The CsLOB1 ‐ CsERF027 Regulatory Module Positively Enhances Citrus Target Spot Disease Resistance by Regulating CsRAP2.3‐CsERF1 Cascade

Citrus target spot disease poses significant threats to the citrus industry. However, the molecular mechanisms underlying citrus resistance to Pseudofabraea citricarpa remain unexplored. In this study, we identified the LBD family gene CsLOB1 and investigated its molecular responses to pathogen infection. Following P. citricarpa inoculation, exogenous methyl jasmonate (MeJA) and ethylene (ET), CsLOB1 expression levels in 'Eureka' citrus were significantly higher than in 'Tarocco' blood orange. OE-CsLOB1 transgenic Citrus sinensis exhibited reduced susceptibility to P. citricarpa concomitant with enhanced expression of systemic acquired resistance marker genes (CsPR1, CsPR2 and CsPR4), maintaining reactive oxygen species (ROS) homeostasis and reductions in H₂O₂ and malondialdehyde (MDA) accumulation. Conversely, CsLOB1-RNAi lines exhibited exacerbated oxidative damage that aggravated the severity of citrus disease. Yeast one-hybrid, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays confirmed that CsLOB1 directly binds to the CsRAP2.3 promoter and the heterodimer formation between CsLOB1 and CsERF027 to upregulate its expression. CsRAP2.3 physically interacts with the CsERF1 promoter, activating the key marker gene CsPDF1.2 in the JA/ET-dependent ERF/ORA59 signal pathway. Notably, exogenous MeJA application increased citrus resistance, and P. citricarpa infection significantly increased the content of JA in citrus. Virus-induced gene silencing (VIGS)-mediated silencing of CsRAP2.3, CsERF1 or CsERF027 enhances the citrus disease incidence. Conversely, overexpression of CsRAP2.3, CsERF1 or CsERF027 in transgenic lines conferred higher disease resistance. Mechanistically, this study establishes that CsLOB1 forms a transcriptional complex with CsERF027 through protein-protein interactions, synergistically activating the CsRAP2.3-CsERF1 regulatory cascade. This coordinated regulation ultimately triggers the JA/ET-dependent ERF/ORA59 branch pathway.