Lucas D. Daurelio

Xanthomonas axonopodis pv. citri uses a plant natriuretic peptide-like protein to modify host homeostasis.

Plant natriuretic peptides (PNPs) are a class of extracellular, systemically mobile molecules that elicit a number of plant responses important in homeostasis and growth. The bacterial citrus pathogen, Xanthomonas axonopodis pv. citri, also contains a gene encoding a PNP-like protein, XacPNP, that shares significant sequence similarity and identical domain organization with plant PNPs but has no homologues in other bacteria. We have expressed and purified XacPNP and demonstrated that the bacterial protein alters physiological responses including stomatal opening in plants. Although XacPNP is not expressed under standard nutrient rich culture conditions, it is strongly induced under conditions that mimic the nutrient poor intercellular apoplastic environment of leaves, as well as in infected tissue, suggesting that XacPNP transcription can respond to the host environment. To characterize the role of XacPNP during bacterial infection, we constructed a XacPNP deletion mutant. The lesions caused by this mutant were more necrotic than those observed with the wild-type, and bacterial cell death occurred earlier in the mutant. Moreover, when we expressed XacPNP in Xanthomonas axonopodis pv. vesicatoria, the transgenic bacteria caused less necrotic lesions in the host than the wild-type. In conclusion, we present evidence that a plant-like bacterial PNP can enable a plant pathogen to modify host responses to create conditions favorable to its own survival.

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.

A plant natriuretic peptide-like molecule of the pathogen Xanthomonas axonopodis pv. citri causes rapid changes in the proteome of its citrus host.

BackgroundPlant natriuretic peptides (PNPs) belong to a novel class of peptidic signaling molecules that share some structural similarity to the N-terminal domain of expansins and affect physiological processes such as water and ion homeostasis at nano-molar concentrations. The citrus pathogen Xanthomonas axonopodis pv. citri possesses a PNP-like peptide (XacPNP) uniquely present in this bacteria. Previously we observed that the expression of XacPNP is induced upon infection and that lesions produced in leaves infected with a XacPNP deletion mutant were more necrotic and lead to earlier bacterial cell death, suggesting that the plant-like bacterial PNP enables the plant pathogen to modify host responses in order to create conditions favorable to its own survival.ResultsHere we measured chlorophyll fluorescence parameters and water potential of citrus leaves infiltrated with recombinant purified XacPNP and demonstrate that the peptide improves the physiological conditions of the tissue. Importantly, the proteomic analysis revealed that these responses are mirrored by rapid changes in the host proteome that include the up-regulation of Rubisco activase, ATP synthase CF1 α subunit, maturase K, and α- and β-tubulin.ConclusionsWe demonstrate that XacPNP induces changes in host photosynthesis at the level of protein expression and in photosynthetic efficiency in particular. Our findings suggest that the biotrophic pathogen can use the plant-like hormone to modulate the host cellular environment and in particular host metabolism and that such modulations weaken host defence.

A Eukaryotic-Acquired Gene by a Biotrophic Phytopathogen Allows Prolonged Survival on the Host by Counteracting the Shut-Down of Plant Photosynthesis

Xanthomonas citri pv. citri, the bacteria responsible for citrus canker posses a biological active plant natriuretic peptide (PNP)-like protein, not present in any other bacteria. PNPs are a class of extracellular, systemically mobile peptides that elicit a number of plant responses important in homeostasis and growth. Previously, we showed that a Xanthomonas citri pv. citri mutant lacking the PNP-like protein XacPNP produced more necrotic lesions in citrus leaves than wild type infections and suggested a role for XacPNP in the regulation of host homeostasis. Here we have analyzed the proteome modifications observed in citrus leaves infected with the wild type and XacPNP deletion mutant bacteria. While both of them cause down-regulation of enzymes related to photosynthesis as well as chloroplastic ribosomal proteins, proteins related to defense responses are up-regulated. However, leaves infiltrated with the XacPNP deletion mutant show a more pronounced decrease in photosynthetic proteins while no reduction in defense related proteins as compared to the wild-type pathogen. This suggests that XacPNP serves the pathogen to maintain host photosynthetic efficiency during pathogenesis. The results from the proteomics analyses are consistent with our chlorophyll fluorescence data and transcript analyses of defense genes that show a more marked reduction in photosynthesis in the mutant but no difference in the induction of genes diagnostic for biotic-stress responses. We therefore conclude that XacPNP counteracts the shut-down of host photosynthesis during infection and in that way maintains the tissue in better conditions, suggesting that the pathogen has adapted a host gene to modify its natural host and render it a better reservoir for prolonged bacterial survival and thus for further colonization.

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

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.

Characterization of Citrus sinensis transcription factors closely associated with the non-host response to Xanthomonas campestris pv. vesicatoria.

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.

Modulating host homeostasis as a strategy in the plant-pathogen arms race

In plant-pathogen interactions, pathogens aim to overcome host defence responses while plants employ a battery of responses to limit pathogen growth and thus disease. In this “arms race” between hosts and pathogens, horizontal gene transfer is a potent source of ‘pathogenic innovation’ for viruses and bacteria. However, bacteria rarely acquire ‘eukaryotic-like’ genes from their hosts, and where they appear to, evidence for a role of the acquired genes remains outstanding. We have recently reported experimental evidence that the citrus canker causing pathogen Xanthomonas axonopodis pv. citri contains a plant natriuretic peptide-like gene (XacPNP) that encodes a protein that modulates host homeostasis to its advantage. We argue that Xanthomonas PNP has been acquired in an ancient horizontal gene transfer, and given that plant and bacterial PNPs trigger a number of similar physiological responses, we make a case of molecular mimicry. Released XacPNP mimics host PNP and results in a suppressed host response, “improved” host tissue health and consequently better pathogen survival in the lesions. Finally, we propose that Xanthomonas axonopodis pv. citri host interactions can serve as model system to study the role of host homeostasis in plant defence against biotrophic pathogens.

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.

Characterization of Citrus sinensis type 1 mitochondrial alternative oxidase and expression analysis in biotic stress.

The higher plant mitochondrial electron transport chain contains an alternative pathway that ends with the AOX (alternative oxidase). The AOX proteins are encoded by a small gene family composed of two discrete gene subfamilies. Aox1 is present in both monocot and eudicot plants, whereas Aox2 is only present in eudicot plants. We isolated a genomic clone from Citrus sinensis containing the Aox1a gene. The orange Aox1a consists of four exons interrupted by three introns and its promoter harbours diverse putative stress-specific regulatory motifs including pathogen response elements. The role of the Aox1a gene was evaluated during the compatible interaction between C. sinensis and Xanthomonas axonopodis pv. citri and no induction of the Aox1a at the transcriptional level was observed. On the other hand, Aox1a was studied in orange plants during non-host interactions with Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria, which result in hypersensitive response. Both phytopathogens produced a strong induction of Aox1a, reaching a maximum at 8 h post-infiltration. Exogenous application of salicylic acid produced a slight increase in the steady-state level of Aox1a, whereas the application of fungi elicitors showed the highest induction. These results suggest that AOX1a plays a role during biotic stress in non-host plant pathogen interaction.

Experimental bio-artificial liver: Importance of the architectural design on ammonia detoxification performance

AIM To determine the influence of the construction design over the biological component’s performance in an experimental bio-artificial liver (BAL) device. METHODS Two BAL models for liver microorgans (LMOs) were constructed. First, we constructed a cylindrical BAL and tested it without the biological component to establish its correct functioning. Samples of blood and biological compartment (BC) fluid were taken after 0, 60, and 120 min of perfusion. Osmolality, hematocrit, ammonia and glucose concentrations, lactate dehydrogenase (LDH) release (as a LMO viability parameter), and oxygen consumption and ammonia metabolizing capacity (as LMO functionality parameters) were determined. CPSI and OTC gene expression and function were measured. The second BAL, a “flat bottom” model, was constructed using a 25 cm2 culture flask while maintaining all other components between the models. The BC of both BALs had the same capacity (approximately 50 cm3) and both were manipulated with the same perfusion system. The performances of the two BALs were compared to show the influence of architecture. RESULTS The cylindrical BAL showed a good exchange of fluids and metabolites between blood and the BC, reflected by the matching of osmolalities, and glucose and ammonia concentration ratios after 120 min of perfusion. No hemoconcentration was detected, the hematocrit levels remained stable during the whole study, and the minimal percentage of hemolysis (0.65% ± 0.10%) observed was due to the action of the peristaltic pump. When LMOs were used as biological component of this BAL they showed similar values to the ones obtained in a Normothermic Reoxygenation System (NRS) for almost all the parameters assayed. After 120 min, the results obtained were: LDH release (%): 14.7 ± 3.1 in the BAL and 15.5 ± 3.2 in the NRS (n = 6); oxygen consumption (μmol/min·g wet tissue): 1.16 ± 0.21 in the BAL and 0.84 ± 0.15 in the NRS (n = 6); relative expression of Cps1 and Otc: 0.63 ± 0.12 and 0.67 ± 0.20, respectively, in the BAL, and 0.86 ± 0.10 and 0.82 ± 0.07, respectively, in the NRS (n = 3); enzymatic activity of CPSI and OTC (U/g wet tissue): 3.03 ± 0.86 and 222.0 ± 23.5, respectively, in the BAL, and 3.12 ± 0.73 and 228.8 ± 32.8, respectively, in the NRS (n = 3). In spite of these similarities, LMOs as a biological component of the cylindrical BAL were not able to detoxify ammonia at a significant level (not detected vs 35.1% ± 7.0% of the initial 1 mM NH4+ dose in NRS, n = 6). Therefore, we built a second BAL with an entirely different design that offers a flat base BC. When LMOs were placed in this “flat bottom” device they were able to detoxify 49.3% ± 8.8% of the initial ammonia overload after 120 min of perfusion (n = 6), with a detoxification capacity of 13.2 ± 2.2 μmol/g wet tissue. CONCLUSION In this work, we demonstrate the importance of adapting the BAL architecture to the biological component characteristics to obtain an adequate BAL performance.