In vivo fluorescent screening for HPPD‐targeted herbicide discovery

4-Hydroxyphenylpyruvate dioxygenase (HPPD), playing a critical role in vitamin E and plastoquinone biosynthesis in plants, has been recognized as one of the most important targets for herbicide discovery for over 30 years. Structure-based rational design of HPPD inhibitors has received more and more research interest. However, a critical challenge in the discovery of new HPPD inhibitors is the common inconsistency between molecular-level HPPD-based bioevaluation and the weed control efficiency in fields, due to the unpredictable biological processes of absorption, distribution, metabolism, and excretion.In this study, we developed a fluorescent-sensing platform of efficient in vivo screening for HPPD-targeted herbicide discovery. The refined sensor has good capability of in situ real-time fluorescence imaging of HPPD in living cells and zebrafish. More importantly, it enabled the direct visible monitoring of HPPD inhibition in plants in a real-time manner.We developed a highly efficient in vivo fluorescent screening method for HPPD-targeted herbicide discovery. This discovery not only offers a promising tool to advance HPPD-targeted herbicide discovery, but it also demonstrates a general path to develop the highly efficient, target-based, in vivo screening for pesticide discovery. © 2022 Society of Chemical Industry.