Fig. 5: PtrVCS2 is recruited to the PtrWOX4a promoter through dimerization with PtrWOX13a.
a–i, BiFC assays in P. trichocarpa stem-differentiating xylem (SDX) protoplasts showing that PtrVCS2 interacts with PtrWOX13a (a), PtrVCS19 (b) and PtrVCS94 (c) but not PtrWOX4b (d) in vivo. Each BiFC pair of constructs was co-transfected with the H2A-1–mCherry nuclear marker construct. Co-transfection of each construct of interest with empty plasmid served as controls (e–i). Scale bars, 10 μm. j, Interaction of PtrWOX13a–PtrVCS2 dimer determined by pull-down assays. k, MEME-ChIP analysis identifies C(A/C)ATCA(A/C) as a statistically defined motif (e-value, 5.2 × 10−10). A total of 6,790 PtrVCS2 binding peaks identified from three biological replicates of ChIP experiments were used for MEME-ChIP analysis. l, Schematic diagram of the WOX13 binding motif in the PtrWOX4a promotor. m, EMSA showing that PtrWOX13a binds to the CAATCAC motif in the PtrWOX4a promoter. n, PtrVCS2 alone fails to bind to the CAATCAC motif in the PtrWOX4a promoter, and PtrWOX13a is required for the association of PtrVCS2 with the PtrWOX4a promoter. In m and n, the nucleotide sequences of the WT M1 and M2 and the mutated M1 (mM1) and M2 (mM2) are shown. The core sequences are shaded in black, and the mutated nucleotide is shaded in grey. Unlabelled PtrWOX4a promoter fragments were used as competitors. Empty vector (pET101-His) was used as a negative control. o,p, ChIP–qPCR assays showing that PtrVCS2 associates with the PtrWOX4a promoter. Transgenic plants overexpressing 3×FLAG (control) or PtrVCS2–3×FLAG were used for the ChIP assays with anti-FLAG antibody in o. WT plants were used for the ChIP analysis with anti-PtrVCS2 antibody in p, and anti-IgG antibody was used as a control. Enrichment of DNA was calculated as the ratio between PtrVCS2–3×FLAG and 3×FLAG or between anti-PtrVCS2 antibody and anti-IgG antibody, normalized to that of the PtrACTIN gene. The data are shown as mean ± s.e.m.; n = 3 biological replicates; two-tailed Student’s t-test; *P < 0.05; NS, not significant. The experiments in a–j,m,n were repeated independently three times, with consistent results.
