ABSTRACT RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of the Leishmania subgenus Viannia , and here we describe the development, optimization, and application of RNAi tools to the study of L. (Viannia) braziliensis . We developed vectors facilitating generation of long-hairpin or “stem-loop” (StL) RNAi knockdown constructs, using Gateway ™ site-specific recombinase technology. A survey of applications of RNAi in L. braziliensis included genes interspersed within multigene tandem arrays such as QDPR , a potential target or modulator of antifolate sensitivity. Other tests include genes involved in cell differentiation and amastigote proliferation ( A600 ), and essential genes of the intraflagellar transport ( IFT ) pathway. We tested a range of stem lengths targeting the L. braziliensis hypoxanthine-guanine phosphoribosyltransferase ( HGPRT ) and reporter firefly luciferase ( LUC ) genes and found that the efficacy of RNAi increased with stem length, and fell off greatly below about 128 nt. We used the StL length dependency to establish a useful ‘hypomorphic’ approach not possible with other gene ablation strategies, with shorter IFT140 stems yielding viable cells with compromised flagellar morphology. We showed that co-selection for RNAi against adenine phosphoryl transferase (APRT1) using 4-aminopyrazolpyrimidine (APP) could increase the efficacy of RNAi against reporter constructs, a useful tool that may facilitate improvements in future work. Thus, for many genes, RNAi provides a useful tool for studying Leishmania gene function with some unique advantages.
