Unknown,Transcriptomics,Genomics,Proteomics

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Phaseolotoxin synthesis is regulated by the GacS/GacA two-component system

ABSTRACT: Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by water-soaked lesions surrounded by a chlorotic halo resulting from the action of a non-host-specific toxin known as phaseolotoxin, that inhibits the enzyme ornithine carbamoyltransferase involved in the arginine biosynthesis pathway. It was previously reported that genes within the Pht cluster were involved in the regulation and synthesis of phaseolotoxin. The GacS/GacA two component signal transduction system controls important pathogenicity and virulence mechanisms in several Gram-negative bacteria. In the present study we hybridized a genomic microarray of P. syringae pv. phaseolicola NPS3121 to compare transcriptional profiles from wild type strain and a gacA- null mutant with a Tox- 11 phenotype. Results show that GacA controls expression of genes within the Pht cluster as well as another group of clustered genes located in a 13 different region in the bacterial chromosome that contains at least one gene unambiguously shown to be directly involved in phaseolotoxin biosynthesis. Results suggest that this cluster is a new pathogenicity island containing genes whose regulation is also under GacA regulatory cascade and it will require further investigation to determine gene functions and their relationship to virulence mechanisms Transcriptional profiles of a P. syringae pv. phaseolicola NPS3121 null gacA mutant were compared to those of the wild-type strain. Cultures were grown in minimal medium at 18Â°C (which is the temperature at which phaseolotoxin is produced and the bacteria achieves full virulence) and harvested during the late log phase of the exponential growth. RNA from three biological replicates and each with two technical replicates were hybridize to control variation that might affect data interpretation.

ORGANISM(S): Pseudomonas syringae pv. phaseolicola

SUBMITTER: JosÃ©-Luis HernÃ¡ndez-Flores

PROVIDER: E-GEOD-16632 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Pseudomonas syringae pv. tomato DC3000 (Pst) is a virulent pathogen, which causes disease on tomato and Arabidopsis. The type III secretion system (TTSS) plays a key role in pathogenesis by translocating virulence effectors from the bacteria into the plant host cell, while the phytotoxin coronatine (COR) contributes to virulence and disease symptom development. Recent studies suggest that both the TTSS and and COR are involved in the suppression of host basal defenses. However, little is known about the interplay between the host gene expression associated with basal defenses and the virulence activities of the TTSS and COR during infection. The global effects of the TTSS and COR on host gene expression associated with other host cellular processes during bacterial infection are also not well characterized. In this study, we used the Affymetrix full genome chip to determine the Arabidopsis transcriptome associated with basal defense to Pst DC3000 hrp mutants and the human pathogenic bacterium Escherichia coli O157:H7. We then used Pst DC3000 virulence mutants to characterize Arabidopsis transcriptional responses to the action of hrp-regulated virulence factors (e.g., TTSS and COR) during bacterial infection. Additionally, we used bacterial fliC mutants to assess the role of the PAMP flagellin in induction of basal defense-associated transcriptional responses. In total, our global gene expression analysis identified more than 5000 Arabidopsis genes that are reproducibly regulated more than 2-fold in three independent biological replicates of at least one type of comparison. Regulation of these genes provides a molecular signature for Arabidopsis basal defense to plant and human pathogenic bacteria, and illustrates both common and distinct global virulence effects of the TTSS, COR, and possibly other hrp-regulated virulence factors during Pst DC3000 infection. Experimenter name = William Underwood; Experimenter phone = 517-353-9182; Experimenter fax = 517-353-9168; Experimenter address = Michigan State University; Experimenter address = 222 Plant Biology Building; Experimenter address = 178 Wilson R.d. Experimenter address = East Lansing, MI; Experimenter zip/postal_code = 48824; Experimenter country = USA Experiment Overall Design: 40 samples were used in this experiment

Project description:P. syringae pv. phaseolicola, the causal agent of halo blight disease in bean, produces a toxin known as phaseolotoxin, whose synthesis involves the product of some of the genes found within the Pht region. This region, considered a pathogenicity island, comprises 23 genes arranged in five transcriptional units; two single-gene units (argK, phtL) and three arranged as operons (phtA, phtD, phtM), most with unknown function. In P. syringae pv. phaseolicola, maximal expression of most of the genes encoded in the Pht region and the synthesis of phaseolotoxin require the product of the phtL gene, which has been proposed to have a regulatory function. In order to evaluate the role of phtL gene in P. syringae pv. phaseolicola, we performed a comparative transcriptional analysis with the wild type and a phtL- mutant strains using microarrays. The microarray data analysis showed that PhtL regulates the expression not only of genes within the Pht region, but also alters the expression of genomic genes related with the iron-acquisition system, pathogenicity, oxidative stress and virulence. This study suggests the possible relation of the PhtL protein with the iron response genes and with the pathogenicity and or virulence of this bacterium. The microarrays used in this study correspond to an assembled DNA microarray of P. syringae pv. phaseolicola NPS3121 previously reported (GEO accession number GPL7115) (HernM-CM-!ndez-Morales et al., 2009). This microarray consist of 1911 probes, with an average length 2.4 Kbp, representing 1X the genome of P. syringae pv. phaseolicola NPS3121, as well as several PCR products corresponding to various known genes, which were printed as controls. Each microarray experiment was repeated six times (two technical replicates with the same RNA samples and three biological replicates using RNA isolated from a different culture). cDNA synthesis, labeling, hybridization, washing, and chip scanning were performed at the Microarray Core Facility at Cinvestav-Langebio. Hybridized microarray slides were scanned (GenePix 4000, Axon Instruments, Inc) at a 10 um resolution adjusting the laser and gain parameters to obtain similar levels of fluorescence intensity in both channels. The spot intensities were quantified using Axon GenePrix Pro 6.0 image analysis software. First, an automatic spot finding and quantification option of the software was used. Subsequently, all spots were inspected individually and in some cases, the spot diameters were corrected or the spots were removed from the analysis. The mean of the signals and the median of backgrounds were used for further analysis. Raw data were imported into the R.2.2.1 software. Background signals were subtracted using the Robust Multichip Analysis (RMA) whereas the normalization of the signal intensities within slides was carried out using M-bM-^@M-^\print-tip loessM-bM-^@M-^] method and the LIMMA package. Normalized data were log2 transformed and then fitted into mixed model ANOVAs using the Mixed procedure. The p-values of the phtL effects were adjusted for by the False Discovery Rate Method M-bM-^@M-^\FDRM-bM-^@M-^]. Changes in signal intensity of M-BM-11.5-fold or higher/lower between phtL- mutant and wild type were highly significant (FDR, p-value M-bM-^IM-$0.05), however we focused only in differential expressed genes that fell within the more traditional criteria, which is the cut-off threshold for up-regulated (M-bM-^IM-%1.8) and down-regulated genes (M-bM-^IM-$0.50).

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Phaseolotoxin synthesis is regulated by the GacS/GacA two-component system

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