Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase...

Area of Science:

• Plant molecular biology
• Biochemistry
• Systems biology

Background:

• Protein-protein interactions (PPIs) are essential for cellular functions and require robust mapping in plants.
• Existing methods for large-scale PPI surveys in plants have limitations.

Purpose of the Study:

• To introduce and validate split firefly luciferase complementation (SFLC) as a sensitive, non-invasive technique for in planta PPI analysis.
• To demonstrate the utility of SFLC for quantitative, high-throughput, and large-scale PPI screening in plants.

Main Methods:

• Split firefly luciferase complementation (SFLC) assay.
• Arabidopsis mesophyll protoplast system for protein expression.
• Microplate luminometer for luciferase activity measurement.
• Testing interactions between Arabidopsis FRB and human FKBP proteins.
• Screening 132 binary PPIs among Arabidopsis auxin response factors (ARFs) and Aux/IAA proteins.

Main Results:

• SFLC assay demonstrated high sensitivity and non-invasiveness for in planta PPI investigation.
• The assay provided quantitative and high-throughput potential using protoplasts and luminometry.
• Near real-time monitoring of dynamic protein interactions was achieved.
• SFLC successfully identified numerous binary PPIs among ARFs and Aux/IAA proteins, validating its power for large-scale screening.

Conclusions:

• SFLC is an ideal method for in vivo quantitative PPI analysis in plant cells.
• The SFLC assay is particularly powerful for large-scale binary PPI screens in plants.
• This technique advances systems biology approaches in plant research.