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. 2022 Aug 30;190(4):2688–2705. doi: 10.1093/plphys/kiac404

Figure 6.

Figure 6

PtoWRKY40 and PtoPHL3 directly regulate expression of PtoPHT1s. A, The structure of vectors constructed for a dual-LUC assay. B, Dual-LUC assay demonstrating that PtoWRKY40 and PtoPHL3 could regulate transcriptional activity governed by the promoters of PtoPHT1;3, PtoPHT1;4, and PtoPHT1;5 (1,500-bp length regions) in N. benthamiana leaves. Error bars indicate SD values from three independent biological replicates. Different letters above bars represent statistically significant differences between groups (P < 0.05) as determined by one-way ANOVA Duncan’s test. C, Distribution of W-box and P1BS elements in 1,500-bp length promoter regions of PtoPHT1;3, PtoPHT1;4, and PtoPHT1;5. Blue and brown boxes represent W-box and P1BS elements, respectively. D, ChIP-qPCR analysis showing the binding of PtoPHL3 and PtoWRKY40 to, respectively, P1BS and W-box in the promoters of PtoPHT1;3, PtoPHT1;4, and PtoPHT1;5 in vivo. Error bars indicate SD values from three independent biological replicates. Asterisks above bars indicate statistically significant differences between the WT and transgenic plants. Student’s t test was used to determine significance statistics; *P < 0.05, **P < 0.01, ***P < 0.001. E, EMSA assay verifying that PtoPHL3 and PtoWRKY40 bind to, respectively, the P1BS and W-box elements in vitro.