The type III secretion system (T3SS) is the main machinery for Pseudomonas savastanoi and other gram-negative bacteria to invade plant cells. HrpR and HrpS form a hetero-hexamer, which activates the expression of HrpL, which induces all T3SS genes by binding to a 'hrp box' in promoters. However, the individual molecular mechanism of HrpR or HrpS has not been fully understood. Through chromatin immunoprecipitation coupled to high-throughput DNA sequencing, we found that HrpR, HrpS, and HrpL had four, 47, and 31 targets on the genome, respectively. HrpS directly bound to the promoter regions of a group of T3SS genes and non-T3SS genes. HrpS independently regulated these genes in a hrpL deletion strain. Additionally, a HrpS-binding motif (GTGCCAAA) was identified, which was verified by electrophoretic mobility shift assay and lux-reporter assay. HrpS also regulated motility and biofilm formation in P. savastanoi. The present study strongly suggests that HrpS alone can work as a global regulator on both T3SS and non-T3SS genes in P. savastanoi. [Formula: see text] Copyright © 2018 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .