Structural complementation of RNAi mutants. TbPFR2tag (panels A–C) or TbPFR2tag-ΔHLA (panels D–F) cells were transfected with GFP dsRNA (first column), CDS dsRNA (second column) or UTRs MIX dsRNAs (last column). At 14 h post-transfection, cells were treated for immunofluorescence with both the ROD-1 antibody (marker for full PFR assembly, yellow) and the BB2 anti-TAG antibody (red). All cells were counterstained with DAPI (blue). Cells of interest are bi-nucleated/bi-flagellated. Transfection of GFP dsRNA (A, D) did not produce any specific phenotype, whatever the recipient cells: the paraflagellar rod could assemble normally (yellow). As expected, TbPFR2-TAG localized to the flagellum (A, red), while TbPFR2-TAG-ΔHLA accumulated in the cytosol (D, red). The CDS dsRNA (B, E) successfully silenced the WT and the tagged TbPFR2 genes on both cell lines: no tagged TbPFR2 was stained with BB2; the paraflagellar rod could not assemble (no yellow signal is visible in the new flagellum). When using the UTRs MIX dsRNAs in TbPFR2tag and TbPFR2tag-ΔHLA cells, the wild-type TbPFR2 gene is silenced (see Figure 3). However, only in the TbPFR2tag cells does TbPFR2-TAG complement the missing TbPFR2 protein: TbPFR2-TAG was stained in the flagellum in (C, red). TbPFR2-TAG-ΔHLA failed to complement in the TbPFR2tag-ΔHLA cells: TbPFR2-TAG-ΔHLA was stained in the cytoplasm in (F, red). Thus, the paraflagellar rod could assemble fully in (C, yellow) but failed to do so in (F, yellow). Scale bar: 10 μm.