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Updated: Aug 30, 2025
Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System
Published on: September 16, 2011
Identification of Feronia-interacting proteins in Arabidopsis thaliana
Jae-Han Choi1, Ji-Woo Kim1, Man-Ho Oh2
1Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, 34134, South Korea.
Background:
Plant growth and development are complex processes modulated by numerous genes, transcription factors, hormones, and peptides. Several reports implicate the membrane-localized Catharanthus roseus receptor-like kinase1 (CrRLK1L) protein, FERONIA (FER), involved in plant development. However, protein targets of FER remain poorly characterized.
Objective:
FER recombinant proteins were analyzed, and FER-interacting proteins were identified, to better understand the function of the Arabidopsis thaliana FER (AtFER) gene in plant development.
Methods:
AtFER-interacting proteins were identified through Yeast-Two Hybrid (Y2H) and validated by bimolecular fluorescence complementation (BiFC). Autophosphorylation activity was evaluated in AtFER site-directed and deletion mutants.
Results:
AtFER cytoplasmic kinase domain (Flag-FER-CD) is autophosphorylated at the Thr residue (s), with T559 and T664 as important sites for AtFER kinase activity. In addition, the carboxy terminal region is essential for AtFER kinase activity. Y2H identified an Armadillo (ARM)-repeat protein (At4g16490) with tandem copies of a degenerate protein sequence motif, a U-BOX 9 (PUB9, At3g07360), IQ-DOMAIN 7 (IQD7, At1g17480), and heteroglycan glucosidase 1 (HGL1, At3g23640) as AtFER-interacting proteins. BiFC confirmed the in vivo interactions between these four proteins and AtFER in tobacco (Nicotiana benthamiana) leaf transient expression assays. The RAPID ALKALINIZATION FACTOR1 (RALF1) peptide, which is a FER ligand, induced the expression of genes encoding the four AtFER-interacting proteins.
Conclusion:
The AtFER-interacting proteins identified in this study are likely involved in FER-mediated intracellular signaling pathways that are essential in plant growth and development, and possibly plant immunity.
