Specific binding of the Xanthomonas campestris pv. vesicatoria AraC-type transcriptional activator HrpX to plant-inducible promoter boxes

Abstract

The pathogenicity of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion system which is encoded by the 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Expression of the hrp operons is strongly induced in planta and in a special minimal medium and depends on two regulatory proteins, HrpG and HrpX. In this study, DNA affinity enrichment was used to demonstrate that the AraC-type transcriptional activator HrpX binds to a conserved cis-regulatory element, the plant-inducible promoter (PIP) box (TTCGC-N(15)-TTCGC), present in the promoter regions of four hrp operons. No binding of HrpX was observed when DNA fragments lacking a PIP box were used. HrpX also bound to a DNA fragment containing an imperfect PIP box (TTCGC-N(8)-TTCGT). Dinucleotide replacements in each half-site of the PIP box strongly decreased binding of HrpX, while simultaneous dinucleotide replacements in both half-sites completely abolished binding. Based on the complete genome sequence of Xanthomonas campestris pv. vesicatoria, putative plant-inducible promoters consisting of a PIP box and a -10 promoter motif were identified in the promoter regions of almost all HrpX-activated genes. Bioinformatic analyses and reverse transcription-PCR experiments revealed novel HrpX-dependent genes, among them a NUDIX hydrolase gene and several genes with a predicted role in the degradation of the plant cell wall. We conclude that HrpX is the most downstream component of the hrp regulatory cascade, which is proposed to directly activate most genes of the hrpX regulon via binding to corresponding PIP boxes.

FIG. 1.

Expression analysis of X. campestris pv. vesicatoria genes with candidate PIP box-regulated promoters by RT-PCR. X. campestris pv. vesicatoria strains 85-10, 85*, and 85*ΔhrpX were grown in NYG medium. 16S rRNA was used as a constitutive control. The DNA samples were separated in a 1.5% agarose gel and stained with ethidium bromide. The results of one representative experiment are shown. XCV refers to the nomenclature used by Thieme et al. (40) and to a locus tag in GenBank. hgi (hrpG induced) refers to the nomenclature used by Noël et al. (31).

FIG. 2.

Complementation of the 85* hrpX deletion mutant by hexahistidine-tagged HrpX. X. campestris pv. vesicatoria strains expressing AvrBs1 were inoculated into an AvrBs1-responsive ECW-10R pepper leaf. The response to the translocation of AvrBs1 was monitored in the wild-type strain (85*) and the isogenic hrpX deletion mutant (85*ΔhrpX) without a plasmid, with plasmid pBBR1MCS-5 (empty vector), and with a pBBR1MCS-5 derivative expressing a hexahistidine-tagged HrpX derivative (HrpX-His). The wild-type and complemented strains induced the hypersensitive response. In contrast, there was no response to 85*ΔhrpX or 85*ΔhrpX with empty vector. X. campestris pv. vesicatoria strains were inoculated at 5 × 10⁸ CFU/ml. Two days after inoculation, the leaf was bleached with ethanol. The dark regions result from phenolic compounds due to the development of the hypersensitive response. The dashed lines indicate the inoculated areas.

FIG. 3.

Genetic organization of the X. campestris pv. vesicatoria hrp gene cluster and DNA fragments used for magnetic DNA affinity enrichment of HrpX. The solid lines at the top indicate the six hrp transcription units, hrpA to hrpF; the thick arrows indicate different genes. hrc genes are shown as open arrows and are labeled with the corresponding letter code, hrp genes are shown in dark gray, hpa genes are shown as striped arrows, and the xopF1 gene is shown in light gray. Perfect PIP boxes (black circles) and the imperfect PIP box of hrpF (open circle) are indicated. DNA fragments used for magnetic DNA affinity enrichment of HrpX are symbolized by black bars below the map (not drawn to scale). Sequences of oligonucleotide primers used for PCR amplification of biotinylated DNA fragments are listed along with the PIP boxes contained by these DNA fragments. Mutant variants of the hrpD PIP box are given at the bottom. Conserved half-sites of the PIP boxes are shown in bold.

FIG. 4.

DNA affinity enrichment of HrpX using a DNA fragment containing a PIP box. (A) Immunoblot analysis of protein eluates after magnetic DNA affinity enrichment. A soluble protein extract of X. campestris pv. vesicatoria strain 85* was applied to DNA fragments with PIP boxes of the divergently transcribed hrpB and hrpC operons (lane 1), of hrpD (lane 2), and of hrpE (lane 3) and to an hrcV DNA fragment lacking a PIP box (lane 4). In a separate experiment, binding of HrpX-His to a DNA fragment with the hrpB-hrpC PIP boxes (lane 5) was compared to binding to a DNA fragment with the imperfect hrpF PIP box (lane 6). Proteins were separated by SDS-PAGE, blotted, and incubated with a His tag-specific antibody. (B) As a control for equal loading of the samples shown in panel A, a second SDS-PAGE gel was stained with silver. Protein samples were as in panel A, lanes 1 to 4. (C) Immunoblot analysis of protein eluates outlined in panel A, using DNA fragments with the hrpD wild-type PIP box (lane 1), a PIP box with TT-to-AG mutations of the first half-site (lane 2), a PIP box with TT-to-AG mutations of the second half-site (lane 3), and a PIP box with TT-to-AG mutations of both half-sites (lane 4). (D) As a loading control for the samples shown in panel C, a second SDS-PAGE gel was stained with Coomassie blue. Since it was expected that the mutant PIP boxes would bind weakly to HrpX, four times more protein was applied in lanes 2 to 4. M, molecular mass marker. The bars correspond to molecular masses of 80, 61, and 48 kDa, from top to bottom.

FIG. 5.

Model of binding of HrpX to PIP boxes of the hrp operons of X. campestris pv. vesicatoria. In cases of class I activation, a HrpX homodimer would bind in a backward orientation with the HTH-1 motif of each subunit contacting the two conserved half-sites of the PIP box. In cases of class II activation, a HrpX homodimer would bind in a forward orientation, thus overlapping with the −35 region and making direct contact with the sigma factor. A schematic DNA double helix illustrating the periodicity of 10.5 bp per helical turn and sequences of the promoter regions of the hrpB to hrpE operons are shown below. Mapped transcriptional start sites are shown in bold (; Bonas, unpublished data).