大麦黄花叶病相关基因WRKY55的克隆及功能研究
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摘要
大麦黄花叶病是我国长江中下游地区大麦种植区域的主要病毒病害,由大麦黄花叶病毒(BaYMV,Barley yellow mosaic virus)及大麦和性花叶病毒(BaMMV,Barley mild mosaic virus)引起,自然条件下病毒寄生于禾谷多黏菌中,通过感染大麦根部导致病害发生。本研究通过分析RNA-seq数据,获得感染BaMMV后上调表达的基因HORVU1Hr1G069640(WRKY55)。荧光定量PCR分析发现WRKY55在感病品种中的表达水平极显著高于抗病材料,说明WRKY55参与到大麦感染黄花叶病的过程。WRKY55全长870 bp,在415～594位核苷酸之间含有WRKY保守结构域,系统进化分析显示该基因位于独立的进化分枝。组织表达分析发现该基因主要在根部和幼嫩组织中表达,而其他成熟部位表达较少甚至没有。农杆菌介导的烟草亚细胞定位实验发现WRKY55位于整个细胞。酵母转录因子活性分析实验未检测到转录激活活性,酵母双杂交实验未检测到WRKY55与感病基因编码蛋白eIF4E和PDIL5-1存在物理相互作用。这些结果显示WRKY55参与到大麦黄花叶病感染。
Barley yellow mosaic virus disease,caused by Barley yellow mosaic virus(BaYMV)and Barley mild mosaic virus(BaMMV),is a major viral disease in barley planting regions of Europe and East Asia countries.In the field,this disease is transmitted y the soil-borne fungus Polymyxa graminis via inoculating the roots.Here,by unlocking the transcriptome datasets,we identified a differentially expressed gene HORVU1Hr1G069640(referred WRKY55)that was significantly up-regulated after infection with b BaMMV.Consistently,by analyzing its expression in a group of naturally-inoculated genotypes,a significantly higher expression of WRKY55 was observed in susceptible relative to resistant genotypes.This gene contains an 870 bp of the coding sequence with a conserved WRKY domain between nucleotides 415′-594′.Phylogenetic analysis revealed an independent branch of this gene,differedto the sequence homologous genes.This gene was highly expressed in roots and young tissues,but rarely expressed in other tissues.By Agrobacterium-mediated transformation into Nicotiana benthamiana Domin,the WRKY55-GFP fusion protein was found in the whole cell.In yeast,no trans-activation activity of WRKY55 was detected and no physical interaction between WRKY55 and either susceptibility factors eIF4 E or PDIL5-1 were observed.This work identified a BaMMV-infected upregulated gene WRKY55,which possibly was involved in infection of BaYMV/BaMMV.
Barley yellow mosaic virus disease,caused by Barley yellow mosaic virus(BaYMV)and Barley mild mosaic virus(BaMMV),is a major viral disease in barley planting regions of Europe and East Asia countries.In the field,this disease is transmitted y the soil-borne fungus Polymyxa graminis via inoculating the roots.Here,by unlocking the transcriptome datasets,we identified a differentially expressed gene HORVU1Hr1G069640(referred WRKY55)that was significantly up-regulated after infection with b BaMMV.Consistently,by analyzing its expression in a group of naturally-inoculated genotypes,a significantly higher expression of WRKY55 was observed in susceptible relative to resistant genotypes.This gene contains an 870 bp of the coding sequence with a conserved WRKY domain between nucleotides 415′-594′.Phylogenetic analysis revealed an independent branch of this gene,differedto the sequence homologous genes.This gene was highly expressed in roots and young tissues,but rarely expressed in other tissues.By Agrobacterium-mediated transformation into Nicotiana benthamiana Domin,the WRKY55-GFP fusion protein was found in the whole cell.In yeast,no trans-activation activity of WRKY55 was detected and no physical interaction between WRKY55 and either susceptibility factors eIF4 E or PDIL5-1 were observed.This work identified a BaMMV-infected upregulated gene WRKY55,which possibly was involved in infection of BaYMV/BaMMV.
引文
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[3]Kashiwazaki S,Minobe Y,Hibino H.Nucleotide sequence of barley yellow mosaic virus RNA2.Journal of General Virology,1991,72(4):995-999
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[6]Plumb R T,Lennon E A,Gutteridge R A.The effects of infectionbybarleyyellowmosaic-virusontheyieldand components of yield of barley.Plant Pathology,1986,35(3):314-318
[7]You Y,Shirako Y.Bymovirus reverse genetics:requirements for RNA2-encoded proteins in systemic infection.Molecular Plant Pathology,2010,11(3):383-394
[8]Yang P,Lupken T,Habekuss A,Hensel G,Steuernagel B,Kilian B,Ariyadasa R,Himmelbach A,Kumlehn J,Scholz U,Ordon F,Stein N. PROTEIN DISULFIDE ISOMERASE LIKE5-1 is a susceptibility factor to plant viruses. Proceedings of the National Academy of Sciences of the United States of America,2014,111(6):2104-2109
[9]Stein N,Perovic D,Kumlehn J,Pellio B,Stracke S,Streng S,OrdonF,Graner A.Theeukaryotictranslationinitiation factor 4E confers multiallelic recessive Bymovirus resistance in Hordeum vulgare.Plant Journal,2005,42(6):912-922
[10]Yang P,Habekuss A,Ordon F,Stein N. Analysis of bymovirus resistance genes on proximal barley chromosome 4HL provides the basis for precision breeding for BaMMV/BaYMV resistance.Theoretical and Applied Genetics,2014,127(7):1625-1634
[11]Yang P,Habekuss A,Hofinger B J,Kanyuka K,Kilian B,Graner A,OrdonF,SteinN.Sequencediversificationin recessiveallelesoftwohostfactorgenessuggestsadaptive selection for bymovirus resistance in cultivated barley from East Asia. Theoretical and Applied Genetics,2017,130(2):331-344
[12]Kalde M,Barth M,Somssich I E,Lippok B.Members of the Arabidopsis WRKY group III transcription factors are part of different plant defense signaling pathways.Molecular Plant and Microbe Interact,2003,16(4):295-305
[13]Wu K L,Guo Z J,Wang H H,Li J.The WRKY family of transcription factors in rice and Arabidopsis and their origins.DNA Research,2005,12(1):9-26
[14]Seo S,Sano H,Ohashi Y.Jasmonate-based wound signal transduction requires activation of WIPK,a tobacco mitogenactivated protein kinase.Plant Cell,1999,11(2):289-298
[15]MaeoK,HayashIS,Kojima-SuzukiH,Morikami A,Nakamura K.Role of conserved residues of the WRKY domain intheDNA-bindingoftobaccoWRKYfamilyproteins.Bioscience Biotechnology and Biochemistry,2001,65(11):2428-2436
[16]Eulgem T,Rushton P J,Schmelzer E,Hahlbrock K,Somssich I E.Early nuclear events in plant defense:rapid gene activation by WRKY transcription factors.EMBO Journal,1999,18(17):4689-4699
[17]Naoumkina M,He X,Dixon R.Elicitor induced transcription factors form metabolic reprogramming of secondary metabolism inMedicagotruncatula.BMCPlantBiology,2008,8(1):132
[18]Turck F,Zhou A,Somssich I E.Stimulus-dependent,promoterspecificbindingoftranscriptionfactor WRKY1toitsnative promoter and the defense related gene PcPR1-1 in Parsley.Plant Cell,2004,16(10):2573-2585
[19]Hinderhofer K,Zentgraf U.Identification of a transcription factorspecificallyexpressedattheonsetofleafsenescence.Planta,2001,213(3):469-473
[20]Derlande L,Oliver J,Peeters N,Feng D X,Khounlotham M,Boucher C,Somssich I,Genin S,Marco Y.Physical interaction betweenRRS1-R,aproteinconferringresistancetobacterial wilt,and PopP2,a type III effector targeted to the plant nucleus.Proceedings of the National Academy of Sciences of the United States of America,2003,100(13):8024-8029
[21]Huang Y,Li M Y,Wu P,Xu Z S,Que F,Wang F,Xiong AS.Membersof WRKYGroupIIItranscriptionfactorsare importantinTYLCVdefensesignalingpathwayintomato(Solanum lycopersicum).BMC Genomics,2016,17(1):788
[22]Pandey S P,Smossich I E.The role of WRKY transcription factorsinplantimmunity.PlantPhysiology,2009,150(4):1648-1655
[23]Liu S A,Kracher B,Ziegler J,Birkenbihl R P,Somssich I E.Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea.eLife,2015,4:e07295
[24]张京,刘旭.大麦种质资源描述规范和数据标准.北京:中国农业出版社,2006:52-53ZhangJ,LiuX.Descriptorsanddatastandardforbarley(Hordeum vulgare L.).Beijing:published by China Agriculture Press,2006:52-53
[25]XiaoYH,LiuDP,ZhangGX,TongHN,ChuC C.Brassinosteroids regulate OFP1,a DLT interacting protein,to modulate plant architecture and grain morphology in rice.Plant Science,2017,8:1698
[26]Rushton P J,Somssich I E,Ringler P,Shen Q J.WRKY transcription factors.Trend in Plant Science,2010,15(5):247-258
[27]王磊,高晓清,朱苓华,周永力,黎志康.植物WRKY转录因子家族基因抗病相关功能的研究进展.植物遗传资源学报,2011,12(1):80-85Wang L,Gao X Q,Zhu L H,Zhou Y L,Li Z K.Advances in researchonfunctionof WRKYtranscriptionfactorgenesin plantresistance.JournalofPlantGeneticResources,2011,12(1):80-85
[28]李颖波,郭桂梅,刘成洪,何婷,高润红,徐红卫,陈志伟,陆瑞菊,黄剑华.大麦HvLECl基因的克隆及其表达特征分析.植物遗传资源学报,2016,17(4):732-737Li Y B,Guo G M,Liu C H,He T,Gao R H,Xu H W,Chen Z W,LuRJ,HuangJH.Cloningandexpressioncharacterization ofHvLEClinHordeumvalgareL..JournalofPlantGenetic Resources,2016,17(4):732-737
[2]陈剑平.大麦黄花叶病及其真菌介体禾谷多粘菌的研究进展.中国病毒学,1992,7(1):1-10ChenJP.Researchadvanceonbarleyyellowmosaicviruses and their fungal vector Polymyxa graminis L.Virologica Sinica,1992,7(1):1-10
[3]Kashiwazaki S,Minobe Y,Hibino H.Nucleotide sequence of barley yellow mosaic virus RNA2.Journal of General Virology,1991,72(4):995-999
[4]Adams M J,Hill S A.Soil-borne mosaic viruses of cereals:The UK situation.London:Home Grown Cereals Authority,1992:1-8
[5]Huth W.Management of yellow mosaic-inducing viruses on barley by selection of resistant cultivars.EPPO Bulletin,1989,19(3):547-553
[6]Plumb R T,Lennon E A,Gutteridge R A.The effects of infectionbybarleyyellowmosaic-virusontheyieldand components of yield of barley.Plant Pathology,1986,35(3):314-318
[7]You Y,Shirako Y.Bymovirus reverse genetics:requirements for RNA2-encoded proteins in systemic infection.Molecular Plant Pathology,2010,11(3):383-394
[8]Yang P,Lupken T,Habekuss A,Hensel G,Steuernagel B,Kilian B,Ariyadasa R,Himmelbach A,Kumlehn J,Scholz U,Ordon F,Stein N. PROTEIN DISULFIDE ISOMERASE LIKE5-1 is a susceptibility factor to plant viruses. Proceedings of the National Academy of Sciences of the United States of America,2014,111(6):2104-2109
[9]Stein N,Perovic D,Kumlehn J,Pellio B,Stracke S,Streng S,OrdonF,Graner A.Theeukaryotictranslationinitiation factor 4E confers multiallelic recessive Bymovirus resistance in Hordeum vulgare.Plant Journal,2005,42(6):912-922
[10]Yang P,Habekuss A,Ordon F,Stein N. Analysis of bymovirus resistance genes on proximal barley chromosome 4HL provides the basis for precision breeding for BaMMV/BaYMV resistance.Theoretical and Applied Genetics,2014,127(7):1625-1634
[11]Yang P,Habekuss A,Hofinger B J,Kanyuka K,Kilian B,Graner A,OrdonF,SteinN.Sequencediversificationin recessiveallelesoftwohostfactorgenessuggestsadaptive selection for bymovirus resistance in cultivated barley from East Asia. Theoretical and Applied Genetics,2017,130(2):331-344
[12]Kalde M,Barth M,Somssich I E,Lippok B.Members of the Arabidopsis WRKY group III transcription factors are part of different plant defense signaling pathways.Molecular Plant and Microbe Interact,2003,16(4):295-305
[13]Wu K L,Guo Z J,Wang H H,Li J.The WRKY family of transcription factors in rice and Arabidopsis and their origins.DNA Research,2005,12(1):9-26
[14]Seo S,Sano H,Ohashi Y.Jasmonate-based wound signal transduction requires activation of WIPK,a tobacco mitogenactivated protein kinase.Plant Cell,1999,11(2):289-298
[15]MaeoK,HayashIS,Kojima-SuzukiH,Morikami A,Nakamura K.Role of conserved residues of the WRKY domain intheDNA-bindingoftobaccoWRKYfamilyproteins.Bioscience Biotechnology and Biochemistry,2001,65(11):2428-2436
[16]Eulgem T,Rushton P J,Schmelzer E,Hahlbrock K,Somssich I E.Early nuclear events in plant defense:rapid gene activation by WRKY transcription factors.EMBO Journal,1999,18(17):4689-4699
[17]Naoumkina M,He X,Dixon R.Elicitor induced transcription factors form metabolic reprogramming of secondary metabolism inMedicagotruncatula.BMCPlantBiology,2008,8(1):132
[18]Turck F,Zhou A,Somssich I E.Stimulus-dependent,promoterspecificbindingoftranscriptionfactor WRKY1toitsnative promoter and the defense related gene PcPR1-1 in Parsley.Plant Cell,2004,16(10):2573-2585
[19]Hinderhofer K,Zentgraf U.Identification of a transcription factorspecificallyexpressedattheonsetofleafsenescence.Planta,2001,213(3):469-473
[20]Derlande L,Oliver J,Peeters N,Feng D X,Khounlotham M,Boucher C,Somssich I,Genin S,Marco Y.Physical interaction betweenRRS1-R,aproteinconferringresistancetobacterial wilt,and PopP2,a type III effector targeted to the plant nucleus.Proceedings of the National Academy of Sciences of the United States of America,2003,100(13):8024-8029
[21]Huang Y,Li M Y,Wu P,Xu Z S,Que F,Wang F,Xiong AS.Membersof WRKYGroupIIItranscriptionfactorsare importantinTYLCVdefensesignalingpathwayintomato(Solanum lycopersicum).BMC Genomics,2016,17(1):788
[22]Pandey S P,Smossich I E.The role of WRKY transcription factorsinplantimmunity.PlantPhysiology,2009,150(4):1648-1655
[23]Liu S A,Kracher B,Ziegler J,Birkenbihl R P,Somssich I E.Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea.eLife,2015,4:e07295
[24]张京,刘旭.大麦种质资源描述规范和数据标准.北京:中国农业出版社,2006:52-53ZhangJ,LiuX.Descriptorsanddatastandardforbarley(Hordeum vulgare L.).Beijing:published by China Agriculture Press,2006:52-53
[25]XiaoYH,LiuDP,ZhangGX,TongHN,ChuC C.Brassinosteroids regulate OFP1,a DLT interacting protein,to modulate plant architecture and grain morphology in rice.Plant Science,2017,8:1698
[26]Rushton P J,Somssich I E,Ringler P,Shen Q J.WRKY transcription factors.Trend in Plant Science,2010,15(5):247-258
[27]王磊,高晓清,朱苓华,周永力,黎志康.植物WRKY转录因子家族基因抗病相关功能的研究进展.植物遗传资源学报,2011,12(1):80-85Wang L,Gao X Q,Zhu L H,Zhou Y L,Li Z K.Advances in researchonfunctionof WRKYtranscriptionfactorgenesin plantresistance.JournalofPlantGeneticResources,2011,12(1):80-85
[28]李颖波,郭桂梅,刘成洪,何婷,高润红,徐红卫,陈志伟,陆瑞菊,黄剑华.大麦HvLECl基因的克隆及其表达特征分析.植物遗传资源学报,2016,17(4):732-737Li Y B,Guo G M,Liu C H,He T,Gao R H,Xu H W,Chen Z W,LuRJ,HuangJH.Cloningandexpressioncharacterization ofHvLEClinHordeumvalgareL..JournalofPlantGenetic Resources,2016,17(4):732-737
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