Figure 4.

OsCAMTAPL interacted with the TIG-ANK domain of OsCAMTA3. (a) The different domains of OsCAMTA3 are shown. OsCAMTA3 contains the CG-1 (the gray area), TIG-ANK(the cyan-yellow area) and IQ (the blue area) domains. The numbers indicate the positions of amino acid sequences. (b) The TIG-ANK domain of OsCAMTA3 interacted with OsCAMTAPL in a Y2H assay. The coding sequence (CDS) of OsCAMTA3^CG−1, OsCAMTA3^TIG-ANK, or OsCAMTA3^IQs was fused to the vector pGADT7 (AD), and the CDS of OsCAMTAPL was fused to pGBKT7 (BD). The empty vectors pGADT7 (AD) and pGBKT7 (BD) were used as negative controls. Yeast cells containing BD-OsCAMTAPL and AD-OsCAMTA3^TIG-ANK plasmids were grown on SD-Leu-Trp-His-Ade medium. (c) Images of N. benthamiana leaves with the indicated constructs in LUC assays. The coding sequence (CDS) of OsCAMTA3^TIG-ANK was fused to the C-terminal fragment of firefly luciferase (cLUC), and the CDS of OsCAMTAPL was fused to the N-terminal fragment of firefly luciferase (nLUC). Green fluorescence protein (GFP)-nLUC and Myc-nLUC were used as negative controls. The indicated constructs were transiently coexpressed in 4-week-old N. benthamiana leaves, and the bioluminescence images were captured by a CCD camera. (d) Co-IP assay was performed by transiently coexpressing OsCAMTA3^TIG-ANK-Myc and OsCAMTAPL-Flag in N. benthamiana leaves. Total protein was extracted and subjected to immunoprecipitation by anti-Flag beads. Immunoblotting analysis was performed with anti-Myc and anti-Flag antibodies. OsCAMTAPL-Flag, GFP-Flag, and OsCAMTA3^TIG-ANK-Myc are indicated by blue, black, and red asterisks. Full-length blots are presented in Supplementary Figure S6.