HrpW Modulates Paracidovorax citrulli Virulence and Plant Immunity via ClRAR1 Interaction in Watermelon.

Bacterial fruit blotch (BFB), caused by Paracidovorax citrulli, severely threatens watermelon production. This study investigates the role of HrpW, an atypical harpin in P. citrulli AAC00-1, in bacterial virulence and host immune modulation. Bioinformatics analysis revealed HrpW harbours a unique signal peptide and structural features distinct from other harpins. Deletion of hrpW impaired bacterial motility, biofilm formation and virulence, while complementation restored these traits. HrpW suppressed reactive oxygen species (ROS) bursts and mitogen-activated protein kinase (MAPK) activation in plants but failed to induce programmed cell death (PCD). Crucially, HrpW inhibited the hypersensitive response (HR) triggered by P. citrulli in non-host tobacco, with ΔhrpW mutant inducing premature HR. RNA-seq analysis demonstrated HrpW downregulated ClRAR1 expression. Silencing ClRAR1 or ClSGT1 compromised watermelon resistance, and notably, ClRAR1-silenced plants exhibited PCD upon HrpW treatment, indicating HrpW-mediated PCD suppression is RAR1-dependent. Importantly, HrpW triggered ubiquitin-dependent degradation of ClRAR1 and independently disrupted ClRAR1-ClSGT1 binding, thereby suppressing effector-triggered immunity (ETI). HrpW translocated into plant cells via the type III secretion system (T3SS), as confirmed by CyaA assays. Intriguingly, low concentrations of HrpW enhanced watermelon resistance to BFB, while high concentrations promoted disease progression, revealing a concentration-dependent duality. This study unveils HrpW as a multifunctional virulence factor that modulates bacterial fitness, suppresses HR and manipulates host immunity via RAR1 targeting. These findings expand our understanding of harpin-mediated pathogenicity and offer insights for sustainable BFB management strategies.