HiBiT In-Locus Tagging in Rice: A Novel Approach for Characterizing Endogenous Protein Dynamics

In this study, we tagged seven endogenous proteins with the HiBiT peptide in-locus and developed a pipeline to investigate the dynamic properties of endogenous proteins in rice. Our results demonstrate that the HiBiT tag is compatible with various analytical modalities such as cell lysis assays, blotting, and bioluminescence imaging, simplifying the characterization of endogenous proteins in plants. Additionally, we demonstrate for the first time that HiBiT-based immunoprecipitation-mass spectrometry (IP-MS) can identify protein-protein interactions. By utilizing IP-MS assays of OsTOR at the endogenous level, we can significantly advance our understanding of cellular endogenous signaling pathways and metabolic regulatory networks. Furthermore, we show that the Agrobacterium-delivered GRAND editing system can also enable HiBiT in-locus labeling in addition to the bombardment delivery of chemically modified donor DNA. Our study demonstrates that HiBiT tagging at OsTB1, OsSKC1, and OsHd3A using the GRAND editing system is feasible. The high throughput and ease of operation of the Agrobacterium delivery system make the HiBiT in-locus tagging based on Agrobacterium delivery a promising approach to characterizing endogenous protein dynamics in the future.