Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae

Pseudomonas syringae uses two-component system RhpRS to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanism and the regulons of RhpRS have yet to be fully elucidated. Here we show that RhpS functions as an autokinase, an RhpR kinase, and a P-RhpR phosphatase. RhpR can also be phosphorylated by small phosphodonor acetyl phosphate. A specific RhpR-binding site containing an inverted repeat (IR) motif GTATC-N6-GATAC, was mapped to its own promoter by DNase I footprint analysis. Electrophoretic mobility shift assay (EMSA) indicated that P-RhpR has higher binding activity than RhpR to the IR motif. To identify additional targets of RhpR in P. syringae, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq), which detected 167 enriched loci including the hrpR promoter, indicating the direct regulation of T3SS cascade genes by RhpR. Genome-wide microarray analysis showed that, besides the T3SS cascade genes, RhpR differentially regulates a large set of genes of various functions in response to different growth conditions. Together, these results suggested that RhpRS is a global regulator that allows P. syringae to sense and respond to environmental changes to coordinate the T3SS and many other biological processes. ChIP-seq analysis of RhpR