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. 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.

Project description:A large-scale characterization of the methylation states of candidate CpG islands (CGIs) throughout the gastric cancer methylome has not previously been conducted. Genome-wide DNA methylation profiles were compared between 4 metastatic and 4 non-metastatic gastric carcinomas (GCs) and their surgical margins (SMs). The GC genome showed significantly higher proportions of hypomethylation in the promoter and exon-1 regions, as well as increased hypermethylation of intragenic fragments when compared to SMs. Differential methylation was observed in CGIs near transcription start sites of 546 genes between GCs and SMs, and 601 genes between metastatic and non-metastatic GCs. From the list of differentially methylated CGIs, 68 candidate genes and 10 known tumor-related genes were selected for further characterization based on their known molecular function using DHPLC. Significant differential methylation was validated in the CGIs of 15 genes between GCs and SMs (Ps<0.05) and confirmed using bisulfite-sequencing. These genes include BMP3, BNIP3, CDKN2A, ECEL1, ELK1, GFRA1, HOXD10, KCNH1, PSMD10, PTPRT, SIGIRR, SRF, TBX5, TFPI2, and ZNF382. Hypomethylation of CGIs correlated with up-regulation of GFRA1 expression in GCs, while hypermethylation of other genes inactivated their transcription. Most importantly, prevalence of GFRA1, SRF, and ZNF382 methylation alterations were inversely and coordinately associated with GC metastasis and the patients’ overall survival throughout discovery and testing cohorts in China as well as independent validation cohorts in Japan and Korea. In conclusion, methylation changes in the CGIs of 15 genes correlated strongly with GC development. GFRA1 hypomethylation and SRF and ZNF382 hypermethylation are potential synergistic biomarkers for the prediction of GC metastasis. To identify differential methylation of CGIs related to GC development and metastasis, genome-wide DNA methylation changes in 8 pairs of GC and SM samples were analysed using the MCAM assay with a 99K custom-designed Agilent oligonucleotide microarray composed of 99,027 probes targeting 6,177 unique protein-coding genes containing at least two methylation-sensitive/insensitive SmaI/ XmaI restriction sites (CCC|GGG/ C|CmCGGG) as described in Shen et al, PLoS Genet 3, 2023-2036 (2007).

Project description:Cooperia oncophora is an economically important gastrointestinal nematode in ruminants. Acquired resistance to Cooperia oncophora infection in cattle develops rapidly as a result of prior infections. Naïve cattle, when given a primary infection of high-dose infective L3 larvae, develop a strong immunity to subsequent reinfection. Compared to primary infection, reinfection resulted in a marked reduction in worm establishment. In order to understand molecular mechanisms underlying the development of acquired resistance, we characterized the transcriptomic responses of the bovine small intestine to a primary infection and reinfection. A total of 23 pathways were significantly impacted during infection. The vitamin D receptor activation was strongly induced only during reinfection, suggesting that this pathway may play an important role in the development of acquired resistance via its potential roles in immune regulation and intestinal mucosal integrity maintenance. The expression of inducible nitric oxide synthase (NOS2) was strongly induced during reinfection but now during primary infection. As a result, several canonical pathways associated with NOS2 were impacted. The genes involved in eicosanoid synthesis, including prostaglandin synthase 2 (PTGS2 or COX2), remained largely unchanged during infection. The rapid development of acquired resistance may help explain the lack of relative pathogenicity by Cooperia oncophora infection in cattle. Our findings will undoubtedly facilitate understanding of molecular mechanisms underlying the development of acquired resistance, which could have an important implication in vaccine design. The transcriptomic profiles of the bovine small intestine in response to both a primary infection and a drug-attenuated reinfection were compared. The data were analyzed using the same condition and procedure. The gene expression profiles of calves 14 days after a primary Cooperia oncophora infection and a drug-attenuated reinfection were compared to their respective age-matched controls (naive controls and drug-drenched worm-free controls)

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Gene expression profiling of a temperature-sensitive strain of Neospora caninum

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