RESUMEN

The CRISPR/Cas9 system is widely used for editing genes in various organisms and is a very useful tool due to its versatility, simplicity, and efficiency. To teach its principles to post-graduate students we designed a laboratory activity to obtain and analyze PDS3 mutants in Arabidopsis thaliana plants consisting of: 1) Design of guide RNAs using bioinformatics tools; 2) plant transformation (which is optional depending on the length of the course); 3) observation and evaluation of the mutant's phenotypes in the Phytoene desaturase (PDS3) gene, which exhibit an albino phenotype and different degrees of mosaicism in the editing events we evaluated; 4) PCR amplification of a fragment that includes the mutated region followed by analysis of single-stranded DNA conformation polymorphisms (SSCP) using native polyacrylamide gel electrophoresis and silver nitrate staining to detect changes in the amplicon sequence due to gene editing. Through SSCP, the students were able to distinguish between homozygous and heterozygous edited plants. A highlight feature of this protocol is the visualization and detection of the mutation/edition without sequencing the edited fragment.

Asunto(s)

Arabidopsis , Sistemas CRISPR-Cas , Arabidopsis/genética , Sistemas CRISPR-Cas/genética , ADN de Cadena Simple , Edición Génica/métodos , Humanos , Plantas Modificadas Genéticamente/genética , ARN Guía de Kinetoplastida/genética

RESUMEN

Ralstonia solanacearum is the causative agent of bacterial wilt disease on a wide range of plant species. Besides the numerous bacterial activities required for host invasion, those involved in the adaptation to the plant environment are key for the success of infection. R. solanacearum ability to cope with the oxidative burst produced by the plant is likely one of the activities required to grow parasitically. Among the multiple reactive oxygen species (ROS)-scavenging enzymes predicted in the R. solanacearum GMI1000 genome, a single monofunctional catalase (KatE) and two KatG bifunctional catalases were identified. In this work, we show that these catalase activities are active in bacterial protein extracts and demonstrate by gene disruption and mutant complementation that the monofunctional catalase activity is encoded by katE. Different strategies were used to evaluate the role of KatE in bacterial physiology and during the infection process that causes bacterial wilt. We show that the activity of the enzyme is maximal during exponential growth in vitro and this growth-phase regulation occurs at the transcriptional level. Our studies also demonstrate that katE expression is transcriptionally activated by HrpG, a central regulator of R. solanacearum induced upon contact with the plant cells. In addition, we reveal that even though both KatE and KatG catalase activities are induced upon hydrogen peroxide treatment, KatE has a major effect on bacterial survival under oxidative stress conditions and especially in the adaptive response of R. solanacearum to this oxidant. The katE mutant strain also exhibited differences in the structural characteristics of the biofilms developed on an abiotic surface in comparison to wild-type cells, but not in the overall amount of biofilm production. The role of catalase KatE during the interaction with its host plant tomato is also studied, revealing that disruption of this gene has no effect on R. solanacearum virulence or bacterial growth in leave tissues, which suggests a minor role for this catalase in bacterial fitness in planta. Our work provides the first characterization of the R. solanacearum catalases and identifies KatE as a bona fide monofunctional catalase with an important role in bacterial protection against oxidative stress.

RESUMEN

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.

Asunto(s)

Apoptosis/efectos de la radiación , Interacciones Huésped-Patógeno/efectos de la radiación , Luz , Nicotiana/fisiología , Pseudomonas syringae/fisiología , Enfermedades de las Plantas/microbiología , Nicotiana/microbiología , Nicotiana/efectos de la radiación

RESUMEN

Nowadays, fertilization and pest control are carried out using chemical compounds that contaminate soil and deteriorate human health. Plant growth promoting bacteria endophytes (PGPBEs), are a well-studied group of bacteria that offers benefits to the host plant, such as phytostimulation, biofertilization, and protection against other microorganisms. The study of Gluconacetobacter diazotrophicus-which belongs to PGPBEs-aids the development of alternative strategies of an integrated approach for crop management practices. Ralstonia solanacearum is responsible for bacterial wilt disease. This phytopathogen is of great interest worldwide due to the enormous economic losses it causes. In this study the action of G. diazotrophicus as a growth promoting bacterium in Arabidopsis thaliana seedlings is analyzed, evaluating the antagonistic mechanisms of this beneficial endophytic bacterium during biotic stress produced by R. solanacearum. Effective colonization of G. diazotrophicus was determined through bacterial counting assays, evaluation of anatomical and growth parameters, and pigments quantification. Biocontrol assays were carried out with Ralstonia pseudosolanacearum GMI1000 model strain and R. solanacearum A21 a recently isolated strain. Inoculation of A. thaliana (Col 0) with G. diazotrophicus Pal 5 triggers a set of biochemical and structural changes in roots, stems, and leaves of seedlings. Discrete callose deposits as papillae were observed at specific sites of root hairs, trichomes, and leaf tissue. Upon R. pseudosolanacearum GMI1000 infection, endophyte-treated plants demonstrated being induced for defense through an augmented callose deposition at root hairs and leaves compared with the non-endophyte-treated controls. The endophytic bacterium appears to be able to prime callose response. Roots and stems cross sections showed that integrity of all tissues was preserved in endophyte-treated plants infected with R. solanacearum A21. The mechanisms of resistance elicited by the plant after inoculation with the endophyte would be greater lignification and sclerosis in tissues and reinforcement of the cell wall through the deposition of callose. As a consequence of this priming in plant defense response, viable phytopathogenic bacteria counting were considerably fewer in endophyte-inoculated plants than in not-inoculated controls. Our results indicate that G. diazotrophicus colonizes A. thaliana plants performing a protective role against the phytopathogenic bacterium R. solanacearum promoting the activation of plant defense system.

RESUMEN

Potato (Solanum tuberosum L.) is one of the main hosts of Ralstonia solanacearum, the causative agent of bacterial wilt. This plant pathogen bacteria produce asymptomatic latent infections that promote its global spread, hindering disease control. A potato breeding program is conducted in Uruguay based on the introgression of resistance from the wild native species S. commersonii Dun. Currently, several backcrosses were generated exploiting the high genetic variability of this wild species resulting in advanced interspecific breeding lines with different levels of bacterial wilt resistance. The overall aim of this work was to characterize the interaction of the improved potato germplasm with R. solanacearum. Potato clones with different responses to R. solanacearum were selected, and colonization, dissemination and multiplication patterns after infection were evaluated. A R. solanacearum strain belonging to the phylotype IIB-sequevar 1, with high aggressiveness on potato was genetically modified to constitutively generate fluorescence and luminescence from either the green fluorescence protein gene or lux operon. These reporter strains were used to allow a direct and precise visualization of fluorescent and luminescent cells in plant tissues by confocal microscopy and luminometry. Based on wilting scoring and detection of latent infections, the selected clones were classified as susceptible or tolerant, while no immune-like resistance response was identified. Typical wilting symptoms in susceptible plants were correlated with high concentrations of bacteria in roots and along the stems. Tolerant clones showed a colonization pattern restricted to roots and a limited number of xylem vessels only in the stem base. Results indicate that resistance in potato is achieved through restriction of bacterial invasion and multiplication inside plant tissues, particularly in stems. Tolerant plants were also characterized by induction of anatomical and biochemical changes after R. solanacearum infection, including hyperplasic activity of conductor tissue, tylose production, callose and lignin deposition, and accumulation of reactive oxygen species. This study highlights the potential of the identified tolerant interspecific potato clones as valuable genetic resources for potato-breeding programs and leads to a better understanding of resistance against R. solanacearum in potato.

RESUMEN

Light is an important environmental signal for almost all living organisms. The light perception is achieved by photoreceptor proteins. As can be observed from the great number of bacterial genomes sequenced, plant pathogenic bacteria encode for a large number of photoreceptor proteins. The physiological implications of these photoreceptors are still poorly characterized. However, recent studies revealed the participation of these photosensory proteins in the pathogenic process. Here, we summarize what is known about these proteins and their role during the virulence process, concluding that the light environment modulates the plant-pathogen interaction.

Asunto(s)

Proteínas Bacterianas/fisiología , Interacciones Huésped-Patógeno , Fotorreceptores Microbianos/fisiología , Plantas/microbiología , Agrobacterium/metabolismo , Agrobacterium/patogenicidad , Luz , Virulencia , Xanthomonas/metabolismo , Xanthomonas/patogenicidad

RESUMEN

Type IV pili (Tfp) are widely distributed adhesins of bacterial surfaces. In plant pathogenic bacteria, Tfp are involved in host colonization and pathogenesis. Xanthomonas citri subsp. citri (Xcc) is the phytopathogen responsible for citrus canker disease. In this work, three Tfp structural genes, fimA, fimA1, and pilA from Xcc were studied. A pilA mutant strain from Xcc (XccΔpilA) was constructed and differences in physiological features, such as motilities, adhesion, and biofilm formation, were observed. A structural study of the purified Tfp fractions from Xcc wild-type and Xcc∆pilA showed that pilins are glycosylated in both strains and that FimA and FimA1 are the main structural components of the pili. Furthermore, smaller lesion symptoms and reduced bacterial growth were produced by Xcc∆pilA in orange plants compared to the wild-type strain. These results indicate that the minor pilin-like gene, pilA, is involved in Tfp performance during the infection process.

Asunto(s)

Proteínas Bacterianas/metabolismo , Citrus/microbiología , Proteínas Fimbrias/metabolismo , Enfermedades de las Plantas/microbiología , Xanthomonas/metabolismo , Proteínas Bacterianas/genética , Proteínas Fimbrias/genética , Eliminación de Gen , Virulencia , Xanthomonas/genética , Xanthomonas/patogenicidad

RESUMEN

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.

Asunto(s)

Catalasa/metabolismo , Citrus sinensis/microbiología , Peróxido de Hidrógeno/metabolismo , Hojas de la Planta/microbiología , Xanthomonas/metabolismo , Biopelículas/crecimiento & desarrollo , Catalasa/genética , Regulación Bacteriana de la Expresión Génica , Estrés Oxidativo/fisiología , Enfermedades de las Plantas/microbiología , Rayos Ultravioleta , Xanthomonas/enzimología , Xanthomonas/efectos de la radiación

RESUMEN

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.

Asunto(s)

Luz , Fotorreceptores Microbianos/metabolismo , Proteínas Quinasas/fisiología , Xanthomonas/fisiología , Histidina Quinasa , Oxígeno/química

RESUMEN

Plants are constantly exposed to stress factors. Biotic stress is produced by living organisms such as pathogens, whereas abiotic stress by unfavourable environmental conditions. In Citrus species, one of the most important fruit crops in the world, these stresses generate serious limitations in productivity. Through biochemical and transcriptomic assays, we had previously characterised the Citrus sinensis (L.) Osbeck nonhost response to Xanthomonas campestris pv. vesicatoria (Doidge), in contrast to Asiatic citrus canker infection caused by Xanthomonas citri subsp. citri (Hasse). A hypersensitive response (HR) including changes in the expression of several transcription factors was reported. Here, a new exhaustive analysis of the Citrus sinensis transcriptomes previously obtained was performed, allowing us to detect the over-representation of photosynthesis, abiotic stress and secondary metabolism processes during the nonhost HR. The broad downregulation of photosynthesis-related genes was correlated with an altered photosynthesis physiology. The high number of heat shock proteins and genes related to abiotic stress, including aquaporins, suggests that stresses crosstalk. Additionally, the secondary metabolism exhibited lignin and carotenoid biosynthesis modifications and expression changes in the cell rescue GSTs. In conclusion, novel features of the Citrus nonhost HR, an important part of the plants' defence against disease that has yet to be fully exploited in plant breeding programs, are presented.

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas/genética , Citrus sinensis/inmunología , Regulación de la Expresión Génica de las Plantas , Fotorreceptores Microbianos/genética , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/inmunología , Xanthomonas/patogenicidad , Proteínas Bacterianas/metabolismo , Citrus sinensis/genética , Citrus sinensis/microbiología , Eliminación de Gen , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Evasión Inmune , Luz , Fotorreceptores Microbianos/metabolismo , Fotosíntesis/fisiología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Proteínas de Plantas/genética , Estructura Terciaria de Proteína , Virulencia , Xanthomonas/genética

RESUMEN

We have solved the structure of ferredoxin-NADP(H) reductase, FPR, from the plant pathogen Xanthomonas axonopodis pv. citri, responsible for citrus canker, at a resolution of 1.5 Å. This structure reveals differences in the mobility of specific loops when compared to other FPRs, probably unrelated to the hydride transfer process, which contributes to explaining the structural and functional divergence between the subclass I FPRs. Interactions of the C-terminus of the enzyme with the phosphoadenosine of the cofactor FAD limit its mobility, thus affecting the entrance of nicotinamide into the active site. This structure opens the possibility of rationally designing drugs against the X. axonopodis pv. citri phytopathogen.

Asunto(s)

Citrus/microbiología , Ferredoxina-NADP Reductasa/química , Flavina-Adenina Dinucleótido/metabolismo , Xanthomonas axonopodis/enzimología , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Homología Estructural de Proteína

RESUMEN

Plants, when exposed to certain pathogens, may display a form of genotype-independent resistance, known as non-host response. In this study, the response of Citrus sinensis (sweet orange) leaves to Xanthomonas campestris pv. vesicatoria (Xcv), a pepper and tomato pathogenic bacterium, was analyzed through biochemical assays and cDNA microarray hybridization and compared with Asiatic citrus canker infection caused by Xanthomonas citri subsp. citri. Citrus leaves exposed to the non-host bacterium Xcv showed hypersensitive response (HR) symptoms (cell death), a defense mechanism common in plants but poorly understood in citrus. The HR response was accompanied by differentially expressed genes that are associated with biotic stress and cell death. Moreover, 58 transcription factors (TFs) were differentially regulated by Xcv in citrus leaves, including 26 TFs from the stress-associated families AP2-EREBP, bZip, Myb and WRKY. Remarkably, in silico analysis of the distribution of expressed sequence tags revealed that 10 of the 58 TFs, belonging to C2C2-GATA, C2H2, CCAAT, HSF, NAC and WRKY gene families, were specifically over-represented in citrus stress cDNA libraries. This study identified candidate TF genes for the regulation of key steps during the citrus non-host HR. Furthermore, these TFs might be useful in future strategies of molecular breeding for citrus disease resistance.

Asunto(s)

Citrus sinensis/metabolismo , Citrus sinensis/microbiología , Interacciones Huésped-Patógeno , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Xanthomonas campestris/fisiología , Alelos , Muerte Celular , Citrus sinensis/citología , Citrus sinensis/genética , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Interacciones Huésped-Patógeno/genética , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/microbiología , Proteínas de Plantas/genética , Estrés Fisiológico/genética

RESUMEN

Xanthomonas axonopodis pv. citri (Xac), the bacterium that causes citrus canker, contains a gene in the hrp [for hypersensitive response (HR) and pathogenicity] cluster that encodes a harpin protein called Hpa1. Hpa1 produced HR in the nonhost plants tobacco, pepper and Arabidopsis, whereas, in the host plant citrus, it elicited a weak defence response with no visible phenotype. Co-infiltrations of Xac with or without the recombinant Hpa1 protein in citrus leaves produced a larger number of cankers in the presence of the protein. To characterize the effect of Hpa1 during the disease, an XacΔhpa1 mutant was constructed, and infiltration of this mutant caused a smaller number of cankers. In addition, the lack of Hpa1 hindered bacterial aggregation both in solution and in planta. Analysis of citrus leaves infiltrated with Hpa1 revealed alterations in mesophyll morphology caused by the presence of cavitations and crystal idioblasts, suggesting the binding of the harpin to plant membranes and the elicitation of signalling cascades. Overall, these results suggest that, even though Hpa1 elicits the defence response in nonhost plants and, to a lesser extent, in host plants, its main roles in citrus canker are to alter leaf mesophyll structure and to aggregate bacterial cells, and thus increase virulence and pathogen fitness. We expressed the N-terminal and C-terminal regions and found that, although both regions elicited HR in nonhost plants, only the N-terminal region showed increased virulence and bacterial aggregation, supporting the role of this region of the protein as the main active domain.

Asunto(s)

Proteínas Bacterianas/metabolismo , Xanthomonas axonopodis/patogenicidad , Amiloide , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas Bacterianas/química , Citrus/inmunología , Citrus/microbiología , Medios de Cultivo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Hojas de la Planta/anatomía & histología , Hojas de la Planta/inmunología , Hojas de la Planta/microbiología , Estructura Terciaria de Proteína , Virulencia , Xanthomonas axonopodis/citología

RESUMEN

Xanthomonas axonopodis 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 (wzt and rfb303) 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, in vivo and in vitro adhesion and biofilm formation. Changes in the expression levels of the LPS biosynthesis genes were observed in a medium that mimics the plant environment. Xacwzt exhibited reduced virulence in host plants compared to Xac wild-type and Xacrfb303. 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.

Asunto(s)

Citrus/microbiología , Lipopolisacáridos/metabolismo , Enfermedades de las Plantas/microbiología , Xanthomonas axonopodis/metabolismo , Xanthomonas axonopodis/patogenicidad , Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Regulación Bacteriana de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno , Mutación , Fenotipo , Enfermedades de las Plantas/inmunología , Hojas de la Planta/genética , Hojas de la Planta/microbiología , Estrés Fisiológico , Virulencia , Xanthomonas axonopodis/genética

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas/metabolismo , Citrus sinensis/microbiología , Interacciones Huésped-Patógeno/fisiología , Xanthomonas axonopodis/crecimiento & desarrollo , Xanthomonas axonopodis/fisiología , Secuencia de Aminoácidos , Adhesión Bacteriana , Proteínas Bacterianas/química , Biopelículas , Recuento de Colonia Microbiana , Biología Computacional , Eliminación de Gen , Genes Bacterianos/genética , Histidina Quinasa , Datos de Secuencia Molecular , Movimiento/fisiología , Procesos Fotoquímicos , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Polisacáridos Bacterianos/biosíntesis , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Xanthomonas axonopodis/enzimología , Xanthomonas axonopodis/genética

RESUMEN

Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified.

Asunto(s)

Proteínas Bacterianas/fisiología , Proteínas de Plantas/fisiología , Xanthomonas/fisiología , Cloroplastos/fisiología , Fotosíntesis , Unión Proteica , Xanthomonas/patogenicidad

RESUMEN

Xanthomonas axonopodis pv. citri is a phytopathogen bacterium that causes severe citrus canker disease. Similar to other phytopathogens, after infection by this bacterium, plants trigger a defense mechanism that produces reactive oxygen species. Ferredoxin-NADP(+) reductases (FNRs) are redox flavoenzymes that participate in several metabolic functions, including the response to reactive oxygen species. Xanthomonas axonopodis pv. citri has a gene (fpr) that encodes for a FNR (Xac-FNR) that belongs to the subclass I bacterial FNRs. The aim of this work was to search for the physiological role of this enzyme and to characterize its structural and functional properties. The functionality of Xac-FNR was tested by cross-complementation of a FNR knockout Escherichia coli strain, which exhibit high susceptibility to agents that produce an abnormal accumulation of (•)O(2)(-). Xac-FNR was able to substitute for the FNR in E. coli in its antioxidant role. The expression of fpr in X. axonopodis pv. citri was assessed using semiquantitative RT-PCR and Western blot analysis. A 2.2-fold induction was observed in the presence of the superoxide-generating agents methyl viologen and 2,3-dimethoxy-1,4-naphthoquinone. Structural and functional studies showed that Xac-FNR displayed different functional features from other subclass I bacterial FNRs. Our analyses suggest that these differences may be due to the unusual carboxy-terminal region. We propose a further classification of subclass I bacterial FNRs, which is useful to determine the nature of their ferredoxin redox partners. Using sequence analysis, we identified a ferredoxin (XAC1762) as a potential substrate of Xac-FNR. The purified ferredoxin protein displayed the typical broad UV-visible spectrum of [4Fe-4S] clusters and was able to function as substrate of Xac-FNR in the cytochrome c reductase activity. Our results suggest that Xac-FNR is involved in the oxidative stress response of Xanthomonas axonopodis pv. citri and performs its biological function most likely through the interaction with ferredoxin XAC1762.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Ferredoxina-NADP Reductasa/química , Ferredoxina-NADP Reductasa/metabolismo , Xanthomonas axonopodis/enzimología , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxina-NADP Reductasa/genética , Prueba de Complementación Genética , Estructura Secundaria de Proteína

RESUMEN

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo2Hex6GalA3Fuc3NAcRha4 and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.

Asunto(s)

Citrus sinensis/inmunología , Citrus sinensis/microbiología , Inmunidad Innata , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Xanthomonas axonopodis/fisiología , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Carbohidratos , Citrus sinensis/anatomía & histología , Citrus sinensis/genética , Regulación de la Expresión Génica de las Plantas/inmunología , Interacciones Huésped-Patógeno , Inmunidad Innata/genética , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/aislamiento & purificación , Datos de Secuencia Molecular , Peróxidos/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/microbiología , Estomas de Plantas/anatomía & histología , Estomas de Plantas/inmunología , Estomas de Plantas/microbiología , Xanthomonas axonopodis/metabolismo

RESUMEN

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.

Asunto(s)

Catalasa/metabolismo , Citrus/microbiología , Xanthomonas axonopodis/enzimología , Xanthomonas axonopodis/patogenicidad , Adaptación Fisiológica/efectos de los fármacos , Catalasa/biosíntesis , Catalasa/genética , Medios de Cultivo , Farmacorresistencia Bacteriana/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/microbiología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/metabolismo , Mutación/genética , Virulencia/efectos de los fármacos , Xanthomonas axonopodis/efectos de los fármacos , Xanthomonas axonopodis/crecimiento & desarrollo