GpaR Drives Type III Secretion and Virulence Through Direct Activation of the HrpG-HrpX Cascade in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak of rice, relies on a functional type III secretion system (T3SS) for full virulence and for induction of the hypersensitive response (HR) in nonhost plants. Although the HrpG-HrpX regulatory cascade is central to T3SS gene expression in Xanthomonas, the upstream regulatory mechanisms controlling this pathway in Xoc remain insufficiently understood. In the present study, we identified GpaR as a previously unrecognised transcriptional activator required for T3SS-associated pathogenicity in Xoc. Deletion of gpaR significantly attenuated virulence in rice and resulted in delayed and weakened HR in Nicotiana benthamiana. Reverse transcription-quantitative PCR analysis showed that transcript levels of hrpG, hrpX, and multiple T3SS-associated hrp genes were markedly reduced in the ΔgpaR mutant. Consistent with these results, β-glucuronidase (GUS) reporter assays demonstrated that promoter activities of hrpG and hrpX were substantially decreased in the absence of GpaR. Furthermore, constitutive expression of hrpG or hrpX in the ΔgpaR background largely restored virulence and HR, indicating that these regulators function downstream of GpaR. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) demonstrated that GpaR binds directly to the promoter regions of hrpG and hrpX in vitro and associates with them in vivo. In addition, in vitro transcription assays showed that GpaR directly activates transcription from both promoters. Collectively, these findings establish GpaR as a direct upstream activator of the HrpG-HrpX cascade and identify a new regulatory layer controlling T3SS expression and virulence in Xoc.