水稻白叶枯病菌毒性表达的负调控因子PXO_02944的分子鉴定
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摘要
【目的】水稻黄单胞水稻致病变种(Xanthomonas oryzae pv.oryzae,简称Xoo)侵染引起水稻白叶枯病,在侵染过程中Xoo可产生胞外多糖(EPS)、胞外酶、黏附因子、T3SS以及其产生的效应因子等毒性因子。细菌第二信使环鸟苷二磷酸(c-di-GMP)的水平在Xoo毒性调控中发挥了重要的作用,而PXO_02944是包含REC、GGDEF和EAL结构域的蛋白,是c-di-GMP信号蛋白家族的成员,研究旨在阐明水稻白叶枯病菌的环鸟苷二磷酸信号蛋白家族成员PXO_02944的基因结构和功能。【方法】根据PXO_02944基因序列信息,以PXO99A基因组DNA为模板,设计引物扩增PXO_02944基因全长,将其GGDEF结构域和EAL结构域分别与已经报道的保守的GGDEF结构域或EAL结构域蛋白进行氨基酸序列比对分析;分别扩增PXO_02944左右臂片段,将其与目的载体pK18mobsacB载体连接,得到质粒pK2944,将pK2944与pMD-GmR载体回收获得的GmR基因片段进行连接,获得重组质粒pK2944-GmR,用于构建基因突变体。将PXO_02944全长基因克隆到载体pHM1上,获得重组质粒pHM1-2944并转化到ΔPXO_02944中,即获得互补菌株ΔPXO_02944::C。对野生型菌株、突变体和互补菌株进行表型测定,分析PXO_02944基因缺失突变对Xoo致病性和EPS产生、生物膜形成、胞外酶活性和鞭毛运动性的影响,并通过qRT-PCR方法测定EPS合成基因和毒性相关基因的表达。【结果】用特异性引物进行PCR扩增,成功地从野生型菌株PXO99A中克隆了PXO_02944;生物信息学分析发现,PXO_02944蛋白具有磷酸化信号接收的REC输入结构域和GGDEF、EAL输出结构域,但GGDEF和EAL结构域保守序列发生了突变。与野生型菌株PXO99A相比,虽然PXO_02944突变体胞外纤维素酶和木聚糖酶活性、鞭毛运动性都无明显变化,但其对水稻的致病性、EPS产生和生物膜形成能力显著增强,T3SS调控基因hrpG和EPS合成基因gumG的转录水平也明显升高。【结论】应答调控因子PXO_02944负调控了水稻白叶枯病菌致病性、EPS产生和生物膜形成这些毒性因子的表达。
【Objective】Xanthomonas oryzae pv. oryzae(Xoo) is the causing pathogen of bacterial blight of rice. Xoo can use extracellular polysaccharide(EPS) and enzymes, T3SS and its effectors for sucessful infection of rice. It has been shown that cyclic di-GMP signaling plays an important role in regulation of virulence in Xoo. PXO_02944, the GGDEF, EAL and REC domain containing protein, belongs to c-di-GMP signaling protein family. The objective of this study is to demonstrate the structure and function of the PXO_02944 in regulation of virulence expression of Xoo. 【Method】 Based on the sequences of PXO_02944, the specific primers were designed to amplify the full length of gene using the genomic DNA of the wildtype strain PXO99A as the template. In silico analysis of the GGDEF and EAL domains of PXO_02944 with the conserved ones from other bacteria were done. Then the upstream and downstream fragments of PXO_02944 were amplified, and ligated to vector pK18mobsacB to get the plasmid pK2944. Then a Gm resistance gene(GmR) was inserted into pK2944 resulting in plasmid pK2944-GmR to construct the gene deletion mutation by marker exchange. The full length of PXO_02944 was cloned and ligated into the vector pHM1, and the plasmid was electroporated into ΔPXO_02944 to get the complementory strain. The virulence, EPS production, enzymatic activities, biofilm formation and motility of ΔPXO_02944 were tested compared with PXO99A, ΔPXO_02944 and complemented strain. And the expression of EPS production and virulence related genes were analyzed.【Result】Bioinformatic analysis indicated that PXO_02944 was a response regulator of two-component regulatory system(TCS), which contained the REC input domain and the GGDEF and EAL output domains. In the mutant, PXO_02944 was deleted by replacing of the GmR. The mutation in PXO_02944 led to significant increases in bacterial virulence on rice, EPS production, biofilm formation, hrpG and gumG gene transcripts compared to PXO99A. Interestingly, no changes in activities of cellulase and xylanase and flagellar motility were observed in ΔPXO_02944. The deficiency above in the mutant could be restored to the wild-type level by in trans expression of the PXO_02944. 【Conclusion】 PXO_02944 might function as a negative regulator in bacterial virulence, EPS production and biofilm formation in Xoo.
【Objective】Xanthomonas oryzae pv. oryzae(Xoo) is the causing pathogen of bacterial blight of rice. Xoo can use extracellular polysaccharide(EPS) and enzymes, T3SS and its effectors for sucessful infection of rice. It has been shown that cyclic di-GMP signaling plays an important role in regulation of virulence in Xoo. PXO_02944, the GGDEF, EAL and REC domain containing protein, belongs to c-di-GMP signaling protein family. The objective of this study is to demonstrate the structure and function of the PXO_02944 in regulation of virulence expression of Xoo. 【Method】 Based on the sequences of PXO_02944, the specific primers were designed to amplify the full length of gene using the genomic DNA of the wildtype strain PXO99A as the template. In silico analysis of the GGDEF and EAL domains of PXO_02944 with the conserved ones from other bacteria were done. Then the upstream and downstream fragments of PXO_02944 were amplified, and ligated to vector pK18mobsacB to get the plasmid pK2944. Then a Gm resistance gene(GmR) was inserted into pK2944 resulting in plasmid pK2944-GmR to construct the gene deletion mutation by marker exchange. The full length of PXO_02944 was cloned and ligated into the vector pHM1, and the plasmid was electroporated into ΔPXO_02944 to get the complementory strain. The virulence, EPS production, enzymatic activities, biofilm formation and motility of ΔPXO_02944 were tested compared with PXO99A, ΔPXO_02944 and complemented strain. And the expression of EPS production and virulence related genes were analyzed.【Result】Bioinformatic analysis indicated that PXO_02944 was a response regulator of two-component regulatory system(TCS), which contained the REC input domain and the GGDEF and EAL output domains. In the mutant, PXO_02944 was deleted by replacing of the GmR. The mutation in PXO_02944 led to significant increases in bacterial virulence on rice, EPS production, biofilm formation, hrpG and gumG gene transcripts compared to PXO99A. Interestingly, no changes in activities of cellulase and xylanase and flagellar motility were observed in ΔPXO_02944. The deficiency above in the mutant could be restored to the wild-type level by in trans expression of the PXO_02944. 【Conclusion】 PXO_02944 might function as a negative regulator in bacterial virulence, EPS production and biofilm formation in Xoo.
引文
[1]White F F,Yang B.Host and pathogen factors controlling the rice-Xanthomonas oryzae interaction.Plant Physiology,2009,150:1677-1686.
[2]Das A,Rangaraj N,Sonti R V.Multiple adhesin-like functions of Xanthomonas oryzae pv.oryzae are involved in promoting leaf attachment,entry,and virulence on rice.Molecular Plant-Microbe Interactions,2009,22(1):73-85.
[3]White F F,Yang B.Host and pathogen factors controlling the rice-Xanthomonas oryzae interaction.Plant Physiology,2009,150(4):1677-1686.
[4]李争,熊鹂,纪志远,邹丽芳,邹华松,陈功友.白叶枯病菌和细菌性条斑病菌多样性的TALE效应蛋白调控水稻抗(感)病性机理与利用策略.中国农业科学,2013,46(14):2894-2901.Li Z,Xiong L,Ji Z Y,Zou L F,Zou H S,Chen G Y.Mechanisms of rice resistance(susceptibility)manipulated by diverse TALEs of Xanthomonas oryzae pv.oryzae and pv.oryzicola and potential utilization in rice breeding.Scientia Agricultura Sinica,2013,46(14):2894-2901.(in Chinese)
[5]Yang F H,Tian F,Sun L,Chen H M,Wu M S,Yang C H,He C Y.A novel two-component system PdeK/PdeR regulates c-di-GMP turnover and virulence of Xanthomonas oryzae pv.oryzae.Molecular Plant-Microbe Interactions,2012,25(10):1361-1369.
[6]He Y W,Wu J,Cha J S,Zhang L H.Rice bacterial blight pathogen Xanthomonas oryzae pv.oryzae produces multiple DSF-family signals in regulation of virulence factor production.BMC Microbiology,2010,10:187.
[7]梁士敏,杨凤环,管文静,吴茂森,陈华民,田芳,许艳丽,何晨阳.水稻白叶枯病菌EAL结构域蛋白VieAxoo基因缺失突变和功能分析.微生物学报,2011,51(1):29-34.Liang S M,Yang F H,Guan W J,Wu M S,Chen H M,Tian F,Xu Y L,He C Y.Gene deletion and functional analysis of the EAL domain protein VieAxoo in Xanthomonas oryzae pv.oryzae.Acta Microbiologica Sinica,2011,51(1):29-34.(in Chinese)
[8]Hengge R.Principles of c-di-GMP signalling in bacteria.Nature Reviews Microbiology,2009,7(4):263-273.
[9]Ryan R P.Cyclic di-GMP signalling and the regulation of bacterial virulence.Microbiology,2013,159:1286-1297.
[10]Schirmer T,Jenal U.Structural and mechanistic determinants of c-di-GMP signalling.Nature Reviews Microbiology,2009,7(10):724-735.
[11]Dey A K,Bhagat A,Chowdhury R.Host cell contact induces expression of virulence factors and VieA,a cyclic di-GMP phosphodiesterase in Vibrio cholerae.Journal of Bacteriology,2013,195(9):2004-2010.
[12]Tamayo R,Schild S,Pratt J T,Camilli A.Role of cyclic Di-GMP during el tor biotype Vibrio cholerae infection:characterization of the in vivo-induced cyclic Di-GMP phosphodiesterase CdpA.Infection and Immunity,2008,76(4):1617-1627.
[13]Roy A B,Petrova O E,Sauer K.The phosphodiesterase DipA(PA5017)is essential for Pseudomonas aeruginosa biofilm dispersion.Journal of Bacteriology,2012,194(11):2904-2915.
[14]Goymer P,Kahn S G,Malone J G,Gehrig S M,Spiers A J,Rainey P B.Adaptive divergence in experimental populations of Pseudomonas fluorescens.II.Role of the GGDEF regulator WspR in evolution and development of the wrinkly spreader phenotype.Genetics,2006,173:515-526.
[15]Bernier S P,Ha D G,Khan W,Merritt J H,O'Toole,G A.Modulation of Pseudomonas aeruginosa surface-associated group behaviors by individual amino acids through c-di-GMP signaling.Research in Microbiology,2011,162:680-688.
[16]Salzberg S L,Sommer D D,Schatz M C,Phillippy A M,Rabinowicz P D,Tsuge S,Furutani A,Ochiai H,Delcher A L,Kelley D,Madupu R,Puiu D,Radune D,Shumway M,Trapnell C,Aparna G,Jha G,Pandey A,Patil P B,Ishihara H,Meyer D F,Szurek B,Verdier V,Koebnik R,Dow J M,Ryan R P,Hirata H,Tsuyumu S,Won Lee S,Seo Y S,Sriariyanum M,Ronald P C,Sonti RV,Van Sluys M A,Leach J E,White F F,Bogdanove A J.Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv.oryzae PXO99A.BMC Genomics,2008,9:204.
[17]Hopkins C M,White F F,Choi S H,Guo A,Leach J E.Identification of a family of avirulence genes from Xanthomonas oryzae pv.oryzae.Molecular Plant-Microbe Interactions,1992,5(6):451-459.
[18]Schfer A,Tauch A,Jger W,Kalinowski J,Thierbach G,Pühler A.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene,1994,145(1):69-73.
[19]Lee S W,Ronald P C.Plant-Pathogen Interaction.Totowa,New Jersey:Humana Press,2007.
[20]Nino-Liu D,Damielle L,Bogdanove A J.A simple method of mass inoculation of rice effective for both pathovars of Xanthomonas oryzae,and the construction of comparable sets of host cDNA libraries spanning early stages of bacterial leaf blight and bacterial leaf streak.Journal of Phytopathology,2005,153:500-504.
[21]He Y W,Ng A Y,Xu M,Lin K,Wang L H,Dong Y H,Zhang L H.Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network.Molecular Microbiology,2007,64(2):281-292.
[22]Kulasakara H,Lee V,Brencic A,Liberati N,Urbach J,Miyata S,Lee D G,Neely A N,Hyodo M,Hayakawa Y,Ausubel F M,Lory S.Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence.Proceedings of the National Academy of Sciences of the United States of America,2006,103(8):2839-2844.
[23]Andro T,Chambost J P,Kotoujansky A,Cattaneo J,Bertheau Y,Barras F,Van Gijsegem F,Coleno A.Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase.Journal of Bacteriology,1984,160(3):1199-1203.
[24]Keen N T,Boyd C,Henrissat B.Cloning and characterization of a xylanase gene from corn strains of Erwinia chrysanthemi.Molecular Plant-Microbe Interactions,1996,9(7):651-657.
[25]Shen Y,Chern M,Silva F G,Ronald P.Isolation of a Xanthomonas oryzae pv.oryzae flagellar operon region and molecular characterization of flhF.Molecular Plant-Microbe Interactions,2001,14(2):204-213.
[26]Tsuge S,Nakayama T,Terashima S,Ochiai H,Furutani A,Oku T,Tsuno K,Kubo Y,Kaku H.Gene involved in transcriptional activation of the hrp regulatory gene hrpG in Xanthomonas oryzae pv.oryzae.Journal of Bacteriology,2006,188(11):4158-4162.
[27]Chao L,Rakshe S,Leff M,Spormann A M.PdeB,a cyclic di-GMP-specific phosphodiesterase that regulates Shewanella oneidensis MR-1 motility and biofilm formation.Journal of Bacteriology,2013,195(17):3827-3833.
[28]Ferreira R B,Antunes L C,Greenberg E P,McCarter L L.Vibrio parahaemolyticus ScrC modulates cyclic dimeric GMP regulation of gene expression relevant to growth on surfaces.Journal of Bacteriology,2008,190(3):851-860.
[29]Sondermann H,Shikuma N J,Yildiz F H.You've come a long way:c-di-GMP signaling.Current Opinion in Microbiology,2012,15(2):140-146.
[30]García Véscovi E,Sciara M I,Castelli M E.Two component systems in the spatial program of bacteria.Current Opinion in Microbiology,2010,13(2):210-218.
[2]Das A,Rangaraj N,Sonti R V.Multiple adhesin-like functions of Xanthomonas oryzae pv.oryzae are involved in promoting leaf attachment,entry,and virulence on rice.Molecular Plant-Microbe Interactions,2009,22(1):73-85.
[3]White F F,Yang B.Host and pathogen factors controlling the rice-Xanthomonas oryzae interaction.Plant Physiology,2009,150(4):1677-1686.
[4]李争,熊鹂,纪志远,邹丽芳,邹华松,陈功友.白叶枯病菌和细菌性条斑病菌多样性的TALE效应蛋白调控水稻抗(感)病性机理与利用策略.中国农业科学,2013,46(14):2894-2901.Li Z,Xiong L,Ji Z Y,Zou L F,Zou H S,Chen G Y.Mechanisms of rice resistance(susceptibility)manipulated by diverse TALEs of Xanthomonas oryzae pv.oryzae and pv.oryzicola and potential utilization in rice breeding.Scientia Agricultura Sinica,2013,46(14):2894-2901.(in Chinese)
[5]Yang F H,Tian F,Sun L,Chen H M,Wu M S,Yang C H,He C Y.A novel two-component system PdeK/PdeR regulates c-di-GMP turnover and virulence of Xanthomonas oryzae pv.oryzae.Molecular Plant-Microbe Interactions,2012,25(10):1361-1369.
[6]He Y W,Wu J,Cha J S,Zhang L H.Rice bacterial blight pathogen Xanthomonas oryzae pv.oryzae produces multiple DSF-family signals in regulation of virulence factor production.BMC Microbiology,2010,10:187.
[7]梁士敏,杨凤环,管文静,吴茂森,陈华民,田芳,许艳丽,何晨阳.水稻白叶枯病菌EAL结构域蛋白VieAxoo基因缺失突变和功能分析.微生物学报,2011,51(1):29-34.Liang S M,Yang F H,Guan W J,Wu M S,Chen H M,Tian F,Xu Y L,He C Y.Gene deletion and functional analysis of the EAL domain protein VieAxoo in Xanthomonas oryzae pv.oryzae.Acta Microbiologica Sinica,2011,51(1):29-34.(in Chinese)
[8]Hengge R.Principles of c-di-GMP signalling in bacteria.Nature Reviews Microbiology,2009,7(4):263-273.
[9]Ryan R P.Cyclic di-GMP signalling and the regulation of bacterial virulence.Microbiology,2013,159:1286-1297.
[10]Schirmer T,Jenal U.Structural and mechanistic determinants of c-di-GMP signalling.Nature Reviews Microbiology,2009,7(10):724-735.
[11]Dey A K,Bhagat A,Chowdhury R.Host cell contact induces expression of virulence factors and VieA,a cyclic di-GMP phosphodiesterase in Vibrio cholerae.Journal of Bacteriology,2013,195(9):2004-2010.
[12]Tamayo R,Schild S,Pratt J T,Camilli A.Role of cyclic Di-GMP during el tor biotype Vibrio cholerae infection:characterization of the in vivo-induced cyclic Di-GMP phosphodiesterase CdpA.Infection and Immunity,2008,76(4):1617-1627.
[13]Roy A B,Petrova O E,Sauer K.The phosphodiesterase DipA(PA5017)is essential for Pseudomonas aeruginosa biofilm dispersion.Journal of Bacteriology,2012,194(11):2904-2915.
[14]Goymer P,Kahn S G,Malone J G,Gehrig S M,Spiers A J,Rainey P B.Adaptive divergence in experimental populations of Pseudomonas fluorescens.II.Role of the GGDEF regulator WspR in evolution and development of the wrinkly spreader phenotype.Genetics,2006,173:515-526.
[15]Bernier S P,Ha D G,Khan W,Merritt J H,O'Toole,G A.Modulation of Pseudomonas aeruginosa surface-associated group behaviors by individual amino acids through c-di-GMP signaling.Research in Microbiology,2011,162:680-688.
[16]Salzberg S L,Sommer D D,Schatz M C,Phillippy A M,Rabinowicz P D,Tsuge S,Furutani A,Ochiai H,Delcher A L,Kelley D,Madupu R,Puiu D,Radune D,Shumway M,Trapnell C,Aparna G,Jha G,Pandey A,Patil P B,Ishihara H,Meyer D F,Szurek B,Verdier V,Koebnik R,Dow J M,Ryan R P,Hirata H,Tsuyumu S,Won Lee S,Seo Y S,Sriariyanum M,Ronald P C,Sonti RV,Van Sluys M A,Leach J E,White F F,Bogdanove A J.Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv.oryzae PXO99A.BMC Genomics,2008,9:204.
[17]Hopkins C M,White F F,Choi S H,Guo A,Leach J E.Identification of a family of avirulence genes from Xanthomonas oryzae pv.oryzae.Molecular Plant-Microbe Interactions,1992,5(6):451-459.
[18]Schfer A,Tauch A,Jger W,Kalinowski J,Thierbach G,Pühler A.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene,1994,145(1):69-73.
[19]Lee S W,Ronald P C.Plant-Pathogen Interaction.Totowa,New Jersey:Humana Press,2007.
[20]Nino-Liu D,Damielle L,Bogdanove A J.A simple method of mass inoculation of rice effective for both pathovars of Xanthomonas oryzae,and the construction of comparable sets of host cDNA libraries spanning early stages of bacterial leaf blight and bacterial leaf streak.Journal of Phytopathology,2005,153:500-504.
[21]He Y W,Ng A Y,Xu M,Lin K,Wang L H,Dong Y H,Zhang L H.Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network.Molecular Microbiology,2007,64(2):281-292.
[22]Kulasakara H,Lee V,Brencic A,Liberati N,Urbach J,Miyata S,Lee D G,Neely A N,Hyodo M,Hayakawa Y,Ausubel F M,Lory S.Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence.Proceedings of the National Academy of Sciences of the United States of America,2006,103(8):2839-2844.
[23]Andro T,Chambost J P,Kotoujansky A,Cattaneo J,Bertheau Y,Barras F,Van Gijsegem F,Coleno A.Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase.Journal of Bacteriology,1984,160(3):1199-1203.
[24]Keen N T,Boyd C,Henrissat B.Cloning and characterization of a xylanase gene from corn strains of Erwinia chrysanthemi.Molecular Plant-Microbe Interactions,1996,9(7):651-657.
[25]Shen Y,Chern M,Silva F G,Ronald P.Isolation of a Xanthomonas oryzae pv.oryzae flagellar operon region and molecular characterization of flhF.Molecular Plant-Microbe Interactions,2001,14(2):204-213.
[26]Tsuge S,Nakayama T,Terashima S,Ochiai H,Furutani A,Oku T,Tsuno K,Kubo Y,Kaku H.Gene involved in transcriptional activation of the hrp regulatory gene hrpG in Xanthomonas oryzae pv.oryzae.Journal of Bacteriology,2006,188(11):4158-4162.
[27]Chao L,Rakshe S,Leff M,Spormann A M.PdeB,a cyclic di-GMP-specific phosphodiesterase that regulates Shewanella oneidensis MR-1 motility and biofilm formation.Journal of Bacteriology,2013,195(17):3827-3833.
[28]Ferreira R B,Antunes L C,Greenberg E P,McCarter L L.Vibrio parahaemolyticus ScrC modulates cyclic dimeric GMP regulation of gene expression relevant to growth on surfaces.Journal of Bacteriology,2008,190(3):851-860.
[29]Sondermann H,Shikuma N J,Yildiz F H.You've come a long way:c-di-GMP signaling.Current Opinion in Microbiology,2012,15(2):140-146.
[30]García Véscovi E,Sciara M I,Castelli M E.Two component systems in the spatial program of bacteria.Current Opinion in Microbiology,2010,13(2):210-218.