Broad application of the ALFA tagging technology for in planta nanobody-based imaging and biochemical characterization of plant proteins

Epitope tags are broadly used for detecting, modifying or purifying proteins of interest, but their breadth of application is often rather poor. Recently, the rationally designed ALFA tag and its ALFA Nanobody complement the repertoire of epitope tags and emerged as a highly versatile epitope tag characterized in various animal models and that outperforms existing tags. Here we evaluated the ALFA tag/ALFA Nanobody technology in plants and showcase its application for in planta protein detection across multiple compartments or cellular structures, protein:protein interaction studies, protein immunoprecipitation, induced-proximity approaches, and super-resolution microscopy. Most importantly, we shed light on the edge of ALFA tagging technology for proteins difficult to tag for topological or functionals reasons. We provide a proof of concept of ALFA tag technology for the detection and functional analysis of the Arabidopsis IRT1 iron transporter. Overall, such versatile and validated toolbox of ALFA tag and ALFA Nanobody applications will serve as a resource of great interest for functional studies in plants.