Essentially all growth and developmental processes in plants require action of the hormone auxin. Auxin regulates gene expression by mediating the degradation of repressor proteins called Aux/IAAs. At low concentrations of auxin, these IAA proteins repress auxin-responsive genes by binding to auxin response factor (ARF) activating transcription factors. A subset of ARF and IAA proteins are involved in the initiation of new lateral roots, which are important for overall root architecture and function. Mutant phenotypes in these ARFs and IAAs fall into two separate classes. Based on this evidence, we hypothesize that the structural properties of specific ARF and IAA proteins cause them to have specific binding preferences. For example, we predict that ARF5 has stronger binding preference for IAA12 than IAA14, while ARF7 has stronger preference for IAA14. To test interactions between these proteins, we are expressing them in the yeast, S. cerevisiae. This allows us to look at individual protein interactions at single cell resolution, which is not possible using a plant based system due to the ubiquity of auxin responses and the presence of multiple IAAs and ARFs. We are using a bimolecular fluorescence complementation approach, in which two complementary halves of fluorescent proteins are fused to two interacting proteins of interest. Specifically, we have fused halves of different fluorescent proteins to IAAs and ARFs, such that each specific IAA-ARF complex exhibits a different color of fluorescence. Using this system, we will compete two different IAA proteins for binding of a single ARF. This approach will allow us to analyze the binding preferences of proteins through quantitative comparison of fluorescence of protein complexes. The results of my research may explain some of the specificities between ARF and IAA mutant phenotypes found in Arabidopsis and how distinct protein properties lead to differences in gene expression and development.