The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli is poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle module subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.

The evolution of complex body plans in land plants has been paralleled by gene duplication and divergence within nuclear auxin-signaling networks. A deep mechanistic understanding of auxin signaling proteins therefore may allow rational engineering of novel plant architectures. Toward that end, we analyzed natural variation in the auxin receptor F-box family of wild accessions of the reference plant and used this information to populate a structure/function map. We employed a synthetic assay to identify natural hypermorphic F-box variants and then assayed auxin-associated phenotypes in accessions expressing these variants. To more directly measure the impact of the strongest variant in our synthetic assay on auxin sensitivity, we generated transgenic plants expressing this allele. Together, our findings link evolved sequence variation to altered molecular performance and auxin sensitivity. This approach demonstrates the potential for combining synthetic biology approaches with quantitative phenotypes to harness the wealth of available sequence information and guide future engineering efforts of diverse signaling pathways.

Thousands of sequenced genomes are now publicly available capturing a significant amount of natural variation within plant species; yet, much of these data remain inaccessible to researchers without significant bioinformatics experience. Here, we present a webtool called ViVa (Visualizing Variation) which aims to empower any researcher to take advantage of the amazing genetic resource collected in the Arabidopsis thaliana 1001 Genomes Project ( http://1001genomes.org ). ViVa facilitates data mining on the gene, gene family, or gene network level. To test the utility and accessibility of ViVa, we assembled a team with a range of expertise within biology and bioinformatics to analyze the natural variation within the well‐studied nuclear auxin signaling pathway. Our analysis has provided further confirmation of existing knowledge and has also helped generate new hypotheses regarding this well‐studied pathway. These results highlight how natural variation could be used to generate and test hypotheses about less‐studied gene families and networks, especially when paired with biochemical and genetic characterization. ViVa is also readily extensible to databases of interspecific genetic variation in plants as well as other organisms, such as the 3,000 Rice Genomes Project ( http://snp-seek.irri.org/ ) and human genetic variation ( https://www.ncbi.nlm.nih.gov/clinvar/ ).

15The corepressor TOPLESS (TPL) is recruited to many promoters, yet the mechanisms 16 by which it inhibits expression of genes is poorly understood. Using a synthetic auxin 17 response circuit in Saccharomyces cerevisiae, we identified two regions of Arabidopsis 18 thaliana TPL that could independently function as repression domains: the LIS1 19 homology domain (LisH) and the CT11-RanBPM (CRA) domain. The CRA repressor 20 domain required direct interaction with the Mediator subunit MED21 for full function. 21 While corepressor multimerization is highly conserved, we found that multimer formation 22 had minimal influence on TPL repression strength in yeast or in plants. Finally, we 23 showed that the LisH domain may have a conserved role in repression in different 24 proteins, as a LisH domain from the human TBL1 protein could replace TPL in synthetic 25 assays. Our work provides insight into the molecular mechanisms of transcriptional 26 repression, while also characterizing short, autonomous repression domains to augment 27 synthetic biology toolkits. 28 29 235