A Hot-Swappable Genetic Switch: Building an Inducible and Trackable Functional Assay for the Essential Gene MEDIATOR...

Essential genes, estimated to be approximately 20% of the Arabidopsis genome, are broadly expressed and required for reproductive success. They are difficult to study, as interfering with their function leads to premature death. Transcription is one of the essential functions of life, and the multiprotein Mediator complex coordinates the regulation of gene expression at nearly every eukaryotic promoter. In this study, we focused on a core mediator component called MEDIATOR21 (MED21), which is required for activation of transcription. Our previous work has also shown a role for MED21 in repression of gene expression through its interaction with a corepressor protein. Here, we sought to differentiate the role MED21 plays in activation versus repression using the model plant Arabidopsis. As mutations in MED21 lead to embryo-lethal phenotypes, we constructed a set of synthetic switches using PhiC31 serine integrases to create an "on-to-off" inducible loss-of-function MED21 in a nonessential tissue. Our technology, which we call Integrase Erasers, enabled med21 mutant plants to survive into adulthood by ablating protein expression selectively in lateral root primordia, allowing quantification and characterization of med21 mutant phenotypes in a postembryonic context. In addition, we engineered chemical induction of the Integrase Eraser to ablate MED21 expression in whole seedlings at a user-specified time point. Finally, we extended this technology to build a hot-swappable Integrase Isoform Switch where expression of the integrase toggled cells from expressing wild-type MED21 to expressing MED21 sequence variants. Our analysis of the entire set of new integrase-based tools demonstrates that this is a highly efficient and robust approach to the study of essential genes.