The SINA E3 Ligase OsDIS1 Negatively Regulates Drought Response in Rice   

Abstract Ubiquitin-regulated protein degradation is a critical regulatory mechanism that controls a wide range of biological processes in plants. Here, we report that OsDIS1 (for Oryza sativa drought-induced SINA protein 1), a C3HC4 RING finger E3 ligase, is involved in drought-stress signal transduction in rice (O. sativa). The expression of OsDIS1 was up-regulated by drought treatment. In vitro ubiquitination assays showed that OsDIS1 possessed E3 ubiquitin ligase activity and that the conserved region of the RING finger was required for the activity. Transient expression assays in Nicotiana benthamiana leaves and rice protoplasts indicated that OsDIS1 was localized predominantly in the nucleus. Overexpression of OsDIS1 reduced drought tolerance in transgenic rice plants, while RNA interference silencing of OsDIS1 enhanced drought tolerance. Microarray analysis revealed that a large number of drought-responsive genes were induced or suppressed in the OsDIS1 overexpression plants under normal and drought conditions. Yeast two-hybrid screening showed that OsDIS1 interacted with OsNek6 (for O. sativa NIMA-related kinase 6), a tubulin complex-related serine/threonine protein kinase. Coexpression assays in N. benthamiana leaves indicated that OsNek6 was degraded by OsDIS1 via the 26S proteasome-dependent pathway and that this degradation was abolished by the OsDIS1(H71Y) mutation, which is essential for its E3 ligase activity. Together, these results demonstrate that OsDIS1 plays a negative role in drought stress tolerance through transcriptional regulation of diverse stress-related genes and possibly through posttranslational regulation of OsNek6 in rice.