Identification and functional characterization of Of-FADD in Ostrinia furnacalis infected with bacteria

Fas-associated protein with death domain (FADD) is a crucial adaptor protein in the insect Immune Deficiency (IMD) signaling pathway. Although its role in regulating antimicrobial peptide (AMP) synthesis has been established in Drosophila, its function in Ostrinia furnacalis remains unclear. In this study, we identified a FADD (designated Of-FADD) with a full-length cDNA of 971 bp, containing a 666 bp open reading frame encoding a 221-residue protein with a characteristic death effector domain (DED) and a death domain (DD). Expression profiling showed the highest transcript levels in fifth-instar larvae, with significant enrichment in the midgut and hemocytes. Bacterial infection experiments demonstrated that both Escherichia coli and Staphylococcus aureus significantly induced Of-FADD expression, with peak upregulation observed at 12 h and 24 h post-infection, respectively. Concurrently, the expression of Relish and AMP genes was also significantly upregulated. RNA interference (RNAi) revealed that knockdown of Of-FADD significantly downregulated the expression of Relish and AMP genes. Following RNAi and subsequent bacterial infection, Of-FADD expression exhibited a compensatory response. Subcellular localization analysis indicated that Of-FADD is distributed in the cytoplasm. Of-FADD can form oligomers but does not assemble into amyloid fibril structures independently under the tested conditions. This study provides transcript-level evidence in a lepidopteran pest that Of-FADD positively modulates AMP-mediated immune responses, likely via the IMD signaling pathway, and that its signal transduction mechanism does not rely on amyloid fibril formation under our experimental conditions. These findings contribute to our understanding of immune mechanisms in lepidopteran pests and offer potential molecular targets for developing novel pest control strategies.