Fig. 3: Bif3 is caused by ectopic expression of ZmWUS1-B.
From: Structural variation at the maize WUSCHEL1 locus alters stem cell organization in inflorescences
a Bif3 revertants identified from a targeted EMS suppressor screen. Three classes of revertants were identified. Revertant1 (Rev1) carries a G-to-A transition occurred at the start codon ATG of ZmWUS1-B. In revertant2 (Rev2), ZmWUS1-B carries a longer promoter (1178 bp from ATG) and lost the 119 bp region. In other revertants (Rev3–20), ZmWUS1-B is missing. b–d CRISPR-Cas9 targeted ZmWUS1 knock-out in Bif3 mutant. b ZmWUS1 gene structure with homeobox domain (HD in dashed white rectangle) highlighted in the first exon. gRNA in red with PAM site underlined. c The tassel (F1) and ear (BC1) of ZmWUS1-B knock-out plants (A619 background). nR, non-resistant to herbicide treatment, R, resistant to herbicide treatment. Scale bars: 1 cm. d CRISPR-Cas9 edits in ZmWUS1-B. gRNA targeted sequence in red. In blue, insertion sequence. e, f qRT-PCR of ZmWUS1 transcripts in wild-type, Rev2, and Rev1 (e) and of ZmWUS1-A and ZmWUS1-B in Rev1 by copy-specific primers based on the G/A SNP (f). Two-tailed Student’s t-test *p < 0.05. n+/+ = 3, nREV1 = 2, nREV2 = 2, nWUS1-A = 3, nWUS1-B = 3 (n independent experiments). g The unique 119 bp region of ZmWUS1-B contains three direct repeats. Yellow boxes with arrow indicate repeat units, and each repeat unit contains the AGATAT motif. h Electrophoretic mobility shift assay (EMSA) with ZmRR8. m119 bp region carries mutations in all repeats. i, j Transient transactivation in maize protoplasts. i Reporter and effector constructs used. The Firefly luciferase is driven by the proximal promoter of ZmWUS1-B (blue arrows), including 444 bp (p444) or 570 bp (p570) including the 119 bp region. The Renilla (Ren) luciferase is used for normalization. j ZmWUS1-B is induced by cytokinin treatments (6-BA), and by type-B ZmRR8 and ZmRR11. Two-tailed Student’s t-test *p < 0.05; **p < 0.01. p570m, 570 bp proximal promoter with AGATAT motifs mutated into TTTTTT. Plotted data represent mean ± standard deviation. Three replicates for each experiment.
