OsRab11 and OsGAP1 are essential for the vesicle trafficking of the vacuolar H(+)-ATPase OsVHA-a1 under high salinity conditions

Abstract

To understand the molecular mechanism of the plant vacuolar H(+)-ATPase in endocytic trafficking and adaptation to high salinity, yeast two-hybrid assay, IP-western hybridization, trafficking assay, RT- and qRT-PCR analyses and growth assay were performed here. To confirm the interaction between OsVHA-a1 and OsGAP1, pull-down assay and Co-IP were performed in vitro and in vivo, respectively. qRT-PCR analysis revealed that the transcription of OsVHA-a1, OsGAP1 and OsRab11 was induced under high salinity. Through the protoplast-based trafficking assay, OsVHA-a1 localized predominantly from the TGN to the PVC under stressed conditions. In addition, both OsGAP1 (R385A) and OsRab11 (S28N) mutants did not interact with OsVHA-a1, and blocked the vesicular trafficking of OsVHA-a1 to the PVC. In a seedling growth assay using the dominant negative OsRab11 (S28N), this mutant was much more sensitive to high salinity than the wild-type. Furthermore, the trafficking assay using isolated vacuoles demonstrated directly that OsGAP1 targeted to the tonoplast of the central vacuole under high salinity. Taken together, it is suggested that OsGAP1 and OsRab11 are essential for the vesicle trafficking of OsVHA-a1 to the PVC and/or the central vacuole under high salinity.