Researchers developed a firefly luciferase complementation imaging (LCI) assay to detect protein-protein interactions in plants. This method uses the firefly luciferase enzyme, which is split into two halves (NLuc and CLuc). When these halves are fused to interacting proteins, they reconstitute active luciferase, which can be visualized using a low-light imaging system. The study tested nine protein pairs known to interact in plants, including bacterial effector proteins, components of the HSP90 chaperone complex, disease resistance proteins, and transcription factors. Strong luciferase complementation was observed for all positive interactions, while mutants that disrupt interactions showed reduced activity. The LCI assay is simple, reliable, and quantitative, suitable for both transient and stable transgenic expression. The LCI assay was tested in protoplasts and intact leaves, showing strong activity for protein pairs like AvrB-RAR1, Pto-NPrf, and AvrPto-Pto. It also detected interactions between HSP90 complex components and WRKY transcription factors. The assay was further validated using Agrobacterium-mediated transient expression in Nicotiana benthamiana, demonstrating its effectiveness in detecting interactions in plant cells. The LCI method is advantageous over other techniques like FRET and BiFC because it is not affected by autofluorescence and can be used without a microscope. It is also suitable for studying interactions at the organismal level and can test multiple protein pairs simultaneously. The study highlights the utility of LCI as a new tool for plant protein-protein interaction studies, offering a simple and effective method for validating protein interactomes.Researchers developed a firefly luciferase complementation imaging (LCI) assay to detect protein-protein interactions in plants. This method uses the firefly luciferase enzyme, which is split into two halves (NLuc and CLuc). When these halves are fused to interacting proteins, they reconstitute active luciferase, which can be visualized using a low-light imaging system. The study tested nine protein pairs known to interact in plants, including bacterial effector proteins, components of the HSP90 chaperone complex, disease resistance proteins, and transcription factors. Strong luciferase complementation was observed for all positive interactions, while mutants that disrupt interactions showed reduced activity. The LCI assay is simple, reliable, and quantitative, suitable for both transient and stable transgenic expression. The LCI assay was tested in protoplasts and intact leaves, showing strong activity for protein pairs like AvrB-RAR1, Pto-NPrf, and AvrPto-Pto. It also detected interactions between HSP90 complex components and WRKY transcription factors. The assay was further validated using Agrobacterium-mediated transient expression in Nicotiana benthamiana, demonstrating its effectiveness in detecting interactions in plant cells. The LCI method is advantageous over other techniques like FRET and BiFC because it is not affected by autofluorescence and can be used without a microscope. It is also suitable for studying interactions at the organismal level and can test multiple protein pairs simultaneously. The study highlights the utility of LCI as a new tool for plant protein-protein interaction studies, offering a simple and effective method for validating protein interactomes.