Figure 4.

SDE15 interacts with CsACD2. A, Y2H assays using SDE15 without the signal peptide as the bait and full-length CsACD2 protein as the prey. SDE15 fused to the GAL4 DNA BD was expressed in combination with CsACD2 fused to the GAL4 AD in the yeast strain Y2HGold. Strains were grown on double dropout medium (DDO) with -Trp and -Leu and screened on quadruple dropout medium (QDO) with -Trp, -Leu, -Ade, and -His supplemented with X-α-Gal and Aureobasidin A (QDO/X/A). The empty BD and AD vectors were used as the negative controls. B, GST pull-down assay. GST-SDE15 and GST EV were expressed in E. coli, immobilized on glutathione sepharose beads, and incubated with E. coli lysate containing MBP-CsACD2. Total cell extract (Input) and eluted protein (Elute) were immunoblotted using the anti-MBP and anti-GST antibody. C, Coimmunoprecipitation (Co-IP) assay. HA-tagged CsACD2 and eYFP-tagged SDE15 were coexpressed in the leaves of N. benthamiana through agroinfiltration. HA tag and eYFP proteins were also expressed in the leaves of N. benthamiana as controls. Co-IP assays were performed using anti-HA and anti-GFP antibodies to determine associations. D, The RCCR domain of CsACD2 interacts with the SDE15 in E. coli lysate. E, The C-terminal of SDE15 interacts with CsACD2. GST-SDE15, GST-SDE15∆N, GST-SDE15∆C, and GST EV were expressed in E. coli, immobilized on glutathione sepharose beads, and incubated with E. coli lysate containing MBP-CsACD2, MBP-RCCR, and free MBP proteins. Total cell extract (Input) and eluted protein (Elute) were immunoblotted using the anti-MBP and anti-GST antibody.