The fly strains used in this study.

The innate immune response requires precise spatiotemporal regulation for organisms to ensure effective pathogen clearance while avoiding detrimental overactivation. Although the core components of the Drosophila Toll pathway are well-established, the post-transcriptional regulatory networks, particularly those involving non-coding RNAs (ncRNAs), remain incompletely understood to date. Here, we elucidate a novel tripartite feedback loop comprising the long noncoding RNA (lncRNA) CR42715, the microRNA (miRNA) miR-965-3p, and the transcription factor (TF) Dif that dynamically modulates Drosophila Toll signaling. Firstly, our results demonstrate that upon Gram-positive bacterial challenge, Dif activates CR42715 expression, which acts as a competitive endogenous RNA (ceRNA) by sponging miR-965-3p to alleviate miR-965-3p-mediated repression of Dif and enhance Dif protein synthesis, thus facilitating Toll signaling immune responses. Secondly, disruption of this feedback loop via genetic manipulation of CR42715 or miR-965-3p leads to dysregulated AMP expression and compromised host survival. Thirdly, the temporal expression analysis reveals that CR42715 is rapidly induced early in infection to boost immunity, while miR-965-3p expression increases later, ensuring timely signal attenuation, which suggests that this dynamic Dif/CR42715/miR-965-3p feedback loop can ensure robust early-phase antimicrobial peptide production while preventing excessive late-phase immunity. Collectively, we unveil a novel TF-lncRNA-miRNA feedback loop that acts as a rheostat to ensure an effective immune response.