The bacterial pathogens Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc) cause leaf blight and leaf streak diseases on rice, respectively. Pathogenesis is largely defined by the virulence genes harboured in the pathogen genome. Recently, we demonstrated that the protein HpaP of the crucifer pathogen Xanthomonas campestris pv. campestris is an enzyme with both ATPase and phosphatase activities, and is involved in regulating the synthesis of virulence factors and the induction of the hypersensitive response (HR). In this study, we investigated the role of HpaP homologues in Xoo and Xoc. We showed that HpaP is required for full virulence of Xoo and Xoc. Deletion of hpaP in Xoo and Xoc led to a reduction in virulence and alteration in the production of virulence factors, including extracellular polysaccharide and cell motility. Comparative transcriptomics and reverse transcription‐quantitative PCR assays revealed that in XVM2 medium, a mimic medium of the plant environment, the expression levels of hrp genes (for HR and pathogenicity) were enhanced in the Xoo hpaP deletion mutant compared to the wild type. By contrast, in the same growth conditions, hrp gene expression was decreased in the Xoc hpaP deletion mutant compared to the wild type. However, an opposite expression pattern was observed when the pathogens grew in planta, where the expression of hrp genes was reduced in the Xoo hpaP mutant but increased in the Xoc hpaP mutant. These findings indicate that HpaP plays a divergent role in Xoo and Xoc, which may lead to the different infection strategies employed by these two pathogens.

VemR is a response regulator of the two‐component signalling systems (TCSs). It consists solely of a receiver domain. Previous studies have shown that VemR plays an important role in influencing the production of exopolysaccharides and exoenzymes, cell motility, and virulence of Xanthomonas campestris pv. campestris (Xcc). However, whether VemR is involved in the essential pathogenicity determinant type III secretion system (T3SS) is unclear. In this work, we found by transcriptome analysis that VemR modulates about 10% of Xcc genes, which are involved in various cellular processes including the T3SS. Further experiments revealed that VemR physically interacts with numerous proteins, including the TCS sensor kinases HpaS and RavA, and the TCS response regulator HrpG, which directly activates the transcription of HrpX, a key regulator controlling T3SS expression. It has been demonstrated previously that HpaS composes a TCS with HrpG or VemR to control the expression of T3SS or swimming motility, while RavA and VemR form a TCS to control the expression of flagellar genes. Mutation analysis and in vitro transcription assay revealed that phosphorylation might be essential for the function of VemR and phosphorylated VemR could significantly enhance the activation of hrpX transcription by HrpG. We infer that the binding of VemR to HrpG can modulate the activity of HrpG to the hrpX promoter, thereby enhancing hrpX transcription. Although further studies are required to validate this inference and explore the detailed functional mechanism of VemR, our findings provide some insights into the complex regulatory cascade of the HpaS/RavA‐VemR/HrpG‐HrpX signal transduction system in the control of T3SS.