Split Nano luciferase complementation for probing protein‐protein interactions in plant cells

Abstract

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 coexpression 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 coreceptor 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.

Applications of bioluminescence in biotechnology and beyond

“…332 Recently, split Nano luciferase has been used to determine protein-protein interactions in plant cells, wherein the PPI between receptor kinase flagellin-sensitive 2 (FLS2) and plant receptor kinase BAK1 (BRI1-associated receptor kinase 1) was found to be induced by bacterial flg22 peptide through a split Nanoluc system. 333 Split luciferase assays have also been used to study viruses, 334,335 as well as to detect protein-protein aggregation in human cells. 336…”

Metabolons, enzyme–enzyme assemblies that mediate substrate channeling, and their roles in plant metabolism

“…Among other methods that use endogenous cell systems for PPI screening, the split-LUC assay ( Varnum et al., 2017 ) and BiFC ( Kudla and Bock, 2016 ; Xing et al., 2016 ; Zhang et al., 2020a ) are common, both of which can detect transient (as well as permanent) PPIs in vivo . In a split-LUC assay, the PPIs of the bait and prey can bring the two separate luciferases together to transiently reconstitute an active luciferase, such as firefly luciferase, nano luciferase, or Renilla luciferase ( Wang et al., 2019 ). Although this method is difficult to use for high-throughput interaction studies, the split-LUC assay is particularly powerful for pinpointing transient and reversible PPIs under native conditions.…”

Glowing Plants Can Light Up the Night Sky? A Review

“…NanoLuc is a small subunit (19 kDa) from shrimp Oplophorous gracilirotris luciferase (Hall et al, 2012). Split NanoLuc was developed to screen PPIs, which avoids imposing steric hindrance caused by huge firefly luciferase halves (Wang et al, 2020). Nano-lantern is an artificially designed luminescent protein fused with enhancedRenilla reniformis luciferase and yellow FP Venus.…”