The N domain is the main functional inhibitory domain. A) Diagrams of dCas9 fused to full-length TEN or different truncated proteins of TEN. N, N-terminal domain; TCP, TCP domain; C, C-terminal domain. B) Relative CsACO1 expression shows that the N domain is the main transcriptional inhibitory domain of TEN. C) Representative photographs showing the phenotype of different transgenic plants harboring the indicated vector transformed in cucumber. All dCas9 variants fused to the N domain (dCas9-TEN, dCas9-N, and dCas9-N+C) and targeting the T4 site resulted in a tendril-less or modified tendril phenotype. Images were digitally extracted for comparison. Scale bars, 10 cm; Arrows point to normal tendrils, and the enlarged images and numbers (1 to 6) in the upper panel originated from the white dashed frames in the bottom panel. D) Diagram of the CsACO1 locus, showing the location of the corresponding sgRNAs (T1, T2, T3, and T4). E) and F) Relative CsACO1 expression when dCas9-N is targeted to 1 of the 4 sites shown in panel D in the cucumber hairy root system (E) and stable cucumber transformations (F). G) Representative photographs showing the phenotype of transgenic plants with dCas9-N targeted to each of the 4 sites in CsACO1. Images were digitally extracted for comparison. Scale bars, 10 cm; red arrows point to normally developed tendrils, and the enlarged images and numbers (7 to 9) in the upper panel originated from the white dashed frames in the bottom panel. Values are shown as means ± Sd (n = 3). Different lowercase letters indicate significant differences among means at P < 0.05 (1-way ANOVA with post hoc Tukey test).