Fig. 3

MdFLP directly binds to the promoters of MdPIN3 and MdPIN10. (a) Y1H assays. MdFLP can bind to the promoter fragments of MdPIN3 and MdPIN10 in a Y1H assay. (b) GUS transient expression assays. Activation assay of MdPIN3 and MdPIN10 promoter activity in tobacco leaves by transient expression of MdFLP together with other effector genes of different combinations, as determined through a GUS activity assay. GUS relative expression level in transgenic leaves by qRT-PCR analysis is shown. At least three independent samples were used for gene expression analyses. Error bars represent ± SE. Significant differences were determined by Student’s t-test (**P < 0.01). (c) Schematic diagram of the promoters in the MdPIN3 and MdPIN10 genes. Red circles indicate AGCCG. The translational start site (ATG) is shown at position 0. Fragment b, e contains the cis-element AGCCG, fragment a contains the cis-element AACGG, fragment c contains the cis-element CGCGG, fragment f contains the cis-element TACCC, and fragment d serves as a negative control. (d) ChIP-qPCR assays were performed using ‘Orin’ apple calli harboring 35S::MdFLP-GFP and probed using anti-GFP antibodies. PCR products that were generated by primer pairs at position ‘b’, ‘e’ shown in c, which include the element AGCCG, are enriched in 35S::MdFLP-GFP transgenic calli. Error bars represent ± SE. Significant differences were determined by Student’s t-test (**P < 0.01). (e-f) EMSA showing that GST-MdFLP directly binds to the promoters of MdPIN3 and MdPIN10. The GST protein was incubated with the labeled probe in the first lane to serve as a negative control. Ten- and 20-fold excess unlabeled probes were used for competition