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. 2015 Mar 13;27(3):839–856. doi: 10.1105/tpc.114.134809

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© 2015 American Society of Plant Biologists. All rights reserved.

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Figure 4. — MPK4 Phosphorylates and Interacts with ASR3.

(A) MAPK-dependent ASR3 phosphorylation. Protoplasts were coexpressed with ASR3-FLAG and MAPK phosphatase MKP or MEKK1. MEK/MKK inhibitor U0126 was added to protoplasts 30 min before flg22 treatment. MAPK activation is shown by immunoblotting with α-pERK antibody (middle panel).

(B) flg22-activated MPK4 phosphorylates ASR3. The individual MAPKs were expressed in protoplasts, activated by flg22 treatment, immunoprecipitated with α-HA antibody, and subjected to an in vitro kinase assay using MBP-ASR3 as a substrate in the presence of [γ-³²P]ATP. Proteins were separated with SDS-PAGE and analyzed by autoradiography (upper panel), and the MAPK expression is shown by immunoblotting (bottom panel).

(C) Time course of flg22-activated MPK4 phosphorylation on ASR3. The experiment was performed as in (B) with 100 nM flg22 treatment for the indicated time. MBP-ASR3 is shown by Coomassie blue staining (CBB).

(D) Thr-189 is required for MPK4-mediated ASR3 phosphorylation. The experiment was performed as in (B) with different MBP-ASR3 mutants as substrates.

(E) The mpk4 mutant abolishes flg22-induced ASR3 phosphorylation. Protoplasts were isolated from Ler and the mpk4 mutant (in Ler background), transfected with ASR3-HA, and treated with 100 nM flg22 for the indicated time.

(F) ASR3 associates with MPK4 in Arabidopsis protoplasts. Protoplasts were cotransfected with ASR3-HA and MPK4-FLAG or an empty vector control (Ctrl). co-IP was performed with α-FLAG antibody (IP: α-FLAG), and the proteins were analyzed using immunoblots with α-HA antibody (IB: α-HA).

(G) ASR3 associates with MPK4 in 35S:ASR3-HA transgenic plants. Ten-day-old seedlings from two independent transgenic lines (OX9 and OX15) were used for co-IP, and transgenic plants carrying an empty vector were used as the control (Ctrl). Co-IP assay was performed with α-HA antibody, and the proteins were analyzed using immunoblots with α-MPK4 antibody (top). The input of ASR3-HA and MPK4 proteins is shown by immunoblots (middle and bottom).

(H) ASR3 directly interacts with MPK4 with an in vitro pull-down assay. GST or GST-MPK4 immobilized on glutathione Sepharose beads was incubated with MBP, MBP-ASR3, MBP-ASR3^T189D, or MBP-ASR3^T189A proteins. The beads were washed and pelleted for immunoblot analysis with α-HA antibody. PD, pull-down.

The above experiments were repeated three times with similar results.