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Related Experiment Video

Updated: Jan 9, 2026

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics
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A quantitative split firefly luciferase complementation assay (SplitLUC) for in planta protein-protein interactions.

Qianwei Liu1, Rainer Kembügler2, Francesc Felipe2

  • 1Joseph Gottlieb Kölreuter Institute for Plant Sciences (JKIP), Karlsruhe Institute of Technology (KIT), 76131, Karlsruhe, Germany.

Protoplasma
|December 10, 2025
PubMed
Summary

This study presents a robust Split firefly luciferase complementation assay (SplitLUC) for detecting plant protein-protein interactions (PPIs) in planta. Fluorescence-based normalisation ensures reliable quantification of these essential molecular interactions.

Keywords:
CCDCooled charge-coupled deviceNightSHADEProtein–protein interactionsSplit firefly luciferase complementation assaySplitLUC

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Area of Science:

  • Plant molecular biology
  • Biochemistry
  • Genetics

Background:

  • Understanding protein-protein interactions (PPIs) is crucial for plant development and stress response.
  • The split firefly luciferase complementation assay (SplitLUC) is a powerful tool for studying PPIs.

Purpose of the Study:

  • To demonstrate and validate the SplitLUC assay for detecting and quantifying PPIs in planta.
  • To investigate the interaction between DDA1 and PYL8 using the SplitLUC assay.

Main Methods:

  • Utilized a cooled CCD-based plant imaging system and microplate reader for SplitLUC.
  • Co-infiltrated Agrobacterium strains with DDA1-nLUC and cLUC-PYL8 constructs.
  • Normalized luminescence signals against TagRFP fluorescence for accurate quantification.

Main Results:

  • A robust luminescent signal confirmed the interaction between DDA1 and PYL8.
  • Control experiments showed no detectable luminescence, confirming assay specificity.
  • Fluorescence-based normalization effectively reduced variability in luminescence signals.

Conclusions:

  • The SplitLUC assay, with fluorescence normalization, provides a reliable quantitative method for studying plant PPIs.
  • This approach is suitable for weak interactions, bridge protein effects, and domain mapping.