ChIP-seq and RNA-seq for RhpR in KB and MM medium

Pseudomonas syringae, the leading cause of crop diseases world-wide, uses type III secretion system (T3SS) to invade host plants. Our previous studies demonstrate that two-component system RhpRS enable P. syringae to coordinate T3SS gene expression, which is dependent on the phosphorylation state of RhpR in different environmental conditions. Homologs of RhpRS are distributed in a wide range of bacterial species, indicating a general regulatory mechanism. However, it remains elusive that how RhpRS relies on external signals and phosphorylation state to exercise its regulatory functions. To this end, we performed ChIP-seq assays to identify specific binding sites of RhpR and RhpRD70A in either King's B (KB, T3SS-inhibiting medium) or minimal medium (MM, T3SS-inducing medium). Through data analysis, we identified 125 KB-dependent binding sites and 188 phosphorylation-dependent binding sites of RhpR. In KB, RhpR directly positively regulated cytochrome c550 production (via ccmA) and alcohol dehydrogenase activity (via adhB), but negatively regulated anthranilate synthase activity (via trpG) and protease activity (via hemB). Besides, phosphorylated RhpR (RhpR-P) directly negatively regulated T3SS (via hrpR and hopR1), swimming motility (via flhA), c-di-GMP level (via PSPPH_2590) and biofilm formation (via algD), while positively regulated twitching motility (via fimA) and lipopolysaccharide production (via PSPPH_2653). Our RNA-seq analyses newly identified 474 and 840 genes that regulated by RhpR in KB and MM respectively. Collectively, the present study revealed that rich nutrient condition allows RhpR to directly regulate the multiple metabolic pathways of P. syringae, while phosphorylation enables RhpR to specifically control virulence and cell envelope. RhpRS governs both virulence and multiple metabolic pathways by tuning its phosphorylation and sensing environmental signals in KB, respectively. Overall design: ChIP-seq for RhpR and RhpR-D70A in KB and MM medium; RNA-seq for RhpS and RhpRS in KB and MM medium