水稻条斑病菌hrpD5基因在致病性中的功能分析
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摘要
水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)成功侵染水稻主要依靠其III型分泌系统(Type III secretion system,T3SS)分泌的效应蛋白。T3SS由hrp-hrc-hpa基因编码,其中主要的hrp和hrc基因突变,病原菌将丧失在寄主水稻上的致病性和非寄主烟草上的过敏性反应(hypersensitive response,HR)。hrpD5基因位于hrp基因簇hrpD操纵单元的第5个基因,在致病性中的功能未知。本研究构建了Xoc的hrpD5缺失突变体RΔhrpD5。植物接种试验显示,RΔhrpD5丧失了对寄主水稻的致病性和在非寄主烟草上激发HR反应的能力;功能互补子虽然能够恢复这2种表型,但是,与野生型菌株相比,其在感病水稻上的毒性显著降低,在非寄主烟草上形成延迟的HR反应;荧光定量PCR结果显示:hrpD5缺失影响其下游hrpD操纵单元基因hrpD6、hrpE和hpaB以及HrpX操纵单元基因hrpF和hrpB1的表达;同时,hrpD5缺失降低了hrpX的mRNA水平,但是不影响hrpX的启动子活性;酵母双杂交结果显示,HrpD5蛋白能够与HrpF蛋白的N端互作。这些结果暗示在Xoc中hrpD5不仅为主要的致病相关基因,而且调控主要的hrp调节基因hrpX的表达。HrpD5新功能的鉴定将为解析T3SS在致病性中的功能提供线索。
Xanthomonas oryzae pv. oryzicola depends on Type III secretion system( T3 SS) to cause disease in host plant rice. T3 SS is encoded by hrp( hypersensitive response and pathogenicity),hrc( hrp conserved) and hpa( hrp associated) genes. Mutations of essential hrp or hrc genes usually abolish the bacterial ability to cause pathogenicity in host rice and hypersensitive response( HR) in non-host tobacco. The hrpD5 gene is the fifth gene with unknown function located in the hrpD operon of the hrp gene cluster. In this work,we constructed a deletion mutant of hrpD5 in Xoc strain RS105( named as RΔhrpD5). Inoculation assays showed that RΔhrpD5 was not able to cause pathogenicity in host rice and failed to trigger HR in non-host tobacco. The lost functions were partially restored in the complementary strain CRΔhrpD5 which had reduced virulence on the susceptible rice and caused delayed HR on non-host tobacco compared to the wild type. Transcriptional analysis showed that mutation of hrpD5 affected the expression of downstream genes hrpD6,hrpE and hpaB in the hrpD operon and genes hrpF and hrpB1 in the HrpX operon,and led to decreased level of hrpX mRNA,but didn’t affect the promoter activity of hrpX. The yeast two-hybrid analysis revealed that HrpD5 interacted with the N terminal ofHrpF. These results indicate that hrpD5 not only is essential for the pathogenicity of Xoc,but also regulates the expression of hrpX,the major hrp regulatory gene. Further functional investigation of HrpD5 may provide some clues to elucidate the roles of T3 SS in pathogenicity of Xoc.
Xanthomonas oryzae pv. oryzicola depends on Type III secretion system( T3 SS) to cause disease in host plant rice. T3 SS is encoded by hrp( hypersensitive response and pathogenicity),hrc( hrp conserved) and hpa( hrp associated) genes. Mutations of essential hrp or hrc genes usually abolish the bacterial ability to cause pathogenicity in host rice and hypersensitive response( HR) in non-host tobacco. The hrpD5 gene is the fifth gene with unknown function located in the hrpD operon of the hrp gene cluster. In this work,we constructed a deletion mutant of hrpD5 in Xoc strain RS105( named as RΔhrpD5). Inoculation assays showed that RΔhrpD5 was not able to cause pathogenicity in host rice and failed to trigger HR in non-host tobacco. The lost functions were partially restored in the complementary strain CRΔhrpD5 which had reduced virulence on the susceptible rice and caused delayed HR on non-host tobacco compared to the wild type. Transcriptional analysis showed that mutation of hrpD5 affected the expression of downstream genes hrpD6,hrpE and hpaB in the hrpD operon and genes hrpF and hrpB1 in the HrpX operon,and led to decreased level of hrpX mRNA,but didn’t affect the promoter activity of hrpX. The yeast two-hybrid analysis revealed that HrpD5 interacted with the N terminal ofHrpF. These results indicate that hrpD5 not only is essential for the pathogenicity of Xoc,but also regulates the expression of hrpX,the major hrp regulatory gene. Further functional investigation of HrpD5 may provide some clues to elucidate the roles of T3 SS in pathogenicity of Xoc.
引文
[1]Nino-Liu D O,Ronald P C,Bogdanove A J.Xanthomonas oryzae pathovars:model pathogens of a model crop[J].Molecular Plant Pathology,2006,7(5):303-324.
[2]Zou L F,Wang X P,Xiang Y,et al.Elucidation of the hrp clusters of Xanthomonas oryzae pv.oryzicola that control the hypersensitive response in nonhost tobacco and pathogenicity in susceptible host rice[J].Applied and Environmental Microbiology,2006,72:6212-6224.
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[4]Weber E,Koebnik R.Positive selection of the Hrp pilin HrpE of the plant pathogen Xanthomonas[J].Journal of Bacteriology,2006,188:1405-1410.
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[8]Buttner D,Nennstiel D,Klusener B,et al.Functional analysis of HrpF,a putative type III translocon protein from Xanthomonas campestris pv.vesicatoria[J].Journal of Bacteriology,2002,184:2389-2398.
[9]Rossier O,Van den Ackerveken G,Bonas U.HrpB2and HrpF from Xanthomonas are type III-secreted proteins and essential for pathogenicity and recognition by the host plant[J].Molecular Microbiology,2000,38:828-838.
[10]Xiao Y L,Li Y R,Liu Z Y,et al.Establishment of the hrp-inducing systems for the expression of the hrp genes of Xanthomonas oryzae pv.oryzicola(in Chinese)[J].Acta Microbiologica Sinica(微生物学报),2007,47:396-401.
[11]Tsuge S,Nakayama T,Terashima S,et al.Gene involved in transcriptional activation of the hrp regulatory gene hrpG in Xanthomonas oryzae pv.oryzae[J].Journal of Bacteriology,2006,188:4158-4162.
[12]Tsuge S,Terashima S,Furutani A,et al.Effects on promoter activity of base substitutions in the cis-acting regulatory element of HrpXo regulons in Xanthomonas oryzae pv.oryzae[J].Journal of Bacteriology,2005,187:2308-2314.
[13]Jiang J,Zou H,Li Y,et al.Expression of the hrcC,hrpE and hpa3 genes is not regulated by the hrpG and hrpX genes in a rice pathogen Xanthomonas oryzae pv.oryzicola(in Chinese)[J].Acta Microbiologica Sinica(微生物学报),2009,49:1018-1025.
[14]Li Y R,Zou H S,Che Y Z,et al.A novel regulatory role of HrpD6 in regulating hrp-hrc-hpa genes in Xanthomonas oryzae pv.oryzicola[J].Molecular PlantMicrobe Interactions,2011,24:1086-1101.
[15]Zeng S Y,Hu J,Huang G X,et al.Identification of a hrp-associated gene hpaB in the role of pathogenicity of Xanthomonas oryzae pv.oryzae(in Chinese)[J].Acta Microbiologica Sinica(微生物学报),2007,47:402-406.
[16]Buttner D,Lorenz C,Weber E,et al.Targeting of two effector protein classes to the type III secretion system by a HpaC-and HpaB-dependent protein complex from Xanthomonas campestris pv.vesicatoria[J].Molecular Microbiology,2006,59:513-527.
[17]Buttner D,Gurlebeck D,Noel L D,et al.HpaB from Xanthomonas campestris pv.vesicatoria acts as an exit control protein in type III-dependent protein secretion[J].Molecular Microbiology,2004,54:755-768.
[18]Cui Y,Zou L,Zou H,et al.HrpE3 is a type III effector protein required for full virulence of Xanthomonas oryzae pv.oryzicola in rice[J].Molecular Plant Pathology,2013,14:678-692.
[19]Zou L F,Li Y R,Chen G Y,et al.A non-marker mutagenesis strategy to generate poly-hrp gene mutants in the rice pathogen Xanthomonas oryzae pv.oryzicola[J].Journal of Integrative Agriculture,2011,10(8):1139-1150.
[20]Hopkins C M,White F F,Choi S H,et al.Identification of a family of avirulence genes from Xanthomonas oryzae pv.oryzae[J].Molecular Plant-Microbe Interactions,1992,5(6):451-459.
[21]Zhao Z H,Xiong L,Shen C W,et al.Construction of two promoter-probe vectors suitable for pathogenicityrelated gene expression analysis in Xanthomonas oryzae pv.oryzicola(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2014,44(5):504-511.
[22]Labes M,Puhler A,Simon R.A new family of RSF1010-derived expression and lac-fusion broadhostrange vectors for gram-negative bacteria[J].Gene,1990,89(1):37-46.
[23]Wang Y Y,Ma W X,Cai L L,et al.Analysis of virulence genes regulating hrp gene expression in Xanthomonas oryzae pv.oryzae(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2016,46(4):492-506.
[24]Evangelista C,Lockshon D,Fields S.The yeast twohybrid system:prospects for protein linkage maps[J].Trends in Cell Biology,1996,6:196-199.
[25]Scheibner F,Schulz S,Hausner J,et al.Type III-dependent translocation of HrpB2 by a nonpathogenic hpaABC mutant of the plant-pathogenic bacterium Xanthomonas campestris pv.vesicatoria[J].Applied and Environmental Microbiology,2016,82:3331-3347.
[26]Hartmann N,Schulz S,Lorenz C,et al.Characterization of HrpB2 from Xanthomonas campestris pv.vesicatoria identifies protein regions that are essential for type III secretion pilus formation[J].Microbiology,2012,158:1334-1349.
[27]Cappelletti P A,dos Santos R F,do Amaral A M,et al.Structure-function analysis of the HrpB2-HrcU interaction in the Xanthomonas citri type III secretion system[J].PLoS One,2011;6:e17614.
[28]Hausner J,Hartmann N,Lorenz C,et al.The periplasmic HrpB1 protein from Xanthomonas spp.binds to peptidoglycan and to components of the type III secretion system[J].Applied and Environmental Microbiology,2013,79:6312-6324.
[2]Zou L F,Wang X P,Xiang Y,et al.Elucidation of the hrp clusters of Xanthomonas oryzae pv.oryzicola that control the hypersensitive response in nonhost tobacco and pathogenicity in susceptible host rice[J].Applied and Environmental Microbiology,2006,72:6212-6224.
[3]Weber E,Koebnik R.Domain structure of HrpE,the Hrp pilus subunit of Xanthomonas campestris pv.vesicatoria[J].Journal of Bacteriology,2005,187:6175-6186.
[4]Weber E,Koebnik R.Positive selection of the Hrp pilin HrpE of the plant pathogen Xanthomonas[J].Journal of Bacteriology,2006,188:1405-1410.
[5]Li Y R,Che Y Z,Zou H S,et al.Hpa2 required by HrpF to translocate Xanthomonas oryzae transcriptional activator-like effectors into rice for pathogenicity[J].Applied and Environmental Microbiology,2011,77:3809-3818.
[6]Buttner D,Noel L,Stuttmann J,et al.Characterization of the nonconserved hpaB-hrpF region in the hrp pathogenicity island from Xanthomonas campestris pv.vesicatoria[J].Molecular Plant-Microbe Interactions,2007,20:1063-1074.
[7]Sugio A,Yang B,White F F.Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv.oryzae[J].Molecular Plant-Microbe Interactions,2005,18:546-554.
[8]Buttner D,Nennstiel D,Klusener B,et al.Functional analysis of HrpF,a putative type III translocon protein from Xanthomonas campestris pv.vesicatoria[J].Journal of Bacteriology,2002,184:2389-2398.
[9]Rossier O,Van den Ackerveken G,Bonas U.HrpB2and HrpF from Xanthomonas are type III-secreted proteins and essential for pathogenicity and recognition by the host plant[J].Molecular Microbiology,2000,38:828-838.
[10]Xiao Y L,Li Y R,Liu Z Y,et al.Establishment of the hrp-inducing systems for the expression of the hrp genes of Xanthomonas oryzae pv.oryzicola(in Chinese)[J].Acta Microbiologica Sinica(微生物学报),2007,47:396-401.
[11]Tsuge S,Nakayama T,Terashima S,et al.Gene involved in transcriptional activation of the hrp regulatory gene hrpG in Xanthomonas oryzae pv.oryzae[J].Journal of Bacteriology,2006,188:4158-4162.
[12]Tsuge S,Terashima S,Furutani A,et al.Effects on promoter activity of base substitutions in the cis-acting regulatory element of HrpXo regulons in Xanthomonas oryzae pv.oryzae[J].Journal of Bacteriology,2005,187:2308-2314.
[13]Jiang J,Zou H,Li Y,et al.Expression of the hrcC,hrpE and hpa3 genes is not regulated by the hrpG and hrpX genes in a rice pathogen Xanthomonas oryzae pv.oryzicola(in Chinese)[J].Acta Microbiologica Sinica(微生物学报),2009,49:1018-1025.
[14]Li Y R,Zou H S,Che Y Z,et al.A novel regulatory role of HrpD6 in regulating hrp-hrc-hpa genes in Xanthomonas oryzae pv.oryzicola[J].Molecular PlantMicrobe Interactions,2011,24:1086-1101.
[15]Zeng S Y,Hu J,Huang G X,et al.Identification of a hrp-associated gene hpaB in the role of pathogenicity of Xanthomonas oryzae pv.oryzae(in Chinese)[J].Acta Microbiologica Sinica(微生物学报),2007,47:402-406.
[16]Buttner D,Lorenz C,Weber E,et al.Targeting of two effector protein classes to the type III secretion system by a HpaC-and HpaB-dependent protein complex from Xanthomonas campestris pv.vesicatoria[J].Molecular Microbiology,2006,59:513-527.
[17]Buttner D,Gurlebeck D,Noel L D,et al.HpaB from Xanthomonas campestris pv.vesicatoria acts as an exit control protein in type III-dependent protein secretion[J].Molecular Microbiology,2004,54:755-768.
[18]Cui Y,Zou L,Zou H,et al.HrpE3 is a type III effector protein required for full virulence of Xanthomonas oryzae pv.oryzicola in rice[J].Molecular Plant Pathology,2013,14:678-692.
[19]Zou L F,Li Y R,Chen G Y,et al.A non-marker mutagenesis strategy to generate poly-hrp gene mutants in the rice pathogen Xanthomonas oryzae pv.oryzicola[J].Journal of Integrative Agriculture,2011,10(8):1139-1150.
[20]Hopkins C M,White F F,Choi S H,et al.Identification of a family of avirulence genes from Xanthomonas oryzae pv.oryzae[J].Molecular Plant-Microbe Interactions,1992,5(6):451-459.
[21]Zhao Z H,Xiong L,Shen C W,et al.Construction of two promoter-probe vectors suitable for pathogenicityrelated gene expression analysis in Xanthomonas oryzae pv.oryzicola(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2014,44(5):504-511.
[22]Labes M,Puhler A,Simon R.A new family of RSF1010-derived expression and lac-fusion broadhostrange vectors for gram-negative bacteria[J].Gene,1990,89(1):37-46.
[23]Wang Y Y,Ma W X,Cai L L,et al.Analysis of virulence genes regulating hrp gene expression in Xanthomonas oryzae pv.oryzae(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2016,46(4):492-506.
[24]Evangelista C,Lockshon D,Fields S.The yeast twohybrid system:prospects for protein linkage maps[J].Trends in Cell Biology,1996,6:196-199.
[25]Scheibner F,Schulz S,Hausner J,et al.Type III-dependent translocation of HrpB2 by a nonpathogenic hpaABC mutant of the plant-pathogenic bacterium Xanthomonas campestris pv.vesicatoria[J].Applied and Environmental Microbiology,2016,82:3331-3347.
[26]Hartmann N,Schulz S,Lorenz C,et al.Characterization of HrpB2 from Xanthomonas campestris pv.vesicatoria identifies protein regions that are essential for type III secretion pilus formation[J].Microbiology,2012,158:1334-1349.
[27]Cappelletti P A,dos Santos R F,do Amaral A M,et al.Structure-function analysis of the HrpB2-HrcU interaction in the Xanthomonas citri type III secretion system[J].PLoS One,2011;6:e17614.
[28]Hausner J,Hartmann N,Lorenz C,et al.The periplasmic HrpB1 protein from Xanthomonas spp.binds to peptidoglycan and to components of the type III secretion system[J].Applied and Environmental Microbiology,2013,79:6312-6324.