ARR1 and GAI interact physically in plants.

(A) Y2H assay of the interaction between ARR1 and truncated versions of GAI. (H, Histidine; 3-AT, 5mM 3-aminotriazol). (B) Y2H assay of the interaction between M5-GAI and truncated versions of ARR1. (H, Histidine; 3-AT, 5mM 3-aminotriazol). (C) Bimolecular Fluorescence Complementation assay of the interaction between GAI and ARR1 in agroinfiltrated N. benthamiana leaves. Size bars, 10μm. (D) Analysis of the interaction between HA-ARR1 or HA-ARR1ΔDDK with YFP-GAI by co-immunoprecipitation (co-IP) with anti-GFP in agroinfiltrated leaves of N. benthamiana. (E) Co-IP assay of the interaction between myc-GAI and HA-ARR1 in Arabidopsis protoplasts. The arrowhead indicates the size of the expected HA-ARR1 band. (F) Co-IP showing the interaction between RGA and ARR1-YFP-HA in Arabidopsis seedlings. Proteins were immunoblotted and consecutively detected with anti-GAI and anti-HA-peroxidase conjugate antibodies. The anti-GAI polyclonal antibody recognizes both GAI and RGA. Forty micrograms of soluble proteins were loaded as input and as unbound control. Soluble proteins from the null mutants gai-t6 and rga-24 grown for 7 days in 0.5 μM PAC plates in continuous light were used as controls. The blue lines in the marker lane indicate the position of the 64 kDa and 98 kDa bands in the upper and middle panels, respectively. Stained bands in the marker lane in the lower panel correspond to 64 kDa and 50 kDa. WT, wild-type Col-0; ARR1, 35S::ARR1-YFP-HA.