Visualization of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in <i>Erwinia amylovora</i> and <i>Pseudomonas syringae</i>

Jin, Qingxu; Hu, Wenqi; Brown, Ian R.; McGhee, Gayle C.; Hart, Patrick; Jones, Alan L.; He, Sheng Yang

doi:10.1046/j.1365-2958.2001.02455.x

Cited by 97 publications

(82 citation statements)

References 57 publications

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“…Similar structures were also observed on Shiga toxin-producing Escherichia coli cells (11). In plant pathogens, longer appendages (hrp pili) have been reported for Pseudomonas syringae (35), Ralstonia solanacearum (39), and Erwinia amylovora (17).…”

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confidence: 71%

NopB, a Type III Secreted Protein of Rhizobium sp. Strain NGR234, Is Associated with Pilus-Like Surface Appendages

et al. 2005

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Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGR⍀rhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.Rhizobia are gram-negative soil inhabitants that induce the formation of highly specialized, nitrogen-fixing organs called nodules on the roots or stems of leguminous plants. Some rhizobial species provoke nodule formation on a limited number of legume genera and are said to have narrow host ranges, e.g., Rhizobium meliloti, which nodulates only three genera of legumes. Other, broad-host-range rhizobia provoke the formation of nodules on many different legumes; e.g., Rhizobium sp. strain NGR234 (hereafter called NGR234) nodulates more than 112 genera of legumes as well as the nonlegume Parasponia andersonii (32,38). The formation of root nodules is a result of an elaborate developmental program directed by signal exchange between the two partners. These signals include flavonoids, Nod factors, surface polysaccharides, and extracellular proteins (7,31,34). After flavonoid induction, NGR234 secretes a number of extracellular proteins called Nops (nodulation outer proteins) via a type III secretion system (TTSS) (24, 41). TTSSs are virulence determinants shared by many diverse gram-negative bacteria that cause disease in plants and animals. The TTSS machinery is highly conserved and encoded by cluster of hrp (hypersensitive response and pathogenesis) or hrc (hrp conserved) genes in phytopathogens (2, 16). Homologues of the hrc genes were found in NGR234 and renamed rhc (Rhizobium conserved) (13,41

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confidence: 71%

NopB, a Type III Secreted Protein of Rhizobium sp. Strain NGR234, Is Associated with Pilus-Like Surface Appendages

et al. 2005

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“…Three minutes later, excess sample was removed using a piece of filter paper. The grid was dried in a fume hood for 5 min, stained with 1% phosphotungstic acid (pH 6.5), air-dried again, and examined using a JEOL 100-CEX transmission electron microscope at an accelerating voltage of 100 kV (18). To investigate the effect of a mutant HrpA protein on the self-assembly of the wild-type HrpA protein, we mixed one part of wild-type HrpA protein with one part of the same concentration (ϳ2 mg/ml) or 10-, 25-, or 50-fold diluted mutant HrpA protein.…”

Section: Methodsmentioning

confidence: 99%

“…The EspA filament is 12 nm in diameter and can be several micrometers long (13,14). The TTSS of plant pathogenic bacteria assembles an extracellular appendage called the Hrp pilus, which is 6 -8 nm wide and several micrometers long (15)(16)(17)(18)(19)(20). The needle, EspA filament, and Hrp pilus are believed to be tunnels linking the type III "secreton" embedded in the bacterial cell wall and a type III "translocon" in the host plasma membrane.…”

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confidence: 99%

Use of Dominant-negative HrpA Mutants to Dissect Hrp Pilus Assembly and Type III Secretion in Pseudomonas syringae pv. tomato

Lee

Kolade²,

Nomura³

et al. 2005

Journal of Biological Chemistry

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The Hrp pilus plays an essential role in the long-distance type III translocation of effector proteins from bacteria into plant cells. HrpA is the structural subunit of the Hrp pilus in Pseudomonas syringae pv. tomato (Pst) DC3000. Little is known about the molecular features in the HrpA protein for pilus assembly or for transporting effector proteins. From previous collections of nonfunctional HrpA derivatives that carry random pentapeptide insertions or single amino acid mutations, we identified several dominant-negative mutants that blocked the ability of wild-type Pst DC3000 to elicit host responses. The dominant-negative phenotype was correlated with the disappearance of the Hrp pilus in culture and inhibition of wild-type HrpA protein self-assembly in vitro. Dominant-negative HrpA mutants can be grouped into two functional classes: one class exerted a strong dominant-negative effect on the secretion of effector proteins AvrPto and HopPtoM in culture, and the other did not. The two classes of mutant HrpA proteins carry pentapeptide insertions in discrete regions, which are interrupted by insertions without a dominant-negative effect. These results enable prediction of possible subunit-subunit interaction sites in the assembly of the Hrp pilus and suggest the usefulness of dominant-negative mutants in dissection of the role of the wild-type HrpA protein in various stages of type III translocation: protein exit across the bacterial cell wall, the assembly and/or stabilization of the Hrp pilus in the extracellular space, and Hrp pilus-mediated long-distance transport beyond the bacterial cell wall. The bacterial type III secretion system (TTSS)1 is a longdistance protein transport system, moving bacterial effector proteins from the bacterial cytoplasm into a eukaryotic cell (1-4). During this long-distance transport, several physical barriers must be traversed: the bacterial cell wall, the host extracellular matrix layer (e.g. plant cell wall or animal mucous layer/glycocalyx), and the host plasma membrane. The TTSS has adapted to such long-distance transport by assembling extracellular needle/pilus-like appendages of various lengths (3, 4). For example, the TTSS of mammalian pathogenic bacteria assembles a needle complex, which consists of a base structure embedded in the bacterial cell wall and a primarily extracellular hollow needle of 6 -8 nm in diameter and 50 -80 nm in length (5-10). In enteropathogenic Escherichia coli, the needle is connected with another extracellular filament called the EspA filament (11,12). The EspA filament is 12 nm in diameter and can be several micrometers long (13,14). The TTSS of plant pathogenic bacteria assembles an extracellular appendage called the Hrp pilus, which is 6 -8 nm wide and several micrometers long (15-20). The needle, EspA filament, and Hrp pilus are believed to be tunnels linking the type III "secreton" embedded in the bacterial cell wall and a type III "translocon" in the host plasma membrane. The secreton allows the exit of effector proteins across the bacte...

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“…Hrpdependent pili have been described in several plant-pathogenic or symbiotic bacteria that also contain a TTS system: Pseudomonas syringae pv. tomato, Ralstonia solanacearum, Erwinia amylovora, and Sinorhizobium fredii (28,32,46,55). The Hrp pilus elongates by the addition of Hrp pilin subunits at the distal end, and also TTS system substrates are secreted only from the pilus tip (27, 35).…”

mentioning

confidence: 99%

Domain Structure of HrpE, the Hrp Pilus Subunit of Xanthomonas campestris pv. vesicatoria

Weber

Koebnik

2005

J Bacteriol

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The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (TTS) system necessary for pathogenicity in susceptible hosts and induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. X. campestris pv. vesicatoria produces filamentous structures, Hrp pili, at the cell surface under hrp-inducing conditions. The Hrp pilus acts as a cell surface appendage of the TTS system and serves as a conduit for the transfer of bacterial effector proteins into the plant cell cytosol. The major pilus component, the HrpE pilin, is unique to xanthomonads and is encoded within the hrp gene cluster. In this study, functional domains of HrpE were mapped by linker-scanning mutagenesis and by reporter protein fusions to an N-terminally truncated avirulence protein (AvrBs3⌬2). Thirteen five-amino-acid peptide insertion mutants were obtained and could be grouped into six phenotypic classes. Three permissive mutations were mapped in the N-terminal half of HrpE, which is weakly conserved within the HrpE protein family. Four dominant-negative peptide insertions in the strongly conserved C-terminal region suggest that this domain is critical for oligomerization of the pilus subunits. Reporter protein fusions revealed that the N-terminal 17 amino acid residues act as an efficient TTS signal. From these results, we postulate a three-domain structure of HrpE with an N-terminal secretion signal, a surface-exposed variable region of the N-terminal half, and a C-terminal polymerization domain. Comparisons with a mutant study of HrpA, the Hrp pilin from Pseudomonas syringae pv. tomato DC3000, and hydrophobicity plot analyses of several nonhomologous Hrp pilins suggest a common architecture of Hrp pilins of different plant-pathogenic bacteria.Many gram-negative bacterial pathogens, including Xanthomonas campestris pv. vesicatoria, possess a unique protein transport system called the type III secretion (TTS) system which transfers so-called effector proteins directly into the host cell. In plant bacterial pathogens, genes encoding the TTS system are referred to as hypersensitive response and pathogenicity (hrp) genes because mutations in these genes abolish induction of the hypersensitive response in resistant host plants and pathogenicity in host plants (36).In X. campestris pv. vesicatoria, the hrp gene cluster is located in a 23-kb chromosomal region and is organized into six operons, designated hrpA to hrpF (6,17,18,24,25,48,58). Twenty-two genes are encoded within the hrp gene cluster, 11 of which are highly conserved among plant and animal pathogens and therefore have been renamed hrc (hrp conserved) genes (5, 56). Eight hrc gene products are associated with bacterial membranes and build up a transenvelope multiprotein complex (21). The remaining three proteins localize in the cytoplasm and are probably involved in energizing the transport process (20,44). The role of the non...

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Visualization of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae

Cited by 97 publications

References 57 publications

NopB, a Type III Secreted Protein of Rhizobium sp. Strain NGR234, Is Associated with Pilus-Like Surface Appendages

NopB, a Type III Secreted Protein of Rhizobium sp. Strain NGR234, Is Associated with Pilus-Like Surface Appendages

Use of Dominant-negative HrpA Mutants to Dissect Hrp Pilus Assembly and Type III Secretion in Pseudomonas syringae pv. tomato

Domain Structure of HrpE, the Hrp Pilus Subunit of Xanthomonas campestris pv. vesicatoria

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