OsCIP1, a secreted protein, binds to and stabilizes OsCR4 to promote aleurone layer development, seed germination and early seedling growth in rice

The receptor kinase CRINKLY 4 (CR4) and its orthologs are known for their essential roles in cell differentiation and their shuttling between plasma membrane and cytoplasmic vesicles, a unique feature tied to their extracellular domain. However, the extracellular regulators of CR4 have been little known. Here we identified an OsCR4 Interacting Protein 1 (OsCIP1) (also named as OsLTPL36 in rice) by a yeast two-hybrid screen using the extracellular domain of OsCR4 (OsCR4E) as bait. OsCIP1/OsLTPL36 harbors a signal peptide and is localized to the outer surface of the plasma membrane. It interacted with the TNFR subdomain of OsCR4, causing an increase in OsCR4 recycling to the plasma membrane. oscip1, in which OsCR4 protein was decreased, exhibited thinner aleurone layer, late germination and delayed growth; while OsCIP1-overexpressing plants, in which OsCR4 protein was increased, displayed enhanced growth at the early seedling stage. OsCIP1 was cleaved between W61 and Q62, and the resulting C-terminal half exhibited a greater affinity for OsCR4E than did its precursor. Abolishing this cleavage site compromises OsCIP1's ability to promote seedling growth. Our results provide valuable clues for the regulation of CR4 activity and its functions in aleurone layer cell differentiation by a secreted small protein in rice.