Asp 52 and Asp 60 in Paracidovorax citrulli HrpG Are Essential for Transcriptional Activation and Hypersensitive Response Induction

Pathogenic bacteria utilize a type III secretion system (T3SS) to inject type III effectors (T3Es) into plant cells, suppressing plant immunity and facilitating colonization. Paracidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of Cucurbitaceae crops, harbors a functional T3SS like many other plant pathogens. The expression of its T3SS and T3Es is regulated by the two-component system response regulators HrpG and HrpX. Here, we demonstrate that the aspartic acid (Asp) residues at positions 52 and 60 in P. citrulli HrpG are essential for its complete function. Plasmid-mediated complementation of the ΔhrpG mutant with hrpG carrying Asp52→alanine (Ala) or Asp60→Ala mutations failed to restore the ability of P. citrulli to induce a hypersensitive response (HR) in tobacco, whereas the Asp46→Ala mutation fully rescued this phenotype. Furthermore, genomic hrpG point mutations generating strains Aac5 (D52A) and Aac5 (D60A) abolish the activation of hrpX transcription, resulting in decreased HrpX accumulation. Collectively, Asp 52 and Asp 60 in P. citrulli HrpG are essential for transcriptional activation activity of hrpX and HR induction, serving as a potential phosphorylation site (Asp 52) for upstream histidine kinases and a Mg2+ coordination site (Asp 60). Given that conserved Asp residues often function as phosphorylation sites in two-component system response regulators, this study provides a foundation for identifying upstream histidine kinases that modulate HrpG activity in P. citrulli.