4具有互作,而与CP、SP不具有互作关系。
利用荧光定量PCR(Real Time PCR)技术检测CB18、NB17基因mRNA在RSV侵染后的水稻叶片和健康水稻叶片中的表达情况。比较mRNA表达量的差异,从而分析RSV侵染及病毒蛋白与寄主蛋白互作对其mRNA表达情况的影响。结果表明,CB18 mRNA在RSV侵染后的水稻中与在健康水稻中的表达量相比表现为上调;NB17 mRNA在RSV侵染后的水稻中与在健康水稻中的表达量相比表现为下调。
借助原核表达载体PGEX-4T-1,构建了CB18的融合蛋白原核表达重组子PGEX-CB18。转化大肠杆菌(Escherichia coli)BL21(DE3)并诱导表达,SDS-PAGE检测其表达产物与预期结果大小一致,表达的融合蛋白GST-CB18相对分子量约为37KD。进一步制备了CB18的抗血清,Western blot结果显示所制备的抗血清能与诱导表达的融合蛋白发生特异性反应,并进一步利用Western blot检测了在RSV侵染后的水稻和健康水稻中CB18蛋白表达量变化,结果发现,CB18在RSV侵染后的水稻中蛋白表达量比在健康水稻中表达量高。
除上述研究之外,本研究还通过RT-PCR扩增获得了水稻条纹病毒(RSV)功能蛋白基因NS2,将其构建到酵母双杂交捕获载体pGADT7和诱饵载体pGBKT7上,通过筛选鉴定获得了重组子pGAD-NS2、pGBK-NS2。将上述重组子分别单独转化到酵母菌AH109中,转化产物涂布不同营养缺陷型培养基,检测NS2自激活活性。将重组子分别与NSVc4、CP、SP酵母表达载体共转化酵母菌AH109,转化产物涂布不同营养缺陷型培养基,从而检测NS2蛋白自身以及与NSVc4、CP、SP的互作情况。结果表明,RSV NS2蛋白对酵母菌AH109未表现出自激活活性,NS2蛋白具有自身互作,而NS2与NSvc4、CP、SP蛋白之间未检测到互作现象。
Rice stripe disease caused by Rice stirpe virus (RSV) is an important disease to rice. It distributes in a large scale of China and brings huge loss to people. In order to further study the function of some viral proteins, pathogenic mechanism of the virus, and molecular mechanisms of plant resistance and susceptibility to RSV, we researched the relationship between viral proteins and host proteins at molecular level. Based on library screening, the yeast two hybrid system was employed to further confirm the interaction of viral protein and host protein. we also studied the potential interactions of the function protein NS2 with other proteins encoded by RSV.
Using rice libraries (“Wuyujing 3”and“KT95-418”), we previourly identified a number of rice proteins that interact with used RSV CP,SP and NSVc4 respectively in the yeast two hybrid system. In this study, 2 interesting clones namely CB18 and NB17 were selected from the yeast two-hybrid screening results. The full-length gene was amplified by RT-PCR, and were then inserted into yeast two-hybrid prey vector pGADT7, thus constructing the recombinants pGAD-CB18 and pGAD-NB17. After that the recombinants pGAD-CB18 or pGAD-NB17 were co-transformed into yeast cell AH109 with bait plasmids pGBK-CP, pGBK-SP and pGBK-NSvc4 respectively, and the co-transformants were plated on different synthetic dropout nutrient medium. The results showed that CB18 interacted with CP weakly but not with NSvc4 and SP; NB17 could interact with NSvc4 but not CP and SP.
The expressions of CB18 and NB17 mRNA in the healthy and RSV-infected rice leaves were examined by Real-Time PCR. The differences of mRNA expression were compared, supposing that any changes in the mRNA expression could be a result of RSV infection or interactions with RSV encoded protein. The results showed: the expression of CB18 mRNA in the RSV-infected rice leaves was up-regulated compared with that in the health rice leaves; The expression of NB17 mRNA in the RSV-infected rice leaves was down-regulated compared with that in the health rice leaves.
Fusion protein expression vector PGEX-CB18 was constructed by using prokaryotic expression vector PGEX-4T-1. The recombinants pGEX-CB18 were transformed into E.coli BL21(DE3) and induced to express. The expression products of GST-fusion protein were detected by SDS-PAGE which showed that it is in same size as expected. Then antiserum of CB18 was prepared using the expressed protein, Western-blot analysis showed that the antiserums could react with the expression products of the fusion genes specifically. Then the antiserum was used to investigate the protein expression difference in RSV infection rice and healthy rice. The results showed that: the protein expression of CB18 in the RSV infected rice is more than that in the healthy rice.
Additionally, RSV NS2 genes was amplified by RT-PCR and inserted into yeast two-hybrid system bait vector pGBKT7 and prey vector pGADT7 respectively. The recombinants were transformed into yeast cell AH109 respectively, and plated on the different synthetic dropout nutrient medium to detect the self-activity .Then, the recombinants were co-transformed into yeast cell AH109 with yeast expression vector CP, SP and NSvc4 respectively and plated on the different synthetic dropout nutrient medium to detect the interactions between NS2 and CP, SP, and NSVc4. The results showed: NS2 have no self-activity .it could interact with itself, but could not interact with CP, SP, and NSVc4.
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54. Hayano-Saito Y,Saito K,Nakamura S.2000.Fine physical mapping of the rice stripe resistance gene locus Stvb-I.Theoretical and Applied Genetics,101(1):59-63
55. Hammamatsu C,Toriyama S,ToyodaT.1993. Ambisense coding strategy of therice stripe virusgenome in vitro translation studies.Journal ofGeneralVirology,74(6):1125-1131
56. Higuchi R,Fockler C.1993.Kinetic PCR analysis real-time monitoring of DNA amplification reactions.Biotechnology,11(9):1026-1030.
57. HaenniA L,deMiranda JR, Falk BW,Fau-quetCM,MayoM A,Maniloff J.2005.Report of the International Committee on Taxonomy of Viruses.Virus taxonomy,ElsevierAcademic Press,San Diego:717-723
58. Hammond K E,Jones J D G.1997.Plant disease resistance genes.Plant Mol.Biol,48:575-607
59. Heath M C.2000.Hypersensitive response-related death. Plant Molecular Biology,44:321-334
60. James P,Halladay J,Craig EA.1996.Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.Genetics,144:1425–1436
61. Koganezawa H,Doi Y and Yora K.1975.Purification of rice stripe virus. Annuals of the Phytopathological.Society of Japan,41:148-154
62. Kiso A ,Yamamoto T.1973.Infection and symptom in rice stripe disease with special referance to disease-specific protein other than virus.Review of Plant Protection Research, 6:75-100
63. Kiso A,Yamamoto T,Kitani K.1974.Studies on rice stripe disease with special reference to the causal virus its lacation in the diseased tissues and the metabolic changes in thediseased plant.Bulletin Shikoku Agricultural Experiment Station,27:1
64. Leister R T,Dahlbeck D,Day B,Li Y.2005.Chesnokova O and Staskawicz B J. Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana .The Plant Cell,17:1268-1278
65. Leon J,Rojo E and Sanchez-Serrano J J.2001.Wound signalling in plants.Journal of Experimental Botany,52 (354):1-9
66. LiangD,MaX Q,Qu Z.2005.Nucleic acid binding property of the gene products ofrice stripe virus.VirusGenes,31(2):203-209
67. Liang D L,Qu Z C,Ma X Q and Hull R.2005.Detection and Localization of Rice stripe virus Gene Products In Vivo.Virus Genes,31 (2):211–221.
68. Melcher U.2000.The“30K”superfamily of viral movement proteins.Journal of General virology,81:257-266
69. Ma J,Ptashne M.1987.A new class of yeast transcriptional activators.Cell,51(1) :113-119.
70. Merits A,Guo D,Jarvekülg L ,Saarma M.1999.Biochemical and genetic evidence for interactions between potato A potyvirus encoded proteins P1 and P3 and proteins of the putative replication complex.Virology ,263:15-22
71. Morin S,Ghanim M and Sobol I.2000.The GroEL protein of the whitefly bemisia tabaci interacts with the coat protein of transmissible and nontransmissible begomoviruses in the yeast two-hybrid system.Virology,276 (2):404-416
72. Ramirez B C and Haenni A L.1994.Molecular biology of tenuiviruses a remarkable of plant viruses.Journal of General Virology,75 (1): 467-475
73. Stewart M G and Banerjee N.1999.Mechanisms of arthropod transmission of plant andanimal viruses.Microbiology and Molecular Reviews,63 (1):128-148
74. Soellick T R,Uhrig J F,Bucher G L,Kellmann J W and Schreier P H.2000.The movement protein NSm of Tomato spotted wilt tospovirus (TSWV): RNA binding, interaction with the TSWV N protein, and identification of interacting plant proteins.Proceedings of the National Academy of Sciences of the United States of America,97 (5):2373-2378
75. Toriyama S,TakahashiM,SanoY.1994.Nucleotide sequence of RNA1, the largestgenomic segment ofrice stripe virus,the proto-type of the tenuiviruses.Journal of General Virology, 75(12): 3569-3579
76. Takahashi M and Toriyama S.1996.Detection of the 22.8k protein encoded by RNA2 of rice stripe virus in infected plant.Annuals of Phytopathological Society of Japan,62 (3):340
77. Takahashi M,Goto C,Matsuda I.1998.expression of rice stripe virus 22.8k protein in insect cells.Annuals of the Phytopathological Society of Japan,65 (3):342
78. Tampo H,Plante D ,Laliberte J F.1999.The cytopIasmic inclusion protein of TuMV interaction with histone H3 of Arabidopsis thaliana.Phytopathology,89 (6):76
79. Uyeda S,Masuta C and Uyeda I.1997.Hypothesis on particle structure and assembly of Rice dwarf phytoreovirus:interactions among multiple structural proteins.Journal of General Virology,78:3135-3140
80. Uyeda S,Masuta C,Uyeda I.1999.The C-terminal region of the P3 structural protein of Rice dwarf phytoreovirus is important for P3-P3 interaction.Archives of Virology,144 (8):1653-1657
81. Visser P B and Bol J F.1999.Nonstructural proteins of tobacco rattle virus which have a role in nematode-transmission: expression pat tern and interact ion with viral coat protein.Journal of General Virology,80:3273-3280
82. Watson M A,Buckholz R,Weiner M P.1996.Vectors encoding alternative antibiotic resistance for use in the yeast two-hybrid system.BioTechniques,21:255–259.
83. Washi O,Ezuka A,Sakurai Y..1968a.Studies on the breeding of rice barieties resistant to stripe deseaseⅡ.Genaetic study on resistance to stripe disease in Japanese upland rice .Japan Journal of Breeding,18:96-101
84. Washi.O,Ezuka A,Sakurai Y..1968b.Studies on the breeding of rice barieties resistant to stripe deseaseⅢ.Genaetic study on resistance to stripe disease in foreign varieties .Japan Journal of Breeding,18:167-172
85. Wong C,Naumovski L.1997.Method to screen for relevant yeast two-hybrid-derived clones by coimmunoprecipitation and colocalization of epitope-tagged fragments application to Bcl-xL.Anal Biochem,252(1):33-9.
86. Xie Q,Sanz-Burgos A P and Guo H.1999.GRAB proteins novel members of the NAC domain family isolated by their interaction with a geminivirus protein.Plant Molecular Biology,39 (4):647-656
87. Yamashita S,Doi Y and Yora K.1985.Intercelluar appearance of rice stripe virus.Annals of the Phytopathological Society of Japan,51 (5):637-641
88. Zhang HM,Yang J,XinX.2007.Genomic analysis ofrice stripe virusZhejiang isolate shows the presence ofan OTU-like domain in the protein encoded by RNA1 and a novel sequence motif con-servedwithin the intergenic regionsofambisense segmentsoftenui-viruses.Archive ofVirology,DOI10.1007/s00705-007-1013-2
89. Zhu S F,Gao F and Li Y.2005.The rice dwarf virus P2 protein interacts with ent-Kaurene Oxidases in Vivo leading to reduced biosynthesis of gibberellins and rice dwarf symptoms..Plant Physiology,139 (4):1935-1945
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53. Hayamo-SaitoY,Tsuji T,Fujiik.1998.Localization of the rice stripe disease resistance gene Stv-bi by graphical genotyping and linkage analysis with molecular markers.Theoretical and Applied Genetics,96 (8):1044-1049
54. Hayano-Saito Y,Saito K,Nakamura S.2000.Fine physical mapping of the rice stripe resistance gene locus Stvb-I.Theoretical and Applied Genetics,101(1):59-63
55. Hammamatsu C,Toriyama S,ToyodaT.1993. Ambisense coding strategy of therice stripe virusgenome in vitro translation studies.Journal ofGeneralVirology,74(6):1125-1131
56. Higuchi R,Fockler C.1993.Kinetic PCR analysis real-time monitoring of DNA amplification reactions.Biotechnology,11(9):1026-1030.
57. HaenniA L,deMiranda JR, Falk BW,Fau-quetCM,MayoM A,Maniloff J.2005.Report of the International Committee on Taxonomy of Viruses.Virus taxonomy,ElsevierAcademic Press,San Diego:717-723
58. Hammond K E,Jones J D G.1997.Plant disease resistance genes.Plant Mol.Biol,48:575-607
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60. James P,Halladay J,Craig EA.1996.Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.Genetics,144:1425–1436
61. Koganezawa H,Doi Y and Yora K.1975.Purification of rice stripe virus. Annuals of the Phytopathological.Society of Japan,41:148-154
62. Kiso A ,Yamamoto T.1973.Infection and symptom in rice stripe disease with special referance to disease-specific protein other than virus.Review of Plant Protection Research, 6:75-100
63. Kiso A,Yamamoto T,Kitani K.1974.Studies on rice stripe disease with special reference to the causal virus its lacation in the diseased tissues and the metabolic changes in thediseased plant.Bulletin Shikoku Agricultural Experiment Station,27:1
64. Leister R T,Dahlbeck D,Day B,Li Y.2005.Chesnokova O and Staskawicz B J. Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana .The Plant Cell,17:1268-1278
65. Leon J,Rojo E and Sanchez-Serrano J J.2001.Wound signalling in plants.Journal of Experimental Botany,52 (354):1-9
66. LiangD,MaX Q,Qu Z.2005.Nucleic acid binding property of the gene products ofrice stripe virus.VirusGenes,31(2):203-209
67. Liang D L,Qu Z C,Ma X Q and Hull R.2005.Detection and Localization of Rice stripe virus Gene Products In Vivo.Virus Genes,31 (2):211–221.
68. Melcher U.2000.The“30K”superfamily of viral movement proteins.Journal of General virology,81:257-266
69. Ma J,Ptashne M.1987.A new class of yeast transcriptional activators.Cell,51(1) :113-119.
70. Merits A,Guo D,Jarvekülg L ,Saarma M.1999.Biochemical and genetic evidence for interactions between potato A potyvirus encoded proteins P1 and P3 and proteins of the putative replication complex.Virology ,263:15-22
71. Morin S,Ghanim M and Sobol I.2000.The GroEL protein of the whitefly bemisia tabaci interacts with the coat protein of transmissible and nontransmissible begomoviruses in the yeast two-hybrid system.Virology,276 (2):404-416
72. Ramirez B C and Haenni A L.1994.Molecular biology of tenuiviruses a remarkable of plant viruses.Journal of General Virology,75 (1): 467-475
73. Stewart M G and Banerjee N.1999.Mechanisms of arthropod transmission of plant andanimal viruses.Microbiology and Molecular Reviews,63 (1):128-148
74. Soellick T R,Uhrig J F,Bucher G L,Kellmann J W and Schreier P H.2000.The movement protein NSm of Tomato spotted wilt tospovirus (TSWV): RNA binding, interaction with the TSWV N protein, and identification of interacting plant proteins.Proceedings of the National Academy of Sciences of the United States of America,97 (5):2373-2378
75. Toriyama S,TakahashiM,SanoY.1994.Nucleotide sequence of RNA1, the largestgenomic segment ofrice stripe virus,the proto-type of the tenuiviruses.Journal of General Virology, 75(12): 3569-3579
76. Takahashi M and Toriyama S.1996.Detection of the 22.8k protein encoded by RNA2 of rice stripe virus in infected plant.Annuals of Phytopathological Society of Japan,62 (3):340
77. Takahashi M,Goto C,Matsuda I.1998.expression of rice stripe virus 22.8k protein in insect cells.Annuals of the Phytopathological Society of Japan,65 (3):342
78. Tampo H,Plante D ,Laliberte J F.1999.The cytopIasmic inclusion protein of TuMV interaction with histone H3 of Arabidopsis thaliana.Phytopathology,89 (6):76
79. Uyeda S,Masuta C and Uyeda I.1997.Hypothesis on particle structure and assembly of Rice dwarf phytoreovirus:interactions among multiple structural proteins.Journal of General Virology,78:3135-3140
80. Uyeda S,Masuta C,Uyeda I.1999.The C-terminal region of the P3 structural protein of Rice dwarf phytoreovirus is important for P3-P3 interaction.Archives of Virology,144 (8):1653-1657
81. Visser P B and Bol J F.1999.Nonstructural proteins of tobacco rattle virus which have a role in nematode-transmission: expression pat tern and interact ion with viral coat protein.Journal of General Virology,80:3273-3280
82. Watson M A,Buckholz R,Weiner M P.1996.Vectors encoding alternative antibiotic resistance for use in the yeast two-hybrid system.BioTechniques,21:255–259.
83. Washi O,Ezuka A,Sakurai Y..1968a.Studies on the breeding of rice barieties resistant to stripe deseaseⅡ.Genaetic study on resistance to stripe disease in Japanese upland rice .Japan Journal of Breeding,18:96-101
84. Washi.O,Ezuka A,Sakurai Y..1968b.Studies on the breeding of rice barieties resistant to stripe deseaseⅢ.Genaetic study on resistance to stripe disease in foreign varieties .Japan Journal of Breeding,18:167-172
85. Wong C,Naumovski L.1997.Method to screen for relevant yeast two-hybrid-derived clones by coimmunoprecipitation and colocalization of epitope-tagged fragments application to Bcl-xL.Anal Biochem,252(1):33-9.
86. Xie Q,Sanz-Burgos A P and Guo H.1999.GRAB proteins novel members of the NAC domain family isolated by their interaction with a geminivirus protein.Plant Molecular Biology,39 (4):647-656
87. Yamashita S,Doi Y and Yora K.1985.Intercelluar appearance of rice stripe virus.Annals of the Phytopathological Society of Japan,51 (5):637-641
88. Zhang HM,Yang J,XinX.2007.Genomic analysis ofrice stripe virusZhejiang isolate shows the presence ofan OTU-like domain in the protein encoded by RNA1 and a novel sequence motif con-servedwithin the intergenic regionsofambisense segmentsoftenui-viruses.Archive ofVirology,DOI10.1007/s00705-007-1013-2
89. Zhu S F,Gao F and Li Y.2005.The rice dwarf virus P2 protein interacts with ent-Kaurene Oxidases in Vivo leading to reduced biosynthesis of gibberellins and rice dwarf symptoms..Plant Physiology,139 (4):1935-1945