The study introduces a firefly luciferase complementation imaging (LCI) assay for detecting protein-protein interactions in plants. The assay utilizes the N-terminal and C-terminal halves of the firefly luciferase enzyme, which only reconstitute active luciferase when fused to interacting proteins. This method is simple, reliable, and quantitative, and can be used for both transient and stable transgenic expression. The authors tested nine pairs of proteins known to interact in plants, including bacterial effectors, the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, disease resistance protein complexes, and transcription factors. The results showed strong luciferase complementation for positive interactions and reduced activity for mutants known to disrupt protein-protein interactions. The LCI assay is particularly useful for studying protein-protein interactions in plants due to its high sensitivity, lack of autofluorescence, and ease of use with low-light imaging systems. The technique is also suitable for large-scale testing of multiple protein pairs and can be applied to both protoplasts and whole plants.The study introduces a firefly luciferase complementation imaging (LCI) assay for detecting protein-protein interactions in plants. The assay utilizes the N-terminal and C-terminal halves of the firefly luciferase enzyme, which only reconstitute active luciferase when fused to interacting proteins. This method is simple, reliable, and quantitative, and can be used for both transient and stable transgenic expression. The authors tested nine pairs of proteins known to interact in plants, including bacterial effectors, the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, disease resistance protein complexes, and transcription factors. The results showed strong luciferase complementation for positive interactions and reduced activity for mutants known to disrupt protein-protein interactions. The LCI assay is particularly useful for studying protein-protein interactions in plants due to its high sensitivity, lack of autofluorescence, and ease of use with low-light imaging systems. The technique is also suitable for large-scale testing of multiple protein pairs and can be applied to both protoplasts and whole plants.