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. 2022 May 30;34(9):3319–3338. doi: 10.1093/plcell/koac161

Figure 5.

Figure 5

RLI1a and RLI1b have different motif recognition properties. A, WebLogos of the R1BS and P1BS motifs. B, EMSA showing that both RLI1a and RLI1b can bind to the R1BS and P1BS cis-elements. DNA fragments containing R1BS and P1BS were incubated with RLI1a-His and RLI1b-His recombinant proteins as indicated. 6xHis proteins were used as negative controls. C, Y1H analysis of the DNA binding ability of RLI1a and RLI1b with the R1BS and P1BS motifs. pLacZ2u was used as a negative control. β-Galactosidase activity was measured to indicate the DNA binding ability of RLI1a and RLI1b with the R1BS and P1BS motifs. Values represent means ± sd of four replicates. (**P < 0.01; Student’s t test). D, Analysis of the transcriptional activation properties of RLI1a and RLI1b in N. benthamiana leaves. A LUC reporter driven by the artificial promoters 4×R1BS-min35S and 4×P1BS-min35S was used to test the transcriptional activity of RLI1a and RLI1b. The different combinations were co-transformed in N. benthamiana leaves via A. tumefaciens-mediated transfection and observed 2 days later; empty vectors with 4×R1BS-min35S and 4×P1BS-min35S were used as the negative control. E, Assays of RLI1a and RLI1b transactivation of the artificial promoters 4×R1BS-min35S and 4×P1BS-min35S using the dual-LUC transient transactivation assay system in N. benthamiana leaves. Data represent means ± SD (n = 4, four repeats in different N. benthamiana leaves in one experiment) (**P < 0.01; Student’s t test).