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. 1997 Apr 1;94(7):3459-64.
doi: 10.1073/pnas.94.7.3459.

Hrp pilus: an hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv. tomato DC3000

Affiliations

Hrp pilus: an hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv. tomato DC3000

E Roine et al. Proc Natl Acad Sci U S A. .

Abstract

Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins share significant similarities with components of the type III secretion apparatus and flagellar assembly apparatus in animal pathogenic bacteria. Here we report that Pseudomonas syringae pv. tomato strain DC3000 (race 0) produces a filamentous surface appendage (Hrp pilus) of 6-8 nm in diameter in a solid minimal medium that induces hrp genes. Formation of the Hrp pilus is dependent on at least two hrp genes, hrpS and hrpH (recently renamed hrcC), which are involved in gene regulation and protein secretion, respectively. Our finding of the Hrp pilus, together with recent reports of Salmonella typhimurium surface appendages that are involved in bacterial invasion into the animal cell and of the Agrobacterium tumefaciens virB-dependent pilus that is involved in the transfer of T-DNA into plant cells, suggests that surface appendage formation is a common feature of animal and plant pathogenic bacteria in the infection of eukaryotic cells. Furthermore, we have identified HrpA as a major structural protein of the Hrp pilus. Finally, we show that a nonpolar hrpA mutant of P. syringae pv. tomato DC3000 is unable to form the Hrp pilus or to cause either an HR or disease in plants.

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Figures

Figure 1
Figure 1
EXPs produced by P. syringae pv. tomato DC3000. Lane 1, EXPs from an hrp-induced culture; lane 2, EXPs from a King’s medium B culture. The gel was stained with 0.025% Coomassie blue R-250. Proteins are named according to their molecular masses (in kDa).
Figure 2
Figure 2
Detection of the Hrp pilus on the surface of P. syringae pv. tomato DC3000. (A) DC3000 grown on King’s medium B agar plates, and (B) DC3000, (C) hrcC, (D) hrpS, (E) pilA, and (F) pilD mutants grown on solid hrp-inducing medium were examined with a transmission electron microscope after staining with 1% potassium phosphotungstic acid (pH 6.5). One to three polar flagella of 15–18 nm in diameter are present on most rod-shaped bacteria (surrounded by dark shadows) in samples (AF); in B, E, and F, many Hrp pili of 6–8 nm in diameter are also present (indicated by arrows). (Scale bars = 200 nm.) pilA and pilD mutants were kindly provided by D. Nunn (University of Illinois).
Figure 3
Figure 3
SDS/PAGE analysis of bacterial surface proteins. An SDS/15% PAGE gel loaded with protein samples prepared from the surface of DC3000 (lane 1), hrcC mutant (lane 2), and hrpS mutant (lane 3) grown on solid hrp-inducing medium, or DC3000 grown on King’s medium B agar plates (lane 4). The gel was stained with 0.025% Coomassie brilliant blue R-250. Lane M, molecular mass markers (Bio-Rad) in kDa. Arrowhead indicates the 10-kDa protein.
Figure 4
Figure 4
Purification of P. syringae pv. tomato DC3000 extracellular appendages (flagella and Hrp pili). (A) Electron micrograph of the pellet fraction containing flagella and Hrp pili (indicated by an arrow). (B) Electron micrograph of a fraction from the middle of the gradient containing only flagella. (C) An SDS/15% PAGE gel of proteins from the pellet fraction (lane 1) or a fraction from the middle of the gradient containing only flagella (lane 2). The gel was stained with 0.025% Coomassie brilliant blue R-250. Lane M, molecular mass markers (Bio-Rad) in kDa.
Figure 5
Figure 5
Surface appendages and associated proteins on P. syringae pv. tomato DC3000 hrpA mutant and hrpA mutant containing pHRPA. (A) hrpA mutant and (B) hrpA mutant containing pHRPA grown on solid hrp-inducing medium were examined with a transmission electron microscope after staining with 1% potassium phosphotungstic acid (pH 6.5). Polar flagella of 15–18 nm in diameter are present on most cells of the hrpA mutant and the hrpA mutant containing pHRPA. Flagella were not seen in the field shown in B. In B, many Hrp pili of 6–8 nm in diameter are present (indicated by arrow). (Scale bars = 200 nm.) (C) An SDS/15% PAGE gel loaded with protein samples prepared from surface of DC3000 (lane 1), hrpA mutant (lane 2), and hrpA mutant containing pHRPA (lane 3) grown on solid hrp-inducing medium. Lane M, molecular weight markers (Bio-Rad) in kDa. The gel was stained with 0.025% Coomassie brilliant blue R-250.

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