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Bioinformatics-Enabled Identification of the HrpL Regulon and Type III Secretion System Effector Proteins of Pseudomonas syringae pv. phaseolicola 1448A

¹Department of Plant Pathology, Cornell University, Ithaca, NY 14853, U.S.A.; ²The Plant Science Initiative and the Department of Plant Pathology, University of Nebraska-Lincoln, 68588, U.S.A.; ³The Institute for Genomic Research, 9712 Medical Center Dr., Rockville, MD 20850, U.S.A.; ⁴United States Department of Agriculture-Agricultural Research Service, Ithaca, NY 14853, U.S.A.; ⁵Department of Botany, University of Toronto, 25 Willcocks St., Toronto, ON M5S 3B2 Canada; ⁶Agricultural Sciences Department, Imperial College at Wye, Ashford, Kent TN25 5AH, United Kingdom; ⁷Cornell Theory Center, Cornell University, Ithaca, NY 14853, U.S.A.

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 hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify 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 recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, 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 Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do 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 host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.

Additional keywords: Avr/Hop proteins .