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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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Expression analysis of Xac catalase genes as a function of the growth phase.(A) Amplified products of the katE, srpA and katG genes by semi-quantitative RT-PCR using RNA preparations from Xac cultures grown in SB medium to early exponential (EE, 4 h), mid-exponential (ME, 8 h), stationary (S, 24 h) and late stationary (LS, 48 h) phases. 16S rRNA was used as a loading control and to quantitate the amount of RNA in RT-PCRs. (B) Expression profiles obtained by densitometric quantification of band intensities. Experiments were performed in triplicate with similar results; error bars indicate ±1 standard deviation of the mean. IOD, integrated optical density; A.U., arbitrary units.
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pone-0010803-g003: Expression analysis of Xac catalase genes as a function of the growth phase.(A) Amplified products of the katE, srpA and katG genes by semi-quantitative RT-PCR using RNA preparations from Xac cultures grown in SB medium to early exponential (EE, 4 h), mid-exponential (ME, 8 h), stationary (S, 24 h) and late stationary (LS, 48 h) phases. 16S rRNA was used as a loading control and to quantitate the amount of RNA in RT-PCRs. (B) Expression profiles obtained by densitometric quantification of band intensities. Experiments were performed in triplicate with similar results; error bars indicate ±1 standard deviation of the mean. IOD, integrated optical density; A.U., arbitrary units.

Mentions: In order to investigate the expression profiles of the Xac catalase genes during growth, we performed semi-quantitative RT-PCR reactions using specific primers designed from the reported gene sequences (Table 1, Figure 3). As a control for constitutive bacterial expression a fragment of 16S rRNA was simultaneously amplified. Expression of katE was hardly detectable at the early and mid-exponential phases of growth, subsequently increasing to reach 5-fold higher levels during the stationary phase. On the other hand, expression of srpA and katG genes was detected throughout all stages of growth, reaching maximal levels in the mid-exponential phase and decreasing gradually towards the stationary phase. The mRNA levels of katG were almost undetectable in the late stationary phase. The catB gene was not included in the figure since no product was observed in the RT-PCR reactions under the conditions tested. To ascertain the absence of contaminating DNA in bacterial RNA samples control PCR reactions where RT was omitted were carried out in parallel for all samples (data not shown).


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)

Expression analysis of Xac catalase genes as a function of the growth phase.(A) Amplified products of the katE, srpA and katG genes by semi-quantitative RT-PCR using RNA preparations from Xac cultures grown in SB medium to early exponential (EE, 4 h), mid-exponential (ME, 8 h), stationary (S, 24 h) and late stationary (LS, 48 h) phases. 16S rRNA was used as a loading control and to quantitate the amount of RNA in RT-PCRs. (B) Expression profiles obtained by densitometric quantification of band intensities. Experiments were performed in triplicate with similar results; error bars indicate ±1 standard deviation of the mean. IOD, integrated optical density; A.U., arbitrary units.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875408&req=5

pone-0010803-g003: Expression analysis of Xac catalase genes as a function of the growth phase.(A) Amplified products of the katE, srpA and katG genes by semi-quantitative RT-PCR using RNA preparations from Xac cultures grown in SB medium to early exponential (EE, 4 h), mid-exponential (ME, 8 h), stationary (S, 24 h) and late stationary (LS, 48 h) phases. 16S rRNA was used as a loading control and to quantitate the amount of RNA in RT-PCRs. (B) Expression profiles obtained by densitometric quantification of band intensities. Experiments were performed in triplicate with similar results; error bars indicate ±1 standard deviation of the mean. IOD, integrated optical density; A.U., arbitrary units.
Mentions: In order to investigate the expression profiles of the Xac catalase genes during growth, we performed semi-quantitative RT-PCR reactions using specific primers designed from the reported gene sequences (Table 1, Figure 3). As a control for constitutive bacterial expression a fragment of 16S rRNA was simultaneously amplified. Expression of katE was hardly detectable at the early and mid-exponential phases of growth, subsequently increasing to reach 5-fold higher levels during the stationary phase. On the other hand, expression of srpA and katG genes was detected throughout all stages of growth, reaching maximal levels in the mid-exponential phase and decreasing gradually towards the stationary phase. The mRNA levels of katG were almost undetectable in the late stationary phase. The catB gene was not included in the figure since no product was observed in the RT-PCR reactions under the conditions tested. To ascertain the absence of contaminating DNA in bacterial RNA samples control PCR reactions where RT was omitted were carried out in parallel for all samples (data not shown).

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