Type B RRs binding sites show additive effect on the expression of ZmWUS1-B.A) SEM images of WT and edited ear primordia. WT, wild-type siblings; +/▵113, heterozygous 113 bp deletion at the enhancer region; +/▵33, heterozygous 33 bp deletion at the enhancer region; +/▵15, heterozygous 15 bp deletion at the enhancer region. Scale bars 500 μm. B) RT-qPCR assays comparing ZmWUS1 expression in 3 enhancer-edited mutants. For each genotype, a minimum of 3 biological replicates were performed. C) SEMs of immature ears of WT and homozygous ▵113 and ▵33 edits. SEM images were digitally extracted for comparison. Scale bars 500 μm. D) Mature ears of WT and homozygous ▵113 and ▵33 edits. Scale bars 2 cm. E to G) Transient transactivation in maize protoplasts. E) Reporter and effector constructs used in this study. The firefly luciferase is driven by the proximal promoter of ZmWUS1-B (denim arrows), including 444 bp (pWUS1), 570 bp of the Bif3 allele (p570), and 3 CRISPR-edited Bif3 alleles of ZmWUS1-B (▵113, ▵33, and ▵15). Two additional promoters were synthesized to carry a deletion of 22bp (between the 1st and 2nd AGATAT motifs; S11) and an insertion of 10 bp (between the 1st and 2nd AGATAT motifs; S43). The Renilla luciferase driven by the CaMV 35S promoter is used for normalization. F and G) Quantification of promoter activities when induced by type B RRs, ZmRR8 and ZmRR11. A minimum of 3 biological replicates were performed. Student's t tests P-value annotation legend: ns, P > 0.05; *0.01 < P < 0.05; **0.001 < P < 0.01; ***0.0001 < P < 0.001; ****P < 0.0001; error bars indicate 95% confidence interval.