Broad-Complex Mediates 20-Hydroxyecdysone-Induced Antimicrobial Peptide Expression in the Silkworm Bombyx mori

The steroid hormone 20-hydroxyecdysone (20E) is a key regulator of insect development and has recently emerged as an important modulator of innate immunity. However, the molecular mechanism by which 20E regulates antimicrobial peptide (AMP) expression in lepidopteran insects remains poorly understood. Here, we investigated the role of the 20E signaling pathway in antibacterial immunity in the silkworm Bombyx mori. Pretreatment with 20E significantly increased larval survival following infection with both Escherichia coli and Bacillus bombyseptieus and accelerated bacterial clearance in vivo. Consistently, 20E rapidly induced the expression of multiple AMP genes in the fat body in a dose- and time-dependent manner. Promoter analysis revealed that Broad-Complex (BrC) binding motifs were highly enriched in most AMP promoters. Functional analyses demonstrated that BrC-Z isoforms selectively activated different AMP promoters, with Leb3 showing the strongest response. Site-directed mutagenesis and electrophoretic mobility shift assays confirmed that BrC-Z directly binds to the Leb3 promoter to activate transcription. We further identified a previously uncharacterized splice variant, BrC-N, which lacks the zinc-finger DNA-binding domain and cannot activate transcription independently but markedly enhances BrC-Z-dependent Leb3 activation, likely through BTB/POZ-mediated protein interactions. Together, these findings establish a 20E-BrC-AMP regulatory axis underlying silkworm antibacterial immunity and reveal a previously unknown mechanism by which a noncanonical BrC isoform potentiates hormone-dependent immune gene expression.