Fig. 5: SMXL6, SMXL7, and SMXL8 directly interact with SPL9 and SPL15.

a Yeast two-hybrid assay shows that SMXL6, SMXL7, and SMXL8 interact with SPL9 and SPL15. LexA and JG served as the negative controls. b LCI assay and quantitative analysis of luminescence intensity showing the interaction between SPL9/SPL15 and SMXL6/7/8 in N. benthamiana epidermal cells. SPL9 and SPL15 were fused to the N-terminal fragment of luciferase (nLuc) while SMXL6/7/8 were fused to the C-terminal fragment of luciferase (cLuc). The interactions between cLuc and SPL9-nLuc or SPL15-nLuc were used as negative controls. Representative images of N. benthamiana leaves 72 h after infiltration were shown. Four independent determinations were assessed. c Pull-down assay shows that SMXL6 directly interacts with both SPL9 and SPL15 in vitro. Purified GST-SPL9SBP or GST-SPL15SBP recombinant proteins were incubated with protein extracts containing His-SMXL6 and further immobilized with glutathione sepharose beads. d Western blot of immunoprecipitated proteins (left panel) and co-immunoprecipitated proteins (right panel) transiently expressed in N. benthamiana. Immunoprecipitation was performed using anti-HA matrix and the co-immunoprecipitated proteins were detected with anti-MYC antibodies. e EMSA assay shows that SMXL6 protein does not inhibit the binding of SPL9SBP and SPL15SBP to the BRC1 promoter fragment. MBP protein is used as the negative control. f Transient expression assay shows that SMXL6, SMXL7, and SMXL8 repress the transactivation activity of both SPL9 and SPL15 on the LUC reporter gene driven by the BRC1 promoter.