Screening DHHCs of S-acylated proteins using an OsDHHC cDNA library and bimolecular fluorescence complementation in rice.

S-acylation is an important lipid modification that primarily involves DHHC proteins (DHHCs) and associated S-acylated proteins. No DHHC-S-acylated protein pair has been reported so far in rice (Oryza sativa L.) and the molecular mechanisms underlying S-acylation in plants are largely unknown. We constructed an OsDHHC cDNA library for screening corresponding pairs of DHHCs and S-acylated proteins using bimolecular fluorescence complementation assays. Five DHHC-S-acylated protein pairs (OsDHHC30-OsCBL2, OsDHHC30-OsCBL3, OsDHHC18-OsNOA1, OsDHHC13-OsNAC9, and OsDHHC14-GSD1) were identified in rice. Among the pairs, OsCBL2 and OsCBL3 were S-acylated by OsDHHC30 in yeast and rice. The localization of OsCBL2 and OsCBL3 in the endomembrane depended on S-acylation mediated by OsDHHC30. Meanwhile, all four OsDHHCs screened complemented the thermosensitive phenotype of an akr1 yeast mutant, and their DHHC motifs were required for S-acyltransferase activity. Overexpression of OsDHHC30 in rice plants improved their salt and oxidative tolerance. Together, these results contribute to our understanding of the molecular mechanism underlying S-acylation in plants.