Project description:Pseudomonas syringae was grown on minimal media and King's B medium. The goal of this project was to generate pilot data in preparation for a summer course on high-throughput sequencing where participants prepared their own RNA-Seq libraries and analyzed the resulting data. The summer course was funded by NIH grant 5R25EB023928-03 "A hands-on, integrative next-generation sequencing course: design, experiment, and analysis".
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, 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: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 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:Many bacteria can transition from a planktonic lifestyle to life attached to a surface. Changes in gene expression have been documented in bacteria in mature biofilms, but few studies have looked at gene expression during the initial stages of surface attachment. To investigate this, we performed RNA-Seq using the model organism Pseudomonas syringae B728a which has been found in rivers and lakes but is known for living on the leaf surface. We compared gene expression of wild-type P. syringae B728a cells attached to a filter for 2 hours to the gene expression of wild-type P. syringae B728a cells in King's medium B broth. We found that certain gene catergories were quickly induced when cells were on a surface such as flagellar synthesis and motility while other gene categories were quickly repressed such as phytotoxin synthesis and transport. These fast changes in gene expression suggest that P. syringae B728a uses surface attachment as a potential cue to better adapt to life on a surface.
Project description:This study evaluates the transcriptome of transgenic Arabidopsis seedlings expressing the effector HopBB1 from the bacteria Pseudomonas syringae
