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. 2019 Apr 18;15(4):e1007728. doi: 10.1371/journal.ppat.1007728

Fig 1. Identification of MKK2 as a new bona fide target of TYLCCNB βC1.

Fig 1

(A) Commassie Brilliant Blue stained SDS-PAGE of immunoprecipiated βC1 complexes prepared from Nicotiana benthamiana for proteomics analysis. Arrows indicate collected bands. (B)-(E) Specific βC1-MKK2 interaction was confirmed by Y2H (B), Co-IP (C), LCI (D), and BiFC (E) assays. In (B), Yeast strain Gold transformed with the indicated plasmid combinations was spotted with 10-fold serial dilutions on synthetic dextrose (SD)/-Leu/-Trp medium and SD/-Leu/-Trp/-His containing 5 mM 3-aminotriazole (3-AT). Interaction between βC1 and NtRFP1 serves as the positive control. In (C), N. benthamiana leaves were infiltrated with A. tumefaciens cells harboring 3Flag-MKK2 with GFP-βC1 or GFP. Co-IP assay was carried out with anti-Flag M2 magnetic beads (Sigma). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblot using anti-GFP and anti-Flag antibody, actin serves as a control. In left panel of (D), Schematic diagram shows the combinations of the infiltrated constructs used for the luciferase complementation assays. In the right panel, the fluorescence signal at different parts of the leaf shows the protein-protein interaction. The combination of NLuc-AGO1 with CLuc-CMV 2b serves as the positive control. In (E), MKK2-nYFP and βC1-cYFP were transiently expressed in the 35S-RFP-H2B transgenic N. benthamiana leaves. Combinations of agro-infiltrated constructs were indicated. Columns from left to right represent YFP fluorescence, RFP fluorescence, bright field and YFP/RFP/bright field overlay. Bars represent 50 μm. (F) Immunocomplex kinase assay shows that βC1 reduces MKK2 mediated MPK4 activation. Flag-MPK4 was transiently expressed in N. benthamiana leaves with HA-tagged MKK2 or its constitutively active form (HA-MKK2/HA-MKK2ac) and GFP-tagged βC1 (GFP-βC1). Flag-MPK4 was immunoprecipitated by α-Flag conjugated beads and subjected for immunoblot using anti-pTEpY antibody. The protein loading of Flag-MPK4, HA-MKK2/MKK2ac, GFP-βC1 and GFP are shown by immunoblot. The asterisk (*) indicates cross-reaction band. Three biological replicates were performed.