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A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization.

Kraiselburd I, Alet AI, Tondo ML, Petrocelli S, Daurelio LD, Monzón J, Ruiz OA, Losi A, Orellano EG - PLoS ONE (2012)

Bottom Line: We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation.Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type.Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process.

View Article: PubMed Central - PubMed

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

ABSTRACT
Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.

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Adhesion of X. axonopodis pv. citri strains to orange leaves.(A) X. axonopodis pv. citri WT, Δlov and Δlov-plov strains were grown in XVM2 medium, cultures were centrifuged, resuspended and placed on the abaxial face of orange leaves. After incubation at 28°C for 6 h under light and dark conditions, surface-attached cells were stained with 0.1% w/v Crystal violet dye. Control: XVM2 medium. The order of inoculation is indicated at the left of the panel. Dashed lines in the leaves indicate the inoculated area (B) Adhesion was quantified by image analysis determining the spot density (intensity/pixel area) of each adhesion region. Data are represented as the mean +/− standard error of three independent biological samples and different letters above the bars indicate significant differences between the corresponding data (p<0.01).
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pone-0038226-g008: Adhesion of X. axonopodis pv. citri strains to orange leaves.(A) X. axonopodis pv. citri WT, Δlov and Δlov-plov strains were grown in XVM2 medium, cultures were centrifuged, resuspended and placed on the abaxial face of orange leaves. After incubation at 28°C for 6 h under light and dark conditions, surface-attached cells were stained with 0.1% w/v Crystal violet dye. Control: XVM2 medium. The order of inoculation is indicated at the left of the panel. Dashed lines in the leaves indicate the inoculated area (B) Adhesion was quantified by image analysis determining the spot density (intensity/pixel area) of each adhesion region. Data are represented as the mean +/− standard error of three independent biological samples and different letters above the bars indicate significant differences between the corresponding data (p<0.01).

Mentions: To study the adhesion of X. axonopodis pv. citri strains to biotic surfaces bacteria were grown in XVM2 medium and placed on the surface of orange leaves. After six hours of incubation at 28°C, the leaves were washed and stained with Crystal violet. When the assay was performed under light conditions, the adhesion ability of the X. axonopodis pv. citri Δlov strain was much lower than for the WT and complemented strains (Figure 8A). In the dark condition, the adhesion of all X. axonopodis pv. citri strains was very low. Bacterial attachment was quantified by digital image analysis (spot density) of the stained leaves (Figure 8B). This quantification demonstrated that under light conditions the adhesion of the Δlov strain was statistically significant lower than that of the WT or complemented strains. In darkness, the differences in the adhesion of the three strains were not statistically significant, but we found a significant reduction in the adhesion of the three X. axonopodis pv. citri strains compared to the corresponding adhesion in the light condition.


A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization.

Kraiselburd I, Alet AI, Tondo ML, Petrocelli S, Daurelio LD, Monzón J, Ruiz OA, Losi A, Orellano EG - PLoS ONE (2012)

Adhesion of X. axonopodis pv. citri strains to orange leaves.(A) X. axonopodis pv. citri WT, Δlov and Δlov-plov strains were grown in XVM2 medium, cultures were centrifuged, resuspended and placed on the abaxial face of orange leaves. After incubation at 28°C for 6 h under light and dark conditions, surface-attached cells were stained with 0.1% w/v Crystal violet dye. Control: XVM2 medium. The order of inoculation is indicated at the left of the panel. Dashed lines in the leaves indicate the inoculated area (B) Adhesion was quantified by image analysis determining the spot density (intensity/pixel area) of each adhesion region. Data are represented as the mean +/− standard error of three independent biological samples and different letters above the bars indicate significant differences between the corresponding data (p<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366940&req=5

pone-0038226-g008: Adhesion of X. axonopodis pv. citri strains to orange leaves.(A) X. axonopodis pv. citri WT, Δlov and Δlov-plov strains were grown in XVM2 medium, cultures were centrifuged, resuspended and placed on the abaxial face of orange leaves. After incubation at 28°C for 6 h under light and dark conditions, surface-attached cells were stained with 0.1% w/v Crystal violet dye. Control: XVM2 medium. The order of inoculation is indicated at the left of the panel. Dashed lines in the leaves indicate the inoculated area (B) Adhesion was quantified by image analysis determining the spot density (intensity/pixel area) of each adhesion region. Data are represented as the mean +/− standard error of three independent biological samples and different letters above the bars indicate significant differences between the corresponding data (p<0.01).
Mentions: To study the adhesion of X. axonopodis pv. citri strains to biotic surfaces bacteria were grown in XVM2 medium and placed on the surface of orange leaves. After six hours of incubation at 28°C, the leaves were washed and stained with Crystal violet. When the assay was performed under light conditions, the adhesion ability of the X. axonopodis pv. citri Δlov strain was much lower than for the WT and complemented strains (Figure 8A). In the dark condition, the adhesion of all X. axonopodis pv. citri strains was very low. Bacterial attachment was quantified by digital image analysis (spot density) of the stained leaves (Figure 8B). This quantification demonstrated that under light conditions the adhesion of the Δlov strain was statistically significant lower than that of the WT or complemented strains. In darkness, the differences in the adhesion of the three strains were not statistically significant, but we found a significant reduction in the adhesion of the three X. axonopodis pv. citri strains compared to the corresponding adhesion in the light condition.

Bottom Line: We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation.Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type.Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process.

View Article: PubMed Central - PubMed

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

ABSTRACT
Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.

Show MeSH
Related in: MedlinePlus