The type III-dependent Hrp pilus is required for productive interaction of Xanthomonas campestris pv. vesicatoria with pepper host plants

Abstract

The plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria expresses a type III secretion system that is necessary for both pathogenicity in susceptible hosts and the 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. Here we show that X. campestris pv. vesicatoria produces filamentous structures, the Hrp pili, at the cell surface under hrp-inducing conditions. Analysis of purified Hrp pili and immunoelectron microscopy revealed that the major component of the Hrp pilus is the HrpE protein which is encoded in the hrp gene cluster. Sequence homologues of hrpE are only found in other xanthomonads. However, hrpE is syntenic to the hrpY gene from another plant pathogen, Ralstonia solanacearum. Bioinformatic analyses suggest that all major Hrp pilus subunits from gram-negative plant pathogens may share the same structural organization, i.e., a predominant alpha-helical structure. Analysis of nonpolar mutants in hrpE demonstrated that the Hrp pilus is essential for the productive interaction of X. campestris pv. vesicatoria with pepper host plants. Furthermore, a functional Hrp pilus is required for type III-dependent protein secretion. Immunoelectron microscopy revealed that type III-secreted proteins, such as HrpF and AvrBs3, are in close contact with the Hrp pilus during and/or after their secretion. By systematic analysis of nonpolar hrp/hrc (hrp conserved) and hpa (hrp associated) mutants, we found that Hpa proteins as well as the translocon protein HrpF are dispensable for pilus assembly, while all other Hrp and Hrc proteins are required. Hence, there are no other conserved Hrp or Hrc proteins that act downstream of HrpE during type III-dependent protein translocation.

FIG. 1.

Genetic organization of the X. campestris pv. vesicatoria hrp gene cluster. The solid lines at the top indicate the six hrp transcription units, A to F; the thick arrows indicate different genes. Conserved hrc genes are shown as black arrows, hrp genes are shown as grey arrows, and hpa genes are shown as open arrows.

FIG. 2.

Detection of Hrp pili at the surface of X. campestris pv. vesicatoria. Bacteria were incubated on coated gold grids for 6 h at 30°C. Transmission electron micrographs of negatively stained specimens are shown. Surface appendages with a diameter of 8 to 10 nm were observed only under hrp-inducing conditions: (A) growth of strain 85E in XVM2 medium; (C) presence of the HrpG* protein in strain 85E*. Neither the hrpG wild-type strain 85E grown in NYG medium (B) nor a TTS mutant (85E* ΔhrcU) (D) produced pili. Arrows indicate Hrp pili. Bars, 200 nm.

FIG. 3.

Purification of Hrp pili. Pili were purified from X. campestris pv. vesicatoria strain 85E grown in XVM2 medium by deoxycholate solubilization and sucrose density gradient centrifugation. Proteins present in the pilus preparations were analyzed by Tricine SDS-PAGE. Lane 1, molecular mass marker; lane 2, sample of Hrp pilus preparation. Arrows point to protein bands for which sequence information has been obtained.

FIG. 4.

Multiple alignment and consensus secondary structure prediction of xanthomonad HrpE proteins. Five HrpE sequences from X. campestris pv. vesicatoria (Xcv), X. oryzae pv. oryzae (Xoo), X. axonopodis pv. glycines (Xag), X. axonopodis pv. citri (Xac), and X. campestris pv. campestris (Xcc) were aligned by using the CLUSTAL X program (57) and subjected to secondary structure prediction (2D). Identical residues are shown in red, and similar residues are shown in blue. Predicted α-helical regions are indicated by a lowercase “h.”

FIG. 5.

Immunogold labeling of X. campestris pv. vesicatoria Hrp pili with anti-HrpE (A), anti-AvrBs3 (B), or anti-HrpF (C) antisera. Bacteria were incubated on EM grids for 6 h in minimal medium, followed by in situ immunogold labeling. The following strains were used for labeling of Hrp pili: 85E* (A and C) and 85* carrying the avrBs3-containing plasmid pDS300F (B). Filled arrows indicate labeled pili, open arrows indicate unlabeled pili, and an arrowhead points to a flagellum. Dark dots along or at Hrp pili are 10-nm-diameter gold particles. Bars, 200 nm.

FIG. 6.

Mapping of the transcriptional start site of hrpE by primer extension analysis. RNAs were extracted from strain 85E(pXV74) grown for 16 h in XVM2 (lane 1) and NYG (lane 2), annealed with oligonucleotide no. 144, and used as templates for reverse transcription. The nucleotide sequence is the reverse complement of the coding strand. The boxed nucleotide refers to the transcriptional start site which is indicated by an arrow.

FIG. 7.

Phenotype and complementation of an hrpE mutant. Bacteria at a concentration of about 2 × 10⁸ CFU/ml were inoculated into a 5-week-old pepper leaf. After 2 days of cultivation, plant reactions were scored. A leaf after bleaching in ethanol is shown.

FIG. 8.

Effects of hrp and hpa mutations on Hrp pilus assembly. Electron micrographs from 85E*-derived bacteria incubated in XVM2 medium are shown. Deletion mutants of hrpF (B), hpaB (C), and hpaE (D) produced pili, whereas the hrpEΔ9-93 mutant does not show any pili (A). Bars, 200 nm.

FIG. 9.

Detection of HrpE protein in X. campestris pv. vesicatoria and effects of hrpE mutations on in vitro type III secretion. Immunoblotting analyses of total protein extracts (TE) and culture supernatants (SN) of an hrpE wild-type strain (85E*) and an hrpEΔ9-93 mutant are shown. Proteins were separated by SDS-PAGE and transferred to nitrocellulose. The blots were probed with HrpE (lanes 1 and 2)-, HrpF (lanes 3 to 5)-, or c-myc (lanes 6 to 8)-specific antibodies, respectively. The anti-c-myc (α-c-myc) antibodies detect triple c-myc-tagged AvrBs1 molecules expressed from plasmid pDSM110. Lanes 1, 3, and 7, 85E*; lanes 2 and 5, 85E* hrpEΔ9-93; lane 4, 85E* ΔhrpF; lane 6, 85E*(pDSM110); lane 8, 85E* hrpEΔ9-93(pDSM110). α-HrpE, anti-HrpE; α-HrpF, anti-HrpF.