Extended Data Fig. 2: MAMP-triggered monoubiquitination of BIK1-family RLCK proteins.
From: Ligand-induced monoubiquitination of BIK1 regulates plant immunity
a, Flg22 induces monoubiquitination of BIK1. Protoplasts from wild-type plants were transfected with BIK1–HA and FLAG–UBQ or a vector (Ctrl), and then treated with 100 nM flg22 for 30 min. After immunoprecipitation with anti-FLAG agarose, ubiquitinated BIK1 was detected by immunoblotting using anti-HA antibody (top). Middle, BIK1–HA proteins; bottom, CBB staining for RuBisCO (RBC). b, Flg22 induces BIK1 monoubiquitination in pBIK1::BIK1-HA transgenic plants. Protoplasts from pBIK1::BIK1-HA/bik1 transgenic plants were transfected with FLAG–UBQ and then treated with 100 nM flg22 for 30 min. After immunoprecipitation with anti-FLAG agarose, ubiquitinated BIK1 was detected by immunoblotting with anti-HA antibody (top). Bottom, BIK1–HA proteins. c, BAK1 is constitutively polyubiquitinated in vivo. Protoplasts from wild-type plants were transfected with BAK1–HA and FLAG–UBQ or control, and then treated with 100 nM flg22 for 30 min. Immunoprecipitation was carried out with anti-FLAG agarose. Ubn-BAK1 proteins were detected as a smear with anti-HA immunoblotting (top). Middle, BAK1–HA proteins; bottom, CBB staining for RBC. d, Flg22 induces FLS2 polyubiquitination. Protoplasts from wild-type plants were transfected with FLS2–HA and FLAG–UBQ and then treated with 100 nM flg22 for 30 min. e, f, Monoubiquitination of BIK1 with UBQ(K0). Protoplasts from pBIK1::BIK1-HA (e) or 35S::BIK1-HA (f) transgenic plants were transfected with FLAG–UBQ(K0) (all lysine residues mutated to arginine) and then treated with 100 nM flg22 for 30 min. The mutations of lysine to arginine in UBQ(K0) are shown at the top of e with amino-acid positions labelled. g, PYR-41 blocks flg22-induced BIK1 monoubiquitination. PYR-41 (50 μM) was added 30 min before flg22 treatment. h, Flg22 induces BIK1 monoubiquitination in the presence of MG132. MG132 (2 μM) was added 1 h or 2.5 h before treatment with flg22. i, Flg22-induced BIK1 monoubiquitination depends on FLS2 and BAK1. Protoplasts isolated from wild-type, fls2 or bak1-4 plants were transfected with BIK1–HA and FLAG–UBQ and then treated with 100 nM flg22 for 30 min. j, elf18, pep1 and chitin induce BIK1 monoubiquitination. 1 μM elf18, 200 nM pep1 or 100 μg/ml chitin was added to protoplasts for 30 min. k, The BIK1 homologue PBL1 is monoubiquitinated upon treatment with flg22. PBL1–HA and FLAG–UBQ were expressed in protoplasts. l, Flg22 induces monoubiquitination of the BIK1-family RLCK PBL10 but not of BSK1. HA-tagged PBL10 or BSK1 was expressed with FLAG–UBQ in wild-type protoplasts. Experiments were repeated at least three times with similar results.
