Project description:We sought to compare and contrast plant host and bacterial transcriptional changes during compatible infections that cause disease (albeit within different symptoms). We investigated the infection by the two Pseudomonas syringae sensu lato strains P. syringae pv. syringae B728a (Psy) and P. amygdali pv. tabaci 11528 (Pta) of Nicotiana benthamiana at an early time point post inoculation to understand how a plant host responds to two related bacteria with different infection strategies. Plant and bacterial transcriptomes were analyzed prior to and five hours post inoculation.
Project description:Pseudomonas syringae is an important plant pathogen that infects a wide variety of crops. The mgo (mangotoxin-generating operon) gene cluster produces an extracellular signaling molecule, leudiazen, and is highly conserved in Pseudomonas syringae strains. Here, we genetically removed mgo in Pseudomonas syringae pv. syringae (Pss) UMAF0158 to interrogate its impacts on bacterial infection. Loss of mgo not only alleviated the chlorosis symptom caused by Pss UMAF0158 infection, but also reduced bacterial population in tomato leaflets. Structure-activity relationship revealed that the diazeniumdiolate group and the isobutyl side chain of leudiazen are critical for its signaling activity. Through global transcriptome analysis, we found that mgo regulates the expression of a new gene cluster in addition to mangotoxin biosynthetic operon, namely RS17235-RS17245. This new gene cluster contributes to in planta survival of Pss UMAF0158 and is widely distributed in Pseudomonas syringae strains. Our results demonstrate that chemical signaling systems in plant pathogens play prominent roles in virulence and population increase and set stages for understanding downstream components of mgo-regulated signaling pathways.
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:Pseudomonas syringae pv. phaseolicola (Pph) is a significant bacterial pathogen of agricultural crops, and phage Φ6 and other members of the dsRNA virus family Cystoviridae undergo lytic (virulent) infection of Pph, using the type IV pilus as the initial site of cellular attachment. Despite the popularity of Pph/phage Φ6 as a model system in evolutionary biology, Pph resistance to phage Φ6 remains poorly characterized. To investigate differences between phage Φ6 resistant Pseudomonas syringae pathovar phaseolicola strains, we performed expression analysis of super and non piliated strains of Pseudomonas syringae to determine the genetic cause of resistance to viral infection.
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:The regulation of transcription is primarily exerted through transcription factors (TFs) binding to genomic DNA. Although molecular mechanisms of TFs have been studied individually for decades, a complete picture of binding profiles of all TFs and their precise targets in the genome are still lacking in the model pathogen Pseudomonas syringae. To this end, we performed a high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) approach on all 301 annotated TFs in P. syringae. Robust enrichment of specific sequences was deduced to 118 SELEX motifs. We identified 12,464 interactions between 100 TFs and their target genes in the genome, for an individual TF ranging from 6 to 1481 sites. It showed that 90% TF binding was of dimeric site type, in which 85% with the head-to-head palindromic binding preference. To further explore the pathogenic mechanism of TFs in P. syringae, we mapped intricate networks of these TFs and their targets in the virulence-associated pathways, many of which were verified by orthologous methods such as ChIP-seq, EMSA, RT-qPCR and a reporter assay of promoter activity. By checking the enrichment of binding sites in pathways, we identified 25 virulence-associated master regulators of which 14 had never been characterized as TFs before. Overall, the present study provides a valuable resource for TF binding specificities in P. syringae and demonstrates a novel and an integrative analysis for seeking the virulence-associated TFs and its target genes. We expect that the results will significantly benefit future studies on the transcriptional regulation in P. syringae, and facilitate the design of drug targets to protect plants from attacks by relevant pathogens.