Introduction: Mitochondria and plastids originate from endosymbiotic events more than one billion years ago in which free-living bacteria were taken up by a host cell and subsequently evolved into organelles that are now fully integrated into the cellular networks of the host. Since the acquisition and evolution of these organelles date far back in time, we use the trypanosomatid Angomonas deanei, which acquired a bacterial endosymbiont (ES) more recently, to study early steps of endosymbiosis. A. deanei harbors a single β-proteobacterial ES whose cell cycle is synchronized with and likely controlled by the host by so-called endosymbiont-targeted proteins (ETPs) that are encoded by the host but transported to the ES.

Goal: Preliminary work revealed that homozygous deletions of several ETP-encoding genes cannot be obtained, suggesting that these genes are essential for survival of the symbiosis. Thus, to study how the ETPs effect the host-ES interaction, we aimed to develop genetic tools to generate inducible homozygous knockout (or knockdown) mutants.

Methods & results: To this end, we generated an A. deanei strain that produces the T7 RNA polymerase (T7RNAP) from a highly expressed locus. To avoid interference with the endogenous expression machinery we identified at least seven potentially silent loci that appeared suitable for transgene insertion and were expected to be transcriptionally inactive under native conditions. Using a blasticidin resistance cassette with T7 promoter as a reporter gene in the T7RNAP background, we demonstrated T7RNAP-dependent gene expression from one of these silent loci. Finally, implementation of the transcriptional regulator TetR enabled us to induce expression in a ligand-depended manner. Furthermore, we are exploring the integration of a blue light-inducible T7RNAP in A. deanei.

Summary: A. deanei is an emerging model system to study early steps of organellogenesis. The conditional knockout system, that enables now inducible perturbations of the system, will be key for the in-depth exploration of the host-ES interaction, and, in particular, to observe effects of ETP losses.