LdTOPIA prevents R-loop formation during polycistronic transcription.A, LtT7TR strain transfected with anti-LtTOPIA construct was either untreated or treated with tetracycline for indicated time points to observe the extent of R-loop formation. Parasites were stained with anti-LdTOPIA-AlexaFluor488, anti-DNA-RNA (S9.6) Alexa Fluor568 antibodies, and counterstained with DRAQ5. Scale Bar, 5 μm. B, graphical representation of the extent of R-loop formation estimated from the fluorescence intensity. [n = 60 (20 nuclei of 3 biological replicates), mean ± SD. p versus 0h]. C, DRIB assay was carried out from genomic DNA isolated from untreated or tetracycline-treated antisense LtTOPIA transfected LtT7TR parasites after indicated time points. D, densitometry of the same samples. [n = 3 and 3 biological replicates, mean ± SD, p values for (+Tet) versus (+Tet +RNaseH) and p versus 0h of (+Tet +RNaseH)]. E, DRIB assay was carried out using genomic DNA isolated from untreated or tetracycline-treated antisense LtTOPIA transfected LtT7TR parasites and complemented with LdTOPIA, LdTOPIAΔNLS or LdTOPIAΔNLS-SV40NLS for indicated time points. F, densitometric analysis of the same samples [n = 3 and 3 biological replicates, mean ± SD, p values for (+Tet) versus (+Tet +LdTOPIA) and p versus 0h of (+Tet +LdTOPIA)].