Project description: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.
Project description: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. Analysis used aerobic steady state RNA from wild-type as control samples for comparison to the experimental samples of hrpL mutant taken 6 h post-inoculation in T3SS inducible minimum media. The microarray results are based on the geomean of ten normalized expression values derived from three biological replicates and two dye-swap experiments with a technology replicate for each array.
Project description:Growth in 0.3 M NaCl M63 minimal medium affects the transcriptome of Dickeya dadantii 3937.in comparison to that of cells grown in M63 medium.
Project description:Growth in 0.3 M NaCl M63 minimal medium affects the transcriptome of Dickeya dadantii 3937.in comparison to that of cells grown in M63 medium. NimbleGen design name 2005-05-31_Ogg_Erwinia_24mer, NimbleGen design ID 2181 A 1:2 expression design for 4597 genes from Dickeya dadantii 3937 with 20 24-mer probe pairs (PM/MM) per gene. Each probe is replicated 2 times. The design includes random GC and other control probes. Protocol: High-density DNA array prepared with Maskless Array Synthesizer (MAS) technology. See manufacturer's website at http://www.nimblegen.com/.
Project description:Investigation of whole genome gene expression level changes in the phytopathogenic Dickeya dadantii wild-type strain 3937 during an acute per os infection of an aphid body, in comparison with a colony grown in standard LB medium. The pathosystem described in this study has been analysed and first published in Grenier et al. 2006, and further detailed in Costechareyre et al. 2011
Project description:We have analysed the global gene expression of the plant pathogen Dickeya dadantii 3937 when grown in vitro under different growth and stress conditions. A multi-factorial design with 32 different experimental conditions was constructed, with two biological replicates for each condition. Cells were grown to exponential phase or stationary phases in four different growth media: M63 supplemented with 0.2% sucrose as carbon source, with or without 0.2% (w/v) polygalacturonate (PGA) and with or without 0.1% Saintpaulia leaf extracts (1 g leaves in 1 L M63). Cells grown in these four media were subjected to different stresses: (i) acid stress, by an incubation of 15 min in the presence of 30 mM malic acid; (ii) oxidative stress, by an incubation of 15 min in the presence of 100 _M H2O2 or (iii) osmotic stress, by an incubation of 15 min in the presence of 300 mM NaCl. The micro-arrays used in this study were custom designed and produced by Roche NimbleGen, Inc. (Madison, WI) based on the annotated sequence of D. dadantii, available at Genbank accession number nM-! CP002038, which comprises 4597 CDS. The 4plex expression micro-arrays consist of 60-mer oligonucleotides, triplicated in three blocks on the array (5 oligonucleotides per CDS).
Project description:Investigation of whole genome gene expression level changes in the phytopathogenic Dickeya dadantii wild-type strain 3937 during an acute per os infection of an aphid body, in comparison with a colony grown in standard LB medium. The pathosystem described in this study has been analysed and first published in Grenier et al. 2006, and further detailed in Costechareyre et al. 2011 A two chip study using total RNA recovered from three separate (aphid-, or in vitro-grown) samples