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The monofunctional catalase KatE of Xanthomonas axonopodis pv. citri is required for full virulence in citrus plants.

Tondo ML, Petrocelli S, Ottado J, Orellano EG - PLoS ONE (2010)

Bottom Line: Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides.The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process.Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Division, Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT

Background: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase).

Methodology/principal findings: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation.

Conclusions: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

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Adaptive response of Xac to hydrogen peroxide treatment.(A) Exponential phase cultures were adapted with the indicated concentrations of H2O2 for 60 min and then exposed to 1 mM H2O2 for 15 min. The number of CFU was determined for each culture before and after the treatment with 1 mM H2O2 by plating of appropriate dilutions. The related survival is defined as the percentage of survival of the pre-adapted culture divided by the percentage of survival of the untreated control. (B) Exponential phase cultures were pre-adapted with 100 µM H2O2 for 60 min. The number of CFU was determined for the preadapted cultures and for the unadapted controls and then H2O2 was added to the final concentrations indicated, followed by an incubation of 15 min. The percentage of survival was calculated as the number of CFU after treatment divided by the number of CFU prior to treatment ×100. Experiments were performed in triplicate; error bars indicate ±1 standard deviation of the mean.
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pone-0010803-g004: Adaptive response of Xac to hydrogen peroxide treatment.(A) Exponential phase cultures were adapted with the indicated concentrations of H2O2 for 60 min and then exposed to 1 mM H2O2 for 15 min. The number of CFU was determined for each culture before and after the treatment with 1 mM H2O2 by plating of appropriate dilutions. The related survival is defined as the percentage of survival of the pre-adapted culture divided by the percentage of survival of the untreated control. (B) Exponential phase cultures were pre-adapted with 100 µM H2O2 for 60 min. The number of CFU was determined for the preadapted cultures and for the unadapted controls and then H2O2 was added to the final concentrations indicated, followed by an incubation of 15 min. The percentage of survival was calculated as the number of CFU after treatment divided by the number of CFU prior to treatment ×100. Experiments were performed in triplicate; error bars indicate ±1 standard deviation of the mean.

Mentions: Based on this observation, the resistance of bacterial cells pre-adapted with sub-lethal levels of H2O2 to a lethal dose of the same agent was assessed. Cultures pre-treated with 10, 30 and 100 µM H2O2 were subsequently challenged with a killing concentration of H2O2 (1 mM, see Figure 2A) and the percentages of survival were determined (Figure 4A). Interestingly, a dose dependent response was observed with these H2O2 concentrations, with a 10-fold increase in resistance after pre-adaptation with 100 µM H2O2. Moreover, Xac cultures pre-treated with 100 µM H2O2 for 1 hour were then incubated with 0.25, 0.5, 1 and 5 mM H2O2 for 15 min (Figure 4B). We found that pre-adapted cells were more resistant than the control cells to all H2O2 concentrations tested, the difference of survival being more pronounced as the H2O2 levels increases. After challenge with 5 mM H2O2 survival of the pre-adapted culture was 100-fold higher than that of the untreated control.


The monofunctional catalase KatE of Xanthomonas axonopodis pv. citri is required for full virulence in citrus plants.

Tondo ML, Petrocelli S, Ottado J, Orellano EG - PLoS ONE (2010)

Adaptive response of Xac to hydrogen peroxide treatment.(A) Exponential phase cultures were adapted with the indicated concentrations of H2O2 for 60 min and then exposed to 1 mM H2O2 for 15 min. The number of CFU was determined for each culture before and after the treatment with 1 mM H2O2 by plating of appropriate dilutions. The related survival is defined as the percentage of survival of the pre-adapted culture divided by the percentage of survival of the untreated control. (B) Exponential phase cultures were pre-adapted with 100 µM H2O2 for 60 min. The number of CFU was determined for the preadapted cultures and for the unadapted controls and then H2O2 was added to the final concentrations indicated, followed by an incubation of 15 min. The percentage of survival was calculated as the number of CFU after treatment divided by the number of CFU prior to treatment ×100. Experiments were performed in triplicate; error bars indicate ±1 standard deviation of the mean.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2875408&req=5

pone-0010803-g004: Adaptive response of Xac to hydrogen peroxide treatment.(A) Exponential phase cultures were adapted with the indicated concentrations of H2O2 for 60 min and then exposed to 1 mM H2O2 for 15 min. The number of CFU was determined for each culture before and after the treatment with 1 mM H2O2 by plating of appropriate dilutions. The related survival is defined as the percentage of survival of the pre-adapted culture divided by the percentage of survival of the untreated control. (B) Exponential phase cultures were pre-adapted with 100 µM H2O2 for 60 min. The number of CFU was determined for the preadapted cultures and for the unadapted controls and then H2O2 was added to the final concentrations indicated, followed by an incubation of 15 min. The percentage of survival was calculated as the number of CFU after treatment divided by the number of CFU prior to treatment ×100. Experiments were performed in triplicate; error bars indicate ±1 standard deviation of the mean.
Mentions: Based on this observation, the resistance of bacterial cells pre-adapted with sub-lethal levels of H2O2 to a lethal dose of the same agent was assessed. Cultures pre-treated with 10, 30 and 100 µM H2O2 were subsequently challenged with a killing concentration of H2O2 (1 mM, see Figure 2A) and the percentages of survival were determined (Figure 4A). Interestingly, a dose dependent response was observed with these H2O2 concentrations, with a 10-fold increase in resistance after pre-adaptation with 100 µM H2O2. Moreover, Xac cultures pre-treated with 100 µM H2O2 for 1 hour were then incubated with 0.25, 0.5, 1 and 5 mM H2O2 for 15 min (Figure 4B). We found that pre-adapted cells were more resistant than the control cells to all H2O2 concentrations tested, the difference of survival being more pronounced as the H2O2 levels increases. After challenge with 5 mM H2O2 survival of the pre-adapted culture was 100-fold higher than that of the untreated control.

Bottom Line: Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides.The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process.Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Division, Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT

Background: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase).

Methodology/principal findings: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation.

Conclusions: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

Show MeSH
Related in: MedlinePlus