The LOV Protein of Xanthomonas citri subsp. citri Plays a Significant Role in the Counteraction of Plant Immune Responses during Citrus Canker

Pathogens interaction with a host plant starts a set of immune responses that result in complex changes in gene expression and plant physiology. Light is an important modulator of plant defense response and recent studies have evidenced the novel influence of this environmental stimulus in the virulence of several bacterial pathogens. Xanthomonas citri subsp. citri is the bacterium responsible for citrus canker disease, which affects most citrus cultivars. The ability of this bacterium to colonize host plants is influenced by bacterial blue-light sensing through a LOV-domain protein and disease symptoms are considerably altered upon deletion of this protein. In this work we aimed to unravel the role of this photoreceptor during the bacterial counteraction of plant immune responses leading to citrus canker development. We performed a transcriptomic analysis in Citrus sinensis leaves inoculated with the wild type X. citri subsp. citri and with a mutant strain lacking the LOV protein by a cDNA microarray and evaluated the differentially regulated genes corresponding to specific biological processes. A down-regulation of photosynthesis-related genes (together with a corresponding decrease in photosynthesis rates) was observed upon bacterial infection, this effect being more pronounced in plants infected with the lov-mutant bacterial strain. Infection with this strain was also accompanied with the up-regulation of several secondary metabolism- and defense response-related genes. Moreover, we found that relevant plant physiological alterations triggered by pathogen attack such as cell wall fortification and tissue disruption were amplified during the lov-mutant strain infection. These results suggest the participation of the LOV-domain protein from X. citri subsp. citri in the bacterial counteraction of host plant defense response, contributing in this way to disease development.Fil: Kraiselburd, Ivana. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Daurelio, Lucas Damian. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Tondo, Maria Laura. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Merelo, Paz. INSTITUT VALENCIÀ D'INVESTIGACIONS AGRÀRIES (IVIA);Fil: Cortadi, Adriana Amalia. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Talón, Manuel. INSTITUT VALENCIÀ D'INVESTIGACIONS AGRÀRIES (IVIA);Fil: Tadeo, Francisco R.. INSTITUT VALENCIÀ D'INVESTIGACIONS AGRÀRIES (IVIA);Fil: Orellano, Elena Graciela. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario

A LOV Protein Modulates the Physiological Attributes of Xanthomonas axonopodis pv. citri Relevant for Host Plant Colonization

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

Functional Characterization of a LOV-Histidine Kinase Photoreceptor from Xanthomonas citri subsp. citri

The blue-light (BL) absorbing protein Xcc-LOV from Xanthomonas citri subsp. citri is composed of a LOV-domain, a histidine kinase (HK) and a response regulator. Spectroscopic characterization of Xcc-LOV identified intermediates and kinetics of the protein's photocycle. Measurements of steady state and time-resolved fluorescence allowed determination of quantum yields for triplet (ΦT = 0.68 ± 0.03) and photoproduct formation (Φ390 = 0.46 ± 0.05). The lifetime for triplet decay was determined as τT = 2.4-2.8 μs. Fluorescence of tryptophan and tyrosine residues was unchanged upon light-to-dark conversion, emphasizing the absence of significant conformational changes. Photochemistry was blocked upon cysteine C76 (C76S) mutation, causing a seven-fold longer lifetime of the triplet state (τT = 16-18.5 μs). Optoacoustic spectroscopy yielded the energy content of the triplet state. Interestingly, Xcc-LOV did not undergo the volume contraction reported for other LOV domains within the observation time window, although the back-conversion into the dark state was accompanied by a volume expansion. A radioactivity-based enzyme function assay revealed a larger HK activity in the lit than in the dark state. The C76S mutant showed a still lower enzyme function, indicating the dark state activity being corrupted by a remaining portion of the long-lived lit state.Fil: Kraiselburd, Ivana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gutt, Alexander. Max‐Planck‐Institute for Chemical Energy Conversion; AlemaniaFil: Losi, Aba. Università di Parma; ItaliaFil: Gärtner, Wolfgang. Max‐Planck‐Institute for Chemical Energy Conversion; AlemaniaFil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

KatG, the bifunctional catalase of xanthomonas citri subsp. citri, responds to hydrogen peroxide and contributes to epiphytic survival on citrus leaves

Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker. This bacterium is exposed to reactive oxygen species (ROS) at different points during its life cycle, including those normally produced by aerobic respiration or upon exposition to ultraviolet (UV) radiation. Moreover, ROS are key components of the host immune response. Among enzymatic ROS-detoxifying mechanisms, catalases eliminate H2O2, avoiding the potential damage caused by this specie. Xcc genome includes four catalase genes. In this work, we studied the physiological role of KatG, the only bifunctional catalase of Xcc, through the construction and characterization of a modified strain (XcckatG), carrying an insertional mutation in the katG gene. First, we evaluated the involvement of KatG in the bacterial adaptive response to H2O2. XcckatG cultures exhibited lower catalase activity than those of the wild-type strain, and this activity was not induced upon treatment with sub-lethal doses of H2O2. Moreover, the KatG-deficient mutant exhibited decreased tolerance to H2O2 toxicity compared to wild-type cells and accumulated high intracellular levels of peroxides upon exposure to sub-lethal concentrations of H2O2. To further study the role of KatG in Xcc physiology, we evaluated bacterial survival upon exposure to UV-A or UV-B radiation. In both conditions, XcckatG showed a high mortality in comparison to Xcc wild-type. Finally, we studied the development of bacterial biofilms. While structured biofilms were observed for the Xcc wild-type, the development of these structures was impaired for XcckatG. Based on these results, we demonstrated that KatG is responsible for Xcc adaptive response to H2O2 and a key component of the bacterial response to oxidative stress. Moreover, this enzyme plays an important role during Xcc epiphytic survival, being essential for biofilm formation and UV resistance.Para citar este articulo: Tondo ML, Delprato ML, Kraiselburd I, Fernández Zenoff MV, Farías ME, Orellano EG (2016) KatG, the Bifunctional Catalase of Xanthomonas citri subsp. citri, Responds to Hydrogen Peroxide and Contributes to Epiphytic Survival on Citrus Leaves. PLoS ONE 11(3): e0151657. doi:10.1371/journal.pone.0151657Fil: Tondo, María Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Delprato, María Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Kraiselburd, Ivana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Fernández Zenoff, María Verónica. Planta Piloto de Procesos Industriales Microbiológicos (PROIMI -CONICET); Argentina.Fil: Farías, María Eugenia. Planta Piloto de Procesos Industriales Microbiológicos (PROIMI -CONICET); Argentina.Fil: Orellano, Elena G. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina

Bacteriophytochromes from Pseudomonas syringae pv. tomato DC3000 modulate the early stages of plant colonization during bacterial speck disease

Living organisms have evolved the ability to perceive and respond to light of different wavelengths within the visible spectrum by the generation of photoreceptor proteins. Recent studies revealed the participation of these proteins in the virulence of plant pathogenic bacteria. Pseudomonas syringae pv. tomato DC3000 (Pto) is responsible for the bacterial speck, which affects tomato crops. Pto genome contains two genes encoding red/far-red light photoreceptors (BphP1: PSPTO_1902 and BphP2: PSPTO_2652). This work demonstrates the participation of Pto phytochromes and light in the bacterial physiology and during the interaction with tomato plants. We found that Pto phytochromes are implicated in the control of some features related with the bacteria capability to enter into the plant apoplast and cause bacterial speck disease, such as motility, biofilm formation, adhesion and emulsification capability. Red light and bacteriophytochromes are important during the early colonization stage of tomato phyllosphere, affecting Pto virulence. In addition, the development of disease symptoms in infiltrated leaflets is affected by light, which may be the consequence of type-two secretion system regulation.Fil: Moyano, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Carrau, Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Petrocelli, Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Kraiselburd, Ivana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gärtner, Wolfgang. Max-Planck-Institute for Chemical Energy Conversion; AlemaniaFil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Functional green-tuned proteorhodopsin from modern stromatolites

The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterologous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-subfamily. Titration down to low pH values (eventually causing partial protein denaturation) indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution) that decayed (τ1 = 3.5 μs) into another red-shifted intermediate. This species decayed in a two-step process (τ2 = 84 μs, τ3 = 11 ms), to which the initial state of E17-PR was reformed with a kinetics of 2 ms. Proton transport capability of the HAAL protein was determined by BLM measurements. Additional blue light irradiation reduced the proton current, clearly identifying a blue light absorbing, M-like intermediate. The apparent absence of this intermediate is explained by closely matching formation and decay kinetics

Pathogenicity and epiphytic fitness of Xcc<i>katG</i> in orange plants.

<p>(A) Growth of Xcc strains in the apoplastic space of orange leaves. Xcc WT, Xcc<i>katG</i> and cXcc<i>katG</i> cells were inoculated at 10⁵ CFU/mL in 10 mM MgCl₂ into the intercellular spaces of fully expanded orange leaves. Bacterial populations in leaf tissues were determined by serial dilution and plating. A representative leaf 20 days after inoculation with the three strains is shown in the lower inset. Left panel, adaxial side; right panel, abaxial side. Dashed lines indicate the infiltrated area. (B) Epiphytic populations of Xcc strains on orange leaves. Bacterial cells were released from the leaf surface by sonication followed by dilution plating. Experiments were performed in triplicate; values are expressed as means ± standard deviations. Statistical significant differences (P < 0.05, ANOVA) between wild-type and <i>katG</i> strains are indicated by an asterisk.</p

Electrometric record of E17R (in 20 mM Hepes, pH 7.4) with the BLM system.

<p>A. Transient currents and B. after the addition of the protonophore FCCP. Black bars indicate illumination with a 75 W XBO long-pass filtered at >495 nm. The grey bar shows the additional excitation of the M-state (>380 nm).</p

Transient absorbance changes of E17R.

<p>A. The kinetics of the absorbance changes are shown for selected wavelengths (399 nm, 517 nm and 598 nm). B. The decay-associated spectra are depicted as obtained from the global fit. The corresponding time constants are given in the figure.</p