HiBiT-Based IP-MS Reveals Novel Interactors of OsTOR in Rice

The Target of Rapamycin (TOR) plays a significant role in energy metabolism and is responsible for regulating crop yield, disease resistance, and stress tolerance. Consequently, the study of OsTOR and its interacting proteins is crucial for understanding the endogenous signaling and metabolic regulatory network in rice. In order to establish a HiBiT-based IP-MS protocol, OsTOR (Os05g0235300) was in situ tagged, which proved to be a challenging task as it is ~26kb in size and difficult to clone. The homozygous T2 HiBiT-tag lines were chosen as the subjects of IP-MS detection, with wild-type plants serving as controls. A total of 772 putative interactors were identified in HiBiT-OsTOR lines through co-immunoprecipitation with anti-HiBiT antibodies, including previously reported RAPTOR1, RAPTOR2, LST8, and UPF1. These results indicate that the HiBiT-tag is capable of IP-grade detections.