Project description:We implemented transcriptional analysis methods using cDNA and high-throughput sequencing data to identify HrpL-regulated genes for six strains of Pseudomonas syringae
Project description:We implemented transcriptional analysis methods using cDNA and high-throughput sequencing data to identify HrpL-regulated genes for six strains of Pseudomonas syringae Each Pseudomonas syringae strains was transformed with either pBAD::EV or pBAD containing native hrpL sequence. Strains were grown in MM media supplemented with arabinose and collected 1, 3, and 5 hours post arabinose treatment. RNA was extracted for each time point and mixed at a 1/3 ratio. After removal of rRNA, double stranded cDNA was generated and library prepared accordeing to Illumina protocols.
Project description:Pseudomonas syringae, a Gram-negative plant pathogen, infects more than 50 crops with its type III secretion system (T3SS) and causes severe economic losses around the world. Although the mechanisms of virulence-associated regulators of P. syringae T3SS have been studied for decades, the crosstalk and network underlying these regulators are still elusive. Previously, we have individually studied a group of T3SS regulators, including AefR, HrpS, and RhpRS. In the present study, we found 4 new T3SS regulator genes (envZ, ompR, tsiS and phoQ) via transposon-mediated mutagenesis. Two-component systems EnvZ and TsiS natively regulate T3SS. In order to uncover the crosstalk between 16 virulence-associated regulators, (including AefR, AlgU, CvsR, GacA, HrpL, HrpR, HrpS, MgrA, OmpR, PhoP, PilR, PsrA, RhpR, RpoN, TsiR and Vfr) in P. syringae, we mapped an intricate network named PSVnet (Pseudomonas syringae Virulence Regulatory Network) by combining differentially expression genes in RNA-seq and binding loci in ChIP-seq of all regulators.
Project description:A ChIP-seq assay was performed to identify the regulons of an ompR-like transcription factor (gene name: 13375, GenBank: AYL80818.1) in Pseudomonas syringae pv. actinidiae. An 13375-overexpressing mutant G1-OE13375, which constitutively express C-terminally Myc-tagged ompR-like gene in the 13375-deletion mutant G1Δ13375, was used in this study. The bacterial cells were cultured either in nutrition-rich KB medium or hrp-derepressing medium (HDM) at 25 C for 24 hours. A PierceTM Magnetic ChIP Kit (Cat. #: 26157, Thermo Fisher Scientific) and a ChIP-grade Myc-Tag Monoclonal Antibody (Myc.A7, Cat. #: MA121316, Thermo Fisher Scientific) were used for sample pretreatment and immunoprecipitation.
Project description:Bacteria relies on two-component systems (TCSs) to respond to a wide range of stimuli or environmental cues for their survival and virulence. However, the functions and synergistic actions of TCSs in genomic level remains unclear. Here, in model phytopathogen Pseudomonas syringae, by integrating multiomics data, we developed a network-based PSTCSome (Pseudomonas syringae two-component systems regulome) to identify functions and crosstalk among global TCSs under either virulence suppressing (King’s B medium, KB) or activating conditions (minimal medium, MM). Transcriptome profiling identifies 2,099 differentially expressed genes (DEGs) in KB and 1,250 DEGs in MM, while ChIP-seq identifies 1,628 target genes across the whole genome. The multiomics results are applied to perform not only gene ontology (GO) analysis to detect the biological processes that TCSs involved in, but also subnetworks and co-expression analysis of 8 virulence-related pathways to decipher the TCSs regulated pathogenic behaviors. The following phenotypic experiments newly confirmed 8 TCSs that regulates type III secretion system (T3SS) (PSPPH_0253, PSPPH_2606, PSPPH_4416, and PSPPH_4451) and surface attachment (PSPPH_0253, PSPPH_3041, PSPPH_3473, PSPPH_3736, PSPPH_4001). We then compute 259 functional genes in KB and 161 in MM for those cluster TCSs. Analysis of cluster TCSs regulons led to the identification of either novel functions or 2 master regulatory TCSs (RhpS/RhpR and PSPPH_4827/4828) toward virulence. Our results show that TCSs in P. syringae dynamically adjust their regulatory networks by sensing the external environment, and then switch bacteria between pathogenic and non-pathogenic states in a sophisticated way. Furthermore, we present an online platform of PSTCSome to facilitate updating, network visualization and user-customized analyses.
Project description:Pseudomonas syringae uses two-component system RhpRS to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanism and the regulons of RhpRS have yet to be fully elucidated. Here we show that RhpS functions as an autokinase, an RhpR kinase, and a P-RhpR phosphatase. RhpR can also be phosphorylated by small phosphodonor acetyl phosphate. A specific RhpR-binding site containing an inverted repeat (IR) motif GTATC-N6-GATAC, was mapped to its own promoter by DNase I footprint analysis. Electrophoretic mobility shift assay (EMSA) indicated that P-RhpR has higher binding activity than RhpR to the IR motif. To identify additional targets of RhpR in P. syringae, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq), which detected 167 enriched loci including the hrpR promoter, indicating the direct regulation of T3SS cascade genes by RhpR. Genome-wide microarray analysis showed that, besides the T3SS cascade genes, RhpR differentially regulates a large set of genes of various functions in response to different growth conditions. Together, these results suggested that RhpRS is a global regulator that allows P. syringae to sense and respond to environmental changes to coordinate the T3SS and many other biological processes. ChIP-seq analysis of RhpR
Project description:Pseudomonas syringae pv. syringae 9644 (Pss9644) is a causal agent of bacterial cherry canker causing necrotic symptoms on leaves, fruits, gummosis and canker in woody tissues of sweet cherry (Prunus avium). To understand which virulent factor genes were expressed in vitro, Pss9644 was grown in rich media (King's B Broth) and minimum media (hrp-inducing minimum media). The latter mimics the in planta environment.
Project description:Pseudomonas syringae uses two-component system RhpRS to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanism and the regulons of RhpRS have yet to be fully elucidated. Here we show that RhpS functions as an autokinase, an RhpR kinase, and a P-RhpR phosphatase. RhpR can also be phosphorylated by small phosphodonor acetyl phosphate. A specific RhpR-binding site containing an inverted repeat (IR) motif GTATC-N6-GATAC, was mapped to its own promoter by DNase I footprint analysis. Electrophoretic mobility shift assay (EMSA) indicated that P-RhpR has higher binding activity than RhpR to the IR motif. To identify additional targets of RhpR in P. syringae, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq), which detected 167 enriched loci including the hrpR promoter, indicating the direct regulation of T3SS cascade genes by RhpR. Genome-wide microarray analysis showed that, besides the T3SS cascade genes, RhpR differentially regulates a large set of genes of various functions in response to different growth conditions. Together, these results suggested that RhpRS is a global regulator that allows P. syringae to sense and respond to environmental changes to coordinate the T3SS and many other biological processes.