Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow...

Area of Science:

• Plant Molecular Biology
• Biochemistry
• Genetics

Background:

• Identifying protein and gene function relies on discovering interaction partners.
• Current methods for finding novel plant protein interactions are limited, often requiring non-plant systems or extensive mutant screens.
• A method to screen for protein interactions directly within living plant cells is highly desirable.

Purpose of the Study:

• To develop and validate a novel random in planta screening method for identifying protein-protein interactions in plants.
• To demonstrate the feasibility of screening for novel protein partners in living Arabidopsis cells.
• To identify novel interaction partners for the Calcium Dependent Protein Kinase 3 (CPK3) protein.

Main Methods:

• Utilized bimolecular complementation of YFP (BiFC) in protoplasted Arabidopsis cells.
• Employed a screen based on fluorescence-activated cell sorting (FACS) to capture YN-bait and YC-cDNA library interactions.
• Confirmed candidate interactions using in planta BiFC and Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) assays.

Main Results:

• Successfully screened a random cDNA library in Arabidopsis protoplasts to identify protein-protein interactions.
• Confirmed novel interaction partners for CPK3, including APX3, HMGB5, ORP2A, and a ricin B-related lectin domain containing protein (At2g39050).
• This represents one of the first successful random in planta screens for protein interactions.

Conclusions:

• The developed method enables random screening for protein-protein interactions directly within living plant cells.
• This approach significantly advances the discovery of novel plant protein interactomes.
• The study identified specific novel interaction partners for CPK3, providing new insights into its function.