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The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence.

ABSTRACT: Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family's response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction.

SUBMITTER: Ficarra FA

PROVIDER: S-EPMC4427454 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence.

Ficarra Florencia A FA Garofalo Cecilia G CG Gottig Natalia N Ottado Jorgelina J

PloS one 20150511 5

Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family's response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for ...[more]

PMID: 25961560

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Project description:Citrus canker is a major disease affecting citrus production in Brazil. It's mainly caused by Xanthomonas citri subsp. citri strain 306 pathotype A (Xac). We analysed the differential expression of proteins secreted by wild type Xac and an asymptomatic mutant for hrpB4 (ΔhrpB4) grown in Nutrient Broth (NB) and a medium mimicking growth conditions in the plant (XAM1). This allowed the identification of 55 secreted proteins, of which 37 were secreted by both strains when cultured in XAM1. In this secreted protein repertoire, the following stand out: Virk, Polyphosphate-selective porin, Cellulase, Endoglucanase, Histone-like protein, Ribosomal proteins, five hypothetical proteins expressed only in the wild type strain, Lytic murein transglycosylase, Lipoprotein, Leucyl-tRNA synthetase, Co-chaperonin, Toluene tolerance, C-type cytochrome biogenesis membrane protein, Aminopeptidase and two hypothetical proteins expressed only in the ΔhrpB4 mutant. Furthermore, Peptidoglycan-associated outer membrane protein, Regulator of pathogenicity factor, Outer membrane proteins, Endopolygalacturonase, Chorismate mutase, Peptidyl-prolyl cis-trans isomerase and seven hypothetical proteins were detected in both strains, suggesting that there was no relationship with the secretion mediated by the type III secretory system, which is not functional in the mutant strain. Also worth mentioning is the Elongation factor Tu (EF-Tu), expressed only the wild type strain, and Type IV pilus assembly protein, Flagellin (FliC) and Flagellar hook-associated protein, identified in the wild-type strain secretome when grown only in NB. Noteworthy, that FliC, EF-Tu are classically characterized as PAMPs (Pathogen-associated molecular patterns), responsible for a PAMP-triggered immunity response. Therefore, our results highlight proteins potentially involved with the virulence. Overall, we conclude that the use of secretome data is a valuable approach that may bring more knowledge of the biology of this important plant pathogen, which ultimately can lead to the establishment of new strategies to combat citrus canker.

Project description:Citrus canker caused by Xanthomonas citri subsp. citri is one of the most important bacterial diseases of citrus, impacting both plant growth and fruit quality. Identifying and elucidating the roles of genes associated with pathogenesis has aided our understanding of the molecular basis of citrus-bacteria interactions. However, the complex virulence mechanisms of X. citri subsp. citri are still not well understood. In this study, we characterized the role of PhoP in X. citri subsp. citri using a phoP deletion mutant, ΔphoP. Compared with wild-type strain XHG3, ΔphoP showed reduced motility, biofilm formation, as well as decreased production of cellulase, amylase, and polygalacturonase. In addition, the virulence of ΔphoP on citrus leaves was significantly decreased. To further understand the virulence mechanisms of X. citri subsp. citri, high-throughput RNA sequencing technology (RNA-Seq) was used to compare the transcriptomes of the wild-type and mutant strains. Analysis revealed 1017 differentially-expressed genes (DEGs), of which 614 were up-regulated and 403 were down-regulated in ΔphoP. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that the DEGs were enriched in flagellar assembly, two-component systems, histidine metabolism, bacterial chemotaxis, ABC transporters, and bacterial secretion systems. Our results showed that PhoP activates the expression of a large set of virulence genes, including 22 type III secretion system genes and 15 type III secretion system effector genes, as well as several genes involved in chemotaxis, and flagellar and histidine biosynthesis. Two-step reverse-transcription polymerase chain reaction analysis targeting 17 genes was used to validate the RNA-seq data, and confirmed that the expression of all 17 genes, except for that of virB1, decreased significantly. Our results suggest that PhoP interacts with a global signaling network to co-ordinate the expression of multiple virulence factors involved in modification and adaption to the host environment during infection.

Project description:BackgroundThe Gram-negative bacterium Xanthomonas citri subsp. citri (Xac) causes citrus canker, one of the most destructive diseases of citrus worldwide. In our previous work, a transposon mutant of Xac strain 306 with an insertion in the XAC3110 locus was isolated in a screening that aimed at identifying genes related to biofilm formation. The XAC3110 locus was named as bdp24 for biofilm-defective phenotype and the mutant was observed to be affected in extracellular polysaccharide (EPS) and lipopolysaccharide (LPS) biosynthesis and cell motility. In this study, we further characterized the bdp24 (XAC3110) gene (designated as gpsX) using genetic complementation assays and expanded the knowledge about the function of the gpsX gene in Xac pathogenesis by investigating the roles of gpsX in EPS and LPS production, cell motility, biofilm formation on host leaves, stress tolerance, growth in planta, and host virulence of the citrus canker bacterium.ResultsThe gpsX gene encodes a putative glycosyltransferase, which is highly conserved in the sequenced strains of Xanthomonas. Mutation of gpsX resulted in a significant reduction of the amount of EPS and loss of two LPS bands visualized on sodium dodecylsulphate- polyacrylamide gels. Biofilm assays revealed that the gpsX mutation affected biofilm formation by Xac on abiotic and biotic surfaces. The gpsX mutant showed delayed bacterial growth and caused reduced development of disease symptoms in susceptible citrus leaves. The gpsX mutant was more sensitive than the wild-type strain to various stresses, including the H(2)O(2) oxidative stress. The mutant also showed attenuated ability in cell motility but not in flagellar formation. Quantitative reverse transcription-PCR assays indicated that mutation of gpsX did not affect the expression of virulence genes such as pthA in Xac strain 306. The affected phenotypes of the gpsX mutant could be complemented to wild-type levels by the intact gpsX gene.ConclusionsTaken together, our data confirm that the gpsX gene is involved in EPS and LPS synthesis and biofilm formation in Xac and suggest that the gpsX gene contributes to the adaptation of Xac to the host microenvironments at early stage of infection and thus is required for full virulence on host plants.

Project description:Xanthomonas citri subsp. citri 306 (XccA) is the causal agent of type A citrus canker (CC), one of the most significant citriculture diseases. Murein lytic transglycosylases (LT), potentially involved in XccA pathogenicity, are enzymes responsible for peptidoglycan structure assembly, remodeling and degradation. They directly impact cell wall expansion during bacterial growth, septum division allowing cell separation, cell wall remodeling allowing flagellar assembly, bacterial conjugation, muropeptide recycling, and secretion system assembly, in particular the Type 3 Secretion System involved in bacterial virulence, which play a fundamental role in XccA pathogenicity. Information about the XccA LT arsenal is patchy: little is known about family diversity, their exact role or their connection to virulence in this bacterium. Among the LTs with possible involvement in virulence, two paralogue open reading frames (ORFs) (one on the chromosome and one in plasmid pXAC64) are passenger genes of the Tn3 family transposon TnXax1, known to play a significant role in the evolution and emergence of pathogenicity in Xanthomonadales and to carry a variety of virulence determinants. This study addresses LT diversity in the XccA genome and examines the role of plasmid and chromosomal TnXax1 LT passenger genes using site-directed deletion mutagenesis and functional characterization. We identified 13 XccA LTs: 12 belong to families 1A, 1B, 1C, 1D (two copies), 1F, 1G, 3A, 3B (two copies), 5A, 6A and one which is non-categorized. The non-categorized LT is exclusive to the Xanthomonas genus and related to the 3B family but contains an additional domain linked to carbohydrate metabolism. The categorized LTs are probably involved in cell wall remodeling to allow insertion of type 3, 4 and 6 secretion systems, flagellum assembly, division and recycling of cell wall and degradation and control of peptidoglycan production. The TnXax1 passenger LT genes (3B family) are not essential to XccA or for CC development but are implicated in peptidoglycan metabolism, directly impacting bacterial fitness and CC symptom enhancement in susceptible hosts (e.g., Citrus sinensis). This underlines the role of TnXax1 as a virulence and pathogenicity-propagating agent in XccA and suggests that LT acquisition by horizontal gene transfer mediated by TnXax1 may improve bacterial fitness, conferring adaptive advantages to the plant-pathogen interaction process.

Project description:BackgroundCitrus canker disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc) has become endemic in areas where high temperature, rain, humidity, and windy conditions provide a favourable environment for the dissemination of the bacterium. Xcc is pathogenic on many commercial citrus varieties but appears to elicit an incompatible reaction on the citrus relative Fortunella margarita Swing (kumquat), in the form of a very distinct delayed necrotic response. We have developed subtractive libraries enriched in sequences expressed in kumquat leaves during both early and late stages of the disease. The isolated differentially expressed transcripts were subsequently sequenced. Our results demonstrate how the use of microarray expression profiling can help assign roles to previously uncharacterized genes and elucidate plant pathogenesis-response related mechanisms. This can be considered to be a case study in a citrus relative where high throughput technologies were utilized to understand defence mechanisms in Fortunella and citrus at the molecular level.ResultscDNAs from sequenced kumquat libraries (ESTs) made from subtracted RNA populations, healthy vs. infected, were used to make this microarray. Of 2054 selected genes on a customized array, 317 were differentially expressed (P < 0.05) in Xcc challenged kumquat plants compared to mock-inoculated ones. This study identified components of the incompatible interaction such as reactive oxygen species (ROS) and programmed cell death (PCD). Common defence mechanisms and a number of resistance genes were also identified. In addition, there were a considerable number of differentially regulated genes that had no homologues in the databases. This could be an indication of either a specialized set of genes employed by kumquat in response to canker disease or new defence mechanisms in citrus.ConclusionFunctional categorization of kumquat Xcc-responsive genes revealed an enhanced defence-related metabolism as well as a number of resistant response-specific genes in the kumquat transcriptome in response to Xcc inoculation. Gene expression profile(s) were analyzed to assemble a comprehensive and inclusive image of the molecular interaction in the kumquat/Xcc system. This was done in order to elucidate molecular mechanisms associated with the development of the hypersensitive response phenotype in kumquat leaves. These data will be used to perform comparisons among citrus species to evaluate means to enhance the host immune responses against bacterial diseases.

Dataset Information

The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence.

Publications

The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence.

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