A visual reporter system for transgene screening and monitoring genome editing in tomato

Ningkai An; Jinbo Wei; Shiliang Chen; Xiaoyue Yang; Kemeng Wu; Xinjie Zhou; Jie Wang; Shasha Wu; Shurong Wu; Chenyi Liu; Yueyue Fu; Jinyu Zhang; Weizhen Yi; Huijun Zhu; Chao Sun; Shuang Wu; Ningkai An; Jinbo Wei; Shiliang Chen; Xiaoyue Yang; Kemeng Wu; Xinjie Zhou; Jie Wang; Shasha Wu; Shurong Wu; Chenyi Liu; Yueyue Fu; Jinyu Zhang; Weizhen Yi; Huijun Zhu; Chao Sun; Shuang Wu

doi:10.48130/vegres-0026-0007

Figure 1.

The vector backbone affects the expression of RUBY system across species and tissues. (a) Different vector backbones affect the expression of RUBY system in Arabidopsis seedlings, scale bar = 1 mm. (b), (c) Different vector backbones affect the expression of RUBY in tobacco (Nicotiana tabacum) tissue culture, scale bar = 1 mm. (d), (e) Different vector backbones induced no obvious pigment deposition in tomato tissue culture, scale bar = 1 mm. (f), (g) The RUBY system's expression in tomato flowers and fruits. (f) Scale bar = 1 mm. (g) Scale bar = 1 cm.

Figure 2.

Copy number detection of the RUBY system in Arabidopsis. (a) Quantification of RUBY's expression in p35S::RUBY-A and p35S::RUBY-B lines was performed to assess positive and false positive rates. Positive, red pigmentation; false positive, green pigmentation. (b) Chi-square analysis: The red dashed line = 3:1 segregation threshold (χ²_0.05 = 3.841); bars show the χ² values; p < 0.05 = significant fit to single-copy segregation.

Figure 3.

Plants' development and epidermal differentiation with the RUBY system. (a), (b) Phenotypic observation and quantification of wild-type (WT) and p35S::RUBY plants, scale bar = 1 cm. (c), (d) Observation and quantification of plant height in WT and p35S::RUBY plants, scale bar = 1 cm. (e) Analysis of leaf morphology in WT and p35S::RUBY plants, scale bar = 1 cm. (f) Analysis of fruit morphology and size in WT and p35S::RUBY plants, scale bar = 1 cm. (g), (h) Observation and quantification of trichome morphology in WT and p35S::RUBY plants, scale bar = 1 mm.

Figure 4.

Design and application of the RUBY system for inducible expression and visualizing genome editing. (a), (c) Schematic of the RUBY-labeled inducible expression and genome editing visualization system. (b) Validation of inducible expression using the RUBY system, scale bar = 1 mm. (d) Transient expression analysis of the RUBY-labeled genome editing visualization system in tobacco (Nicotiana benthamiana), scale bar = 1 cm. (e) A dual-phase CRISPR workflow enabling simultaneous gene editing and subsequent Cas9 elimination in tomato.