RNAi-based gene silencing using a GFP sentinel system in Histoplasma capsulatum

RNA interference (RNAi) has revolutionized reverse genetics in eukaryotic organisms, particularly those in which homologous recombination is inefficient or impractical. The ability to deplete or knock-down a targeted gene product without requiring genetic disruption provides a rapid means of analyzing mutant phenotypes and defining gene functions. In Histoplasma capsulatum, in vivo-produced RNA stem-loop molecules are effective in triggering RNAi of the targeted gene and the RNAi effect is both heritable and stable. The use of a green fluorescent protein (GFP) sentinel for RNAi, in which cosilencing of GFP fluorescence is used as an indicator of target gene depletion, rapidly identifies RNAi lines of H. capsulatum. Here, we describe the construction of RNAi-triggering vectors, generation of silenced lines, and utilization of the GFP sentinel RNAi system in H. capsulatum.