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Fructose-Bisphophate Aldolase Exhibits Functional Roles between Carbon Metabolism and the hrp System in Rice Pathogen Xanthomonas oryzae pv. oryzicola  [PDF]
Wei Guo, Li-fang Zou, Yu-rong Li, Yi-ping Cui, Zhi-yuan Ji, Lu-lu Cai, Hua-song Zou, William C. Hutchins, Ching-hong Yang, Gong-you Chen
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031855
Abstract: Fructose-bisphophate aldolase (FbaB), is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique fbaB gene of Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak, led the pathogen not only unable to use pyruvate and malate for growth and delayed its growth when fructose was used as the sole carbon source, but also reduced extracellular polysaccharide (EPS) production and impaired bacterial virulence and growth in rice. Intriguingly, the fbaB promoter contains an imperfect PIP-box (plant-inducible promoter) (TTCGT-N9-TTCGT). The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, the expression of fbaB in X. oryzae pv. oryzicola RS105 strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3 were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3 reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in RΔfbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator.
Establishment of the hrp-inducing systems for the expression of the hrp genes of Xanthomonas oryzae pv.oryzicola

XIAO You-lun,LI Yu-rong,LIU Zhi-yang,XIANG Yong,CHEN Gong-you,

微生物学报 , 2007,
Abstract: 水稻条斑病细菌(Xanthomonas oryzae pv.oryzicola,Xooc)决定在非寄主植物上激发过敏反应(hypersensitive response)和在寄主水稻上具致病性(pathogenicity)的hrp基因簇是诱导表达的。为研究hrp基因的功能,利用hpa1和hrpX基因的启动子与gfp基因进行融合,构建了hrp基因诱导表达系统。绿色荧光蛋白表达揭示,Xooc的hrp基因在营养丰富的NB培养基上不能有效表达,在hrp诱导培养基XOM3上可有效表达。以hrpX和hrpG突变体为参照,RT-PCR研究结果提示,Xooc野生型菌株hpa1基因在NB上不能有效表达,在XOM3培养基上可有效表达。相应地,hrpX突变体中hpa1基因不能被诱导表达,而在hrpG突变体中hpa1基因转录表达水平低于野生菌。研究结果还证实,水稻悬浮细胞能高效诱导Xooc的hrp基因表达。Xooc hrp基因诱导表达系统的建立为研究hrp基因功能、发掘T3SS效应分子以及开展Xooc致病性研究奠定了基础。
Detection and Identification of Xanthomonas oryzae pv.oryzae and Xanthomonas oryzae pv.oryzicola by Real-time Fluorescent PCR

Liao Xiaolan Zhu Shuifang,Zhao Wenjun Luo Kuan Qi Yanxiang,

微生物学报 , 2003,
Abstract: A novel and sensitive real time PCR was developed to detection \%Xanthomonas ory zae\% pv.\%oryzae\% and \%Xanthomonas oryzae\% pv.\%oryzicola\%, which cause the bacteria leaf blight (BLB) and leaf streak respectively, Universal and specific TaqMan probes, which were designed based on the sequence of Putative siderophor e receptor gene cds were used to detect 13 bacteria and one phytoplasmas, only in \%X.oryzae \%pv. \%oryzae\% and \%X.oryzae \%pv. \%oryzicola\%, fluorescen t signal can be collected with their specific probes respectively. The level of detection of the probe was 30.6fg plasmid, roughly equaling to one cell and 100 times sensit ive than PCR gel electrophoresis detection.\% X.oryzae \%pv. \%oryzae\% and \% X.oryzae \%pv \%oryzicola\% were detected from seed washes and DNA extracted fro m the seed washes of naturally infected seeds and in fected leaves as small as 10g naturally infected seeds or 0.3g leaf. This method is little time consumption (only 2h) and without contamination from PCR product .
Identification of 17 HrpX-Regulated Proteins Including Two Novel Type III Effectors, XOC_3956 and XOC_1550, in Xanthomonas oryzae pv. oryzicola  [PDF]
Xiao-bo Xue, Li-fang Zou, Wen-xiu Ma, Zhi-yang Liu, Gong-you Chen
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0093205
Abstract: The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9≤X≤25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism.
Identification of extracellular polysaccharide-associated genes in Xanthomonas oryzae pv. oryzicola

Dan Zhou,Lifang Zou,Huasong Zou,Gongyou Chen,

微生物学报 , 2011,
Abstract: Objective] Previously,seventeen extracellular polysaccharide-associated mutants of Xanthomonas oryzae pv.oryzicola(Xoc) were acquired from our randomly Tn5-inserted mutant library.To know the Tn5-inserted genes of these mutants and their contribution to EPS production and virulence in rice,Methods] in this study,we first identified and characterized the Tn5-targeted genes in these mutants and then inoculated them in susceptible rice for virulence assessment.Results] Tn5 transposon was inserted in genes o...
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

Jia Jiang,Huasong Zou,Yurong Li,Gongyou Chen,

微生物学报 , 2009,
Abstract: 摘要:【目的】决定水稻条斑病菌(Xanthomonas oryzae pv. oryzicola)在非寄主植物上激发过敏反应(hypersensitive response, HR)和在寄主水稻上致病性(pathogenicity)的hrp基因簇是受hrpG和hrpX基因调控的,但还不清楚hrpG和hrpX基因是否共同决定着所有hrp基因的表达。【方法】本文通过基因敲除方式获得了水稻条斑病菌的hrpG和hrpX基因的双突变体。【结果】烟草和水稻上测定结果显示,双突变体与单突变体一样,均在烟草上失去HR激发能力和丧失在水稻上的致病性;相应地,功能互补后双突变体恢复至野生表型。细菌在水稻悬浮细胞、hrp诱导培养基XOM3和营养丰富的培养基NB中生长后的RT-PCR结果显示,NB中hrp基因低水平表达,XOM3和水稻细胞能够高水平诱导hrp基因表达。无论何种生长条件,hrpG单突变体中hrcC、hrcT、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcJ和hrpG基因不表达;hrpX单突变体中hpa2、hrcC、hpa3、hrpE和hrpG基因表达,而hpa1、hrcT、hpaB和hrcJ基因不表达;hrpG和hrpX双突变体中hrcC、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcT、hrcJ和hrpG基因不表达。【结论】这提示,水稻条斑病菌的hrcC、hrpE和hpa3基因不受hrpG和hrpX基因单独或同时调控,而hrcT基因受HrpG调控。由此推测,水稻条斑病菌III型分泌系统关键组份的表达有可能通过另外的信号途径进行调控,这为进一步分析III型分泌途经的形成提供了线索。
The HD-GYP Domain Protein RpfG of Xanthomonas oryzae pv. oryzicola Regulates Synthesis of Extracellular Polysaccharides that Contribute to Biofilm Formation and Virulence on Rice  [PDF]
Yuanbao Zhang, Chao Wei, Wendi Jiang, Lei Wang, Churui Li, Yunyue Wang, John Maxwell Dow, Wenxian Sun
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0059428
Abstract: Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important diseases in rice. However, little is known about the pathogenicity mechanisms of Xoc. Here we have investigated the function of three HD-GYP domain regulatory proteins in biofilm formation, the synthesis of virulence factors and virulence of Xoc. Deletion of rpfG resulted in altered production of extracellular polysaccharides (EPS), abolished virulence on rice and enhanced biofilm formation, but had little effect on the secretion of proteases and motility. In contrast, mutational analysis showed that the other two HD-GYP domain proteins had no effect on virulence factor synthesis and tested phenotypes. Mutation of rpfG led to up-regulation of the type III secretion system and altered expression of three putative glycosyltransferase genes gumD, pgaC and xagB, which are part of operons directing the synthesis of different extracellular polysaccharides. The pgaABCD and xagABCD operons were greatly up-regulated in the Xoc ΔrpfG mutant, whereas the expression of the gum genes was unaltered or slightly enhanced. The elevated biofilm formation of the Xoc ΔrpfG mutant was dramatically reduced upon deletion of gumD, xagA and xagB, but not when pgaA and pgaC were deleted. Interestingly, only the ΔgumD mutant, among these single gene mutants, exhibits multiple phenotype alterations including reduced biofilm and EPS production and attenuated virulence on rice. These data indicate that RpfG is a global regulator that controls biofilm formation, EPS production and bacterial virulence in Xoc and that both gumD- and xagB-dependent EPS contribute to biofilm formation under different conditions.
Cloning, sequencing and fuctional study of gacA gene from Xanthomonas oryzae pv. oryzicola

YANG Wan-feng,CHEN Lei,LIU Hong-xi,HU Bai-shi,LIU Feng-quan,

微生物学报 , 2007,
Abstract: 根据黄单胞菌gacA基因的同源性设计简并引物,采用PCR方法从水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)中克隆了gacA同源基因,命名为gacAXooc。序列比较显示,该基因在黄单胞菌中是相对保守的。通过同源重组的方法,构建了gacAXooc的插入突变株。对0.1% Tryptone的趋化应答能力检测发现,gacA突变株的趋化能力明显降低,证明gacA与Xooc的趋化性相关。
Function of a two-component system RpfCxoc/RpfGxoc in Xanthomonas oryzae pv.oryzicola

Yancun Zhao,Chunhui Liu,Guoliang Qian,Fangqun Yin,Yijing Zhou,Zhiwei Song,Fengquan Liu,

微生物学报 , 2012,
Abstract: Objective] To elucidate the biological functions of a two-component system RpfCxoc/RpfGxoc in Xanthomonas oryzae pv.oryzicola(Xoc).Method] Based on the genome template from Xoc wild-type strain Rs105,the rpfCxoc and rpfGxoc genes were amplified by polymerase chain reaction(PCR).The in-frame deletion mutations of rpfCxoc,rpfGxoc and rpfGCxoc(rpfCxoc and rpfGxoc double genes) were performed by the suicide vector pK18mobsacB,and determined diffusible signal factor(DSF) biosynthesis,pathogenicity in host rice,biofilm,extracellular polysaccharide(EPS) production and cell morphology.Result] rpfCxoc and rpfGxoc were cloned from the genomic DNA of Rs105.PCR analysis demonstrated that the rpfCxoc,rpfGxoc and rpfGCxoc genes were in-frame deleted successfully.Compared to the wild-type strain Rs105,DSF were overproduced in ΔrpfCxoc and ΔrpfGCxoc,but DSF production was remarkably decreased in ΔrpfGxoc.The DSF production of these mutants was restored by introducing the complemented cosmid pUFR-rpfCxoc,pUFR-rpfGxoc and pUFR-rpfGCxoc,respectively.Subsequent experimental results indicated that mutation of rpfCxoc,rpfGxoc and rpfGCxoc resulted in pathogenicity loss of Xoc in host rice,and decreased biosynthesis level of EPS at 34.1%-48.5% compared to that of Rs105.In L medium(Tryptoen,10 g/L;yeast extract,5 g/L;sodium chloride,5 g/L;D-glucose,1 g/L;pH7.0),Rs105 was growing at planktonic pattern,but the mutation of rpfCxoc and rpfGxoc led to Xoc cell aggregation at the wall of the flaks at the air-liquid interfaces,and ΔrpfGxoc generated reticulation biofilm at the bottom of the flaks.But ΔrpfGCxoc only generated reticulation biofilm at the bottom of the flaks.Conclusion] The two-component system RpfCxoc/RpfGxoc modulated DSF biosynthesis,EPS production and biofilm dispersal of Xoc,which was required for the pathogenicity of Xoc in host rice.
Analysis of the flgD and flgE genes regulated by diffusible signal factor in Xanthomonas oryzae pv. oryzicola
水稻细菌性条斑病菌中受DSF 调控的鞭毛基因flgD、flgE 的功能分析

Fangqun Yin,Yancun Zhao,Chunhui Liu,Gouliang Qian,Jiaqin Fan,Baishi Hu,Fengquan Liu,

微生物学报 , 2011,
Abstract: 摘要:【目的】为了阐明可扩散性信号分子( diffusible signal factor,DSF) 调控的鞭毛基因对水稻细菌性条斑病菌(Xanthomonas oryzae pv. oryzicola,Xoc)Rs105 的致病性等方面的影响。【方法】采用PCR 的方法克隆靶标基因flgDxoc 和flgExoc,用同源重组法构建缺失突变体,测定突变体及其互补菌株的菌体形态、运动性、致病性及过敏性反应等表型,用反转录PCR(RT-PCR) 的方法验证Rs105 和ΔrpfFxoc( rpfFxoc 基因
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