A multiplex hextuple luciferase assay for advancing cellular signaling analysis in plant cells

Plant cell-based assays offer substantial cost and throughput advantages over in planta testing. Current protoplast-based cellular activity assays are single biological measurements that require rigorous control to normalize inherent experimental variations when comparing across experiments. Commonly used assays incorporate two colorimetric, fluorescent, or luminescent probes, or a combination thereof, to measure one cellular response normalized against a control. Recently, multiplex hextuple luciferase assaying was developed to interrogate five signaling pathways against a control at once in mammalian cells. Inspired by this work, we explored, optimized, and adopted this strategy for plant protoplast applications, enabling the simultaneous measurement of up to five different cellular activities against a control from a single plant sample. As a proof of principle, we engineered inducible promoter-driven luciferase reporters to observe five different promoter activities activated by the exogenous application of stimuli against a constitutive control luciferase reporter. The strength of exogenous inducers, small-molecule inhibitors, or genetic small interfering RNA is indicated by luminescence light emission from the expression of luciferase driven by the different reporter activities. We measured inducible promoter activities by simultaneously reading multiple luciferases per sample, which significantly reduced the sample size and improved across experimental comparisons. The results of inducible promoter activation and inhibition using multiplexed luciferase reporters were comparable to those of conventional single luciferase reporters. The protoplast-based multiplex luciferase assay multiplies the number of testing agents in a single experiment by five, improving detection sensitivity through light emission measurements and enhancing comparability across experiments.