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Comparing gene expression profiles of wild-type and the ∆phcA mutant strains of Ralstonia solanacearum during tomato colonization

ABSTRACT: Purpose: To comprehensively identify traits controlled by the PhcA quorum sensing system, and to better understand how this regulator affects pathogen behavior in its natural host environment Method: 21 day-old tomato plants were inoculated with approximately 2000 cells of either wild-type Rs strain GMI1000 or ΔphcA through a cut petiole. After 72 h in a 28°C growth chamber (12h day/night cycle), 0.1 g of stem tissue spanning the petiole was harvested and transferred immediately to a tube containing 900 µl of pre-chilled transcription stop solution (5% [vol/vol] water-saturated phenol in ethanol) and ground using a Powerlyzer. RNA was isolated from these ground samples using hot phenol method. Total RNA was treated with RiboZero kits to reduce bacterial and plant rRNA, followed by tomato mRNA reduction using polyA selection and used to make libraries with the Illumina TruSeq strand-specific mRNA sample preparation system. After RNA fragmentation, strand-specific libraries were constructed by first-strand cDNA synthesis using random primers, sample cleanup and second-strand synthesis using DNA Polymerase I and RNase H. A single 'A' base was added to the cDNA fragments followed by ligation of the adapters. The final cDNA library was further purified and enriched with PCR, and quality checked using the Bioanalyzer 2100. The libraries were sequenced using the Illumina HiSeq2500, yielding ~20 million 1x50bp reads per sample Conclusion: Defining the PhcA in planta regulon identified traits that make R. solanacearum successful at high cell density during bacterial wilt disease, but also identified traits that help it survive at low cell densities

ORGANISM(S): Ralstonia solanacearum

PROVIDER: GSE98074 | GEO | 2017/09/08

SECONDARY ACCESSION(S): PRJNA383862

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study is to complare the transcitpional profiles of freshly isolated, splenic, murin GCBC and other activated and naive non-germinal center B cell populations. Methods: Cells were freshly isolated, bead purified and FACS Ari sorted . Sorted cells (1.5-3x106 cells per sample) were washed twice in cold PBS and RNA was prepared using shredder-columns (Qiagen; QIAshredder 79656) and the RNeasy Plus Mini kit (Qiagen) following the manufacturer’s instructions. 3 independent RNA libraries per cell population were prepared using Illumina TruSeq Stranded mRNA sample preparation kit. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by using dUTP in the Second Strand Marking Mix, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The products are then purified and enriched with PCR to create the final cDNA library. The cDNA libraries are validated using KAPA Biosystems primer premix kit with Illumina-compatible DNA primers and Qubit 2.0 fluorometer. Quality is examined using Agilent Tapestation 2200.The cDNA libraries were pooled at a final concentration 1.8pM. Samples were sequenced using Illumina NextSeq 500 with 75bp paired-end reads and aligned to the mm10 genome using the STAR aligner. The number of uniquely aligned reads ranged from 31 to 77 million. Using an optimized data analysis workflow, Gene-level counts were determined using featureCounts and raw counts were analyzed for differential expression using the “voom” method in the “limma” R package. Results: After determining genes that were differentially expressed between splenic GCBC and activated B cells, we performed gene set enrichment analysis (GSEA). Differentially expressed genes were compared to several previously defined hypoxic gene signatures.

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Comparing gene expression profiles of wild-type and the ∆phcA mutant strains of Ralstonia solanacearum during tomato colonization

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