Figure 2.

flg22 Perception Induces ASR3 Phosphorylation.

(A) ASR3 displays a mobility shift upon flg22 treatment. Protoplasts were transfected with HA-tagged ASR3 for 12 h and treated with 100 nM flg22 for the indicated amounts of time. RBC, Rubisco.

(B) The flg22 treatment induces ASR3 mobility shift in 35S:ASR3-HA transgenic plants. The leaves from 4-week-old ASR3-HA transgenic plants were hand-inoculated with 100 nM flg22 or water for 30 min. Control “-” denotes the leaves without inoculation.

(C) Multiple MAMPs trigger ASR3 mobility shift. Protoplasts were transfected with ASR3 for 12 h and treated with 100 nM flg22, 100 nM elf18, 50 μg/mL chitin, or 5 μg/mL LPS for 15 min.

(D) AvrRpt2 or AvrRps4 does not induce ASR3 mobility shift. Protoplasts were cotransfected with ASR3-FLAG and AvrRpt2-HA or AvrRps4-HA.

(E) Cold or heat treatment does not induce ASR3 mobility shift. Protoplasts were transfected with ASR3-HA for 8 h at 23°C and incubated for additional 2 h at 23, 4, or 42°C.

(F) The flg22-induced ASR3 mobility shift depends on functional FLS2/BAK1 receptor complex. Protoplasts isolated from fls2 or bak1-4 mutants were cotransfected with ASR3-HA and empty vector control (Ctrl), FLAG-tagged FLS2, FLS2 kinase mutant (FLS2km), or BAK1.

(G) Kinase inhibitor K252a blocks flg22-induced ASR3 mobility shift. K252a was applied 30 min before 100 nM flg22 treatment. The controls were nontreatment (-) or solvent (DMSO).

(H) λPP removes the flg22-induced mobility shift of ASR3. Protein extracts from protoplasts transfected with ASR3-HA or BIK1-HA were treated with λPP following the standard protocol. NaF, a phosphatase inhibitor, compromised λPP phosphatase activity.

(I) CIP treatment abolishes the mobility shift of BIK1 but not ASR3. Protoplasts were transfected with ASR3-HA or BIK1-HA and treated with or without 100 nM flg22 for 30 min.

The above experiments were repeated three times with similar results.