Genetic Screen in Drosophila Larvae Links ird1 Function to Toll Signaling in the Fat Body and Hemocyte Motility.

Martin R Schmid,Mika Rämet,Jesper Kronhamn,Hoa T M Vo,Tor Erik Rusten,Susanna Valanne,Hairu Yang,Ines Anderl,Dan Hultmark

doi:10.1371/journal.pone.0159473

Abstract

To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response.

Highlights

The core components of the Toll signaling pathway were initially identified in a classical genetic screen for mutations that affect Drosophila embryonic development, where Toll signaling was found to control dorsal-ventral polarity [1,2,3]
With a limited number of genetic crosses this approach makes it possible to screen the major part of the Drosophila genome for the effect of a 50% reduction of gene dosage
The low pH quenches GFP, acting as a reporter for autophagy activity and autophagymediated flux to the lysosome [54,55]. We expressed this marker with the HmlΔ-GAL4 driver, in effect limiting our study to plasmatocytes, as we found this driver to be down-regulated in lamellocytes

Summary

Introduction

The core components of the Toll signaling pathway were initially identified in a classical genetic screen for mutations that affect Drosophila embryonic development, where Toll signaling was found to control dorsal-ventral polarity [1,2,3]. The Toll pathway was shown to activate a set of genes that code for antimicrobial peptides in Drosophila [4,5]. These peptides are effectors of the humoral arm of the phylogenetically highly conserved defense system of innate immunity (reviewed in [6,7]), serving as a first line of defense after infection in all higher organisms. The Toll pathway is named after the Toll gene, which encodes a transmembrane receptor. Toll homologs were later identified in the mouse as well as in humans [12,13], where Toll-like receptors (TLRs) are crucial for activating and coordinating both innate and acquired immunity

Methods

Results

Conclusion