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

Area of Science:

• Plant molecular biology
• Biochemistry
• Cell signaling

Background:

• Protein-protein interactions (PPIs) are crucial for plant signal transduction.
• The split firefly luciferase (Fluc) complementation (SLC) assay is commonly used for PPI analysis.
• Large Fluc fragments can potentially interfere with protein function.

Purpose of the Study:

• To develop a novel, sensitive assay for detecting plant PPIs.
• To overcome limitations associated with the traditional Fluc-based SLC assay.
• To validate a new Nano luciferase (Nluc)-based dual system for PPI and protein allostery studies.

Main Methods:

• Developed a dual-use system combining Nluc-based SLC and co-immunoprecipitation assays.
• Coexpressed proteins of interest fused to HA- or FLAG-tagged Nluc halves.
• Validated the system by reproducing known PPIs in protoplasts and Agrobacterium-transformed plants.
• Applied the system to study Arabidopsis CERK1 homodimerization and heterodimerization, and allosteric changes in CNGC2/CNGC4.

Main Results:

• Successfully validated the Nluc-based system by confirming known plant PPIs.
• Demonstrated the system's utility in analyzing homodimerization and heterodimerization of Arabidopsis CERK1.
• Showcased the system's capability to detect allosteric changes in plant calcium channels induced by elicitors.
• The Nluc assay proved effective for probing constitutive and inducible PPIs and protein allostery.

Conclusions:

• The Nluc-based split complementation assay is a robust and sensitive tool for studying plant PPIs.
• This system offers advantages over traditional Fluc-based methods due to Nluc's smaller size and improved properties.
• The developed dual system facilitates the investigation of diverse protein interactions and allosteric regulation in plants.