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

Abstract

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