The Optimized HaloTag Technology Provides a Versatile Tool for Cell Imaging in Plants

• DOI: 10.1111/pce.15491
• Publication Date: 2025-04-04T10:33:39Z
• Authors: Hongping Qian; Changwen Xu; Xinxiu Zuo; Yuan Zhang; Ruohan Geng; Jinyu Wang; Xi Zhang; Guangchao Wang; Ruili Li; Xiaojuan Li; Xinwei Wang; Yaning Cui; Jinxing Lin

ABSTRACTThe innovative HaloTag is a labeling technology that plays a crucial role in advanced fluorescence imaging. However, due to the complexity of plant materials, the application of HaloTag technology in the field of plants is still in its infancy. To expand the application of HaloTag technology in plant cells, we constructed six eukaryotic gene expression vectors with different localizations carrying HaloTag labels, then transformed Arabidopsis thaliana and Nicotiana benthamiana, and finally stained the living cells using HaloTag ligands. Live‐cell imaging showed that HaloTag did not affect the localization pattern and biological functions of the target protein, and by improving the experimental conditions, the optimized HaloTag technology could be more effectively applied to plants. Remarkably, we first displayed that the optimized HaloTag technology exhibits superior labeling performance compared to conventional fluorescent proteins, including photostability, monomer properties, multicolor imaging and spatio‐temporal differentiated labeling. More importantly, the combination of HaloTag technology with single‐particle tracking reveals the specific dynamics of membrane proteins and the changes in the rearrangement of ER‐PM connectivity, providing a powerful tool for analyzing protein dynamics. Taken together, this promising HaloTag technology enriches the labeling toolkit for studying spatiotemporal dynamics and various biological processes.