ShadowAuxin: Optimization of Quenching FRET Pairs for a Fluorescent Auxin Biosensor

Auxin is a hormone crucial in nearly every aspect of plant growth and development. Advancing our understanding of auxin signaling in maize and other crop plants may provide needed tools for agricultural scientists to help feed a growing population in the face of global climate change. However, existing tools for detecting and measuring auxin level and location have significant shortcomings that can limit their utility in plants. We are working to build and test a novel auxin biosensor, ShadowAuxin, capable of accurate quantification and tracking of auxin in live plants. Our biosensor design relies on the dimerization of two fluorescent proteins (a donor and an acceptor) capable of Förster Resonance Energy Transfer (FRET) when in close proximity. Our biosensor utilizes a quenching fluorescent protein acceptor in the FRET pair; when this pair are held in close proximity by fused dimerization domains, the donor fluorescent signal should dim. Pilot experiments in yeast cells have shown that quenching FRET can be measured via fluorescence flow cytometry, and have revealed the importance of dimer-domain choice and carefully controlled protein expression level. A new pair of human-hormone inducible promoters has enabled us to precisely control the expression level of donor and acceptor proteins in order to maximize FRET quenching. This system allowed observation of quenching FRET without significant cellular toxicity; however we observed a maximum of only 12.9% decrease in fluorescent signal. This is likely due to off-target dimerization and imperfect intracellular stoichiometry of the fluorescent proteins. Therefore, current and future experiments focus on increasing FRET efficiency by adjusting promoter strength and testing new sets of heterodimerization domains. Ultimately, this quenching FRET system will be coupled to an auxin-sensing domain to generate a biosensor that relies on single-color fluorescence measurements and provides rapid response and wide dynamic range.