Figure 2: ABI1 can interact with PUB12 and PUB13.

(a) ABI1 interacts with PUB12 and PUB13 in a yeast two-hybrid assay. AD: Gal4 activation domain; BD: Gal4 DNA-binding domain. 2D: synthetic dropout medium without Trp and Leu; 3D: synthetic dropout interaction medium without Trp, Leu and His. (b) The expression of PUB12 and PUB13 is induced by ABA treatment. RNAs were isolated from 7-day-old seedlings treated with 50 μM ABA for different times. Three independent experiments were done, each with three replicates. (c) ABI1, but not ABI2, interacts with PUB12 and PUB13 in a Co-IP assay. ABI1-Myc and PUB12- or PUB13-Flag plasmids, or ABI2-Myc and PUB12- or PUB13-Flag plasmids, were co-transfected into Arabidopsis protoplasts. Co-IP was carried out with anti-Myc agarose from total isolated proteins, and immunoblotting analysis was done with anti-Flag and anti-Myc antibody. (d) ABI1 does not co-immunoprecipitate PUB9 but co-immunoprecipitates PUB13 in the Arabidopsis protoplast transient expression assay. ABI1-Myc and PUB9-Flag or PUB13-Flag plasmids were co-expressed in protoplasts. Co-IP and immunoblotting analysis were done as in c. (e) The domain composition of PUB12/13 proteins. UND, U-box N-terminal domain; U, U-box domain; ARM, Armadillo repeat domain. (f) ABI1 co-immunoprecipitates the ARM domain of PUB12 but not UND-U-box in the Arabidopsis protoplast transient expression assay. ABI1-Myc and ARM-Flag, ABI1-Myc and UND-U-box-Flag, or ABI1-Myc and PUB12-Flag plasmids were co-expressed in protoplasts. Co-IP and immunoblotting analysis were done as in c. (g) ABI1 interacts with the ARM domain of PUB13. The assay was similar to that in f. (h) ABI1 can interact with PUB12 or PUB13 as indicated by the split firefly luciferase complementation imaging (LCI) assay. ABI1 translationally fused with the C terminus of LUC (ABI1-cLUC) was co-expressed with PUB13 translationally fused with the N-terminus of LUC (PUB13-nLUC) by co-infiltrating Agrobacterium carrying different plasmids into Nicotiana benthamiana leaves. Images were collected 3 d after Agrobacterium infiltration. (i) ABI1 can interact with the ARM domain of PUB12 or PUB13 as indicated by the LCI assay as in h. The interaction of FLS2-cLUC with PUB13 ARM-nLUC was used as a positive control.