Red light delays programmed cell death in non-host interaction between Pseudomonas syringae pv tomato DC3000 and tobacco plants

Light modulates almost every aspect of plant physiology, including plant-pathogen interactions. Among these, the hypersensitive response (HR) of plants to pathogens is characterized by a rapid and localized programmed cell death (PCD), which is critical to restrict the spread of pathogens from the infection site. The aim of this work was to study the role of light in the interaction between Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) and non-host tobacco plants. To this end, we examined the HR under different light treatments (white and red light) by using a range of well-established markers of PCD. The alterations found at the cellular level included: i) loss of membrane integrity and nuclei, ii) RuBisCo and DNA degradation, and iii) changes in nuclease profiles and accumulation of cysteine proteinases. Our results suggest that red light plays a role during the HR of tobacco plants to Pto DC3000 infection, delaying the PCD process.Fil: Moyano, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Lopez Fernandez, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; 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: Nannini, Julian Maria. 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; ArgentinaFil: 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; ArgentinaFil: Maldonado, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentin

Light modulates important physiological features of Ralstonia pseudosolanacearum during the colonization of tomato plants

Ralstonia pseudosolanacearum GMI1000 (Rpso GMI1000) is a soil-borne vascular phytopathogen that infects host plants through the root system causing wilting disease in a wide range of agro-economic interest crops, producing economical losses. Several features contribute to the full bacterial virulence. In this work we study the participation of light, an important environmental factor, in the regulation of the physiological attributes and infectivity of Rpso GMI1000. In silico analysis of the Rpso genome revealed the presence of a Rsp0254 gene, which encodes a putative blue light LOV-type photoreceptor. We constructed a mutant strain of Rpso lacking the LOV protein and found that the loss of this protein and light, influenced characteristics involved in the pathogenicity process such as motility, adhesion and the biofilms development, which allows the successful host plant colonization, rendering bacterial wilt. This protein could be involved in the adaptive responses to environmental changes. We demonstrated that light sensing and the LOV protein, would be used as a location signal in the host plant, to regulate the expression of several virulence factors, in a time and tissue dependent way. Consequently, bacteria could use an external signal and Rpsolov gene to know their location within plant tissue during the colonization process.Fil: Tano, María Josefina. 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: Ripa, Maria Belén. 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: Tondo, Maria Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - 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. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Rodriguez, María Victoria. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Ferreira, Virginia. Universidad de la República. Facultad de Química; UruguayFil: Siri, María Inés. Universidad de la República. Facultad de Química; UruguayFil: Piskulic, Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: 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

The Monofunctional Catalase KatE of Xanthomonas axonopodis pv. citri Is Required for Full Virulence in Citrus Plants

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

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

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

Modifications of <em>Xanthomonas axonopodis</em> pv. citri Lipopolysaccharide Affect the Basal Response and the Virulence Process during Citrus Canker

<div><p><em>Xanthomonas axonopodis</em> pv. citri (Xac) is the phytopathogen responsible for citrus canker, one of the most devastating citrus diseases in the world. A broad range of pathogens is recognized by plants through so-called pathogen-associated molecular patterns (PAMPs), which are highly conserved fragments of pathogenic molecules. In plant pathogenic bacteria, lipopolisaccharyde (LPS) is considered a virulence factor and it is being recognized as a PAMP. The study of the participation of Xac LPS in citrus canker establishment could help to understand the molecular bases of this disease. In the present work we investigated the role of Xac LPS in bacterial virulence and in basal defense during the interaction with host and non host plants. We analyzed physiological features of Xac mutants in LPS biosynthesis genes (<em>wzt</em> and <em>rfb30</em>3) and the effect of these mutations on the interaction with orange and tobacco plants. Xac mutants showed an increased sensitivity to external stresses and differences in bacterial motilities, <em>in vivo</em> and <em>in vitro</em> adhesion and biofilm formation. Changes in the expression levels of the LPS biosynthesis genes were observed in a medium that mimics the plant environment. Xac<em>wzt</em> exhibited reduced virulence in host plants compared to Xac wild-type and Xac<em>rfb303</em>. However, both mutant strains produced a lower increase in the expression levels of host plant defense-related genes respect to the parental strain. In addition, Xac LPS mutants were not able to generate HR during the incompatible interaction with tobacco plants. Our findings indicate that the structural modifications of Xac LPS impinge on other physiological attributes and lead to a reduction in bacterial virulence. On the other hand, Xac LPS has a role in the activation of basal defense in host and non host plants.</p> </div

Coronary flow reserve and myocardial diastolic dysfunction in arterial hypertension

The aim of this study was to assess the relation between coronary blood flow and left ventricular (LV) myocardial diastolic dysfunction in arterial hypertension. The study population included 30 hypertensive patients who were free of coronary artery disease and pharmacologic therapies. They underwent standard Doppler echocardiography and color tissue Doppler of the middle posterior septum at baseline and with high-dose dobutamine, and second-harmonic Doppler flow analysis of the distal left anterior descending coronary artery at baseline and after vasodilation by dipyridamole (0.56 mg/kg IV in 4'). Coronary flow reserve (CFR) was estimated as the ratio of hyperemic and baseline diastolic flow velocities. According to CFR, hypertensives were divided into 2 groups: 15 patients with normal CFR (>/=2) and 15 patients with reduced CFR (<2). The 2 groups were comparable for sex, age, body mass index, baseline heart rate, and blood pressure. LV mass index was greater in hypertensives with reduced CFR (p <0.01). By color tissue Doppler, baseline and high-dose dobutamine septal systolic velocities did not differ between the 2 groups. The ratio between myocardial velocities in early diastole and at atrial contraction (E(m)/A(m) ratio) was lower in patients with reduced CFR, both at baseline (p <0.05) and with high-dose dobutamine (p <0.00001). After adjusting for age, body mass index, LV mass index, and both high-dose dobutamine diastolic blood rate and heart rate by a multiple linear regression analysis, E(m)/A(m) ratio at high-dose dobutamine was independently associated with CFR in the overall population (beta 0.62, p <0.0005) (cumulative R(2) 0.38, p <0.0005). In conclusion, this study provides evidence of an independent association between CFR and myocardial diastolic function. In hypertensive patients without coronary artery stenosis, CFR alteration may be a determinant of myocardial diastolic dysfunction or diastolic impairment that should be taken into account as possibly contributing to coronary flow reduction

Cell membrane injuries of orange and tobacco leaves produced by bacterial infection.

<p>The leaves were inoculated with Xac wild-type, Xac<i>wzt</i> and Xac<i>rfb303</i> at 10⁷ CFU ml^-1 and the assays were performed at 7 (orange) or 2 (tobacco) days post-infiltration.</p>a<p>Data represent mean ± standard deviation of three independent experiments.</p

Biofilm formation.

<p>GFP-labeled Xac strains were grown on chambered cover slides and visualized under CLS microscopy after 2 and 5 days of bacterial growth. For each time period the left panels show cell aggregation at the bottom of the chambered cover slides with a magnification of 40× and the right panels show a 2× zoom of the regions marked in the previous panels. Scale bars, 50 µm.</p

Interaction of Xac strains with non host plants.

<p>(A) Phenotype developed on tobacco leaves inoculated with Xac wild-type, Xac<i>wzt</i> and Xac<i>rfb303</i> at 10⁷ CFU ml⁻¹ in 10 mM MgCl₂. Representative leaves are shown 24 hpi. (B) Bacterial growth of Xac wild-type, Xac<i>wzt</i> and Xac<i>rfb303</i> in tobacco leaves during 9 days. Values represent the mean ± standard deviation of three independent experiments. (C) Effect of pre-inoculation of tobacco leaves with LPS from Xac wild-type. A tobacco leaf area was inoculated with LPS (100 µg ml⁻¹) and 20 h later, Xac strains at 10⁷ CFU ml^–1 were inoculated into the same area. A representative 24 hpi leaf and bacterial growth curves during 9 days are shown. Scale bars, 1 cm.</p