Figure 8.

ABA signal transduction factors MdbZIP23 and MdbZIP46 interact with MdWRKY9 in vivo and vitro to further regulate the activity of MdSWEET9b. A, B) Y2H assays. (A) Screening for self-activating regions of MdWRKY9-BD vector that F₁ segment was self-activated. Thus, we used MdWRKY9-F5-PGBKT7 as the bait for subsequent Y2H assays. (B) MdWRKY9 interacted with MdbZIP23 and MdbZIP46. The empty pGADT7 vector (AD) served as a negative control. Blue lines indicate interactions between MdWRKY9 and MdbZIP23/46. C, D) MdWRKY9 interacted with MdbZIP23 and MdbZIP46 in pull-down assays. The “+” and “−” indicate the presence and absence of the indicated protein, respectively. E) MdWRKY9 interacted with MdbZIP23 and MdbZIP46 in BiFC assays. F) LUC experiments showed that MdbZIP23 and MdbZIP46 interacted with MdWRKY9 to promote the regulation of MdWRKY9 on MdSWEET9b. Error bars represent the ±SD of three independent biological replicates. Asterisks indicate statistical significance (**P < 0.01, Student's t-test). G, H) EMSA experiments showed that MdbZIP23 and MdbZIP46 interacted with MdWRKY9 to promote the regulation of MdWRKY9 on MdSWEET9b. The “+” and “−” indicate the presence and absence of the indicated probe or protein, respectively.