Characterization ofavrPphE,a Gene for Cultivar-Specific Avirulence fromPseudomonas syringaepv.phaseolicolaWhich Is Physically Linked tohrpY,a NewhrpGene Identified in the Halo-Blight Bacterium

Mansfıeld, John W.; Jenner, Carol E.; Hockenhull, R; Bennett, Mark A.; Stewart, R B

doi:10.1094/mpmi-7-0726

Cited by 89 publications

(88 citation statements)

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“…As expected, the cytoplasmic DnaK protein was almost exclusively detected in the soluble fraction. Similar results were obtained with E. coli MC4100 harboring the complete P. syringae phaseolicola TTS regulon on the low copy number cosmid pPPY430 and the hrpL factor under lac control on a separate plasmid (23,38). MC4100/ pPPY430/pHrpL grown in the presence (Fig.…”

Section: Resultssupporting

confidence: 83%

“…Intracellular HrcN production is coincident with biosynthesis of the extracellular TTS pilus (Fig. 2E, arrows) (9) and with the acquisition of pathogenic potential (data not shown) (23). TTS pili were not present in noninduced cells (Fig.…”

Section: Resultsmentioning

confidence: 94%

“…Bacterial Strains and Plasmids-Escherichia coli strains and plasmids were manipulated as described (22,23). P. syringae phaseolicola NPS3121 was grown at 30°C in King's B medium (24) or hrp-derepressing fructose minimal medium (25) containing rifamycin (100 g/ml).…”

Section: Methodsmentioning

confidence: 99%

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Type III Protein Translocase

Pozidis

Chalkiadaki

Gomez-Serrano

et al. 2003

Journal of Biological Chemistry

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Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F 1 /V 1 -ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, crosslinking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, ϳ300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, ϳ3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 94%

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Type III Protein Translocase

Pozidis

Chalkiadaki

Gomez-Serrano

et al. 2003

Journal of Biological Chemistry

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“…The clone pPPY430 contains the hrp gene cluster from Psph race 6 except for part of hrpRS (33). Comparison of the region of PPY430 containing hrpZ (subclone pPPY438; GenBank accession no.…”

Section: Resultsmentioning

confidence: 99%

HrpZPsph from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore invitro

Lee

Klüsener²,

Tsiamis³

et al. 2000

Proceedings of the National Academy of Sciences

120

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The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZPsph), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZPsph was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZ Psph to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZPsphrelated proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZPsph. The HrpZPsphmediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl ؊ . Such pore-forming activity may allow nutrient release and͞or delivery of virulence factors during bacterial colonization of host plants.

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“…However, direct interaction of HrpL with Hrp box sequences and determination of essential nucleotides within Hrp box sequences by site-specific mutagenesis have not been reported. Furthermore, avr genes have been found with Hrp promoters containing variations in four positions in the canonical motif (19,21), and we have little knowledge of the extent of the Hrp regulon beyond the Hrp secretion system and a handful of effector genes. Thus, methods for defining functional Hrp boxes and identifying them globally in a P. syringae genome would advance the search for effector genes and other factors in a regulon that is central to plant pathogenicity.…”

Section: Dc3000 This Process Involved (I) Identification Of 9 New VImentioning

confidence: 99%

Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor

Fouts

Abramovitch

Alfano

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

276

241

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The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpLresponsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved (i) identification of 9 new virulenceimplicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpLdependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N -(indole-3-acetyl)-L-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL. P seudomonas syringae pv. tomato DC3000 has emerged as an important model organism in molecular plant pathology because of its genetic tractability, its pathogenicity on both tomato and Arabidopsis thaliana, and its ability to deliver virulence effector proteins into host cells via a type III protein secretion system. To support worldwide functional genomic investigations of P. s. tomato DC3000, we are determining the complete sequence of the DC3000 genome. A draft sequence is now available (http:͞͞www.tigr.org͞tdb͞mdb͞ mdbinprogress.html), and we have initiated a genomewide investigation of potential virulence factors with a search for virulencerelated genes in the same regulon as the type III secretion system.Virulence effector proteins delivered to or into host cells by type III secretion systems are key factors in the pathogenicity of many bacteria, including animal pathogens in the genera Salmonella, Yersinia, Shigella, and Escherichia, and plant pathogens in the genera Pseudomonas, Erwinia, Xanthomonas, Ralstonia, and Pantoea (1). In plant pathogens, the type III secretion machinery is referred to as the hypersensitive response and pathogenicity (Hrp) system ...

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Characterization ofavrPphE,a Gene for Cultivar-Specific Avirulence fromPseudomonas syringaepv.phaseolicolaWhich Is Physically Linked tohrpY,a NewhrpGene Identified in the Halo-Blight Bacterium

Cited by 89 publications

References 0 publications

Type III Protein Translocase

Type III Protein Translocase

HrpZPsph from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore invitro

Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor

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