Key message <p>The Radiation Sensitive 23B (RAD23B) protein functions upstream of the E3 ubiquitin ligase Embryo Sac Development Arrest 40 (EDA40), collaborating to regulate root architecture and affects the length of the root apical meristem.</p> Abstract <p>An optimal root system architecture plays a crucial role in water and mineral uptake in plants, primarily governed by the regulation of primary and lateral root development. Radiation Sensitive 23B (RAD23B), an ubiquitin-like/ubiquitin-associated (UBL/UBA) shuttle protein, mediates protein degradation through the ubiquitin/26S proteasome system (UPS) and is essential for plant root development. However, the mechanisms by which RAD23B regulates root development and whether this regulation depends on the UPS, are still unclear. In this study, an activation tagging-based suppressor screen was performed in the <i>rad23b</i> background, leading to the identification of Embryo Sac Development Arrest 40 (EDA40) as a new root growth regulator. <i>EDA40</i> encodes an E3 ubiquitin ligase, which serves as a part of UPS. The overexpression of <i>EDA40</i> rescues the root growth defects of <i>rad23b,</i> while the <i>eda40</i> null mutant exhibits obvious root developmental defects. However, the overexpression of RAD23B is unable to suppress the root defects of <i>eda40</i>. Notably, the <i>rad23b eda40</i> double mutant displays a root defect as severe as <i>rad23b</i>, suggesting that RAD23 may function upstream of EDA40. Together, these findings uncover a novel genetic relationship between RAD23B and EDA40 in root development and offer new insights into the role of the UPS in shaping root system architecture.</p>