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Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

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Identification of a plasma membrane complex that interacts with phyB to regulate ROS production.

Devasantosh Mohanty¹, Yosef Fichman², María Ángeles Peláez-Vico¹

¹Division of Plant Sciences and Technology, College of Agriculture Food and Natural Resources, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, United States.

Plant Physiology

|April 15, 2026

Summary

This summary is machine-generated.

Plant stress responses involve reactive oxygen species (ROS) regulated by FERONIA, phytochrome B (phyB), and RBOHD. These proteins form a complex essential for managing ROS levels during excess light stress in Arabidopsis.

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Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

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Published on: May 29, 2010

Area of Science:

Plant molecular biology
Plant signaling pathways
Reactive oxygen species metabolism

Background:

Reactive oxygen species (ROS) are crucial signaling molecules in plants, influencing growth, development, and environmental responses.
RBOHD-mediated ROS production is modulated by PHYTOCHROME B (phyB), which is itself regulated by FERONIA phosphorylation.
This suggests a potential interaction network involving FERONIA, phyB, and RBOHD in stress-induced ROS regulation.

Purpose of the Study:

To investigate the interactions between FERONIA, phyB, and RBOHD under excess light (EL) stress in Arabidopsis thaliana.
To elucidate the roles of FERONIA kinase activity and phyB phosphorylation in regulating RBOHD-driven ROS production.
To identify other potential interacting partners involved in this stress response pathway.

Main Methods:

Immunoprecipitation and proximity labeling techniques were employed to identify protein-protein interactions.
Split-luciferase assays were used to confirm interactions in vivo.
Functional validation assays assessed the necessity of identified proteins for ROS production under EL stress.

Main Results:

Direct interactions were confirmed between phyB, RBOHD, and FERONIA.
FERONIA's kinase activity and its phosphorylation of phyB are essential for RBOHD-dependent ROS production during EL stress.
CYSTEINE-RICH RECEPTOR LIKE KINASE 10 (CRK10) and PLASMA MEMBRANE INTRINSIC PROTEIN 2;6 (PIP2;6) were identified as interacting partners of RBOHD and phyB, and are required for EL-induced ROS production.

Conclusions:

A novel plasma membrane complex involving FERONIA, RBOHD, CRK10, and PIP2;6, which interacts with phyB, regulates ROS production in Arabidopsis under stress.
This protein complex integrates multiple signaling pathways, playing a key role in plant stress adaptation.
The findings provide new insights into the molecular mechanisms governing ROS homeostasis and signaling in plants.