A genome-wide and transcriptome-integrated identification of CsWRKY40 as a candidate regulator associated with B. subtilis-mediated reduction of anthracnose symptoms in sweet orange

Sweet orange ( Citrus sinensis ) is an important commercial fruit tree, and anthracnose associated with Colletotrichum spp. severely affects yield and fruit quality. Bacillus subtilis is an important biocontrol bacterium with the potential to suppress pathogens and induce host defense; however, the host transcriptional mechanisms associated with B. subtilis -related reduction of anthracnose symptoms in sweet orange remain unclear. In this study, 55 CsWRKY family members were identified from the sweet orange reference genome DVS_A1.0. By integrating anthracnose infection and B. subtilis intervention treatments, we systematically analyzed the structural characteristics, expression responses, and candidate regulatory networks of the CsWRKY family. Quantitative phenotypic analysis showed that leaf lesion diameter was 8.72 ± 0.84 mm in the BT group and 5.47 ± 0.66 mm in the BST group, corresponding to a 37.3% reduction after B. subtilis treatment. Transcriptomic analysis identified 28 differentially expressed CsWRKY genes in BT vs CK and 27 in BST vs BT, indicating extensive WRKY involvement in transcriptional reprogramming during anthracnose infection and biocontrol intervention. Integrating expression patterns, predicted PPI network analysis, and RT-qPCR validation prioritized CsWRKY40 as a candidate regulatory node; its expression was downregulated under BT treatment and restored/upregulated under BST treatment. CsWRKY40 contained multiple potential phosphorylation sites predicted by NetPhos 3.1. A genome-wide promoter scan integrated with differential expression analysis identified 5143 W-box-containing candidates responsive in BT vs CK and/or BST vs BT. Integrated GO, KEGG/KO, NR, Swiss-Prot, and protein-domain annotations further classified 1736 as defense-associated candidates involved in ROS/redox homeostasis, defense responses, hormone and MAPK signaling, cell wall remodeling, secondary metabolism, and pathogenesis-related functions. These findings support CsWRKY40 as a candidate regulatory node associated with post-infection B. subtilis treatment and the accompanying reduction of anthracnose symptoms in sweet orange, providing a basis for future functional validation and disease-resistance research. Not applicable.