欧美杨细菌性溃疡病菌双组分信号转导系统LqHK1基因的功能
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摘要
【目的】明确欧美杨细菌性溃疡病病原菌中双组分信号转导系统LqHK1基因的生物学功能,以阐明该病原菌的致病机制。【方法】采用生物信息学方法鉴定双组分系统LqHK1,利用同源重组方法获得该基因缺失突变体菌株△LqHK1,分析突变体及其互补菌株的生长速率、游动性、生物膜形成、致病性等生物学特性。同时,采用qRT-PCR检测与病原菌游动性相关的基因flgB、flgC、flgE的表达水平,并定量分析接种病原菌后在接种点部位杨树组织内病原菌的DNA含量。【结果】鉴定出病原细菌的双组分系统基因LqHK1,通过同源重组获得缺失突变体△LqHK1。表型分析显示:△LqHK1在1年生107杨茎干上的致病力显著下降;突变体在寄主体内定殖的菌量明显少于野生型菌株;突变体菌株的游动能力比野生型显著降低,游动性相关基因flgB、flgC、flgE在突变体中的表达量显著下降;LqHK1突变体生物膜的形成能力显著降低,生长速率与野生型无显著差异。【结论】LqHK1基因缺失突变体在107杨上的致病能力显著下降,欧美杨细菌性溃疡病病原菌双组分信号转导系统LqHK1基因与病原菌的致病性密切相关。
【Objective 】Poplar bacterial canker caused by Lonsdalea quercina subsp. populi is a disease,which is serious harm to poplar industry. In this study,the biological function of the LqHK1 gene in L. quercina subsp. populi was investigated to provide viable knowledge for further understanding the pathogenic mechanism of pathogen.【Method】The bioinformatics method was used to indentify the two component systems LqHK1. The gene deletion mutant strain △LqHK1 was constructed by homologous recombination. Verifying PCR and Southern blot were used to investigate the biological characteristics of the mutant strains and their complementary strains,such as growth rate,motility,biofilm formation,and pathogenicity. At the same time,qRT-PCR was used to test the expression levels of the motility related genes flgB,flgC,flgE,and quantitatively analyze DNA content of poplar tissue pathogen in the vaccination site after pathogen inoculation.【Result】The two-component system gene LqHK1 was identified and the deletion mutant △LqHK1 had been obtained by homologous recombination. Phenotypic analysis showed that pathogenicity test on annual poplar branches was significantly less virulent than wide-type,while the complemented mutant HB LqHK1 restored the virulence to the wild-type level. The mutant had significantly less colonization in host than the wild type strain. Mutant strain had reduced swimming ability compared with the wild-type. The motility related genes flgB,flgC,flgE expression significantly decreased in mutant;forming ability LqHK1 mutant biofilms significantly decreased,but the growth rate of LqHK1 mutant strains had no significant difference from wild type. 【Conclusion】Studies indicate that the bacterial poplar canker Lonsdalea quercina subsp. populi two-component signal transduction system LqHK1 gene is closely related to pathogenpathogenicity.
【Objective 】Poplar bacterial canker caused by Lonsdalea quercina subsp. populi is a disease,which is serious harm to poplar industry. In this study,the biological function of the LqHK1 gene in L. quercina subsp. populi was investigated to provide viable knowledge for further understanding the pathogenic mechanism of pathogen.【Method】The bioinformatics method was used to indentify the two component systems LqHK1. The gene deletion mutant strain △LqHK1 was constructed by homologous recombination. Verifying PCR and Southern blot were used to investigate the biological characteristics of the mutant strains and their complementary strains,such as growth rate,motility,biofilm formation,and pathogenicity. At the same time,qRT-PCR was used to test the expression levels of the motility related genes flgB,flgC,flgE,and quantitatively analyze DNA content of poplar tissue pathogen in the vaccination site after pathogen inoculation.【Result】The two-component system gene LqHK1 was identified and the deletion mutant △LqHK1 had been obtained by homologous recombination. Phenotypic analysis showed that pathogenicity test on annual poplar branches was significantly less virulent than wide-type,while the complemented mutant HB LqHK1 restored the virulence to the wild-type level. The mutant had significantly less colonization in host than the wild type strain. Mutant strain had reduced swimming ability compared with the wild-type. The motility related genes flgB,flgC,flgE expression significantly decreased in mutant;forming ability LqHK1 mutant biofilms significantly decreased,but the growth rate of LqHK1 mutant strains had no significant difference from wild type. 【Conclusion】Studies indicate that the bacterial poplar canker Lonsdalea quercina subsp. populi two-component signal transduction system LqHK1 gene is closely related to pathogenpathogenicity.
引文
郭利民,张鸣放,王洪轩.2008.濮阳市发现杨树新病害.河南林业科技,28(3):36-36.(Guo L M,Zhang M F,Wang H X.2008.New poplar disease wa discovered in Puyang of China.Journal of Henan Forestry Scienc and Technology,28(3):36-36.[in Chinese])
李宾,李爱宁,魏强,等.2015.欧美杨细菌性溃疡病菌hrc J基因的功能分析.林业科学,51(12):71-78.(Li B,Li A N,Wei Q,et al.2015.Functional analysis of hrc J gene in Lonsdalea quercina subsp.populi.Scientia Silvae Sinicae,51(12):71-78.[in Chinese])
任飞娟.2011.欧美杨溃疡病病原鉴定.北京:北京林业大学硕士学位论文.(Ren F J.2011.Pathogen identifiction of Populus euramericana canke disease.Beijing:MS thesis of Beijing Forestry University.[in Chinese])
单世平,郭照辉,张德元,等.2014.贪铜菌6-5双组分信号系统czcR2-czc S2的克隆和生物信息学分析.中国农学通报,30(36):148-154.(Shan S P,Guo Z H,Zhang D Y,et al.2014.Gene cloning and bioinformatics analysis of two-cczcR2-czc S2 from Cupria vidus.Chinese Agricultural Science Bulletin,30(36):148-154.[in Chinese])
商靖.2014.欧美杨细菌性溃疡病菌侵染循环及病害流行研究.北京:北京林业大学博士学位论文.(Shang J.2014.Study on the infection cycle and field epidemic o Lonsdalea quercina subsp.populi on Populus×euramericana.Beijing:Ph D thesis of Beijing Forestry University.[in Chinese])
宋菲,向军,陆树良.2010.激光共聚焦显微镜和改良微孔板法观察细菌生物膜形成.中国感染与化疗杂志,10(5):368-372.(Song F,Xiang J,Lu S L.2010.The formation of bacterial biofilm studied by using confocal laser scanning microscopy and modified microtiter-plate test.Chinese Journal of Infection and Chemotherapy,10(5):368-372.[in Chinese])
王梁燕.2008.耐辐射球菌中反应调节蛋白的研究.杭州:浙江大学博士学位论文.(Wang L Y.2008.Study on response regulators in Deinococcu radiodurans.Hangzhou:Ph D thesis of Zhejiang University.[in Chinese])
王薇,胡丹丹,李槿年,等.2014.拟态弧菌Omp U蛋白的黏附功能及所介导的致病作用.水产学报,38(5):731-740.(Wang W,Hu D D,Li J N,et al.2014.Adhension function of Omp Uprotein from Vibrio mimicus and its role in virulence.Journal o Fisheries of China,38(5):731-740.[in Chinese])
杨莉,张力群,贺伟,等.2014.Ⅲ型分泌系统是欧美杨溃疡病菌Lonsdalea quercina重要的致病因子.植物病理学报,44(5):512-520.(Yang L,Zang L Q,He W,et al.2014.Type III secretion system is an essential pathogenic factor in poplar canker pathogen Lonsdalea quercina.Acta Phytopathologica Sinica,44(5):512-520.[in Chinese])
Bontemps G S,Madec E,Lacroix J M,et al.2015.The two-component system Cpx AR is essential for virulence in the phytopathogen bacteria Dickeya dadantii EC3937.Environmental Microbiology,17(11):4415-4428.
Charles T C,Nester E W.1993.A chromosomally encoded twocomponent sensory transduction system is required for virulence of Agrobacterium tumefaciens.Journal of Bacteriology,175(20):6614-6625.
Hoang T T,Karkhoff-Schweizer R A R,Kutchma A J,et al.1998.Abroad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences:application for isolation of unmarked Pseudomonas aeruginosa mutants.Gene,212(1):77-86.
Hoch J A.2000.Two-component and phosphorelay signal,transduction.Current Opinion In Microbiol,3(2):165-170.
Kessler B,Lorenzo V,Timmis K N.1992.A general system to integratelac Z fusions into the chromosomes of gram-negative eubacteria:regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.Molecular and General Genetics MGG,233(1):293-301.
Khajanchi B K,Sha J,Kozlova E V,et al.2009.N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system,biofilm formation,protease production,and in vivo virulence in a clinical isolate of Aeromonas hydrophila.Microbiology,155(11):3518-3531.
Kovach M E,Elzer P H,Hill D S,et al.1995.Four new derivatives of the broad-host-range cloning vector p BBR1MCS,carrying different antibiotic-resistance cassettes.Gene,166(1):175-176.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod.Methods,25(4):402-408.
Li Y,He W,Ren F J,et al.2014.A canker disease of Populus×euramericana in China caused by Lonsdalea quercina subsp populi.Plant Disease,98(3):368-378.
Mizuno T.1997.Compilation of all genes encoding two-component phosphotransfer signal transducers in the genome of Escherichia coli.DNA Research,4(2):161-168.
Morohoshi T,Nakamura Y,Yamazaki G,et al.2007.The plant pathogen Pantoea ananatis produces N-acylhomoserine lactone and causes center rot disease of onion by quorum sensing.Journal of Bacteriology,189(22):8333-8338.
Ninfa A J,Magasanik B.1986.Covalent modification of the gln Gproduct,NRI,by the gln L product,NRII,regulates the transcription of the gln ALG operon in Escherichia coli.Proceedings of the National Academy of Sciences,83(16):5909-5913.
Pan S Q,Charles T,Jin S,et al.1993.Preformed dimeric state of the sensor protein Vir A is involved in plant Agrobacterium signal transduction.Proceedings of The National Academy of Sciences,90(21):9939-9943.
Shang J,Liu B L,He W.2015.A new method to detect Lonsdalea quercina in infected plant tissues by real-time PCR.Forest Pathology,45(1):28-35.
Yang L,Xu B,He W,et al.2014.The Hrp W protein of Lonsdalea quercina N-5-1 has pectate lyase activity and is required for full bacterial virulence.Journal of Basic Microbiology,54(10):1126-1135.
Yang W,Liu Y,Chen L,et al.2007.Zinc up-ake regulator(zur)gene involved inzinc homeostasis and virulence of Xanthomonas oryzae pv.oryzae in rice.Curr Microbiol,54:307-314.
Zhao Y,Wang D,Nakka S,et al.2009.Systems level analysis of twocomponent signal transduction systems in Erwinia amylovora:role in virulence,regulation of amylovoran biosynthesis and swarming motility.BMC Genomics,10(1):245.
李宾,李爱宁,魏强,等.2015.欧美杨细菌性溃疡病菌hrc J基因的功能分析.林业科学,51(12):71-78.(Li B,Li A N,Wei Q,et al.2015.Functional analysis of hrc J gene in Lonsdalea quercina subsp.populi.Scientia Silvae Sinicae,51(12):71-78.[in Chinese])
任飞娟.2011.欧美杨溃疡病病原鉴定.北京:北京林业大学硕士学位论文.(Ren F J.2011.Pathogen identifiction of Populus euramericana canke disease.Beijing:MS thesis of Beijing Forestry University.[in Chinese])
单世平,郭照辉,张德元,等.2014.贪铜菌6-5双组分信号系统czcR2-czc S2的克隆和生物信息学分析.中国农学通报,30(36):148-154.(Shan S P,Guo Z H,Zhang D Y,et al.2014.Gene cloning and bioinformatics analysis of two-cczcR2-czc S2 from Cupria vidus.Chinese Agricultural Science Bulletin,30(36):148-154.[in Chinese])
商靖.2014.欧美杨细菌性溃疡病菌侵染循环及病害流行研究.北京:北京林业大学博士学位论文.(Shang J.2014.Study on the infection cycle and field epidemic o Lonsdalea quercina subsp.populi on Populus×euramericana.Beijing:Ph D thesis of Beijing Forestry University.[in Chinese])
宋菲,向军,陆树良.2010.激光共聚焦显微镜和改良微孔板法观察细菌生物膜形成.中国感染与化疗杂志,10(5):368-372.(Song F,Xiang J,Lu S L.2010.The formation of bacterial biofilm studied by using confocal laser scanning microscopy and modified microtiter-plate test.Chinese Journal of Infection and Chemotherapy,10(5):368-372.[in Chinese])
王梁燕.2008.耐辐射球菌中反应调节蛋白的研究.杭州:浙江大学博士学位论文.(Wang L Y.2008.Study on response regulators in Deinococcu radiodurans.Hangzhou:Ph D thesis of Zhejiang University.[in Chinese])
王薇,胡丹丹,李槿年,等.2014.拟态弧菌Omp U蛋白的黏附功能及所介导的致病作用.水产学报,38(5):731-740.(Wang W,Hu D D,Li J N,et al.2014.Adhension function of Omp Uprotein from Vibrio mimicus and its role in virulence.Journal o Fisheries of China,38(5):731-740.[in Chinese])
杨莉,张力群,贺伟,等.2014.Ⅲ型分泌系统是欧美杨溃疡病菌Lonsdalea quercina重要的致病因子.植物病理学报,44(5):512-520.(Yang L,Zang L Q,He W,et al.2014.Type III secretion system is an essential pathogenic factor in poplar canker pathogen Lonsdalea quercina.Acta Phytopathologica Sinica,44(5):512-520.[in Chinese])
Bontemps G S,Madec E,Lacroix J M,et al.2015.The two-component system Cpx AR is essential for virulence in the phytopathogen bacteria Dickeya dadantii EC3937.Environmental Microbiology,17(11):4415-4428.
Charles T C,Nester E W.1993.A chromosomally encoded twocomponent sensory transduction system is required for virulence of Agrobacterium tumefaciens.Journal of Bacteriology,175(20):6614-6625.
Hoang T T,Karkhoff-Schweizer R A R,Kutchma A J,et al.1998.Abroad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences:application for isolation of unmarked Pseudomonas aeruginosa mutants.Gene,212(1):77-86.
Hoch J A.2000.Two-component and phosphorelay signal,transduction.Current Opinion In Microbiol,3(2):165-170.
Kessler B,Lorenzo V,Timmis K N.1992.A general system to integratelac Z fusions into the chromosomes of gram-negative eubacteria:regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.Molecular and General Genetics MGG,233(1):293-301.
Khajanchi B K,Sha J,Kozlova E V,et al.2009.N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system,biofilm formation,protease production,and in vivo virulence in a clinical isolate of Aeromonas hydrophila.Microbiology,155(11):3518-3531.
Kovach M E,Elzer P H,Hill D S,et al.1995.Four new derivatives of the broad-host-range cloning vector p BBR1MCS,carrying different antibiotic-resistance cassettes.Gene,166(1):175-176.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod.Methods,25(4):402-408.
Li Y,He W,Ren F J,et al.2014.A canker disease of Populus×euramericana in China caused by Lonsdalea quercina subsp populi.Plant Disease,98(3):368-378.
Mizuno T.1997.Compilation of all genes encoding two-component phosphotransfer signal transducers in the genome of Escherichia coli.DNA Research,4(2):161-168.
Morohoshi T,Nakamura Y,Yamazaki G,et al.2007.The plant pathogen Pantoea ananatis produces N-acylhomoserine lactone and causes center rot disease of onion by quorum sensing.Journal of Bacteriology,189(22):8333-8338.
Ninfa A J,Magasanik B.1986.Covalent modification of the gln Gproduct,NRI,by the gln L product,NRII,regulates the transcription of the gln ALG operon in Escherichia coli.Proceedings of the National Academy of Sciences,83(16):5909-5913.
Pan S Q,Charles T,Jin S,et al.1993.Preformed dimeric state of the sensor protein Vir A is involved in plant Agrobacterium signal transduction.Proceedings of The National Academy of Sciences,90(21):9939-9943.
Shang J,Liu B L,He W.2015.A new method to detect Lonsdalea quercina in infected plant tissues by real-time PCR.Forest Pathology,45(1):28-35.
Yang L,Xu B,He W,et al.2014.The Hrp W protein of Lonsdalea quercina N-5-1 has pectate lyase activity and is required for full bacterial virulence.Journal of Basic Microbiology,54(10):1126-1135.
Yang W,Liu Y,Chen L,et al.2007.Zinc up-ake regulator(zur)gene involved inzinc homeostasis and virulence of Xanthomonas oryzae pv.oryzae in rice.Curr Microbiol,54:307-314.
Zhao Y,Wang D,Nakka S,et al.2009.Systems level analysis of twocomponent signal transduction systems in Erwinia amylovora:role in virulence,regulation of amylovoran biosynthesis and swarming motility.BMC Genomics,10(1):245.