Finding Molecular Targets for Engineering Auxin Signaling with Synthetic Interaction Domains

Food security is in jeopardy due to climate change and the demands of population growth. A deep mechanistic understanding of auxin signaling proteins may allow rational crop engineering to accelerate improvements in agricultural efficiency. The auxin pathway is an ideal target for engineering since it regulates a plethora of developmental processes. Auxin is perceived by the TIR1/AFB receptor proteins which then bind to AUX/IAA repressors and relieve inhibition of ARF transcription factors to allow gene expression. We call this an auxin response circuit. However, the mechanism by which auxin signaling proteins regulate gene expression and physiology remains unclear. ARF transcription factor activity is typically regulated by interaction with AUX/IAA repressors through PB1 domains, which form high-order multimeric complexes. We hypothesize that the identity of ARF-Aux/IAA regulatory complexes determine promoter specificity and dynamics of gene regulation. However, this hypothesis is experimentally challenging to address due to the presence of numerous family members and the promiscuous, multimeric interactions between ARF and Aux/IAA proteins.

To untangle this complex regulatory pathway, we designed synthetic ARF and AUX/IAA constructs swapping the nonspecific PB1 interaction domains with highly specific interaction domains of tunable affinity using modular cloning methods. We expressed combinations of these constructs in yeast containing an auxin responsive promoter and TIR1/AFB auxin receptor to determine the impact of protein-protein binding specificity and affinity on gene expression dynamics. Steady-state activation assays performed on strains with only synthetic ARFs and an auxin responsive promoter showed elevated reporter activation which confirms that these synthetic transcription factors are functional in vivo. Strains containing the full auxin response circuit showed an auxin response similar to the native yeast ARC. Comparison of gene expression data between our ARF-Aux/IAA synthetic pairs vs. native pairs will allow us to refine our understanding of the mechanism by which auxin regulates numerous genes.