2007 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 This report documents research conducted as part of project 1907-21000-027-00D, “Pseudomonas syringae Systems Biology”, initiated February 26, 2007. The focus of the project is on iron-dependent gene regulation. We have completed the characterization of the binding sites for PvdS, one of the key genes involved in transcriptional regulation in response to iron bioavailability in the DC3000 genome, and have constructed a computational model for identifying putative binding sites in related genomes. We have conducted initial genetic experiments on two additional regulators in preparation for detailed investigations. We have also identified global responses to iron depletion using microarrays (see bridging report). We found iron-dependent gene expression of many virulence-related genes and other previously uncharacterized genes with possible regulatory function. The messenger RNA sequencing effort also yielded many candidate genes involved in regulation, including some “small RNAs” that have biological function as RNA species rather than coding for protein. We have confirmed expression for several small RNAs and identified similar genes in related genomes. An essential gene in iron uptake regulation is the ferric uptake regulator, Fur. We have cloned this gene and have isolated and purified this protein in preparation for conducting protein-DNA binding experiments. Integrating sequence and expression information is a challenging aspect of systems biology. We evaluated several database technologies for combining computational predictions, experimental data on gene expression, and textual descriptions of gene function for the entire genome. Finally, we made significant progress towards characterizing the highly repetitive DNA sequences that frequently appear between genes. While the function of these repetitive sequences is not known, we have identified comparable regions in other closely related strains of Pseudomonas syringae. Research is also conducted under the following Specific Cooperative Agreements between ARS and Cornell University: "Gene Regulation in Pseudomonas syringae" 1907-21000-027-05S. Progress this year included the isolation of a mutant in pvdS, a gene encoding a key regulatory protein, and the identification of transcription start sites for seven genes controlled by PvdS. "Modeling Gene Expression in Pseudomonas syringae" 1907-21000-027-04S. Investigations were conducted to study iron-dependent gene regulation in chemostat cultures and to construct a simplified, prototype metabolic model. "Functional Organization of Biomolecular Networks" 1907-21000-017-03S. During the reporting period, computational analyses were employed to identify genomic regions with distinct statistical characteristics. "Genetics of Gene Expression in Pseudomonas syringae" 1907-21000-017-01S. This agreement terminated September 16, 2006 and was inactive during this reporting period. "Global Assessment of Protein Expression in Pseudomonas syringae" 1907-21000-017-02S. This agreement terminated September 29, 2007 and was inactive during this reporting period; Additional details can be found in the specific reports for each of these agreements. 4.Accomplishments Global assessment of gene expression by direct sequencing of messenger RNA. Dramatic improvements in genome sequencing technology have made it feasible to directly capture the entire messenger RNA population in the cell under different environmental conditions. We have used this to enumerate the Pseudomonas syringae genes expressed under iron-limited conditions under culture. This technical advance has led to the discovery of novel genes and may represent cost-effective and sensitive alternative to existing high-throughput technologies such as microarrays. 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 5