A quantitative split firefly luciferase complementation assay (SplitLUC) for in planta protein-protein interactions

Understanding protein-protein interactions (PPIs) in planta is essential for deciphering the molecular mechanisms underlying plant development and responses to environmental stresses. Here, we demonstrate the application of the split firefly luciferase complementation assay (SplitLUC) using a cooled charge-coupled device (CCD)-based plant imaging system and a microplate reader to detect and quantify PPIs in planta. As an example, we investigated the previously reported interaction between DET1- and DDB1-ASSOCIATED 1 (DDA1), a component of the CULLIN4 (CUL4)-E3 ubiquitin ligase complex, and PYR1-like 8 (PYL8), a known substrate of the same complex. Co-infiltration of Agrobacterium strains carrying DDA1-nLUC and cLUC-PYL8 constructs resulted in a robust luminescent signal upon addition of D-luciferin, which was visualised and quantified using the NightSHADE evo Plant Imaging System. Control combinations lacking either fusion partner or containing only empty vectors did not produce detectable luminescence, confirming the specificity of the interaction. To account for infiltration efficiency and variability in transgene expression, the luminescence values were normalised against fluorescence from co-infiltrated TagRFP, measured using a Tecan Spark microplate reader. This normalisation strategy effectively mitigated leaf-to-leaf variation in luminescence signals and demonstrated that the SplitLUC assay, when combined with fluorescence-based normalisation, provides a robust and reliable quantitative method for studying PPIs in planta. We propose that this approach is well-suited for investigating weaker interactions, assessing the influence of additional (bridge) proteins, and mapping interaction domains within the proteins of interest.