Elucidating The Molecular Mechanisms Of SPIN6/SPL11Involving In Plant Programmed Cell Death And Defense Response Regulation In Rice

The rice U-box E3ligase SPL11and its Arabidopsis ortholog PUB13negatively regulate plant programmed cell death （PCD） and defense response. However, what components involve in the SPL11/PUB13-mediated PCD and defense signaling pathways remains unknown. In this study, we report that SPL11-interacting Protein6（SPIN6）, a Rho GTPase-activating protein （RhoGAP）,interacts with both SPL11and the small GTPase OsRAC1to negatively modulates plant PCD and innate immunity in rice. The results are summaried as following.1. SPIN6encodes an entire cellular localized plant specific RhoGAP protein and interacts with SPL11in vitro and in vivo.2. Knock-down and knock-out of SPIN6in rice displays plant cell death and enhances the resistance to both rice blast Manaporthe oryzae and bacterial blight Xanthomonas oryzae pv. oryzae （Xoo） pathogens, indicating SPIN6is a negative regulator in controlling plant PCD and defense response.3. Knock-down and knock-out of SPIN6in rice elevates the ROS accumulation when treated with the PAMPs chitin and flg22, and also significantly up-regulates a series of defense related genes expression, suggesting that SPIN6involves in chitin-and flg22-dependent PTI signaling.4. By the analysis of yeast two-hybrid, BiFC and Co-immunoprecipitation （Co-IP）, we also found that SPIN6intreacts with a small GTPase OsRAC1,a critical regulator involving in rice ETI and PTI signaling. SPIN6also possesses the GAP activity to hydrolysis GTP-bound OsRACl. The expression pattern analysis revealed that OsRACl is significantly induced in SPIN6RNAi silencing rice when treated with chitin and flg22. These results indicated that SPIN6involves in OsRAC1-mediated defense signaling.5. By a yeast two-hybrid screening using SPIN6as a bait,32SPIN6interacting candidate genes are identified, named as RhoGAP SPIN6-associated proteins_RAPs.Insterestingly, we also found that SPIN6forms dimer in vitro and in vivo.Conclusively, our results demonstrated that SPIN6is a novel RhoGAP protein that connects SPL11-mediated ubiquitination with OsRAC1-modulated defensome to negatively regulate plant PCD and innate immune signaling.