The Contribution of the Predicted Sorting Platform Component HrcQ to Type III Secretion in Xanthomonas campestris pv. vesicatoria Depends on an Internal Translation Start Site

Pathogenicity of the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. T3S systems are conserved in plant- and animal-pathogenic bacteria and consist of at least nine structural core components, which are designated Sct (secretion and cellular translocation) in animal-pathogenic bacteria. Sct proteins are involved in the assembly of the membrane-spanning secretion apparatus which is associated with an extracellular needle structure and a cytoplasmic sorting platform. Components of the sorting platform include the ATPase SctN, its regulator SctL, and pod-like structures at the periphery of the sorting platform consisting of SctQ proteins. Members of the SctQ family form a complex with the C-terminal protein domain, SctQC, which is translated as separate protein and likely acts either as a structural component of the sorting platform or as a chaperone for SctQ. The sorting platform has been intensively studied in animal-pathogenic bacteria but has not yet been visualized in plant pathogens. We previously showed that the SctQ homolog HrcQ from X. campestris pv. vesicatoria assembles into complexes which associate with the T3S system and interact with components of the ATPase complex. Here, we report the presence of an internal alternative translation start site in hrcQ leading to the separate synthesis of the C-terminal protein region (HrcQC). The analysis of genomic hrcQ mutants showed that HrcQC is essential for pathogenicity and T3S. Increased expression levels of hrcQ or the T3S genes, however, compensated the lack of HrcQC. Interaction studies and protein analyses suggest that HrcQC forms a complex with HrcQ and promotes HrcQ stability. Furthermore, HrcQC colocalizes with HrcQ as was shown by fluorescence microscopy, suggesting that it is part of the predicted cytoplasmic sorting platform. In agreement with this finding, HrcQC interacts with the inner membrane ring protein HrcD and the SctK-like linker protein HrpB4 which contributes to the docking of the HrcQ complex to the membrane-spanning T3S apparatus. Taken together, our data suggest that HrcQC acts as a chaperone for HrcQ and as a structural component of the predicted sorting platform.

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XopC and XopJ, Two Novel Type III Effector Proteinsfrom Xanthomonas campestris pv.vesicatoria

Journal of Bacteriology ◽

10.1128/jb.185.24.7092-7102.2003 ◽

2003 ◽

Vol 185 (24) ◽

pp. 7092-7102 ◽

Cited By ~ 65

Author(s):

Laurent Noël ◽

Frank Thieme ◽

Jana Gäbler ◽

Daniela Büttner ◽

Ulla Bonas

Keyword(s):

Pseudomonas Syringae ◽

Plant Cell ◽

Type Iii Secretion ◽

Xanthomonas Campestris ◽

Pathogenic Bacteria ◽

Effector Proteins ◽

Type Iii ◽

Genome Wide ◽

A Genome ◽

Xanthomonas Campestris Pv.Vesicatoria

ABSTRACT Pathogenicity of the gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria depends on a type III secretion (TTS) system which translocates bacterial effector proteins into the plant cell. Previous transcriptome analysis identified a genome-wide regulon of putative virulence genes that are coexpressed with the TTS system. In this study, we characterized two of these genes, xopC and xopJ. Both genes encode Xanthomonas outer proteins (Xops) that were shown to be secreted by the TTS system. In addition, type III-dependent translocation of both proteins into the plant cell was demonstrated using the AvrBs3 effector domain as a reporter. XopJ belongs to the AvrRxv/YopJ family of effector proteins from plant and animal pathogenic bacteria. By contrast, XopC does not share significant homology to proteins in the database. Sequence analysis revealed that the xopC locus contains several features that are reminiscent of pathogenicity islands. Interestingly, the xopC region is flanked by 62-bp inverted repeats that are also associated with members of the Xanthomonas avrBs3 effector family. Besides xopC, a second gene of the locus, designated hpaJ, was shown to be coexpressed with the TTS system. hpaJ encodes a protein with similarity to transglycosylases and to the Pseudomonas syringae pv. maculicola protein HopPmaG. HpaJ secretion and translocation by the X. campestris pv. vesicatoria TTS system was not detectable, which is consistent with its predicted Sec signal and a putative function as transglycosylase in the bacterial periplasm.

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The Type III Secretion System of Xanthomonas fuscans subsp. fuscans Is Involved in the Phyllosphere Colonization Process and in Transmission to Seeds of Susceptible Beans

Applied and Environmental Microbiology ◽

10.1128/aem.02906-07 ◽

2008 ◽

Vol 74 (9) ◽

pp. 2669-2678 ◽

Cited By ~ 42

Author(s):

A. Darsonval ◽

A. Darrasse ◽

D. Meyer ◽

M. Demarty ◽

K. Durand ◽

...

Keyword(s):

Type Iii Secretion ◽

Xanthomonas Campestris ◽

Pathogenic Bacteria ◽

Type Iii Secretion System ◽

Secretion System ◽

Regulatory Genes ◽

Population Densities ◽

Host Colonization ◽

Plant Pathogenic Bacteria ◽

Type Iii

ABSTRACT Understanding the survival, multiplication, and transmission to seeds of plant pathogenic bacteria is central to study their pathogenesis. We hypothesized that the type III secretion system (T3SS), encoded by hrp genes, could have a role in host colonization by plant pathogenic bacteria. The seed-borne pathogen Xanthomonas fuscans subsp. fuscans causes common bacterial blight of bean (Phaseolus vulgaris). Directed mutagenesis in strain CFBP4834-R of X. fuscans subsp. fuscans and bacterial population density monitoring on bean leaves showed that strains with mutations in the hrp regulatory genes, hrpG and hrpX, were impaired in their phyllospheric growth, as in the null interaction with Escherichia coli C600 and bean. In the compatible interaction, CFBP4834-R reached high phyllospheric population densities and was transmitted to seeds at high frequencies with high densities. Strains with mutations in structural hrp genes maintained the same constant epiphytic population densities (1 × 105 CFU g−1 of fresh weight) as in the incompatible interaction with Xanthomonas campestris pv. campestris ATCC 33913 and the bean. Low frequencies of transmission to seeds and low bacterial concentrations were recorded for CFBP4834-R hrp mutants and for ATCC 33913, whereas E. coli C600 was not transmitted. Moreover, unlike the wild-type strain, strains with mutations in hrp genes were not transmitted to seeds by vascular pathway. Transmission to seeds by floral structures remained possible for both. This study revealed the involvement of the X. fuscans subsp. fuscans T3SS in phyllospheric multiplication and systemic colonization of bean, leading to transmission to seeds. Our findings suggest a major contribution of hrp regulatory genes in host colonization processes.

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The YscU/FlhB homologue HrcU from Xanthomonas controls type III secretion and translocation of early and late substrates

Microbiology ◽

10.1099/mic.0.075176-0 ◽

2014 ◽

Vol 160 (3) ◽

pp. 576-588 ◽

Cited By ~ 7

Author(s):

Jens Hausner ◽

Daniela Büttner

Keyword(s):

Substrate Specificity ◽

Type Iii Secretion ◽

Xanthomonas Campestris ◽

Pathogenic Bacteria ◽

Effector Proteins ◽

Effector Protein ◽

Amino Acid Residues ◽

Type Iii ◽

Cytoplasmic Region ◽

Essential Components

The majority of Gram-negative plant- and animal-pathogenic bacteria employ a type III secretion (T3S) system to deliver effector proteins to eukaryotic cells. Members of the YscU protein family are essential components of the T3S system and consist of a transmembrane and a cytoplasmic region that is autocatalytically cleaved at a conserved NPTH motif. YscU homologues interact with T3S substrate specificity switch (T3S4) proteins that alter the substrate specificity of the T3S system after assembly of the secretion apparatus. We previously showed that the YscU homologue HrcU from the plant pathogen Xanthomonas campestris pv. vesicatoria interacts with the T3S4 protein HpaC and is required for the secretion of translocon and effector proteins. In the present study, analysis of HrcU deletion, insertion and point mutant derivatives led to the identification of amino acid residues in the cytoplasmic region of HrcU (HrcUC) that control T3S and translocation of the predicted inner rod protein HrpB2, the translocon protein HrpF and the effector protein AvrBs3. Mutations in the vicinity of the NPTH motif interfered with HrcU cleavage and/or the interaction of HrcUC with HrpB2 and the T3S4 protein HpaC. However, HrcU function was not completely abolished, suggesting that HrcU cleavage is not crucial for pathogenicity and T3S. Given that mutations in HrcU differentially affected T3S and translocation of HrpB2 and effector proteins, we propose that HrcU controls the secretion of different T3S substrate classes by independent mechanisms.

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Membrane topology of conserved components of the type III secretion system from the plant pathogen Xanthomonas campestris pv. vesicatoria

Microbiology ◽

10.1099/mic.0.039248-0 ◽

2010 ◽

Vol 156 (7) ◽

pp. 1963-1974 ◽

Cited By ~ 21

Author(s):

Carolin Berger ◽

Guillaume P. Robin ◽

Ulla Bonas ◽

Ralf Koebnik

Keyword(s):

Plant Pathogen ◽

Type Iii Secretion ◽

Xanthomonas Campestris ◽

Pathogenic Bacteria ◽

Membrane Topology ◽

Host Cells ◽

Reporter Protein ◽

Inner Membrane ◽

Bacterial Membranes ◽

Type Iii

Type III secretion (T3S) systems play key roles in the assembly of flagella and the translocation of bacterial effector proteins into eukaryotic host cells. Eleven proteins which are conserved among Gram-negative plant and animal pathogenic bacteria have been proposed to build up the basal structure of the T3S system, which spans both inner and outer bacterial membranes. We studied six conserved proteins, termed Hrc, predicted to reside in the inner membrane of the plant pathogen Xanthomonas campestris pv. vesicatoria. The membrane topology of HrcD, HrcR, HrcS, HrcT, HrcU and HrcV was studied by translational fusions to a dual alkaline phosphatase–β-galactosidase reporter protein. Two proteins, HrcU and HrcV, were found to have the same membrane topology as the Yersinia homologues YscU and YscV. For HrcR, the membrane topology differed from the model for the homologue from Yersinia, YscR. For our data on three other protein families, exemplified by HrcD, HrcS and HrcT, we derived the first topology models. Our results provide what is believed to be the first complete model of the inner membrane topology of any bacterial T3S system and will aid in elucidating the architecture of T3S systems by ultrastructural analysis.

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Functional Characterization of the Type III Secretion ATPase HrcN from the Plant Pathogen Xanthomonas campestris pv. vesicatoria

Journal of Bacteriology ◽

10.1128/jb.01446-08 ◽

2008 ◽

Vol 191 (5) ◽

pp. 1414-1428 ◽

Cited By ~ 47

Author(s):

Christian Lorenz ◽

Daniela Büttner

Keyword(s):

Type Iii Secretion ◽

Xanthomonas Campestris ◽

Pathogenic Bacteria ◽

Functional Characterization ◽

Effector Proteins ◽

Host Cells ◽

Dependent Manner ◽

Phosphate Binding ◽

Type Iii

ABSTRACT Many gram-negative plant and animal pathogenic bacteria employ a type III secretion (T3S) system to inject effector proteins into the cytosol of eukaryotic host cells. The membrane-spanning T3S apparatus is associated with an ATPase that presumably provides the energy for the secretion process. Here, we describe the role of the predicted ATPase HrcN from the plant pathogenic bacterium Xanthomonas campestris pathovar vesicatoria. We show that HrcN hydrolyzes ATP in vitro and is essential for T3S and bacterial pathogenicity. Stability of HrcN in X. campestris pv. vesicatoria depends on the conserved HrcL protein, which interacts with HrcN in vitro and in vivo. Both HrcN and HrcL bind to the inner membrane protein HrcU and specifically localize to the bacterial membranes under T3S-permissive conditions. Protein-protein interaction studies revealed that HrcN also interacts with the T3S substrate specificity switch protein HpaC and the global T3S chaperone HpaB, which promotes secretion of multiple effector proteins. Using an in vitro chaperone release assay, we demonstrate that HrcN dissociates a complex between HpaB and the effector protein XopF1 in an ATP-dependent manner, suggesting that HrcN is involved in the release of HpaB-bound effectors. Effector release depends on a conserved glycine residue in the HrcN phosphate-binding loop, which is crucial for enzymatic activity and protein function during T3S. There is no experimental evidence that T3S can occur in the absence of the ATPase, in contrast to recent findings reported for animal pathogenic bacteria.

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