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. 2002 Apr 15;21(8):1909-15.
doi: 10.1093/emboj/21.8.1909.

The Hrp pilus of Pseudomonas syringae elongates from its tip and acts as a conduit for translocation of the effector protein HrpZ

Chun-Mei Li  1 , Ian Brown, John Mansfield, Conrad Stevens, Tristan Boureau, Martin Romantschuk, Suvi Taira
Affiliations

The Hrp pilus of Pseudomonas syringae elongates from its tip and acts as a conduit for translocation of the effector protein HrpZ

Chun-Mei Li et al. EMBO J. .

Abstract

The type III secretion system (TTSS) is an essential requirement for the virulence of many Gram-negative bacteria infecting plants, animals and man. Pathogens use the TTSS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cell, where the effectors subvert host defences. Plant pathogens have to translocate their effector proteins through the plant cell wall barrier. The best candidates for directing effector protein traffic are bacterial appendages attached to the membrane-bound components of the TTSS. We have investigated the protein secretion route in relation to the TTSS appendage, termed the Hrp pilus, of the plant pathogen Pseudomonas syringae pv. tomato. By pulse expression of proteins combined with immunoelectron microscopy, we show that the Hrp pilus elongates by the addition of HrpA pilin subunits at the distal end, and that the effector protein HrpZ is secreted only from the pilus tip. Our results indicate that both HrpA and HrpZ travel through the Hrp pilus, which functions as a conduit for the long-distance translocation of effector proteins.

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Figures

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Fig. 1. Immunogold localization of the FLAG epitope (20 nm gold particles) in a Hrp pilus composed entirely of FLAG-HrpA. The FLAG tag was inserted after amino acid 23 of HrpA and cloned into pDN18. The hrpA mutant of DC3000 harbouring the FLAG-HrpA-encoding pDN18 derivative was used to examine pilus production after 18 h incubation in Hrp-inducing minimal medium. Bar = 0.5 µm.
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Fig. 2. Immunoblot showing production and secretion of FLAG-HrpA by DC3000 harbouring pMerFLAGHrpA with anti-HrpA antiserum. Bacteria were grown for 18 h before extraction of proteins, which were separated by 16% SDS–PAGE and probed with anti-HrpA antibodies. Lanes 1 and 2 are cellular fractions; lanes 3–6 are extracellular fractions. Lanes 1 and 3, King’s B medium; lanes 2 and 4, King’s B with HgCl2; lane 5, Hrp-inducing medium; lane 6, Hrp-inducing medium with HgCl2.
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Fig. 3. Pili incorporating induced FLAG-HrpA are decorated by immunogold labelling (20 nm gold particles) of the FLAG epitope at the distal end of the appendage. Bacteria (DC3000 pMerFLAGHrpA) were grown for 8 h in minimal medium to allow pilus formation and then examined after the addition of HgCl2 to induce FLAG-HrpA. (A) Pilus before addition of HgCl2; note the absence of immunogold label; (B–D) 15, 30 and 60 min, respectively, after FLAG-HrpA induction. Bars: (A) and (B) = 0.25 µm; (C) and (D) = 0.5 µm.
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Fig. 4. Quantitative analysis of the development of the Hrp pilus. The length of the unlabelled proximal (open column) and immunogold- labelled distal (shaded column) portion of the Hrp pilus incorporating the FLAG-HrpA subunit was recorded from at least 30 pili at each time point after induction of FLAG-HrpA (means ± SEM are given). Immunogold labelling was achieved using anti-FLAG monoclonal antibodies.
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Fig. 5. Immunogold localization of HrpZ in DC3000ΔhrpZ harbouring pMerHrpZ, which allows mercury-driven HrpZ expression. (A and B) Emergence of HrpZ (immunogold label) at the tip of the pilus 30 and 60 min, respectively, after the addition of HgCl2 to induce HrpZ following 8 h growth in minimal medium to allow pilus formation. (C) Labelling along the proximal base of the pilus if bacteria were grown continuously in minimal medium + HgCl2 for 8 h. Bars = 0.5 µm.
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