The RING-Type Ubiquitin Ligase XBAT35.2 Positively Modulates Plant Immunity by Promoting FLS2 Protein Accumulation

Pattern-triggered immunity (PTI) serves as a critical frontline defense in plants, initiated by the recognition of pathogen- or microbe-associated molecular patterns by cell-surface pattern recognition receptors (PRRs). The PRR FLAGELLIN-SENSING 2 (FLS2), which perceives bacterial flagellin (flg22), is essential for plant defense against bacterial pathogens. In this study, we demonstrate that the Arabidopsis ubiquitin ligase (E3) XBAT35.2 positively regulates FLS2-mediated PTI by modulating FLS2 protein stability. XBAT35 belongs to an Arabidopsis E3 ligase family that features an ankyrin-repeat (ANK)-RING domain architecture and is highly homologous to tomato XBSL35 (XB3 ortholog 5 in Solanum lycopersicum), an interactor of the tomato E2 enzyme Fni3 that has been implicated in Lys63-linked ubiquitination and plant immunity. The XBAT35 transcript undergoes alternative splicing, giving rise to two isoforms, XBAT35.1 and XBAT35.2. Unlike XBAT34 and XBAT35.1, XBAT35.2 was shown to function in plant immunity against the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000. Overexpression of XBAT35.2 increases FLS2 protein accumulation without affecting BAK1 levels, and the xbat35 null mutant exhibits enhanced flg22-induced FLS2 degradation. XBAT35.2 is localized to the plasma membrane (PM) and the Golgi apparatus, interacting with FLS2, BAK1, and BIK1 at the PM in vivo via its ANK domain. Treatment with flg22 strengthens XBAT35.2 interactions with FLS2 and BAK1 but reduces its association with BIK1. Notably, XBAT35.2 does not ubiquitinate FLS2 in vitro. These findings suggest that XBAT35.2 contributes to plant PTI by modulating FLS2 protein stability, presumably without directly ubiquitinating FLS2, and likely also facilitates early FLS2-mediated immune signaling. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.