Table 1.
All strains used in this study.
Figure 1.
(A) HrpL-FLAG is recognized by anti-FLAG antibody in a Western Blot. (B) hrpL-FLAG and WT DC3000 strains evoke the hypersensitive response while it is abolished in the ΔhrpL strain. Bacteria were infiltrated via a blunt syringe into three independent leaves at 3×108 CFU/ml. Photos were taken after 3 days. Symptoms are identical among three replicates. (C) Expression of a plasmid-based (pBS181) β-glucuronidase (GUS) reporter driven by a hrp promoter (hrpJ) in hrpL-FLAG and WT DC3000 backgrounds. Three different media were used: HMM (hrp-minimal medium for highest induction of hrp promoters), MG (Mannitol-Glutamate medium for intermediate induction of hrp promoters but better bacterial growth), and KB (King’s B rich medium for repression of HrpL regulon expression). All plates contain appropriate antibiotics to maintain plasmids and X-Gluc, a substrate of GUS. (D-E) Relative fold change of transcript levels for hrpL and hopQ1-1 (PSPTO_0877) after medium shift from KB to MG (supplemented with ferric iron at 50 µM final concentration) over 9 hours.
Table 2.
Read mapping statistics.
Table 3.
Data for new hrp promoters.
Figure 2.
hrp promoter sequence alignment.
(A). Motif logo for annotated hrp promoter sequences. (B). Motif logo for all hrp promoters including the newly identified set. The –35 region is highly conserved but two cytosines in the –10 region show variability. (C). Alignment of individual motifs sequences. Motif ID is the central position (genome coordinate) for the associated ChIP-Seq zone of enrichment. Genes downstream of hrp promoters are identified by PSPTO numbers. (*): Candidate hrp promoter is oriented in an antisense direction relative to PSPTO_4750. PSPTO_3948–9: candidate hrp promoter is between PSPTO_3948 and PSPTO_3949, which are oriented covergently. Motif logos were created by Weblogo [75]. Sequences were aligned and visualized using SeaView [76].
Figure 3.
Associated sequence read counts at HrpL-binding sites.
(A) The height of each bar corresponds to the number of sequence reads associated with each site enriched by ChIP-Seq, normalized by the number of reads surrounding the peak region (see Materials and Methods). Heights for hrpL-FLAG and ΔhrpL strains are plotted for comparison. (B) Transcription start site clusters at hrp promoters upstream of PSPTO_5633, PSPTO_0371, PSPTO_1843, and PSPTO_2691. Red arrows depict hrp promoters. Read counts for captured 5′-ends are shown at positions downstream from each promoter. The mini-scales on the x-axis are positioned so that the leftmost vertical bar corresponds to the 3′ nucleotide of the –10 region in Figure 2 for the corresponding promoter. Dots indicate individual nucleotide coordinates.
Figure 4.
Validation of new hrp promoters.
(A). ChIP_qPCR experiments to test enrichment of DNA fragments at putative HrpL binding sites. Values for each gene were normalized to results for gyrA (DNA gyrase subunit A). gap-1 (glyceraldehyde 3-phosphate dehydrogenase, type I), not predicted to be HrpL-regulated, was used as a negative control. All fold changes above the expression value for gyrA are classified as enriched (above the horizontal line). (B). Induction of cloned hrp promoter-gfp fusions. Induction was measured by relative fluorescence normalized by OD600 (GFP fluorescence/OD) in hrp-inducing and hrp-repressing conditions. The hrp promoter::gfp fusion constructs were expressed in the DC3000 ΔpvsA siderophore mutant. The promoter trap vector without a promoter insert was used as a negative control (NC). GFP was measured using a Synergy 2 plate reader (Biotech) with excitation from 475 to 495 nm and emission from 506 to 526 nm. OD was measured at 600 nm using the same plate reader. A kinetics reading procedure was used, and a single data point at 5 hours was plotted for all strains, which is the time at which they show a peak value. (C). qRT-PCR analysis showing HrpL-dependent differential expression of transcripts downstream from hrp promoters in WT DC3000 and ΔhrpL strains. The relative fold change was measured after 1.5 hours on MG supplemented with iron (50 µM final concentration) normalized to gyrA. For determination of the relative expression, expression of each gene in the ΔhrpL mutant was set to 1. Expression of each gene in the WT strain was then normalized to the corresponding gene in the ΔhrpL mutant. All data points are the averages of 3 replicates with standard deviations.
Figure 5.
Summary of data for PSPTO_5633.
(A). ChIP-Seq, RNA-Seq and promoter motif at PSPTO_5633 locus. The transcription start site mapped by 5′ capture in RNA-Seq and its location relative to the predicted motif are consistent with the presence of a genuine hrp promoter. The profiles, along with genome annotation, are shown using Artemis. Red and green traces correspond to sequence read counts on the positive and negative strands, respectively. The sequence containing the hrp promoter motif is enclosed in a box. (B) Evidence that PSPTO_5633 is translocated through the DC3000 T3SS. N. benthamiana leaves were infiltrated with 5×107 CFU/ml of the indicated DC3000 strains carrying plasmids in which PSPTO_5633 was fused to the Cya translocation reporter, or an AvrPto-Cya control. Total cAMP produced as a result of Cya activity in leaf extracts 6 hours after infiltration is shown for all the strains. PSPTO_5633 is translocated into leaf cells from wild-type DC3000 (T3SS+) and from a DC3000ΔgspD (T2SS− mutant. No translocation was observed in the DC3000ΔhrcQ-U (T3SS− mutant) background. The data represent the average cAMP (pmol) with standard deviations computed using data from 3 plants. The experiment was repeated 3–5 times for all strains except for PSPTO_5633(DC3000 T2SS−), which was repeated twice. (C) SignalP analysis showing C, S and Y scores for each position in the sequence of PSPTO_5633, where C-score is the raw cleavage site score, S-score is the signal peptide score and Y-score is the combined cleavage site score. Similar analyses for avrPto1 (a T3SS-translocated effector), PSPTO_1766 (lipase, generally known to target the Sec pathway), and a housekeeping gene (gyrase, generally known to function inside bacterial cells) are shown for comparison.
Figure 6.
Orthologs and hrp promoter motifs for DC3000 HrpL regulon orthologs in the P. syringae subgroup.
A blank (white) cell indicates that no ortholog was detected. “ = = ” indicates that an orthologous gene was identified but no upstream sequence could be extracted (due to incomplete sequence information and segmentation in draft genomes). For cases in which orthologs were detected and upstream sequences recovered, the color represents the –logarithm (base 10) of the HMM E-value for the best motif matching the hrp promoter model in the upstream sequence. A continuous color scheme is used where blue represents a poor match (E-value = 1), dark red indicates an intermediate match (E-value = 1e-02), and bright red indicates a good match (E-value 1e-05). Most verified hrp promoters in DC3000 match with values above 3. The leftmost gene column represents orthologs for the HrpL sigma factor, PSPTO_1404. In DC3000, this sigma factor is transcribed from a RpoN-responsive promoter [77]. Genes immediately downstream of hrp promoters are shown in columns, as they appear in CEL, hrp/hrc cluster, followed by type III effectors, chaperones and helpers, and non-type III function genes. Newly found members are in green background. 3-color scale is used: Color: Blue …… dark red …… light red Value: 0…….…………2………….…….5.
Figure 7.
Ortholog inventory of HrpL regulon in Pseudomonadales.
Green represents newly found members; black represents previously annotated regulon members. The values shown represent counts of orthologs of HrpL regulon members across 1060 species.