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

Epitope tags are widely used for detecting, modifying, or purifying proteins of interest, but their range of application is often limited. Recently, the rationally designed ALFA tag and its ALFA nanobody have expanded the repertoire of epitope tags and emerged as a highly versatile system characterized in various animal models, outperforming existing tags. Here, we evaluated the ALFA tag/ALFA nanobody technology in plants and demonstrated its application for in planta protein detection across multiple compartments and cellular structures, protein-protein interaction studies, protein immunoprecipitation, induced-proximity approaches, and super-resolution microscopy. Most importantly, we highlight the potential of the ALFA tagging technology for proteins that are difficult to tag due to topological or functional constraints. We provide proof of concept for the ALFA tag technology in the detection and functional analysis of the Arabidopsis IRT1 Fe transporter. Overall, this versatile and validated toolbox of ALFA tag and ALFA nanobody applications will serve as a valuable resource for functional studies in plants.