A split GAL4 RUBY assay for visual in planta detection of protein-protein interactions

Area of Science:

• Plant molecular biology
• Biochemistry
• Genetics

Background:

• Protein-protein interactions (PPIs) are crucial for cellular functions.
• Detecting PPIs in living plants in real-time is challenging.
• Existing methods often require complex sample processing.

Purpose of the Study:

• To develop a novel, visually detectable assay for protein-protein interactions in plant leaves.
• To enable real-time, in planta assessment of PPIs.
• To validate the assay's accuracy and utility in studying plant-pathogen interactions.

Main Methods:

• Developed a split GAL4 RUBY assay using yeast GAL4 and VP16 transcription factors.
• Fused candidate proteins to transcription factor domains.
• Transiently expressed fusions in *Nicotiana benthamiana* leaves via *Agrobacterium* infiltration.
• Utilized a RUBY reporter gene activated by PPIs to produce visual betalain pigment.

Main Results:

• The assay allows macroscopic, real-time visual detection of PPIs in living plant leaves.
• The split GAL4 RUBY assay is adaptable for both qualitative and quantitative assessments.
• Successfully detected interactions between wheat Sr27 resistance protein and *AvrSr27* effectors, including a weaker interaction with *avrSr27-3*.

Conclusions:

• The split GAL4 RUBY assay provides a powerful tool for studying PPIs in plants.
• The assay revealed insights into pathogen virulence strategies by detecting differential interactions.
• This method can advance research on plant immunity and disease resistance.