Leaf and flower metagenome
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA472855 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7503221_1.fastq.gz | Fastqsanger.gz | |||
| SRR7503221_2.fastq.gz | Fastqsanger.gz | |||
| SRR7503222_1.fastq.gz | Fastqsanger.gz | |||
| SRR7503222_2.fastq.gz | Fastqsanger.gz | |||
| SRR7503223_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 16 |
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Project description:Analysis of gene expression in Arabidopsis thaliana leaf, stem and flower tissues. Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5 inch square pot either in a growth chamber or a green house set to 25*C by day, 20*C by night. Days were set to a 16hr photoperiod with 125 micro mol m-2s-1 fluorescent irradiation. Expanding leaves were harvested 15 days post germination in the middle of the photoperiod (3 replicates). Expanding upper 2" of the stem with siliques and pedicels removed were harvested 29 days post germination in the middle of the photoperiod (4 replicates). Developed flowers and unopened buds were harvested 29 days post germination in the middle of the photoperiod (4 replicates). RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method (Invitrogen, Ramonell et al. (2002) Mol. Plant Pathol. 3: 301) and purified with a silica membrane column (Qiagen, RNeasy). Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer's protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying signal intensities by ratio figure which set GapC (ID_REF = 258588_s_at) to 8500. Keywords: other
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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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Project description:Analysis of gene expression in Arabidopsis thaliana leaf, stem and flower tissues.,Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5 inch square pot either in a growth chamber or a green house set to 25*C by day, 20*C by night. Days were set to a 16hr photoperiod with 125 micro mol m-2s-1 fluorescent irradiation.,Expanding leaves were harvested 15 days post germination in the middle of the photoperiod (3 replicates).,Expanding upper 2 inch of the stem with siliques and pedicels removed were harvested 29 days post germination in the middle of the photoperiod (4 replicates).,Developed flowers and unopened buds were harvested 29 days post germination in the middle of the photoperiod (4 replicates).,RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method (Invitrogen, Ramonell et al. (2002) Mol. Plant Pathol. 3: 301) and purified with a silica membrane column (Qiagen, RNeasy). Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer's protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying signal intensities by ratio figure which set GapC (ID_REF = 258588_s_at) to 8500.
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