Genome-Wide Identification of HrpL-Regulated Genes in the Necrotrophic Phytopathogen Dickeya dadantii 3937

BACKGROUND: Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. METHODOLOGY/PRINCIPAL FINDINGS: To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens

Genetic improvement of tomato by targeted control of fruit softening

Controlling the rate of softening to extend shelf life was a key target for researchers engineering genetically modified (GM) tomatoes in the 1990s, but only modest improvements were achieved. Hybrids grown nowadays contain 'non-ripening mutations' that slow ripening and improve shelf life, but adversely affect flavor and color. We report substantial, targeted control of tomato softening, without affecting other aspects of ripening, by silencing a gene encoding a pectate lyase

The Gene Ontology in 2010: extensions and refinements

The Gene Ontology (GO) Consortium (http://www.geneontology.org) (GOC) continues to develop, maintain and use a set of structured, controlled vocabularies for the annotation of genes, gene products and sequences. The GO ontologies are expanding both in content and in structure. Several new relationship types have been introduced and used, along with existing relationships, to create links between and within the GO domains. These improve the representation of biology, facilitate querying, and allow GO developers to systematically check for and correct inconsistencies within the GO. Gene product annotation using GO continues to increase both in the number of total annotations and in species coverage. GO tools, such as OBO-Edit, an ontology-editing tool, and AmiGO, the GOC ontology browser, have seen major improvements in functionality, speed and ease of use.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by Oxford University Press. The published article can be found at: http://nar.oxfordjournals.org/

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

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

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

<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

All strains used in this study.

<p>All strains used in this study.</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