Development of RNAi tools for studying Leishmania (Viannia) and their application to the study of the Leishmania flagellum

• DOI: 10.1096/fasebj.26.1_supplement.951.2
• Publication Date: 2021-06-21T15:26:24Z
• Authors: Lon-Fye Lye; Katherine Owens; Timothy Notton; Erin Acino; Joseph Marcus; Stephen M. Beverley
• Keywords: Argonaute

RNA interference (RNAi) is a powerful tool for genetic analysis. While many Leishmania species lost the RNAi pathway deep in evolution, within subgenus Viannia retain genes. Correspondingly, introduction of constructs expressing dsRNA L. (V.) braziliensis resulted substantial down regulation all target mRNAs tested. We are optimizing and developing protocols that will facilitate use applied them to study flagellum. developed vectors allowing rapid generation stem‐loop (StL) using Gateway (Invitrogen®) technology, working on methods inducible maximization activity. The promastigote flagellum contains paraflagellar rod (PFR); genes encoding two major proteins PFR1 or PFR2 yielded parasites lacking PFR exhibiting motility defects. probed role intraflagellar transport (IFT) by targeting four implicated anterograde retrograde trafficking. were unable recover transfectants bearing respective StL constructs. same transfected into an Argonaute null mutant which lacks functional pathway. All IFT now successfully introduced mutant. These data suggest essential viability promastigotes.