Flagellin induces BAK1-dependent FLS2-PUB13 complex association. (A) BAK1 interacts with PUB13 in a Co-IP assay. Protoplasts were co-expressed with BAK1-HA and PUB13-FLAG or a control vector. The Co-IP was carried out with an anti-FLAG antibody (IP: α-FLAG), and the proteins were analyzed using Western blot with an anti-HA antibody (WB: α-HA). The top panel shows that BAK1 co-immunoprecipitates with PUB13. The middle and bottom panels show the expression of BAK1-HA and PUB13-FLAG proteins. Protoplasts were stimulated with 1 μM flg22 for 10 min. (B) flg22 induces FLS2-PUB13 association in protoplasts. (C) flg22 induces FLS2-PUB13 association in Arabidopsis seedlings. Twelve-day-old seedlings of pPUB13::PUB13-FLAG transgenic plants were treated with 50 μM MG132 for 1 hr before H₂O or 1 μM flg22 treatment for 10 min. * indicates the specific band of PUB13-FLAG in pPUB13::PUB13-FLAG transgenic plants. (D) flg22-induced FLS2-PUB13 association depends on BAK1. (E) flg22 stimulates rapid association of FLS2-PUB13. The above experiments were repeated at least three times with similar results.

(A) BAK1 phosphorylates PUB12 and PUB13 in vitro. An in vitro kinase assay was performed by incubating MBP fusion protein of BAK1 cytosolic domain (BAK1) or BAK1Km together with MBP, MBP-PUB12 or MBP-PUB13. Phosphorylation was analyzed by autoradiography (top panel), and the protein loading control was shown by Coomassie blue staining (bottom panel). (B) flg22 enhances PUB13 phosphorylation by BAK1. BAK1-FLAG or BAK1Km-FLAG was expressed in WT or fls2 protoplasts for 8 hr followed by 1 μM flg22 treatment for 10 min. BAK1-FLAG was immunoprecipitated with an anti-FLAG antibody, and subjected to a kinase assay with MBP or MBP-PUB13 as substrate. The top band is phosphorylated PUB13 as indicated with an asterisk *, and the bottom band is BAK1 autophosphorylation. (C) Kinase inhibitor K252a suppresses FLS2 and PUB13 association. The above experiments were repeated at least three times with similar results.

(A) PUB12 and PUB13 ubiquitinate FLS2. The HA-tagged FLS2 cytosolic domain was purified as MBP fusion protein. The ubiquitination of FLS2 was detected by an anti-HA antibody. The overall ubiquitination was detected by an anti-FLAG antibody. (B) C262 and W289 are required for PUB13 E3 ligase activity. (C) PUB13 ubiquitinates FLS2, but not BAK1 or BIK1. The above experiments were repeated three times with similar results.

(A) ROS burst in Arabidopsis leaves triggered by flg22. Leaf discs were treated with H₂O (Ctrl) or 100 nM flg22. The data are shown as means ± standard errors from 40 leaf discs. (B) flg22-induced callose deposition. Leaves were treated with 1μM flg22 for 6 and 12 hr. (C) flg22-induced gene induction. Twelve-day-old Arabidopsis seedlings were treated with 10 nM flg22 for 0.5 or 6 hr. The data are shown as means ± standard errors from 3 independent biological repeats. (D) The bacterial growth assay of DC3000 and Psm. Four-week-old Arabidopsis plants were inoculated with bacteria at a concentration of 5×10⁵ cfu/ml. The data are shown as means ± standard errors from 3 replicates. * indicates a significant difference with p<0.05 when compared with data from WT plants. (E) PUB12/13 promote FLS2 degradation. Twelve-day-old seedlings were treated with or without 50 μM MG132 for 1 hr before 1 μM flg22 treatment for 0.5 hr. The equal protein loading was shown by Coomassie blue staining (CBS) for RuBisCo (bottom panel). The above experiments were repeated three to four times with similar results.