One Primer Pair for All: A Standardized Vector Toolbox for Protein‐Protein Interactions and Protein Localization in Plants

Understanding protein-protein interactions (PPIs) is crucial for elucidating the molecular mechanisms underlying plant growth, development, and stress responses. While techniques for analyzing PPIs have been well developed, validating a PPI typically requires a combination of four to five techniques. For each technique, specific primers are designed to clone the gene of interest (GOI) into a given vector. As a result, validating a PPI requires the synthesis of four to five primer pairs, making the process both complex and costly. To overcome these limitations, we have optimized and functionally validated an In-Fusion-based vector toolbox for the flexible construction of PPI vectors in plants. The standardized toolbox facilitates the cloning of a GOI into different PPI vectors using only one primer pair. Furthermore, we extended one of the standardized vectors for protein expression and localization studies. With the same primer pair, a GOI can be easily cloned into vectors with different tags or fluorophores. Our optimized vector toolbox will significantly simplify the assembly of PPI vectors as well as other plant expression vectors for subcellular localization and protein expression.