scholarly journals Erwinia carotovora Quorum Sensing System Regulates Host-Specific Virulence Factors and Development Delay in Drosophila melanogaster

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Filipe J. D. Vieira ◽  
Pol Nadal-Jimenez ◽  
Luis Teixeira ◽  
Karina B. Xavier
Keyword(s):  
Cell Wall ◽  
Drosophila Melanogaster ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Plant Cell Wall ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Cell Wall Degrading Enzymes ◽  
Content Type ◽  
Pectolytic Enzymes

ABSTRACT Multihost bacteria have to rapidly adapt to drastic environmental changes, relying on a fine integration of multiple stimuli for an optimal genetic response. Erwinia carotovora spp. are phytopathogens that cause soft-rot disease. Strain Ecc15 in particular is a model for bacterial oral-route infection in Drosophila melanogaster as it harbors a unique gene, evf, that encodes the Erwinia virulence factor (Evf), which is a major determinant for infection of the D. melanogaster gut. However, the factors involved in the regulation of evf expression are poorly understood. We investigated whether evf could be controlled by quorum sensing as, in the Erwinia genus, quorum sensing regulates pectolytic enzymes, the major virulence factors needed to infect plants. Here, we show that transcription of evf is positively regulated by quorum sensing in Ecc15 via acyl-homoserine lactone (AHL) signal synthase ExpI and AHL receptors ExpR1 and ExpR2. We also show that the load of Ecc15 in the gut depends upon the quorum sensing-mediated regulation of evf. Furthermore, we demonstrate that larvae infected with Ecc15 suffer a developmental delay as a direct consequence of the regulation of evf via quorum sensing. Finally, we demonstrate that evf is coexpressed with plant cell wall-degrading enzymes (PCWDE) during plant infection in a quorum sensing-dependent manner. Overall, our results show that Ecc15 relies on quorum sensing to control production of both pectolytic enzymes and Evf. This regulation influences the interaction of Ecc15 with its two known hosts, indicating that quorum sensing signaling may impact bacterial dissemination via insect vectors that feed on rotting plants. IMPORTANCE Integration of genetic networks allows bacteria to rapidly adapt to changing environments. This is particularly important in bacteria that interact with multiple hosts. Erwinia carotovora is a plant pathogen that uses Drosophila melanogaster as a vector. To interact with these two hosts, Ecc15 uses different sets of virulence factors: plant cell wall-degrading enzymes to infect plants and the Erwinia virulence factor (evf) to infect Drosophila. Our work shows that, despite the virulence factors being specific for each host, both sets are coactivated by homoserine lactone quorum sensing and by the two-component GacS/A system in infected plants. This regulation is essential for Ecc15 loads in the gut of Drosophila and minimizes the developmental delay caused by the bacteria with respect to the insect vector. Our findings provide evidence that coactivation of the host-specific factors in the plant may function as a predictive mechanism to maximize the probability of transit of the bacteria between hosts.

scholarly journals Quorum sensing regulation in Erwinia carotovora affects development of Drosophila melanogaster infected larvae

2019 ◽  
Author(s):  
Filipe J. D. Vieira ◽  
Pol Nadal-Jimenez ◽  
Luis Teixeira ◽  
Karina B. Xavier
Keyword(s):  
Drosophila Melanogaster ◽  
Quorum Sensing ◽  
Developmental Delay ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Plant Cell Wall ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Pectolytic Enzymes ◽  
Two Component

AbstractMulti-host bacteria must rapidly adapt to drastic environmental changes, relying on integration of multiple stimuli for an optimal genetic response. Erwinia spp. are phytopathogens that cause soft-rot disease in plants. Erwinia carotovora Ecc15 is used as a model for bacterial oral-route infection in Drosophila melanogaster as it harbors a gene, the Erwinia virulence factor (Evf), which has been previously shown to be a major determinant for infection of D. melanogaster gut. However, the factors involved in regulation of evf expression are poorly understood. We investigated whether evf could be controlled by quorum sensing since, in the Erwinia genus, quorum sensing regulates pectolytic enzymes, the major virulence factors needed to infect plants. Here, we show that transcription of evf is positively regulated by quorum sensing in Ecc15 via the acyl-homoserine lactone (AHL) signal synthase ExpI, and the AHL receptors ExpR1 and ExpR2. Moreover, we demonstrate that the GacS/A two-component system is partially required for evf expression. We also show that the load of Ecc15 in the gut depends upon the quorum sensing-mediated regulation of evf. Furthermore, we demonstrate that larvae infected with Ecc15 suffer a developmental delay as a direct consequence of the regulation of evf via quorum sensing. Overall, our results show that Ecc15 relies on quorum sensing to control production of both pectolytic enzymes and Evf. This regulation influences the interaction of Ecc15 with its two known hosts, indicating that quorum sensing and GacS/A signaling systems may impact bacterial dissemination via insect vectors that feed on rotting plants.SignificanceIntegration of genetic networks allows bacteria to rapidly adapt to changing environments. This is particularly important in bacteria that interact with multiple hosts. Erwinia carotovora Ecc15 is a plant pathogen that uses Drosophila melanogaster as a vector. To interact with these two hosts, Ecc15 uses two different sets of virulence factors: plant cell wall-degrading enzymes to infect plants and the Erwinia virulence factor (evf) to infect Drosophila. Our work shows that, despite the virulence factors being different, both are regulated by homoserine lactone quorum sensing and the two component GacS/A system. Moreover, we show that these pathways are essential for Ecc15 loads in the gut of Drosophila and that this interaction carries a cost to the vector in the form of a developmental delay. Our findings provide evidence for the importance of quorum sensing regulation in the establishment of multi-host interactions.

scholarly journals Phloretin, an Apple Phytoalexin, Affects the Virulence and Fitness of Pectobacterium brasiliense by Interfering With Quorum-Sensing

2021 ◽  
Vol 12 ◽  
Author(s):  
Manoj Pun ◽  
Netaly Khazanov ◽  
Ortal Galsurker ◽  
Michal Weitman ◽  
Zohar Kerem ◽  
...  
Keyword(s):  
Cell Wall ◽  
Quorum Sensing ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Soft Rot ◽  
Homoserine Lactone ◽  
Cell Wall Degrading Enzymes ◽  
Docking Simulations ◽  
E Coli ◽  
Degrading Enzymes

The effects of phloretin a phytoalexin from apple, was tested on Pectobacterium brasiliense (Pb1692), an emerging soft-rot pathogen of potato. Exposure of Pb1692 to 0.2 mM phloretin a concentration that does not affect growth, or to 0.4 mM a 50% growth inhibiting concentration (50% MIC), reduced motility, biofilm formation, secretion of plant cell wall-degrading enzymes, production of acyl–homoserine lactone (AHL) signaling molecules and infection, phenotypes that are associated with bacterial population density-dependent system known as quorum sensing (QS). To analyze the effect of growth inhibition on QS, the activity of ciprofloxacin, an antibiotic that impairs cell division, was compared to that of phloretin at 50% MIC. Unlike phloretin, the antibiotic hardly affected the tested phenotypes. The use of DH5α, a QS-negative Escherichia coli strain, transformed with an AHL synthase (ExpI) from Pb1692, allowed to validate direct inhibition of AHL production by phloretin, as demonstrated by two biosensor strains, Chromobacterium violaceaum (CV026) and E. coli (pSB401). Expression analysis of virulence-related genes revealed downregulation of QS-regulated genes (expI, expR, luxS, rsmB), plant cell wall degrading enzymes genes (pel, peh and prt) and motility genes (motA, fim, fliA, flhC and flhD) following exposure to both phloretin concentrations. The results support the inhibition of ExpI activity by phloretin. Docking simulations were used to predict the molecular associations between phloretin and the active site of ExpI, to suggest a likely mode of action for the compound’s inhibition of virulence.

scholarly journals A Metabolic Regulator Modulates Virulence and Quorum Sensing Signal Production in Pectobacterium atrosepticum

2013 ◽  
Vol 26 (3) ◽  
pp. 356-366 ◽  
Author(s):  
Marion F. Cubitt ◽  
Peter E. Hedley ◽  
Neil R. Williamson ◽  
Jenny A. Morris ◽  
Emma Campbell ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Plant Cell Wall ◽  
Homoserine Lactone ◽  
Insertion Mutation ◽  
Virulence Determinants ◽  
Methionine Biosynthesis ◽  
Cell Wall Degrading Enzymes ◽  
Pectobacterium Atrosepticum ◽  
Small Regulatory Rna ◽  
The Impact

Plant cell wall–degrading enzymes (PCWDE) are key virulence determinants in the pathogenesis of the potato pathogen Pectobacterium atrosepticum. In this study, we report the impact on virulence of a transposon insertion mutation in the metJ gene that codes for the repressor of the methionine biosynthesis regulon. In a mutant strain defective for the small regulatory RNA rsmB, PCWDE are not produced and virulence in potato tubers is almost totally abolished. However, when the metJ gene is disrupted in this background, the rsmB– phenotype is suppressed and virulence and PCWDE production are restored. Additionally, when metJ is disrupted, production of the quorum-sensing signal, N-(3-oxohexanoyl)-homoserine lactone, is increased. The metJ mutant strains showed pleiotropic transcriptional impacts affecting approximately a quarter of the genome. Genes involved in methionine biosynthesis were most highly upregulated but many virulence-associated transcripts were also upregulated. This is the first report of the impact of the MetJ repressor on virulence in bacteria.

scholarly journals Transgenic Plants Producing the Bacterial Pheromone N-Acyl-Homoserine Lactone Exhibit Enhanced Resistance to the Bacterial Phytopathogen Erwinia carotovora

2001 ◽  
Vol 14 (9) ◽  
pp. 1035-1042 ◽  
Author(s):  
Andres Mäe ◽  
Marcos Montesano ◽  
Viia Koiv ◽  
E. Tapio Palva
Keyword(s):  
Quorum Sensing ◽  
Transgenic Tobacco ◽  
Plant Cell Wall ◽  
Ectopic Expression ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Defense Responses ◽  
Dependent Manner ◽  
Wild Type ◽  
Enhanced Resistance

Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.

scholarly journals Oligogalacturonide-Mediated Induction of a Gene Involved in Jasmonic Acid Synthesis in Response to the Cell-Wall-Degrading Enzymes of the Plant Pathogen Erwinia carotovora

1999 ◽  
Vol 12 (7) ◽  
pp. 640-644 ◽  
Author(s):  
Cecilia Norman ◽  
Sabina Vidal ◽  
E. Tapio Palva
Keyword(s):  
Cell Wall ◽  
Plant Cell Wall ◽  
Extracellular Enzymes ◽  
Erwinia Carotovora ◽  
Acid Synthesis ◽  
Allene Oxide ◽  
Cell Wall Degrading Enzymes ◽  
Degrading Enzymes ◽  
High Sequence Homology ◽  
Oxide Synthase

Identification of Arabidopsis thaliana genes responsive to plant cell-wall-degrading enzymes of Erwinia carotovora subsp. carotovora led to the isolation of a cDNA clone with high sequence homology to the gene for allene oxide synthase, an enzyme involved in the biosynthesis of jasmonates. Expression of the corresponding gene was induced by the extracellular enzymes from this pathogen as well as by treatment with methyl jasmonate and short oligogalacturonides (OGAs). This suggests that OGAs are involved in the induction of the jasmonate pathway during plant defense response to E. carotovora subsp. carotovora attack.

scholarly journals Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels

2017 ◽  
Vol 83 (22) ◽  
Author(s):  
Doris Tisch ◽  
Kyle R. Pomraning ◽  
James R. Collett ◽  
Michael Freitag ◽  
Scott E. Baker ◽  
...  
Keyword(s):  
Cell Wall ◽  
Gene Regulation ◽  
Carbon Source ◽  
Trichoderma Reesei ◽  
Plant Cell Wall ◽  
Female Fertility ◽  
Mating Types ◽  
Carbon Source Utilization ◽  
Cell Wall Degrading Enzymes ◽  
Content Type

ABSTRACT The filamentous fungus Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis. IMPORTANCE Trichoderma reesei is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertility-specific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance.

scholarly journals Interacting Signal Pathways Control Defense Gene Expression in Arabidopsis in Response to Cell Wall-Degrading Enzymes from Erwinia carotovora

2000 ◽  
Vol 13 (4) ◽  
pp. 430-438 ◽  
Author(s):  
Cecilia Norman-Setterblad ◽  
Sabina Vidal ◽  
E. Tapio Palva
Keyword(s):  
Gene Expression ◽  
Cell Wall ◽  
Target Genes ◽  
Plant Cell Wall ◽  
Erwinia Carotovora ◽  
Signal Pathways ◽  
Cell Wall Degrading Enzymes ◽  
Defense Gene Expression ◽  
Defense Gene ◽  
Degrading Enzymes

We have characterized the role of salicylic acid (SA)-independent defense signaling in Arabidopsis thaliana in response to the plant pathogen Erwinia carotovora subsp. carotovora. Use of pathway-specific target genes as well as signal mutants allowed us to elucidate the role and interactions of ethylene, jasmonic acid (JA), and SA signal pathways in this response. Gene expression studies suggest a central role for both ethylene and JA pathways in the regulation of defense gene expression triggered by the pathogen or by plant cell wall-degrading enzymes (CF) secreted by the pathogen. Our results suggest that ethylene and JA act in concert in this regulation. In addition, CF triggers another, strictly JA-mediated response inhibited by ethylene and SA. SA does not appear to have a major role in activating defense gene expression in response to CF. However, SA may have a dual role in controlling CF-induced gene expression, by enhancing the expression of genes synergistically induced by ethylene and JA and repressing genes induced by JA alone.

scholarly journals Identification of a New Quorum-Sensing-Controlled Virulence Factor in Erwinia carotovora subsp. atroseptica Secreted via the Type II Targeting Pathway

2005 ◽  
Vol 18 (4) ◽  
pp. 334-342 ◽  
Author(s):  
Mark Corbett ◽  
Sam Virtue ◽  
Kenneth Bell ◽  
Paul Birch ◽  
Tom Burr ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Virulence Factors ◽  
Gel Electrophoresis ◽  
Xanthomonas Campestris ◽  
Signal Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Type Ii ◽  
Plant Assays

Two-dimensional polyacrylamide gel electrophoresis of the secreted proteins of Erwinia carotovora subsp. atroseptica revealed alow-abundance protein that was identified by mass spectrometry as a homologue of a Xanthomonas campestris avirulence protein with unknown function. The predicted Svx protein has an N-terminal signal sequence and zinc binding-region signature, and the mature protein is post-translationally modified. A 2D difference gel electrophoresis (DIGE) showed that the protein is secreted by the type II (out) secretion apparatus, which is also responsible for the secretion of the major known virulence factors, PelC and CelV. Transcription of the svx gene is under Nacyl- homoserine lactone-mediated quorum-sensing control. The svx gene was inactivated by transposon insertion. The mutant showed a decrease in virulence in potato plant assays, demonstrating a role for Svx in the pathogenicity of E. carotovora subsp. atroseptica. These results show that Svx is a previously unidentified virulence determinant which is secreted by the out machinery and is regulated by quorum sensing, two systems employed by several other virulence factors. Thus, the type II secretory machine is a conduit for virulence factors other than the main pectinnases and cellulase in E. carotovora subsp. atroseptica.

scholarly journals The Trk Potassium Transporter Is Required for RsmB-Mediated Activation of Virulence in the Phytopathogen Pectobacterium wasabiae

2015 ◽  
Vol 198 (2) ◽  
pp. 248-255 ◽  
Author(s):  
Rita S. Valente ◽  
Karina B. Xavier
Keyword(s):  
Cell Wall ◽  
Environmental Factor ◽  
Plant Cell ◽  
Plant Pathogen ◽  
Plant Cell Wall ◽  
Cell Wall Degrading Enzymes ◽  
Content Type ◽  
Regulatory Pathways ◽  
Potassium Transporter ◽  
Degrading Enzymes

ABSTRACTPectobacterium wasabiae(previously known asErwinia carotovora) is an important plant pathogen that regulates the production of plant cell wall-degrading enzymes through anN-acyl homoserine lactone-based quorum sensing system and through the GacS/GacA two-component system (also known as ExpS/ExpA). At high cell density, activation of GacS/GacA induces the expression of RsmB, a noncoding RNA that is essential for the activation of virulence in this bacterium. A genetic screen to identify regulators of RsmB revealed that mutants defective in components of a putative Trk potassium transporter (trkHandtrkA) had decreasedrsmBexpression. Further analysis of these mutants showed that changes in potassium concentration influencedrsmBexpression and consequent tissue damage in potato tubers and that this regulation required an intact Trk system. Regulation ofrsmBexpression by potassium via the Trk system occurred even in the absence of the GacS/GacA system, demonstrating that these systems act independently and are both required for full activation of RsmB and for the downstream induction of virulence in potato infection assays. Overall, our results identified potassium as an essential environmental factor regulating the Rsm system, and the consequent induction of virulence, in the plant pathogenP. wasabiae.IMPORTANCECrop losses from bacterial diseases caused by pectolytic bacteria are a major problem in agriculture. By studying the regulatory pathways involved in controlling the expression of plant cell wall-degrading enzymes inPectobacterium wasabiae, we showed that the Trk potassium transport system plays an important role in the regulation of these pathways. The data presented further identify potassium as an important environmental factor in the regulation of virulence in this plant pathogen. We showed that a reduction in virulence can be achieved by increasing the extracellular concentration of potassium. Therefore, this work highlights how elucidation of the mechanisms involved in regulating virulence can lead to the identification of environmental factors that can influence the outcome of infection.

scholarly journals Regulatory Network Controlling Extracellular Proteins in Erwinia carotovora subsp. carotovora: FlhDC, the Master Regulator of Flagellar Genes, Activates rsmB Regulatory RNA Production by Affecting gacA and hexA (lrhA) Expression

2008 ◽  
Vol 190 (13) ◽  
pp. 4610-4623 ◽  
Author(s):  
Yaya Cui ◽  
Asita Chatterjee ◽  
Hailian Yang ◽  
Arun K. Chatterjee
Keyword(s):  
Plant Cell Wall ◽  
Rna Binding ◽  
Pectate Lyase ◽  
Extracellular Enzyme ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Hypersensitive Reaction ◽  
Extracellular Proteins ◽  
Cell Wall Degrading Enzymes ◽  
Negative Effect

ABSTRACT Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC− mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (HarpinEcc) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC+ plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC− mutant are responsible for the inhibition of rsmB RNA production, a condition conducive to the accumulation of free RsmA. Indeed, studies with an RsmA− FlhDC− double mutant and multiple copies of rsmB + DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation of fliA has no bearing on exoprotein production in E. carotovora subsp. carotovora. Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA, and rsmB RNA in the context of the exoprotein production and virulence of E. carotovora subsp. carotovora.

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