Genome-Wide Identification of Transcriptional Start Sites in the Plant Pathogen Pseudomonas syringae pv. tomato str. DC3000

RNA-Seq has provided valuable insights into global gene expression in a wide variety of organisms. Using a modified RNA-Seq approach and Illumina's high-throughput sequencing technology, we globally identified 5′-ends of transcripts for the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. A substantial fraction of 5′-ends obtained by this method were consistent with results obtained using global RNA-Seq and 5′RACE. As expected, many 5′-ends were positioned a short distance upstream of annotated genes. We also captured 5′-ends within intergenic regions, providing evidence for the expression of un-annotated genes and non-coding RNAs, and detected numerous examples of antisense transcription, suggesting additional levels of complexity in gene regulation in DC3000. Importantly, targeted searches for sequence patterns in the vicinity of 5′-ends revealed over 1200 putative promoters and other regulatory motifs, establishing a broad foundation for future investigations of regulation at the genomic and single gene levels

Genomic plasticity enables phenotypic variation of Pseudomonas syringae pv. tomato DC3000.

Whole genome sequencing revealed the presence of a genomic anomaly in the region of 4.7 to 4.9 Mb of the Pseudomonas syringae pv. tomato (Pst) DC3000 genome. The average read depth coverage of Pst DC3000 whole genome sequencing results suggested that a 165 kb segment of the chromosome had doubled in copy number. Further analysis confirmed the 165 kb duplication and that the two copies were arranged as a direct tandem repeat. Examination of the corresponding locus in Pst NCPPB1106, the parent strain of Pst DC3000, suggested that the 165 kb duplication most likely formed after the two strains diverged via transposition of an ISPsy5 insertion sequence (IS) followed by unequal crossing over between ISPsy5 elements at each end of the duplicated region. Deletion of one copy of the 165 kb region demonstrated that the duplication facilitated enhanced growth in some culture conditions, but did not affect pathogenic growth in host tomato plants. These types of chromosomal structures are predicted to be unstable and we have observed resolution of the 165 kb duplication to single copy and its subsequent re-duplication. These data demonstrate the role of IS elements in recombination events that facilitate genomic reorganization in P. syringae

Global Analysis of the HrpL Regulon in the Plant Pathogen <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 Reveals New Regulon Members with Diverse Functions

<div><p>The type III secretion system (T3SS) is required for virulence in the gram-negative plant pathogen <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000. The alternative sigma factor HrpL directly regulates expression of T3SS genes via a promoter sequence, often designated as the “<i>hrp</i> promoter.” Although the HrpL regulon has been extensively investigated in DC3000, it is not known whether additional regulon members remain to be found. To systematically search for HrpL-regulated genes, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and bulk mRNA sequencing (RNA-Seq) to identify HrpL-binding sites and likely <i>hrp</i> promoters. The analysis recovered 73 sites of interest, including 20 sites that represent new <i>hrp</i> promoters. The new promoters lie upstream of a diverse set of genes encoding potential regulators, enzymes and hypothetical proteins. PSPTO_5633 is the only new HrpL regulon member that is potentially an effector and is now designated HopBM1. Deletions in several other new regulon members, including PSPTO_5633, PSPTO_0371, PSPTO_2130, PSPTO_2691, PSPTO_2696, PSPTO_3331, and PSPTO_5240, in either DC3000 or Δ<i>hopQ1-1</i> backgrounds, do not affect the hypersensitive response or <i>in planta</i> growth of the resulting strains. Many new HrpL regulon members appear to be unrelated to the T3SS, and orthologs for some of these can be identified in numerous non-pathogenic bacteria. With the identification of 20 new <i>hrp</i> promoters, the list of HrpL regulon members is approaching saturation and most likely includes all DC3000 effectors.</p></div

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Validation of new <i>hrp</i> promoters.

<p>(A). ChIP_qPCR experiments to test enrichment of DNA fragments at putative HrpL binding sites. Values for each gene were normalized to results for <i>gyrA</i> (DNA gyrase subunit A). <i>gap-1</i> (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 <i>gyrA</i> are classified as enriched (above the horizontal line). (B). Induction of cloned <i>hrp</i> promoter-<i>gfp</i> fusions. Induction was measured by relative fluorescence normalized by OD₆₀₀ (GFP fluorescence/OD) in <i>hrp</i>-inducing and <i>hrp</i>-repressing conditions. The <i>hrp</i> promoter::<i>gfp</i> fusion constructs were expressed in the DC3000 Δ<i>pvsA</i> 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 <i>hrp</i> promoters in WT DC3000 and <i>ΔhrpL</i> strains. The relative fold change was measured after 1.5 hours on MG supplemented with iron (50 µM final concentration) normalized to <i>gyrA.</i> 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.</p

Summary of data for PSPTO_5633.

<p>(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 <i>hrp</i> 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 <i>hrp</i> promoter motif is enclosed in a box. (B) Evidence that PSPTO_5633 is translocated through the DC3000 T3SS. <i>N. benthamiana</i> 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 DC3000Δ<i>gspD</i> (T2SS⁻ mutant. No translocation was observed in the DC3000Δ<i>hrcQ-U</i> (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.</p

<i>hrp</i> promoter sequence alignment.

<p>(A). Motif logo for annotated <i>hrp</i> promoter sequences. (B). Motif logo for all <i>hrp</i> 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 <i>hrp</i> promoters are identified by PSPTO numbers. (*): Candidate <i>hrp</i> promoter is oriented in an antisense direction relative to PSPTO_4750. PSPTO_3948–9: candidate <i>hrp</i> promoter is between PSPTO_3948 and PSPTO_3949, which are oriented covergently. Motif logos were created by Weblogo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Crooks1" target="_blank">[75]</a>. Sequences were aligned and visualized using SeaView <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Galtier1" target="_blank">[76]</a>.</p

Ortholog inventory of HrpL regulon in <i>Pseudomonadales</i>.

<p>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.</p

Data for new <i>hrp</i> promoters.

<p>Operon: PSPTO identifier for gene immediately downstream from promoter.</p><p>Function: annotated function for operon-identifying gene.</p><p>Coordinate: DC3000 genome coordinate for the region bracketing the –35 and –10 regions of the promoter. “c” designates that the promoter is found on the complementary strand.</p><p>Evidence: Experimental evidence for <i>hrp</i> promoters from this study.</p><p>• H: <i>hrp</i> promoter motif found;</p><p>• B: Binding activity for HrpL (ChIP-qPCR) observed;</p><p>• I: Induction observed in promoter fusion (threshold = 2.4; 2×negative control);</p><p>• S: mRNA 5′-end captured (TSS) within 10 bps from the 3′ end of −10 promoter element. No threshold is applied. Absolute values for read counts appear in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115.s009" target="_blank">Table S5</a>. TSSs from Filiatrault <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Filiatrault2" target="_blank">[46]</a> were also taken into consideration.</p><p>qRT-PCR: Transcript abundance for regions downstream from <i>hrp</i> promoters in DC3000 compared to that in a <i>ΔhrpL</i> strain. Values in brackets indicate that abundance was measured upstream from coding region. Values are average of one or two biological replicates with three technical replicates and standard deviation.</p><p>Published data:</p><p>• Mucyn <i>et al.</i>: ‘+’ indicates that the gene was classified as differentially expressed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone-0106115-t003" target="_blank">Table 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115.s007" target="_blank">Table S3</a> by Mucyn <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Mucyn1" target="_blank">[26]</a>.</p><p>• Promoters: symbols indicate whether <i>hrp</i> promoters were reported by Chang <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Chang1" target="_blank">[22]</a> (C), Fouts <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Fouts1" target="_blank">[21]</a> (F), or by Ferreira <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Ferreira1" target="_blank">[23]</a> (M).</p><p>• Genes: symbols indicate whether genes downstream from <i>hrp</i> promoters were tested and/or reported by Chang <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Chang1" target="_blank">[22]</a> (C), Fouts <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Fouts1" target="_blank">[21]</a> (F), or by Ferreira <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Ferreira1" target="_blank">[23]</a> (M). Genes that showed no differential expression, or which were not tested by Ferreira <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Ferreira1" target="_blank">[23]</a> are designated (M_o) and (M₁), respectively.</p><p>No binding evidence for HrpL or upstream <i>hrp</i> promoter motifs were observed in association with 12 of the 14 genes proposed by Mucyn <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106115#pone.0106115-Mucyn1" target="_blank">[26]</a> as putative novel HrpL regulon members. These are PSPTO_0829 (clpB protein), PSPTO_0851 (hypothetical protein), PSPTO_1371 (effector locus protein), PSPTO_2129 (sensory box histidine kinase/response regulator), PSPTO_2208 (heat shock protein HtpG), PSPTO_3148 (magnesium chelatase subunit ChII), PSPTO_4210 (ATP-dependent protease La), PSPTO_4332 (hypothetical protein), PSPTO_4376 (chaperonin, 60 kDa), PSPTO_4505 (dnaK protein), PSPTO_4716 (hypothetical protein), and PSPTO_4723 (hypothetical protein).</p><p>Data for new <i>hrp</i> promoters.</p