Project description:Some strains of the foliar pathogen Pseudomonas syringae are adapted for growth and survival on leaf surfaces and in the leaf interior. Global transcriptome profiling was used to evaluate if these two habitats offer distinct environments for bacteria and thus present distinct driving forces for adaptation. Further transcriptome profiling was performed to understand the various environmental conditions that P. syringae cells encounter during their association with plants. RNA was collected from P. syringae pv. syringae strain B728a cells that were exposed to seven treatments. The treatments included five in vitro treatments, namely exposing cells to a basal medium, sodium chloride to confer an osmotic stress, hydrogen peroxide to confer an apoplastic growth, iron limitation, nitrogen limitation. They also included two in planta treatments, namely recovering cells from epiphytic sites after surface inoculation and 72 h of growth on bean (Phaseolus vulgaris L.) leaves and recovering cells from apoplastic sites after infiltration and 48 h of growth in bean leaves. The results suggested that B728a cells experience vastly different environments when growing on the surface versus the interior of leaves and identified distinct traits that are likely used for persistence and growth in these environments. RNA was collected from B728a cells that were exposed to seven treatments, each with two biological replicates in each of three laboratories. Experimental methods were standardized across the three laboratories. For the in vitro treatments, exponential cells from two independent cultures were each exposed to the five treatments. For each treatment, the cells originating from the two cultures were pooled and the RNA was extracted; this was repeated in its entirety and the two RNA pools were combined. The resulting RNA representing four independent cultures served as a single biological replicate. Two biological replicates for each treatment were generated in this manner in each of three separate laboratories. Epiphytic B728a populations were established following spray inoculation onto bean leaves and subsequent incubation; the epiphytic cells recovered from 400 to 600 leaves inoculated at one time served as a biological replicate, and the procedure was repeated to provide two biological replicates. Due to the availability of facilities, all of the epiphytic treatments were conducted at as single laboratory, with cultures provided from each of the other laboratories, and the cell pellets were returned to those laboratories for RNA extraction and analysis. Apoplastic B728a populations were established following vacuum infiltration into bean leaves and subsequent incubation; the apoplastic cells recovered from 40 to 80 leaves inoculated at one time served as a biological replicate, and two biological replicates were generated at each of the three laboratories. The RNA from all treatments was submitted to Roche Nimblegen, Inc where it was labeled and hybridized to an ORF-based microarray that included 5,071 ORFs and 61 putative sRNAs, with each ORF represented by 14 60-mer nucleotide probes. The fluorescence intensity for each probe was measured and subjected to robust multiarray averaging, which included adjustment for the background intensity, log2 transformation, quantile normalization and median polishing, and a robust estimated mean value was determined for each ORF and putative sRNA on the array.

Project description:The plant pathogen Pseudomonas syringae pv. syringae B728a grows and survives on leaf surfaces and in the leaf apoplast of its host, bean. Global transcriptome profiling was used to understand the contribution of distinct regulators to B728a fitness and pathogenicity. We performed a transcriptome analysis of B728a and nine regulator mutants recovered from the surface and interior of leaves and exposed to various environmental stresses in culture. These mutants were nonpolar deletion mutants lacking a single target regulator gene: ahlR, aefR, gacS, retS, salA, rpoS, algU, hrpL and rpoN. RNA was collected from cells of these strains that were exposed to five treatments in vitro, namely a basal medium, sodium chloride to confer an osmotic stress, hydrogen peroxide to confer an oxidative stress, iron limitation, and nitrogen limitation, and were recovered from two bean (Phaseolus vulgaris L.) leaf sites, namely epiphytic sites after leaf surface inoculation and 72 h of growth and apoplastic sites after leaf infiltration and 48 h of growth. The results indicated that the quorum-sensing regulators AhlR and AefR influenced few genes in planta or in vitro. In contrast, GacS and a downstream regulator SalA formed a large regulatory network that included a branch that regulated diverse traits and was independent of plant-specific environmental signals, and a signal-dependent branch that positively regulated secondary metabolite genes and negatively regulated the type III secretion system. RetS functioned almost exclusively to repress secondary metabolite genes in cells in culture but not on leaves. SalA functioned as a central regulator of iron status, based on its reciprocal regulation of pyoverdine and achromobactin genes, and also sulfur uptake, suggesting a role in the iron-sulfur balance. Among the sigma factors examined, AlgU influenced many more genes than RpoS, and most AlgU-regulated genes depended on RpoN. RpoN differentially impacted many AlgU- and GacS-activated genes in cells recovered from apoplastic versus epiphytic sites, suggesting differences in environmental signals or bacterial stress status in these two habitats. Collectively, our findings illustrate a central role for GacS, SalA, RpoN and AlgU in global regulation in B728a in planta and a high level of plasticity in their response to distinct environmental signals.

Project description:The plant pathogen Pseudomonas syringae pv. syringae B728a grows and survives on leaf surfaces and in the leaf apoplast of its host, bean. Global transcriptome profiling was used to understand the contribution of distinct regulators to B728a fitness and pathogenicity. We performed a transcriptome analysis of B728a and nine regulator mutants recovered from the surface and interior of leaves and exposed to various environmental stresses in culture. These mutants were nonpolar deletion mutants lacking a single target regulator gene: ahlR, aefR, gacS, retS, salA, rpoS, algU, hrpL and rpoN. RNA was collected from cells of these strains that were exposed to five treatments in vitro, namely a basal medium, sodium chloride to confer an osmotic stress, hydrogen peroxide to confer an oxidative stress, iron limitation, and nitrogen limitation, and were recovered from two bean (Phaseolus vulgaris L.) leaf sites, namely epiphytic sites after leaf surface inoculation and 72 h of growth and apoplastic sites after leaf infiltration and 48 h of growth. The results indicated that the quorum-sensing regulators AhlR and AefR influenced few genes in planta or in vitro. In contrast, GacS and a downstream regulator SalA formed a large regulatory network that included a branch that regulated diverse traits and was independent of plant-specific environmental signals, and a signal-dependent branch that positively regulated secondary metabolite genes and negatively regulated the type III secretion system. RetS functioned almost exclusively to repress secondary metabolite genes in cells in culture but not on leaves. SalA functioned as a central regulator of iron status, based on its reciprocal regulation of pyoverdine and achromobactin genes, and also sulfur uptake, suggesting a role in the iron-sulfur balance. Among the sigma factors examined, AlgU influenced many more genes than RpoS, and most AlgU-regulated genes depended on RpoN. RpoN differentially impacted many AlgU- and GacS-activated genes in cells recovered from apoplastic versus epiphytic sites, suggesting differences in environmental signals or bacterial stress status in these two habitats. Collectively, our findings illustrate a central role for GacS, SalA, RpoN and AlgU in global regulation in B728a in planta and a high level of plasticity in their response to distinct environmental signals. RNA was collected from cells of B728a and nine regulator mutants following exposure to seven treatments. Each treatment was performed using two biological replicates, with B728a, M-bM-^HM-^FrpoS, M-bM-^HM-^FalgU, M-bM-^HM-^FhrpL and M-bM-^HM-^FrpoN examined in one laboratory, designated lab A, B728a, M-bM-^HM-^FahlR and M-bM-^HM-^FaefR examined in lab B, and B728a, M-bM-^HM-^FretS, M-bM-^HM-^FgacS and M-bM-^HM-^FsalA examined in lab C. Experimental methods were standardized across the three laboratories. For the in vitro treatments, exponential cells from two independent cultures were each exposed to the five treatments. For each treatment, the cells originating from the two cultures were pooled and the RNA was extracted; this was repeated in its entirety and the two RNA pools were combined. The resulting RNA representing four independent cultures served as a single biological replicate. Two biological replicates for each strain in each treatment were generated in this manner, with B728a included as a strain in each of the laboratories. Epiphytic populations of B728a and the nine regulator mutants were established following spray inoculation onto bean leaves and subsequent incubation; the epiphytic cells recovered from 400 to 600 leaves inoculated at one time served as a biological replicate, and the procedure was repeated to provide two biological replicates. Due to the availability of facilities, all of the epiphytic treatments were conducted at a single laboratory, lab B, with cultures provided from each of the other laboratories, and the cell pellets were returned to those laboratories for RNA extraction and analysis. Apoplastic B728a populations were established following vacuum infiltration into bean leaves and subsequent incubation; the apoplastic cells recovered from 40 to 80 leaves inoculated at one time served as a biological replicate, and two biological replicates were generated at each of the three laboratories. The RNA from all treatments was submitted to Roche Nimblegen, Inc where it was labeled and hybridized to an ORF-based microarray that included 5,071 ORFs and 61 putative sRNAs, with each ORF represented by 14 60-mer nucleotide probes. The fluorescence intensity for each probe was measured and subjected to robust multiarray averaging, which included adjustment for the background intensity, log2 transformation, quantile normalization and median polishing, and a robust estimated mean value was determined for each ORF and putative sRNA on the array.

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Pseudomonas syringae B728a responses to environmental stresses and growth in and on bean leaves

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