|Buell, C. Robin|
Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/14/2006
Publication Date: 11/1/2006
Citation: Vencato, M., Tian, F., Alfano, J.R., Buell, C., Cartinhour, S.W., Declerck, G.A., Guttman, D.S., Stavrinides, J., Joardar, V., Lindeberg, M., Bronstein, P., Mansfield, J.W., Myers, C.R., Collmer, A., Schneider, D.J. 2006. Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector proteins of Pseudomonas syringae pv. phaseolicola 1448A. Molecular Plant-Microbe Interactions. 19(11):1193-1206. Interpretive Summary: The bacteria Pseudomonas syringae pv. phaseolicola is known to cause halo blight disease on beans. During infection, the type III secretion system (T3SS) is used to secrete proteins from the bacterial cell into the host cell. This process is regulated by HrpL, an alternative sigma factor. Using bioinformatics methods developed for a closely related pathovar, P. syringae pv. tomato DC3000, we identified 45 putative promoters recognized by HrpL in the Pseudomonas syringae pv. phaseolicola 1448A genome. Thirteen of these promoters were tested experimentally and shown to function as HrpL-dependent promoters. The genes downstream of the verified and predicted promoters include the core T3SS infrastructure, 27 candidate effectors with potential virulence function and 10 genes with no apparent relation to the T3SS. Six of the novel candidate effectors were shown to be translocated into host cells in a HrpL-dependent manner. In addition, mutations in two of the non-T3SS related genes lead to reduced virulence on Arabidopsis thaliana. These results establish the utility of the bioinformatic/candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.
Technical Abstract: The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the Hrp type III secretion system (T3SS). To identity genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which was recently sequenced to completion. We identified 45 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related TTSS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time PCR, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Cya (adenylate cyclase from Bordetella pertussis) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function but have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in Arabidopsis leaves. These results establish the utility of the bioinformatic/candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.