Functional Dissection of an Innate Immune Response by a Genome-Wide RNAi Screen

Abstract

The innate immune system is ancient and highly conserved. It is the first line of defense and the only recognizable immune system in the vast majority of metazoans. Signaling events that convert pathogen detection into a defense response are central to innate immunity. Drosophila has emerged as an invaluable model organism for studying this regulation. Activation of the NF-κB family member Relish by the caspase-8 homolog Dredd is a central, but still poorly understood, signaling module in the response to gram-negative bacteria. To identify the genes contributing to this regulation, we produced double-stranded RNAs corresponding to the conserved genes in the Drosophila genome and used this resource in genome-wide RNA interference screens. We identified numerous inhibitors and activators of immune reporters in a cell culture model. Epistatic interactions and phenotypes defined a hierarchy of gene action and demonstrated that the conserved gene sickie is required for activation of Relish. We also showed that a second gene, defense repressor 1, encodes a product with characteristics of an inhibitor of apoptosis protein that inhibits the Dredd caspase to maintain quiescence of the signaling pathway. Molecular analysis revealed that Defense repressor 1 is upregulated by Dredd in a feedback loop. We propose that interruption of this feedback loop contributes to signal transduction.

Methods to study Drosophila immunity

“…While the patchy efficiency of first generation RNAis relegated in vivo screens to fishing expeditions, improved second-generation RNAi libraries promise optimized throughput in RNAi-based screens. S2-based RNAi screens have successfully identified novel Toll, Imd and JNK pathway components [56][57][58][59], determinants of intracellular pathogen resistance [60] or phagocytic receptors on hemocytes [61,62], and in vivo RNAi screens have contributed to our understanding of Drosophila hemocyte development or response to intestinal infection [63,64].…”

Caspase inhibitors: viral, cellular and chemical

“…Similarly, in Drosophila, proteins such as dBruce, a BIR bearing protein and the RING protein Defense repressor 1 (Dnr1) might also be capable of regulating caspases via as yet undetermined mechanisms. 56 …”

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing

“…The residual uncleaved dsRNA was removed from the esiRNA preparation using Microcon 100 microconcentrators (Millipore, Billerica, MA). The Drosophila RNAi library has been previously described 15 . Genes involved in RNAi were identified using an RNAi of RNAi approach 13,14 .…”