Adapting the Split GAL4 RUBY assay for Golden Gate cloning: applications in Nicotiana benthamiana protein interaction studies.

Studying protein-protein interactions (PPIs) in their native cellular environment remains challenging in plant systems. Here at TSL, we have adapted the split GAL4 RUBY assay (Chen et al., 2023) for Golden Gate compatibility, creating an alternative workflow for plant PPI research. Our contribution focuses on technical optimization for compatibility with the increasingly adopted Golden Gate cloning system (Engler et al., 2008). This adaptation provides researchers with additional cloning options, enabling straightforward assembly of test constructs within modular Golden Gate cloning methods. We validated our Golden Gate components using the established mCherry/LaM-4 nanobody interaction pair, confirming functionality through betalain pigment accumulation in Nicotiana benthamiana leaves. Transition of the original method to the Golden Gate cloning system now enables the incorporation of this visual PPI detection system into modular cloning workflows, particularly benefiting labs using Type IIS restriction-based assembly cloning methods. Our presentation focuses on the practical aspects of implementing this Golden Gate-compatible system, providing detailed protocols and discussing the optimization parameters which influence detection sensitivity. This technical adaptation aims to support broader adoption of the split GAL4 RUBY system by researchers studying plant immune receptors, signalling components, and pathogen effector targets.