Citrus PR4A is involved in the defense responses against Xanthomonas citri subsp. citri.

1. Overexpression of CmPR4A improved resistance to Xanthomonas citri subsp. citri ( Xcc ) in sweet orange and resistance to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis . 2. The major effector pthA4 secreted by Xcc interacts with CmWRKY75, a positive regulator of CmPR4A , and up-regulates CmPR4A expression in Citron C-05 leaves. 3. CmSMU2 interacts with CmPR4A to increase ROS accumulation, thereby enhancing resistance to Xcc in citrus. Citrus canker, caused by Xanthomonas citri subsp. citri ( Xcc ), is a globally quarantine disease that infect nearly all Citrus cultivars. Remarkably, Citron C-05 has been identified with complete and active resistance to Xcc . However, the mechanism underlying Citron C-05’s resistance to Xcc remains elusive. In this study, we identified a gene cluster on the chromosome 8 of citrus genome comprising five pathogenesis-related 4-like genes. PR4A was up-regulated in Citron C-05 leaves under Xcc infection, exhibiting the highest expression among these PR4-like genes. In addition, PR4A expression was higher in leaves of disease-resistant genotypes compared to susceptible genotypes under Xcc invasion. Bimolecular fluorescence complementation (BiFC) and Split-Luc assays indicated that CmWRKY75, a <pg=>positive regulator of PR4A , interacted with pthA4 and up-regulated expression of PR4A in Citron C-05 leaves. Segmentation of the CmPR4A promoter revealed that P CmPR4A -P -516 may be involved in regulating PR4A expression. Transient overexpression of CmPR4A improved resistance to Xcc in sweet orange, and three transgenic lines of OE-CmPR4A exhibited resistance to Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000) in Arabidopsis . Furthermore, CmSMU2 was identified through yeast two-hybrid library using CmPR4A as bait, BiFC and Split-Luc assays further verified their interaction. Transient overexpression of CmSMU2 in the sweet orange increased resistance to Xcc . Co-expression of CmSMU2 and CmPR4A enhanced accumulation of reactive oxygen species compared to CmSMU2 or CmPR4A , indicating that they may synergistically enhance resistance to Xcc in citrus. These findings lay the groundwork for a theoretical analysis of the mechanism underlying the resistance of Citron C-05 against citrus canker.