Immune responses are triggered when pattern recognition receptors (PRRs) recognize microbial molecular patterns. The Arabidopsis (Arabidopsis thaliana) receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE1 (BIK1) acts as a signaling hub of plant immunity. BIK1 homeostasis is maintained by a regulatory module in which CALCIUM-DEPENDENT PROTEIN KINASE28 (CPK28) regulates BIK1 turnover via the activities of two E3 ligases. Immune-induced alternative splicing of CPK28 attenuates CPK28 function. However, it remained unknown whether CPK28 is under proteasomal control. Here, we demonstrate that CPK28 undergoes ubiquitination and 26S proteasome-mediated degradation, which is enhanced by flagellin treatment. Two closely related ubiquitin ligases, ARABIDOPSIS TÓXICOS EN LEVADURA31 (ATL31) and ATL6, specifically interact with CPK28 at the plasma membrane; this association is enhanced by flagellin elicitation. ATL31/6 directly ubiquitinate CPK28, resulting in its proteasomal degradation. Furthermore, ATL31/6 promote the stability of BIK1 by mediating CPK28 degradation. Consequently, ATL31/6 positively regulate BIK1-mediated immunity. Our findings reveal another mechanism for attenuating CPK28 function to maintain BIK1 homeostasis and enhance immune responses.

Disulfide bonds are essential for the folding of the eukaryotic secretory and membrane proteins in the endoplasmic reticulum (ER), and ER oxidoreductin-1 (Ero1) and its homologs are the major disulfide donors that supply oxidizing equivalents in the ER. Although Ero1 homologs in yeast (Saccharomyces cerevisiae) and mammals have been extensively studied, the mechanisms of plant Ero1 functions are far less understood. Here, we found that both Arabidopsis (Arabidopsis thaliana) ERO1 and its homolog AtERO2 are required for oxidative protein folding in the ER. The outer active site, the inner active site, and a long-range noncatalytic disulfide bond are required for AtERO1's function. Interestingly, AtERO1 and AtERO2 also exhibit significant differences. The ero1 plants are more sensitive to reductive stress than the ero2 plants. In vivo, both AtERO1 and AtERO2 have two distinct oxidized isoforms (Ox1 and Ox2), which are determined by the formation or breakage of the putative regulatory disulfide. AtERO1 is mainly present in the Ox1 redox state, while more AtERO2 exists in the Ox2 state. Furthermore, AtERO1 showed much stronger oxidative protein-folding activity than AtERO2 in vitro. Taken together, both AtERO1 and AtERO2 are required to regulate efficient and faithful oxidative protein folding in the ER, but AtERO1 may serves as the primary sulfhydryl oxidase relative to AtERO2. R (2007) An Arabidopsis quiescin-sulfhydryl oxidase regulates cation homeostasis at the root symplast-xylem interface. EMBO J 26: 3203-3215 Alon A, Grossman I, Gat Y, Kodali VK, DiMaio F, Mehlman T, Haran G, Baker D, Thorpe C, Fass D (2012) The dynamic disulphide relay of quiescin sulphydryl oxidase. Nature 488: 414-418