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Fructose-bisphophate aldolase exhibits functional roles between carbon metabolism and the hrp system in rice pathogen Xanthomonas oryzae pv. oryzicola.

Guo W, Zou LF, Li YR, Cui YP, Ji ZY, Cai LL, Zou HS, Hutchins WC, Yang CH, Chen GY - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education of China, College of Plant Protection, Nanjing Agricultural University, Nanjing, China.

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-N(9)-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.

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The expression of fabB in X. oryzae pv. oryzicola is negatively by hrpX and hrpG in rice suspension cells.(A) Schematic map of the promoter region containing PIP-box and -10 box-like motif of fabB fused with a promoterless gusA gene. * stands for base substitutions. The constructs are listed on the right. (B) Expression analysis of fbaB by real-time quantitative RT-PCR. RNAs were isolated from cultures of the wild-type RS105, the hrpG deletion mutant RΔhrpG and the hrpX deletion mutant RΔhrpX strains which were grown in NB medium and rice suspension cells for 16 h, respectively. The relative mRNAs level of fbaB was calculated with respect to the level of the corresponding transcript in the wild-type RS105. (C) Effects of the mutated PIP-box on gusA transcript. The gusA transcript level by the wild-type PIP-box promoter (a) and three base-substituted PIP-box promoter (b, c, d) in the wild-type RS105, the hrpX mutant RΔhrpX and the hrpG mutant RΔhrpX were investigated, respectively. All the reporter strains above were cultured in rice suspension cells for 16 h and gusA transcript levels were then determined by real-time PCR. The transcript of gusA in the wild-type was taken as one unit. Data are the mean ± SD of triplicate measurements from a representative experiment; and similar results were obtained in two other independent experiments. The asterisks in each horizontal data column indicate significant differences at P = 0.01 by t test.
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pone-0031855-g003: The expression of fabB in X. oryzae pv. oryzicola is negatively by hrpX and hrpG in rice suspension cells.(A) Schematic map of the promoter region containing PIP-box and -10 box-like motif of fabB fused with a promoterless gusA gene. * stands for base substitutions. The constructs are listed on the right. (B) Expression analysis of fbaB by real-time quantitative RT-PCR. RNAs were isolated from cultures of the wild-type RS105, the hrpG deletion mutant RΔhrpG and the hrpX deletion mutant RΔhrpX strains which were grown in NB medium and rice suspension cells for 16 h, respectively. The relative mRNAs level of fbaB was calculated with respect to the level of the corresponding transcript in the wild-type RS105. (C) Effects of the mutated PIP-box on gusA transcript. The gusA transcript level by the wild-type PIP-box promoter (a) and three base-substituted PIP-box promoter (b, c, d) in the wild-type RS105, the hrpX mutant RΔhrpX and the hrpG mutant RΔhrpX were investigated, respectively. All the reporter strains above were cultured in rice suspension cells for 16 h and gusA transcript levels were then determined by real-time PCR. The transcript of gusA in the wild-type was taken as one unit. Data are the mean ± SD of triplicate measurements from a representative experiment; and similar results were obtained in two other independent experiments. The asterisks in each horizontal data column indicate significant differences at P = 0.01 by t test.

Mentions: Previous reports have demonstrated that the PIP-box of HrpX regulons serves as a cis-regulated element in a HrpX-dependent manner [37], [38], [44]. Analysis of the promoter region of fbaB of X. oryzae pv. oryzicola BLS256 (Figure S1) by searching the existence of similar PIP-box sequence and by using a promoter-prediction software (http://www.fruitfly.org/seq_tools/promoter.html) revealed an imperfect PIP-box (TTCGT-N9-TTCGT) interval by 30 bp sequence with a -10 box-like motif (CAGCAT) upstream of the fbaB start codon (Figure 3A), suggesting that the expression of fbaB may be regulated by HrpX and HrpG, the latter controls the expression of hrpX[34], [35]. To investigate this, a real-time quantitative RT-PCR was employed to assay the action of the fbaB transcript with hrpX and hrpG. The fbaB relative transcript level displayed a significant increase (P = 0.01, t test) in the hrpX mutant RΔhrpX and the hrpG mutant RΔhrpG than that of the wild-type RS105 when the strains grew in rice suspension cells for 16 h. The expression of fbaB in RΔhrpG was higher than that in RΔhrpX, whereas there were no obvious difference of the fbaB expression among these three tested strain when they grew in NB medium (Figure 3B). These results demonstrate that the expression of fbaB is inducible in planta and repressed by HrpX and HrpG when the pathogen infects the host rice rather than in necrotrophic growth. The negative regulation of fbaB with HrpG and HrpX and the higher expression of fbaB in the hrpG mutant than in the hrpX mutant imply that other unkown factor(s) may involve in the regution of fbaB.


Fructose-bisphophate aldolase exhibits functional roles between carbon metabolism and the hrp system in rice pathogen Xanthomonas oryzae pv. oryzicola.

Guo W, Zou LF, Li YR, Cui YP, Ji ZY, Cai LL, Zou HS, Hutchins WC, Yang CH, Chen GY - PLoS ONE (2012)

The expression of fabB in X. oryzae pv. oryzicola is negatively by hrpX and hrpG in rice suspension cells.(A) Schematic map of the promoter region containing PIP-box and -10 box-like motif of fabB fused with a promoterless gusA gene. * stands for base substitutions. The constructs are listed on the right. (B) Expression analysis of fbaB by real-time quantitative RT-PCR. RNAs were isolated from cultures of the wild-type RS105, the hrpG deletion mutant RΔhrpG and the hrpX deletion mutant RΔhrpX strains which were grown in NB medium and rice suspension cells for 16 h, respectively. The relative mRNAs level of fbaB was calculated with respect to the level of the corresponding transcript in the wild-type RS105. (C) Effects of the mutated PIP-box on gusA transcript. The gusA transcript level by the wild-type PIP-box promoter (a) and three base-substituted PIP-box promoter (b, c, d) in the wild-type RS105, the hrpX mutant RΔhrpX and the hrpG mutant RΔhrpX were investigated, respectively. All the reporter strains above were cultured in rice suspension cells for 16 h and gusA transcript levels were then determined by real-time PCR. The transcript of gusA in the wild-type was taken as one unit. Data are the mean ± SD of triplicate measurements from a representative experiment; and similar results were obtained in two other independent experiments. The asterisks in each horizontal data column indicate significant differences at P = 0.01 by t test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285194&req=5

pone-0031855-g003: The expression of fabB in X. oryzae pv. oryzicola is negatively by hrpX and hrpG in rice suspension cells.(A) Schematic map of the promoter region containing PIP-box and -10 box-like motif of fabB fused with a promoterless gusA gene. * stands for base substitutions. The constructs are listed on the right. (B) Expression analysis of fbaB by real-time quantitative RT-PCR. RNAs were isolated from cultures of the wild-type RS105, the hrpG deletion mutant RΔhrpG and the hrpX deletion mutant RΔhrpX strains which were grown in NB medium and rice suspension cells for 16 h, respectively. The relative mRNAs level of fbaB was calculated with respect to the level of the corresponding transcript in the wild-type RS105. (C) Effects of the mutated PIP-box on gusA transcript. The gusA transcript level by the wild-type PIP-box promoter (a) and three base-substituted PIP-box promoter (b, c, d) in the wild-type RS105, the hrpX mutant RΔhrpX and the hrpG mutant RΔhrpX were investigated, respectively. All the reporter strains above were cultured in rice suspension cells for 16 h and gusA transcript levels were then determined by real-time PCR. The transcript of gusA in the wild-type was taken as one unit. Data are the mean ± SD of triplicate measurements from a representative experiment; and similar results were obtained in two other independent experiments. The asterisks in each horizontal data column indicate significant differences at P = 0.01 by t test.
Mentions: Previous reports have demonstrated that the PIP-box of HrpX regulons serves as a cis-regulated element in a HrpX-dependent manner [37], [38], [44]. Analysis of the promoter region of fbaB of X. oryzae pv. oryzicola BLS256 (Figure S1) by searching the existence of similar PIP-box sequence and by using a promoter-prediction software (http://www.fruitfly.org/seq_tools/promoter.html) revealed an imperfect PIP-box (TTCGT-N9-TTCGT) interval by 30 bp sequence with a -10 box-like motif (CAGCAT) upstream of the fbaB start codon (Figure 3A), suggesting that the expression of fbaB may be regulated by HrpX and HrpG, the latter controls the expression of hrpX[34], [35]. To investigate this, a real-time quantitative RT-PCR was employed to assay the action of the fbaB transcript with hrpX and hrpG. The fbaB relative transcript level displayed a significant increase (P = 0.01, t test) in the hrpX mutant RΔhrpX and the hrpG mutant RΔhrpG than that of the wild-type RS105 when the strains grew in rice suspension cells for 16 h. The expression of fbaB in RΔhrpG was higher than that in RΔhrpX, whereas there were no obvious difference of the fbaB expression among these three tested strain when they grew in NB medium (Figure 3B). These results demonstrate that the expression of fbaB is inducible in planta and repressed by HrpX and HrpG when the pathogen infects the host rice rather than in necrotrophic growth. The negative regulation of fbaB with HrpG and HrpX and the higher expression of fbaB in the hrpG mutant than in the hrpX mutant imply that other unkown factor(s) may involve in the regution of fbaB.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education of China, College of Plant Protection, Nanjing Agricultural University, Nanjing, China.

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-N(9)-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.

Show MeSH
Related in: MedlinePlus