Functional And Mechanistic Analysis Of The SINA E3Ligase OsDIS1in Rice

Stresses seriously affect crop yields and quality, and constrain the global agricultural production, which has become the main challenges for sustainable development of agriculture with the growing population. Ubiquitin-protease pathway (UPS) is one of the most important regulation mechanisms for protein modification in plants. It functions in cell cycle and light cycle regulation, hormone signal transduction, metabolic regulation, DNA repair and responses to biotic and abiotic stresses. In this study, by combining biochemistry, plant molecular biology and genetics methods, we characterized the biological and biochemical functions of the SINA E3ubiquitin ligase OsDIS1(O. sativa drought induced SINA protein1) and elucidated the relationship with its interacting proteins in rice.In vitro ubiquitination assays showed that OsDIS1possessed time-dependent E3ubiquitin ligase activity, and that the RING finger conserved region was required for the activity. Nicotiana benthamiana transient expression assays and rice protoplast transfection indicated that OsDIS1was localized predominantly in the nucleus, as well as some localized in the cytoplasm and cytomembrane. Both OsDIS1overexpresson and RNAi transgenic lines were produced. Drought treatments showed that overexpression of OsDIS1reduced drought tolerance in transgenic rice plants while RNAi silencing of OsDIS1enhanced drought tolerance. Microarray analysis revealed that many drought responsive genes were induced or suppressed in the OsDIS1overexpression plants under normal and drought conditions. Yeast two-hybrid (Y2H) screening showed that OsDIS1interacted with OsNek6, a serine/threonine protein kinase and the interaction was confirmed by GST pull-down and in vivo co-immunoprecipitation assays. Co-sublocalization of OsDIS1and OsNek6showed that OsDIS1was not able to coexist with OsNek6while the mutant OsDIS1(H71Y) did. OsNek6in vivo degradation assay demonstrated that OsDIS1promoted OsNek6degradation while the OsDIS1(H71Y) mutant did not. The MG132treatment showed that OsNek6was degraded via the26S proteosome-dependent pathway. It seems that OsNek6degradtion via OsDIS1-mediated ubiquitination and OsNek6is the substrate of OsDIS1in rice.Y2H assays also showed that OsDIS1interacted with the drought positive regulator OsSKIPa, which was confirmed by in vivo co-immunoprecipitation assays. The in vivo degradation and expression assays in the OsDISl overexpression and RNAi transgenic plants revealed that OsDIS1was not able to promote OsSKIPa protein degradation in rice. Together, these results demonstrate that OsDIS1plays a negative role in drought stress responses through transcriptional regulation of diverse stress-related genes and post-translational regulation of OsNek6and OsSKIPa in rice.