Browsing by Author "Titov, Soubir"

Protein-protein interactions (PPIs) regulate many different cellular processes including transcription, translation, cell division, signal transduction, and oncogenic transformation. It is therefore important to develop sensitive and versatile techniques for the detection of these protein-protein interactions in order to fully understand protein functions. The most commonly used and traditional technique, the yeast two-/three hybrid (Y2H/Y3H) method, often results in false positives and false negatives, and other widely used techniques, such as bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), and bimolecular fluorescence complementation (BiFC) require extensive instrumentation. When compared with other PPI detection methods, the luciferase-based complementation assay specially split luciferase is believed to deliver the most sensitive and highest dynamic range, making it ideal for large-scale analysis. Therefore, for testing PPIs in planta, split Renilla luciferase complementation assay was chosen. In order to conduct this experiment, a series of plasmid constructs were made to enable the transient expression of fusion proteins. A well known protein pair, Arabidopsis nuclear Histone 2A and 2B, was tested initially as a proof of concept, and then three more proteins of the Gerbera MADS-box B class were investigated. For Arabidopsis Histone 2A and 2B, the intensity in all combinations was on average 9.4-fold higher in Relative Luminescence Units (RLUs) than the mock treated protoplasts. Moreover, in the case of Gerbera MADS-box B class proteins, the protein pairs GDEF1-GDEF2, GDEF1-GGLO1, and GDEF2-GGLO1 showed 8.4-19.4, 9.5-15.8, and 8.3-9.1-fold higher signals than the mock treated protoplasts. These results suggest that various complexes formed from different combinations of these three B class MADS-box proteins may increase the complexity of their regulatory functions, thus specifying the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in Gerbera. Finally, it was concluded that split Renilla luciferase can be a simple, reliable, fast, and effective method for examining PPIs in planta.