The RhoGAP SPIN6 Associates with SPL11 and OsRac1 and Negatively Regulates Programmed Cell Death and Innate Immunity in Rice

<div><p>The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its <i>Arabidopsis</i> ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 <i>in vitro</i> and <i>in vivo</i>. SPL11 ubiquitinates SPIN6 <i>in vitro</i> and degrades SPIN6 <i>in vivo</i> via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of <i>Spin6</i> in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen <i>Magnaporthe oryzae</i> and the bacterial blight pathogen <i>Xanthomonas oryzae</i> pv. <i>oryzae</i>. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the <i>Spin6</i> RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state <i>in vitro</i> and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.</p>