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

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.

Figure 1. Catalase activity in Xac as influenced by the growth phase.

(A) Xac cultures were grown aerobically 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, and soluble extracts were prepared as described in Materials and Methods . Total catalase activity was assayed as described by Beers and Sizer with 10 mM H₂O₂ at 25°C. (B) Equal amounts of protein (25 µg) were separated by 8% non-denaturing PAGE and stained for catalase activity by the method of Scandalios . A simultaneously run Coomassie-stained gel (not shown) indicated equal protein loadings between samples. The positions of the electrophoretically discernible catalase species Kat1, Kat2, and Kat3 are indicated.

Figure 2. Hydrogen peroxide resistance of Xac cultures in different growth stages.

Cells in early exponential (A) or stationary (B) phase of growth were exposed to the indicated concentrations of H₂O₂ for 15 min. The number of CFU was determined for each culture before and after the peroxide treatment by plating of appropriate dilutions. The percentage of survival is defined as the number of CFU after treatment divided by the number of CFU prior to treatment ×100. Data are expressed as the mean ± standard deviation of three independent experiments.

Figure 3. 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.

Figure 4. 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.

Figure 5. Expression of Xac catalase genes in the plant-mimicking XVM2 medium.

(A) Amplified products of the catalase genes by semi-quantitative RT-PCR using RNA preparations from early exponential Xac cultures grown in NB and in XVM2. As a control for constitutive bacterial expression a fragment of 16S rRNA was simultaneously amplified. (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.

Figure 6. Detection of catalase activity in Xac cultures grown in NB and XVM2 media.

Xac cultures were grown aerobically in NB and XVM2 media to early exponential (EE, 7 h), and stationary (S, 16 h) phases, and soluble extracts were prepared as described in Materials and Methods . Equal amounts of protein (40 µg) were separated by 8% non-denaturing PAGE and stained for catalase activity by the method of Scandalios . A simultaneously run Coomassie-stained gel (not shown) indicated equal protein loadings between samples.

Figure 7. Catalase activity and hydrogen peroxide resistance in the XackatE mutant.

(A) Xac wild-type (WT), XackatE (katE⁻) and cXackatE (ckatE) strains were grown aerobically in SB medium to early exponential (EE, 4 h) and stationary (S, 24 h) phases, and soluble extracts were prepared as described in Materials and Methods . Equal amounts of protein (25 µg) were separated by 8% non-denaturing PAGE and stained for catalase activity by the method of Scandalios . (B) Cells in early exponential (EE) or stationary (S) phase of growth were exposed to the indicated concentrations of H₂O₂ for 15 min. The number of CFU was determined for each culture before and after the peroxide treatment by plating of appropriate dilutions. The percentage of survival is defined as the number of CFU after treatment divided by the number of CFU prior to treatment ×100. Data are expressed as the mean ± standard deviation of three independent experiments.

Figure 8. Effect of katE disruption on pathogenicity.

Xac WT (WT), XackatE (katE ⁻) and cXackatE (ckatE) cells were inoculated at 10⁵ CFU ml⁻¹ in 10 mM MgCl₂ into the intercellular spaces of fully expanded orange leaves. (A) A representative leaf 20 days after inoculation is shown. Left panel, adaxial side; right panel, abaxial side. Dashed lines indicate the infiltrated area. (B) Bacterial growth of Xac cells in orange leaves. Values represent means of three independent samples; error bars represent standard deviations.