Figure 2.

PUB22 Interacts with and Is Phosphorylated by MPK3.

(A) Interaction between PUB22 and MPK3 detected by bimolecular fluorescence complementation in Arabidopsis protoplasts. nYFP-MPK3 or nYFP-MPK11 was coexpressed with cYFP-PUB22 W40A as indicated. Free mCherry was coexpressed to label the cytoplasm and nucleus. Shown are representative pictures. Arrows indicate chloroplast autofluorescence. Bar = 50 µm.

(B) UBQ10:GFP-PUB22/pub22 pub23 pub24 transgenic seedlings were treated with flg22 (1 µM) for 20 and 60 min and total protein (input) was subjected to IP with anti-GFP beads. Endogenous coimmunoprecipitated MPK3 was detected with MPK3-antibodies. The experiment was repeated three times with similar results.

(C) MBP-PUB22 pull-down (PD) assay using purified GST-MPK3 and GST-MPK6 on glutathione agarose beads as baits. Asterisk indicates a potential GST cleavage product.

(D) PUB22 mobility shift after flg22 treatment. UBQ10:GFP-PUB22/pub22 pub23 pub24 transgenic seedlings were treated with DMSO (ctrl), flg22 (1 µM), or okadaic acid (OA; 1 µM) for 20 min. Total protein samples were resolved by Phos-tag PAGE. The experiment was repeated with similar results. Mobility shift is indicated by the arrow.

(E) PUB22 mobility shift after flg22 treatment in the wild-type Col-0 and mpk3 backgrounds. HA-PUB22 was transiently expressed and treated with DMSO or flg22 (100 nM) for 20 min. Total protein samples were resolved by Phos-tag PAGE. The experiment was repeated three times with similar results. Mobility shift is indicated by the arrow.

(F) PUB22 mobility shift induction by MKK5 in wild-type Col-0 and mpk3 backgrounds. HA-PUB22 was transiently coexpressed with MKK5 LysArg (inactive) or AspAsp (constitutively active). After overnight incubation, total protein samples were resolved by Phos-tag PAGE. The experiment was repeated with similar results. Mobility shift is indicated by the arrow.

(G) GST-PUB22 was incubated alone or with activated GST-MPK3, GST-MPK4, and untagged MPK6 (white arrowheads). Phosphorylation was visualized with ProQ Diamond stain.

(H) Cartoon depicting the localization of the in vitro phosphorylated sites by MPK3 on PUB22 identified by LC-MS/MS.