Genome-wide screening approach to find novel Wolbachia-host interactions in Wolbachia-infected JW18 Drosophila cells.

(A) Schematic of screen layout. Wolbachia-infected JW18 cells are seeded into 384-well plates pre-arrayed with the DRSC version 2.0 whole genome RNAi library designed to include dsRNAs that target the whole Drosophila genome. All plates were screened in triplicate. Cells and dsRNA were incubated for 5 days before processing for an automated high-throughput RNA-FISH assay to detect changes in Wolbachia 23s rRNA levels. (B) Representative image of Wolbachia detection at 20x with the 23s rRNA Wolbachia FISH probe (magenta) in JW18 cells containing a GFP-Jupiter transgene labeling microtubules (grey). (C) Negative control dsRNA against LacZ not present in our system. (D-F) RNAi control against GFP-Jupiter shows efficient knockdown by 90.2% visually (D, E) as well as 97.9% reduction in protein levels by Western blot (F). (G, H) Positive control for increasing Wolbachia levels using efficient RNAi-mediated silencing of host gene RpL40. (I) Positive control for decreasing Wolbachia levels through treatment with doxycycline for 5 days. (J) Quantification of Wolbachia FISH intensity for controls. (K) Quantification of Wolbachia level fold-change relative to untreated JW18 cells using DNA qPCR. Note: Scale bars in B-D, G, and I represent 20μm.