Silencing of Toll pathway components by direct injection of double-stranded RNA into Drosophila adult flies

Abstract

Double-stranded RNA (dsRNA) gene interference is an efficient method to silence gene expression in a sequence-specific manner. Here we show that the direct injection of dsRNA can be used in adult Drosophila flies to disrupt function of endogenous genes in vivo. As a proof of principle, we have used this method to silence components of a major signaling cascade, the Toll pathway, which controls fruit fly resistance to fungal and Gram-positive bacterial infections. We demonstrate that the knockout is efficient only if dsRNA is injected in 4- or more day-old flies and that it lasts for at least 1 week. Furthermore, we report dsRNA-based epistatic gene analysis via injection of a mixture of two dsRNAs and propose that injection of dsRNA represents a powerful method for rapid functional analysis of genes in Drosophila melanogaster adults, particularly of those whose mutations are lethal during development.

Figure 1

dsRNA silencing of the Drs-GFP transgene. Six-day-old DD1 females were injected with dsRNA against the Drs-GFP transgene (dsGFP), the immune-unrelated gene oda (dsOPA) or injection buffer (buffer) and 4 days later were either infected with Gram-positive bacteria (M.luteus) or left unchallenged as a control (N.I.). (A) GFP expression was analyzed 1 day later using fluorescence microscopy. (B) The expression of Drs-GFP (GFP), Drs and ribosomal protein gene rp49 as a loading control, was monitored by northern blotting. (C) dsGFP was injected into 1–10-day-old flies and the level of Drs-GFP mRNA after M.luteus infection was monitored. (D) The graph represents relative percentages of Drs-GFP expression normalized by rp49 in two independent experiments. 100% of expression corresponds to Drs-GFP expression in buffer-injected flies.

Figure 2

Time course of Drs expression in dsCACT-injected flies. DD1 females were injected with buffer or dsCACT and Drs expression was monitored over 10 days. The graph represents results of two independent experiments and shows relative Drs expression normalized by rp49. The lower panel shows representative northern blotting data for Drs and rp49.

Figure 3

dsRNA silencing of other components of Toll pathway. Four days after dsOPA or dsSPN43Ac injection into 6-day-old DD1 females. Efficiency of silencing was analyzed by fluorescence microscopy (A) or by northern blotting using probes specific for Drs and ribosomal protein rp49 (B). The white arrow in (A) points to a necrotic spot appearing after dsSPN43Ac injection. (C) Level of Drs expression 1 day after M.luteus challenge of buffer-, dsTOLL-, dsDIF- or dsDRS-injected flies. The graph shows relative percentages of Drs expression normalized by rp49. (D) RT–PCR analysis of dsRNA silencing of Dif (dsDIF) and Toll (dsTOLL). Actin 5C was used as an internal control. (E) Four days after dsOPA, dsPKC, dsREF(2)P, dsPKC+dsREF(2)P or dsDRS injection, flies were infected with the Gram-positive bacterium M.luteus or the fungus B.bassiana. The expression of Drs and rp49 was analyzed by northern blotting 1 day after infection with M.luteus and 2 days after infection with B.bassiana.

Figure 4

dsRNA-based epistatic analysis in adult flies. Drs expression in double knockouts (DKO) was monitored 4 days after concomitant injection of dsRNA corresponding to the serpin Spn43Ac gene (dsSPN43Ac) in combination with dsRNA against either a control unrelated gene (dsOPA), Dif (dsDIF), the atypical protein kinase C (dsPKC) or Ref(2)P [dsREF(2)P]. Levels of Drs expression in the double knockouts were compared with those of buffer-injected flies before (C) or after challenge with M.luteus (M.l.).