利用gusA基因在水稻组织中示踪和定量水稻白叶枯病菌的方法
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摘要
水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,Xoo)从自然孔口水孔或者伤口处侵入水稻叶片,在维管束中定殖、繁殖和扩展,引起典型的叶枯症状。可视化这个动态的病程过程和快速定量水稻组织中细菌的群体是Xoo-水稻互作研究中亟待突破的技术难点。本研究在PXO99~A菌株中表达gusA基因,将其置于lacZ启动子、hrpX启动子和不含有(T_1)_4终止子的hrpX启动子下,构建了3个示踪菌株PXO99~A AGUSRBS、PXO99_(GUS)~A X和PXO99GUSX-。水稻上毒性测定结果显示,这3个示踪菌株与野生型PXO99~A展现相同的毒性。利用示踪菌株,通过注射接种法,能够观察到Xoo在水稻维管束中定殖和扩展的动态变化;通过喷雾接种法,能够观察到Xoo从叶尖或者叶缘侵入水稻叶片引起发病的特性;发现PXO99_(GUS)~A X和PXO99~A AGUSX-比PXO99_(GUS)~A RBS能够更加灵敏地反映这些病程。同时发现,PXO99_(GUS)~ARBS的细菌数量与受其侵染的水稻组织的GUS活性显著正相关。本研究也评价了利用GUS活性测定法精确和快速地定量水稻组织中细菌群体的可行性。以上结果表明这套GUS系统是非常有效的,能够示踪病原菌的侵染过程和监测细菌在水稻组织中的群体数量,这将为Xoo-水稻互作研究提供有力的技术支持。
Xanthomonas oryzae pv. oryzae infects rice leaves through natural openings such as hydathodes or wounds and colonizes the vascular tissues,then bacteria multiply and spread into the whole leaf through the xylem leading to a typical leaf blight symptom. However,it is still bothered to visualize symptom progress and to determine bacterial population rapidly in rice tissues in Xoo-rice interaction system. In this study,we constructed three GUS tracking strains PXO99_(GUS)~ARBS,PXO99_(GUS)~AX and PXO99_(GUS)~AX-in PXO99 Aexpressing gusA as reporter gene under control of the lacZ promoter,hrpX promoter and the hrpX promoter without( T_1)_4 terminators,respectively. Pathogenicity assays showed that three strains displayed same virulence as the wild type strain PXO99 A. By using the tracking strains,the dynamic changes of localization and propagation of Xoo were successfully detected from rice vascular system when injection inoculation was applied. Meanwhile,the invasion through hydathodes at the leaf tip and expansion along leaf margin were observed in the spraying inoculation method. PXO99_(GUS)~AX and PXO99_(GUS)~AX-were found to be more sensitive than PXO99_(GUS)~ARBS in tracing the developing progress of blight symptom. Based on statistical analysis,bacterial population of PXO99_(GUS)~ARBS showed significantly positive correlation with the GUS activity of infected rice tissues,implying that the GUS activity was truly derived from Xoo cell numbers. As a conclusion,a GUS marker system was developed for accurate and rapid determination of bacterial growth in rice tissues in this study. This system is useful for tracking the bacterial infection and monitoring bacterial population in rice,which will provide an efficient method for the research on Xoo-rice interaction.
Xanthomonas oryzae pv. oryzae infects rice leaves through natural openings such as hydathodes or wounds and colonizes the vascular tissues,then bacteria multiply and spread into the whole leaf through the xylem leading to a typical leaf blight symptom. However,it is still bothered to visualize symptom progress and to determine bacterial population rapidly in rice tissues in Xoo-rice interaction system. In this study,we constructed three GUS tracking strains PXO99_(GUS)~ARBS,PXO99_(GUS)~AX and PXO99_(GUS)~AX-in PXO99 Aexpressing gusA as reporter gene under control of the lacZ promoter,hrpX promoter and the hrpX promoter without( T_1)_4 terminators,respectively. Pathogenicity assays showed that three strains displayed same virulence as the wild type strain PXO99 A. By using the tracking strains,the dynamic changes of localization and propagation of Xoo were successfully detected from rice vascular system when injection inoculation was applied. Meanwhile,the invasion through hydathodes at the leaf tip and expansion along leaf margin were observed in the spraying inoculation method. PXO99_(GUS)~AX and PXO99_(GUS)~AX-were found to be more sensitive than PXO99_(GUS)~ARBS in tracing the developing progress of blight symptom. Based on statistical analysis,bacterial population of PXO99_(GUS)~ARBS showed significantly positive correlation with the GUS activity of infected rice tissues,implying that the GUS activity was truly derived from Xoo cell numbers. As a conclusion,a GUS marker system was developed for accurate and rapid determination of bacterial growth in rice tissues in this study. This system is useful for tracking the bacterial infection and monitoring bacterial population in rice,which will provide an efficient method for the research on Xoo-rice interaction.
引文
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[5]Yu C,Chen H,Tian F,et al.RpoN2-and Fli A-regulated fliTX is indispensible for flagellar motility and virulence in Xanthomonas oryzae pv.oryzae[J].BM CMicrobiology,2017,17(1):171.
[6]Fan S,Tian F,Li J,et al.Identification of phenolic compounds that suppress the virulence of Xanthomonas oryzae on rice via the type III secretion system[J].Molecular Plant Pathology,2017,18(4):555-568.
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[10]Wang K M,Zhang X P.Study on the competitiveness of peanut bradyrhizobial strains using gusA(in Chinese)[J].Journal of Anhui Agricultural Sciences(安徽农业科学).2008,36(2):621-622.
[11]Huang D L,Tang D J,Liao Q,et al.The Zur of Xanthomonas campestris is involved in hypersensitive response and positively regulates the expression of the hrp cluster via hrpX but not hrpG[J].Molecular Plant-Microbe Interactions,2009,22(3):321-329.
[12]Fan X,Guo J,Zhou Y,et al.The ColRS-regulated membrane protein gene XAC1347 Is involved in copper homeostasis and hrp gene expression in Xanthomonas citri subsp.citri[J].Frontiers in Microbiology,2018,9:1171.
[13]Zhou X,Teper D,Andrade M O,et al.A Phosphorylation Sw itch on Lon Protease Regulates Bacterial Type III Secretion System in Host[J].BM C Microbiology,2008,8(1):164.
[14]Andrade M O,Farah C S,Wang N.The post-transcriptional regulatorrsmA/csrA activates T3SS by stabilizing the 5'UTR of hrpG,the master regulator of hrp/hrc genes,in Xanthomonas[J].PLoS Pathogens,2014,10(2):e1003945.
[15]Li Y R,Zou H S,Che Y Z,et al.A novel regulatory role of Hrp D6 in regulating hrp-hrc-hpa genes in Xanthomonas oryzae pv.oryzicola[J].Molecular PlantMicrobe Interactions,2011,24(9):1086-1101.
[16]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.
[17]Li Y R,Che Y Z,Zou H S,et al.Hpa2 required by Hrp F to translocate Xanthomonas oryzae transcriptional activator-like effectors into rice for pathogenicity[J].Applied and Environmental Microbiology,2011,77(11):3809-3818.
[18]Labes M,Puhler A,Simon R.A new family of RSF1010-derived expression and lac-fusion broadhost-range vectors for gram-negative bacteria[J].Gene 1990,89(1):37-46.
[19]Wang M X,Liu Z Y,Dong Q C,et al.Identification of genes regulating hrpG expression in Xanthomonas oryzae pv.oryzae(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2015,45(2):130-138.
[20]Smith M A,Bidochka M J.Bacterial fitness and plasmid loss:the importance of culture conditions and plasmid size[J].Canadian Journal of Microbiology,1998,44(4):351-355.
[21]Bird L E,Rada H,Verma A,et al.Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli[J].Journal of Visualized Experiments,2015,(95):e52357.
[22]Newman K L,Almeida R P,Purcell A H,et al.Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera[J].Applied and Environmental Microbiology,2003,69(12):7319-7327.
[23]Yu Q H,Dong S M,Zhu W Y,et al.Use of green fluorescent protein to monitor Lactobacillus in the gastro-intestinal tract of chicken[J].FEM S Microbiology Letters,2007,275(2):207-213.
[24]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.
[25]Makino S,Sugio A,White F,et al.Inhibition of resistance gene-mediated defense in rice by Xanthomonas oryzae pv.oryzicola[J].Molecular PlantMicrobe Interactions,2006,19(3):240-249.
[26]Yang B,Sugio A,White F F.Avoidance of host recognition by alterations in the repetitive and C-terminal regions of AvrXa7,a type III effector of Xanthomonas oryzae pv.oryzae[J].Molecular Plant-Microbe Interactions,2005,18(2):142-149.
[27]Yang B,Zhu W,Johnson L B,et al.The virulence factor AvrXa7 of Xanthomonas oryzae pv.oryzae is a type III secretion pathway-dependent nuclear-localized double-stranded DNA-binding protein[J].Proceedings of the National Academy of Sciences.USA,2000,97(17):9807-9812.
[28]Zhu W,Yang B,Wills N,et al.The C terminus of AvrXa10 can be replaced by the transcriptional activation domain of VP16 from the herpes simplex virus[J].The Plant Cell,1999,11(9):1665-1674.
[29]Zhu W,Yang B,Chittoor J M,et al.AvrXa10 contains an acidic transcriptional activation domain in the functionally conserved C terminus[J].Molecular Plant-Microbe Interactions,1998,11(8):824-832.
[30]Zhu W,Ma Gbanua M M,White F F.Identification of two novel hrp-associated genes in the hrp gene cluster of Xanthomonas oryzae pv.oryzae[J].Journal of Bacteriology,2000,182(7):1844-1853.
[31]Kametani-Ikawa Y,Tsuge S,Furutani A,et al.An H-NS-like protein involved in the negative regulation of hrp genes in Xanthomonas oryzae pv.oryzae[J].FEM S Microbiology Letters,2011,319(1):58-64.
[32]Seo Y S,Sriariyanun M,Wang L,et al.A two-genome microarray for the rice pathogens Xanthomonas oryzae pv.oryzae and X.oryzae pv.oryzicola and its use in the discovery of a difference in their regulation of hrp genes[J].BM C Microbiology,2008,8:99.
[33]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(11):4158-4162.
[34]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(3):396-401.
[35]Mole B M,Baltrus D A,Dangl J L,et al.Global virulence regulation networks in phytopathogenic bacteria[J].Trends in Microbiology,2007,15(8):363-371.
[2]Verdier V,Vera Cruz C,Leach J E.Controlling rice bacterial blight in Africa:needs and prospects[J].Journal of Biotechnology,2012,159(4):320-328.
[3]Salzberg S L,Sommer D D,Schatz M C,et al.Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv.oryzae PXO99A[J].BMCGenomics,2008,9:204.
[4]Han S W,Park C J,Lee S W,et al.An efficient method for visualization and growth of fluorescent Xanthomonas oryzae pv.oryzae in planta[J].BM C Microbiology,2008,8(1):164.
[5]Yu C,Chen H,Tian F,et al.RpoN2-and Fli A-regulated fliTX is indispensible for flagellar motility and virulence in Xanthomonas oryzae pv.oryzae[J].BM CMicrobiology,2017,17(1):171.
[6]Fan S,Tian F,Li J,et al.Identification of phenolic compounds that suppress the virulence of Xanthomonas oryzae on rice via the type III secretion system[J].Molecular Plant Pathology,2017,18(4):555-568.
[7]Tornero P,Dangl J L.A high-throughput method for quantifying growth of phytopathogenic bacteria in Arabidopsis thaliana[J].The Plant Journal,2001,28(4):475-481.
[8]Fan J,Crooks C,Lamb C.High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens lux CDABE[J].The Plant Journal,2008,53(2):393-399.
[9]Viazovaia A A,Limeshchenko E V,Buren V M.Biological properties of the wild rhizosphere strain Pseudomonas fluorescens 2137 and its derivatives marked with the gusA gene[J].Microbiology,2006,75(5):689-695.
[10]Wang K M,Zhang X P.Study on the competitiveness of peanut bradyrhizobial strains using gusA(in Chinese)[J].Journal of Anhui Agricultural Sciences(安徽农业科学).2008,36(2):621-622.
[11]Huang D L,Tang D J,Liao Q,et al.The Zur of Xanthomonas campestris is involved in hypersensitive response and positively regulates the expression of the hrp cluster via hrpX but not hrpG[J].Molecular Plant-Microbe Interactions,2009,22(3):321-329.
[12]Fan X,Guo J,Zhou Y,et al.The ColRS-regulated membrane protein gene XAC1347 Is involved in copper homeostasis and hrp gene expression in Xanthomonas citri subsp.citri[J].Frontiers in Microbiology,2018,9:1171.
[13]Zhou X,Teper D,Andrade M O,et al.A Phosphorylation Sw itch on Lon Protease Regulates Bacterial Type III Secretion System in Host[J].BM C Microbiology,2008,8(1):164.
[14]Andrade M O,Farah C S,Wang N.The post-transcriptional regulatorrsmA/csrA activates T3SS by stabilizing the 5'UTR of hrpG,the master regulator of hrp/hrc genes,in Xanthomonas[J].PLoS Pathogens,2014,10(2):e1003945.
[15]Li Y R,Zou H S,Che Y Z,et al.A novel regulatory role of Hrp D6 in regulating hrp-hrc-hpa genes in Xanthomonas oryzae pv.oryzicola[J].Molecular PlantMicrobe Interactions,2011,24(9):1086-1101.
[16]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.
[17]Li Y R,Che Y Z,Zou H S,et al.Hpa2 required by Hrp F to translocate Xanthomonas oryzae transcriptional activator-like effectors into rice for pathogenicity[J].Applied and Environmental Microbiology,2011,77(11):3809-3818.
[18]Labes M,Puhler A,Simon R.A new family of RSF1010-derived expression and lac-fusion broadhost-range vectors for gram-negative bacteria[J].Gene 1990,89(1):37-46.
[19]Wang M X,Liu Z Y,Dong Q C,et al.Identification of genes regulating hrpG expression in Xanthomonas oryzae pv.oryzae(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2015,45(2):130-138.
[20]Smith M A,Bidochka M J.Bacterial fitness and plasmid loss:the importance of culture conditions and plasmid size[J].Canadian Journal of Microbiology,1998,44(4):351-355.
[21]Bird L E,Rada H,Verma A,et al.Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli[J].Journal of Visualized Experiments,2015,(95):e52357.
[22]Newman K L,Almeida R P,Purcell A H,et al.Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera[J].Applied and Environmental Microbiology,2003,69(12):7319-7327.
[23]Yu Q H,Dong S M,Zhu W Y,et al.Use of green fluorescent protein to monitor Lactobacillus in the gastro-intestinal tract of chicken[J].FEM S Microbiology Letters,2007,275(2):207-213.
[24]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.
[25]Makino S,Sugio A,White F,et al.Inhibition of resistance gene-mediated defense in rice by Xanthomonas oryzae pv.oryzicola[J].Molecular PlantMicrobe Interactions,2006,19(3):240-249.
[26]Yang B,Sugio A,White F F.Avoidance of host recognition by alterations in the repetitive and C-terminal regions of AvrXa7,a type III effector of Xanthomonas oryzae pv.oryzae[J].Molecular Plant-Microbe Interactions,2005,18(2):142-149.
[27]Yang B,Zhu W,Johnson L B,et al.The virulence factor AvrXa7 of Xanthomonas oryzae pv.oryzae is a type III secretion pathway-dependent nuclear-localized double-stranded DNA-binding protein[J].Proceedings of the National Academy of Sciences.USA,2000,97(17):9807-9812.
[28]Zhu W,Yang B,Wills N,et al.The C terminus of AvrXa10 can be replaced by the transcriptional activation domain of VP16 from the herpes simplex virus[J].The Plant Cell,1999,11(9):1665-1674.
[29]Zhu W,Yang B,Chittoor J M,et al.AvrXa10 contains an acidic transcriptional activation domain in the functionally conserved C terminus[J].Molecular Plant-Microbe Interactions,1998,11(8):824-832.
[30]Zhu W,Ma Gbanua M M,White F F.Identification of two novel hrp-associated genes in the hrp gene cluster of Xanthomonas oryzae pv.oryzae[J].Journal of Bacteriology,2000,182(7):1844-1853.
[31]Kametani-Ikawa Y,Tsuge S,Furutani A,et al.An H-NS-like protein involved in the negative regulation of hrp genes in Xanthomonas oryzae pv.oryzae[J].FEM S Microbiology Letters,2011,319(1):58-64.
[32]Seo Y S,Sriariyanun M,Wang L,et al.A two-genome microarray for the rice pathogens Xanthomonas oryzae pv.oryzae and X.oryzae pv.oryzicola and its use in the discovery of a difference in their regulation of hrp genes[J].BM C Microbiology,2008,8:99.
[33]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(11):4158-4162.
[34]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(3):396-401.
[35]Mole B M,Baltrus D A,Dangl J L,et al.Global virulence regulation networks in phytopathogenic bacteria[J].Trends in Microbiology,2007,15(8):363-371.