Figure 5

Interspecies structural complementation of RNAi mutants. TbPFR2i-derived trypanosomes, constitutively expressing GFP (panel A; PCGFP cells) or TcPFR2 (panel B; PCTcPFR2 cells) were cultured for 48 h in the absence (-TET) or in the presence (+TET) of tetracycline. In these TbPFR2i-derived cells, tetracyline-induction triggers RNAi-silencing of the endogenous TbPFR2 gene. The cells were then treated for immunofluorescence using L8C4 as an anti-TbPFR2 (right panel, black background) and counterstained with DAPI (left panel, merged with phase contrast image). Non-induced PCGFP cells show normal flagellar staining (A, -TET) while induced cells show an almost total loss of flagellar signal (A, +TET) due to silencing of TbPFR2. Non-induced PCTcPFR2 cells show an intense L8C4 signal in the flagellum and sometimes in the cytoplasm, due to overexpression (B, -TET). Upon tetracycline-induction of these cells, the flagellar L8C4 decoration did not disappear, indicating that TcPFR2 was not subject to RNAi and that TcPFR2 could successfully localize to the flagellum (B, +TET).