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

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 ?phcA 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 Overall design: Bacterial mRNA profiles of wild-type and ?phcA mutant were generated by using the Illumina HiSeq2500, yielding ~20 million 1x50bp reads per sample. There were biological replicates per treatment.