2008 Annual Report 1a.Objectives (from AD-416) Identify promoters and ECF sigma factors that control expression of known and suspected virulence factors. Characterize the subset of the transcriptome related to growth in defined medium, the induction of virulence factors, and response to iron bioavailability. Elucidate mechanisms leading to iron-dependent expression of operons encoding virulence factors and regulators. 1b.Approach (from AD-416) Research will employ an interdisciplinary approach involving computational biology and laboratory methods for high-throughput functional genomics and genetics. Expression studies including the use of microarrays and high-throughput reporter screens will be used to characterize the components and behavior of virulence-related pathways, especially those related to iron homeostasis. Mutants in key regulatory proteins and gene reporter systems will be used to elucidate regulatory interactions. Computational methods will be used to identify regulatory motifs, detect statistically significant correlations in gene expression, and model selected pathways. We aggressively integrate laboratory and computational approaches to genome-scale problems in order to design and implement the most effective experiments and analytical methods. 3.Progress Report Significant advances were made in several areas. First, the availability of complete genome sequences for three P. syringae strains and an expanded collection of other closely related pseudomonads made it possible to develop a high-resolution map of core and variable regions of the P. syringae DC3000 genome. Many virulence-related genes are located in variable regions, suggesting that horizontal gene transfer is an important mechanism in the evolution of pathogenic capacity. Second, the sequencing-based strategy for characterization gene expression patterns was extended by the development and validation of a strand-specific protocol. A high-coverage transcript map was produced from RNA isolated from bacteria grown in iron-limited conditions. This transcript map was used to identify putative start sites for over 200 transcriptional units in the genome, 31 of which have been validated by conventional techniques. Careful manual analysis of these data, in conjunction with comparative genomics methods, resulted in the prediction or preliminary confirmation of over 60 additional putative sRNA genes and riboswitches. Computational methods were used to identify promoters or repressor binding sites immediately upstream of a subset of these genes. Many of these genes and associated regulatory motifs are also present in syntenic regions in other Pseudomonas spp. of agricultural interest (e.g., P. fluorescens Pf-5). Finally, a significant portion of the iron-responsive genes identified in microarray experiments were assigned to specific regulatory pathways controlled by sigma factors (HrpL, PvdS, and RpoD) or the high-level regulators (e.g., Fur). Where data exist, these putative promoters are consistent with the global transcript map obtained under comparable conditions. The research addresses the Action Plan for National Program 303: Plant Diseases, Component 2: Biology, Ecology, Epidemiology and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors and Problem Statement 2A: Pathogen Biology, Virulence Determinants and Genetics of the Pathogen. 4.Accomplishments 1. High-throughput RNA sequencing strategies for direct characterization of gene expression. A detailed analysis of gene expression requires the ability to associate every RNA in the cell with its corresponding gene. To address this need, we developed a new RNA sequencing protocol in collaboration with Illumina, Inc. The sequence data were analyzed using novel statistical procedures and custom software and was carefully validated by other experiments. The final result is a “map” that shows the RNA levels corresponding to every position in the genome and provides a global view of the biochemical activities in the cell. The new method is applicable to any other bacterial species that can be grown in the laboratory, including many pathogens of plants and animals, and will help identify genes that are important in the disease process. The research addresses the Action Plan for National Program 303: Plant Diseases, Component 2: Biology, Ecology, Epidemiology and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors and Problem Statement 2A: Pathogen Biology, Virulence Determinants and Genetics of the Pathogen. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings 7 Review Publications Lindeberg, M., Myers, C., Collmer, A., Schneider, D.J. 2008. Roadmap to new virulence determinants in Pseudomonas syringae: Insights from Comparative Genomics and Genome Organization. Molecular Plant-Microbe Interactions. 21:685-700. Swingle, B.M., Thete, D., Moll, M., Myers, C., Schneider, D.J., Cartinhour, S.W. 2008. Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato DC3000 reveals regulon members and insights regarding PvdS function in other pseudomonads. Molecular Microbiology. 68(4):871-889. Munkvold, K.R., Martin, M.E., Bronstein, P., Collmer, A. 2008. A survey of the Pseudomonas syringae pv. tomato DC3000 type III secretion system effector repertoire reveals several effectors that are deleterious when expressed in Saccharomyces cerevisiae. Molecular Plant-Microbe Interactions. 21(4):490-502.