Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/2/2006
Publication Date: 11/1/2006
Citation: Fereirra, A.O., Myers, C.R., Gordon, J., Martin, G.B., Vencato, M., Collmer, A., Wehling, M.D., Alfano, J.R., Moreno-Hagelsieb, G., Lamboy, W.F., Declerck, G.A., Schneider, D.J., Cartinhour, S.W. 2006. Whole-genome expression profiling defines the hrpl regulon of pseudomonas syringae pv. tomato dc3000, allows de novo reconstruction of the hrp cis element, and identifies novel co-regulated genes. Molecular Plant-Microbe Interactions. 19(11):1167-1179. Interpretive Summary: Pseudomonas syringae pv. tomato DC3000 is a model bacterial pathogen of tomato and Arabidopsis that deploys a type III secretion system (a highly sophisticated secretion system used to introduce specific bacterial proteins called effectors into the plant host cells) to disrupt plant cell defenses during pathogenesis. Accordingly, elucidation of the regulatory mechanisms in the bacteria that control the expression of the type III secretion system and associated effector proteins is a prerequisite for understanding the molecular basis of plant pathogenesis. In this study, we describe an integrated computational-experimental approach to identifying all genes coordinately regulated with the type III secretion system and associated effectors. In particular, whole genome microarray experiments were conducted to identify genes that are differentially expressed in the presence of HrpL, a so-called sigma factor that was previously known to control the expression of numerous effectors. Gibbs sampling and hidden Markov models were used, respectively, to identify the DNA motif recognized by HrpL from the regions upstream of differentially expressed genes and to use these sequences to construct a sensitive method for searching for similar sequences throughout the P. syringae DC3000 genome. Discrepancies were resolved by further experimental testing using real time PCR and additional sequence analysis to identify putative unannotated genes. This work represents two major advances. First, an essential complete inventory of the genes involved in type III secretion now available. Second, the resulting model can be used to identify HrpL regulated genes in other pathovars of P. syringae.
Technical Abstract: Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a Hrp (hypersensitive response and pathogenicity) type III secretion system (TTSS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an ORF-specific whole genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and hrpL knockout strains. Hierarchical clustering of the expression patterns revealed a subset of genes that were up-regulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated operons revealed the Hrp box as the only identifiable regulatory motif and facilitated an interative refinement process involving real-time PCR testing of additional candidate HrpL-activated genes. This iterative bioinformatic-experimental approach to a comprehensive analysis of the Hrp regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in uncharacterized biosynthetic or metabolic pathways. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that are likely important to the host-specific virulence of P. syringae.