OsEHD1 and OsEHD2, two EH domain proteins targeted by E3 ubiquitin ligase OsBBI1, negatively modulate rice immunity against blast and bacterial blight diseases

Posttranslational modifications (PTMs) are essential regulatory mechanisms that play a critical role in plant immunity. Previously, we demonstrated that OsBBI1, a RING finger type E3 ligase, contributes to rice resistance against blast disease. In this study, we identified two Eps15 homology domain (EHD)- containing proteins, OsEHD1 and OsEHD2, as substrates of OsBBI1 and investigated their roles in rice immunity against Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo). We found that OsBBI1 ubiquitinated and promoted the degradation of OsEHD1 and OsEHD2 via ubiquitin/26S proteasome system (UPS) pathway. CRISPR/Cas9-mediated knockout of OsEHD1 and OsEHD2 led to enhanced immunity against M. oryzae and Xoo, improved expression of pathogen-induced immunity-associated genes, and strengthened pattern-triggered immunity (PTI), while overexpression of OsEHD1 resulted in opposite phenotypes. Additionally, OsEHD1 and OsEHD2 interacted with three SUMO proteins, OsSUMO3, OsSUMO5, or OsSUMO6, with SUMOylation sites in OsEHD1 and OsEHD2 being critical for these interactions. OsSUMO6 enhanced the stability of OsEHD1 and OsEHD2 to promote their negative immune regulation, whereas OsBBI1 reversed these negative immune functions. This study delineates a regulatory network of OsEHD1 and OsEHD2 proteins in rice immunity, highlighting the balance between OssBBI1-mediated ubiquitination and SUMOylation.