Novel assays to monitor gene expression and protein-protein interactions in rice using the bioluminescent protein, NanoLuc

Abstract

Luciferases have been widely utilized as sensitive reporters to monitor gene expression and protein-protein interactions. Compared to firefly luciferase (Fluc), a recently developed luciferase, Nanoluciferase (NanoLuc or Nluc), has several superior properties such as a smaller size and stronger luminescence activity. We compared the reporter properties of Nluc and Fluc in rice (Oryza sativa). In both plant-based two-hybrid and split luc complementation (SLC) assays, Nluc activity was detected with higher sensitivity and specificity than that with Fluc. To apply Nluc to research involving the photoperiodic regulation of flowering, we made a knock-in rice plant in which the Nluc coding region was inserted in-frame with the OsMADS15 gene, a target of the rice florigen Hd3a. Strong Nluc activity in response to Hd3a, and in response to change in day length, was detected in rice protoplasts and in a single shoot apical meristem, respectively. Our results indicate that Nluc assay systems will be powerful tools to monitor gene expression and protein-protein interaction in plant research.

The rice OsERF101 transcription factor regulates the NLR Xa1-mediated perception of TAL effectors and Xa1-mediated immunity

“…To analyze the strength of the interaction between Xa1 and the TAL effectors, we carried out a split NanoLuc luciferase complementation assay (Taoka et al, 2021).…”

“…DNA fragments of Xa1 , BED , NB , LRR , AvrXa7 , and Xoo1132 were transferred into p35S-LgBiT-T7-GW or p35S-SmBiT-T7-GW using the Gateway system with LR clonase reactions (Taoka et al , 2021). The Firefly Luciferase gene controlled by the CaMV 35S promoter was used as an internal control.…”

Liquid-liquid phase separation of florigen activation complex induces flowering

“…Hd3apro:Hd3a-GFP, rolCpro:Hd3a-GFP, rolCpro:GFP and Ubqpro:H2B-GFP transgenic rice plants were described previously 7,19 . Transgenic rice cell lines derived from the OsMADS15-nanoLuc gene targeting line (M15NL-KI) were described previously 28 . Transgenic rice plants were generated using Agrobacterium (Agrobacterium tumefaciens)-mediated transformation of rice calli, as previously described, and hygromycin-resistant plants were regenerated from the transformed calli 7 .…”

“…Transformation of rice protoplasts was performed as described previously 2,28 . Five micrograms of Hd3a and OsFD1 effector constructs and 0.5 µg of pUbqFluc reporter plasmid 2 were transfected into 0.3-1.0 × 10 protoplasts per ml by the PEG-mediated transformation method.…”

“…Luciferase activity was measured as described previously 28 FAC formation induces condensate formation, which requires the interaction of Hd3a with 14-3-3 via the Hd3a residues R64, F103 and R132. G173 in the C-terminal disordered region and the internal R121 are essential for condensate formation after FAC formation.…”