Split Nano Luciferase Complementation for Probing Protein‐Protein Interactions in Plant Cells

: Deciphering protein-protein interactions (PPIs) is fundamental for understanding signal transduction pathways in plants. The split firefly luciferase (Fluc) complementation (SLC) assay has been widely used for analyzing PPIs. However, concern has risen about the bulky halves of Fluc interfering with the functions of their fusion partners. Nano luciferase (Nluc) is the smallest substitute for Fluc with improved stability and luminescence. Here, we developed a dual-use system enabling the detection of PPIs through the Nluc-based SLC and co-immunoprecipitation assays. This was realized by co-expression of two proteins under investigation in fusion with the HA- or FLAG-tagged Nluc halves, respectively. We validated the robustness of this system by reproducing multiple previously documented PPIs in protoplasts or Agrobacterium-transformed plants. We next applied this system to evaluate the homodimerization of Arabidopsis CERK1, a co-receptor of fungal elicitor chitin, and its heterodimerization with other homologs in the absence or presence of chitin. Moreover, split fragments of Nluc were fused to two cytosolic ends of Arabidopsis calcium channels CNGC2 and CNGC4 to help sense the allosteric change induced by the bacterial elicitor flg22. Collectively, these results demonstrate the usefulness of the Nluc-based SLC assay for probing constitutive or inducible PPIs and protein allostery in plant cells. This article is protected by copyright. All rights reserved.