Figure 1.

SOS2 Interacts with 14-3-3 λ in Planta.

(A) SOS2 kinase assay. SOS2 was immunoprecipitated from sos2-2 Pro35S:6×Myc-SOS2 plants grown in the absence (−) or presence (+) of salt and used in in vitro kinase assays with GST-SCaBP8 as substrate. IB, immunoblot; CBB, Coomassie Brilliant Blue; Autorad, autoradiograph. Experimental details are provided in Methods.

(B) Analysis of the SOS2 and 14-3-3 λ interaction in Arabidopsis leaf protoplasts. Purified Pro35S:6×Myc-SOS2 and Pro35S:Flag-HA-14-3-3 λ plasmids were cotransformed into Arabidopsis leaf protoplasts. Transiently expressed proteins were immunoprecipitated with anti-C-Myc antibody–conjugated agarose. Immunoblot assays with anti-C-Myc and anti-Flag antibodies were used to detect Myc-SOS2 and SOS2-interacting 14-3-3 λ, respectively. As a control, Myc-SOS2 or Flag-14-3-3 λ was transiently expressed in Arabidopsis leaf protoplasts and purified with anti-C-Myc or anti-Flag antibody–conjugated agarose, respectively. Anti-C-Myc and anti-Flag antibodies were used to detect immunoprecipitated Myc-SOS2 and Flag-14-3-3 λ, respectively. Experimental details are provided in Methods. IP, immunoprecipitation.

(C) In vivo pull-down assay to analyze the SOS2 and 14-3-3 λ interaction. Ten-day-old Pro35S:Flag-HA-SOS2 transgenic seedlings were treated with water or 100 mM NaCl for 16 h, and then Flag-HA-SOS2 was immunoprecipitated with anti-HA antibody–conjugated agarose. The immunoprecipitated proteins were analyzed by immunoblot using anti-Flag and anti-14-3-3 λ antibodies.

(D) Bimolecular fluorescence complementation analysis in N. benthamiana. Plasmids containing Pro35S:YFP^N-SOS2 and Pro35S:YFP^C-14-3-3 λ or Pro35S:YFP^N-SOS2 and Pro35S:YFP^C were transiently cotransformed into N. benthamiana leaves. The YFP fluorescence signal was detected using a Zeiss LSM 510 META confocal microscope. Experimental details are provided in Methods. Bar = 50 μm.