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 PCmPR4A-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.
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