Double-stranded RNA can mediate the suppression of uracil phosphoribosyltransferase expression in Toxoplasma gondii

Abstract

Double-stranded RNA (dsRNA) homologous to the Toxoplasma gondii uracil phosphoribosyltransferase (TgUPRT) gene is able to modulate the UPRT gene expression in T. gondii. The dsRNA, which was produced either from a constructed plasmid or from an in vitro transcription reaction, was capable of down-regulating the expression of TgUPRT. Stably transformed T. gondii expressing the dsRNA, which was capable of growing in the presence of the prodrug 5-fluoro-2(')-deoxyuridine (FDUR), appeared to maintain the engineered plasmid as an extra-chromosomal DNA. When cultured in the absence of the selection pressure, the FDUR resistant parasites slowly reverted to the FDUR sensitive phenotype. The level of the dsRNA necessary to confer FDUR resistance was estimated at 2-8 copies per parasite. More importantly the introduction of the in vitro synthesized dsRNA homologous to the TgUPRT gene into T. gondii can also induce the specific mRNA degradation, resulting in a lowered UPRT activity.