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 typically involve single biological measurements that require rigorous controls to normalize inherent experimental variation when comparing across experiments. Commonly used assays incorporate two colorimetric, fluorescent, or luminescent probes, or a combination thereof, to measure one cellular response normalized to a control. Recently, multiplex hextuple luciferase assays were developed in mammalian cells to simultaneously interrogate five signaling pathways against a control. Inspired by this work, we explored, optimized, and adapted 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 monitor five different promoter activities activated by the exogenous application of stimuli relative to a constitutive control luciferase reporter. The strength of exogenous inducers, small-molecule inhibitors, or small interfering RNA (siRNA)-mediated genetic perturbation is indicated by light emission from luciferase expression driven by different reporter activities. We measured inducible promoter activities by simultaneously reading multiple luciferases per sample, which significantly reduced sample size and improved comparisons across experiments. The results of inducible promoter activation and inhibition using multiplexed luciferase reporters were comparable to those obtained using conventional single luciferase reporters. The protoplast-based multiplex luciferase assay enables the simultaneous testing of up to five agents in a single experiment, while improving detection sensitivity through light emission measurements and enhancing comparability across experiments.