<p>Cysteine proteases are proteolytic enzymes that are widely present in various plant tissues and organs and are involved in diverse cellular and physiological processes. Cystatin is a specific inhibitor of cysteine proteases. It restricts their activity by interacting with them within cells. Conserved cystatin inhibitor structures are typically located in the middle and C-terminal regions of the peptide. However, the mechanism by which cysteine proteases interact with specific cystatins remains unclear. In this study, the cold responsive rice cysteine protease OsCysP7 was used as bait to screen for interacting cystatins. Of the 12 rice cystatins tested, none could interact with the precursor of OsCysP7, which bears the self-inhibitory domain. Only OsCPI2 was found to interact with the mature OsCysP7 via yeast two-hybrid screening. This interaction was further confirmed by a bimolecular fluorescence complementation (BiFC) test. Analysis of the interaction domains showed that only the N-terminal sequence of OsCPI2 could interact with mature OsCysP7. A fragment comprising the first 57 amino acids in the N-terminal region was sufficient for interaction with mature OsCysP7. Although the C-terminus of OsCPI2 comprises two conserved inhibitory domains, these domains alone were insufficient for interaction with mature OsCysP7. Further experiments with variants of OsCPI2 revealed that the residue at position 57 was crucial for the specific interaction between the two proteins. This suggests that the N-terminal region of OsCPI2 is the key domain responsible for determining its interaction with mature OsCysP7.</p>