Assessment of interkingdom TIR domain interactions using a modular yeast tripartite split-GFP system

Thanks to its genetic resourcefulness, Saccharomyces cerevisiae has been traditionally used as a model organism to detect and study protein-protein interactions. The recent development of synthetic biology toolkits brings an opportunity to expand such utilities. Here, we coupled the modular cloning (MoClo) system to the tripartite split-GFP strategy to systematically study protein interactions in particular cellular localizations. To provide proof-of-principle of this yeast-based tripartite split-GFP (ytpGFP), we challenged interactions of bacterial TIR domain-containing proteins ( Brucella BtpA and BtpB) between themselves, as well as against human TIR adaptors of the myddosome and triffosome innate immune signaling complexes. While fluorescence microscopy provided clues about which member of the interacting pair drives subcellular localization of the complex, interactions could be readily tracked and quantified by flow cytometry. The ytpGFP system detected known homotypic or heterotypic complexes and defined previously unknown interkingdom TIR-TIR interactions. Human MyD88, TIRAP, TRAM, and TRIF were capable of interacting with both BtpA and BtpB TIR domains. Furthermore, our results suggest that the N-terminal non-TIR extensions of both BtpA and, especially, BtpB limit the ability of their TIR domains to interact. This strategy is a useful addition to other yeast-based tools to study protein complexes.