Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/25/2011
Publication Date: 12/28/2011
Citation: Filiatrault, M.J., Stodghill, P., Myers, C.R., Bronstein, P.A., Butcher, B.G., Lam, H., Grills, G., Schweitzer, P., Wang, W., Schneider, D.J., Cartinhour, S.W. 2011. Genome-wide identification of transcriptional start sites in the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. PLoS One. 6(12). Available: http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029335. Interpretive Summary: Bacteria transcribe genomic DNA into RNA or transcripts. Locating where the synthesis of RNA begins is a difficult task. Since transcription is tightly regulated, determining the locations in the DNA that correspond to the start of transcription can provide important insight into how bacteria respond to their environment and cause disease. Here, we developed a method to specifically locate this site and sequenced the molecules using ultra-high-throughput sequencing technology. We then accurately identified the first bases representing the beginning of the transcripts and mapped them to their corresponding genomic location. We applied this method to the RNA of Pseudomonas syringae DC3000, producing the first large-scale inventory of RNA start sites in this important plant pathogen. We discovered over 1200 start sites and further computational analyses allowed for the assignment of putative regulatory regions to many of these transcripts. Not only has this study contributed to the annotation of the P. syringae genome, but it has also provided immense insight into how the expression of a large number of genes may be controlled in P. syringae.
Technical Abstract: RNA-Seq has provided valuable insights into global gene expression in a number of organisms. Using a modified RNA-Seq approach and Illumina’s high-throughput sequencing technology, we globally identified 5’-ends of transcripts for the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. A substantial fraction of 5’-ends obtained by this method were consistent with results obtained using global RNA-Seq and 5’RACE. As expected, many 5’-ends are positioned a short distance upstream of annotated genes. We also captured 5’-ends within intergenic regions, providing evidence for the expression of un-annotated genes and non-coding RNAs, and detected numerous examples of antisense transcription, demonstrating additional levels of complexity in gene regulation in DC3000. Importantly, targeted searches for sequence patterns in the vicinity of 5’-ends revealed over 1200 putative promoters and other regulatory motifs, establishing a broad foundation for future investigations of regulation at the genomic and single gene levels.