Fig. 6

CsGRAS9 mutants of Citrus sinensis cv. 'Anliu' sweet orange generated by genome editing of the promoter of CsGRAS9. A Chromatograms of PCR products from wild-type (WT) C. sinensis cv. 'Anliu' sweet orange serve as the negative control. Colony sequencing chromatograms from the T0 proCsGRAS9-edited C. sinensis cv. 'Anliu' line #18–2 show three types of insertions and deletions at the target site: Type I (+ 1 bp insertion), Type II (+ 1 bp different insertion), and Type III (−86 bp deletion). Representative chromatograms are shown. In line #23, mutations in both alleles of the CsGRAS9 promoter are shown, with a 62 bp deletion as the only type of mutation. Colony sequencing chromatograms from line #9 show four types of mutations: Type I (+ 1 bp insertion), Type II (−62 bp deletion), Type III (−86 bp deletion), and Type IV (WT). EBE regions are highlighted in red. “–” indicates deletions, “ + ” indicates insertions. The underlined black (CCT) indicates PAM. B Schematic representation of the CRISPR construct containing the CsGRAS9 gRNA. The purple block indicates the sgRNA scaffold, and red nucleotides were chosen for the gRNA in the EBE region of the CsGRAS9 promoter; the protospacer-adjacent motif (PAM) is underlined. C-D GUS staining and expression in N. benthamiana leaves transformed with effector and reporter combinations via transient expression assays. The CDS regions of pthA5 and pthA6 were cloned into the pHB vector as effectors. Truncated promoter fragments of the 604-bp Type III (−86 bp) promoter from T0 proCsGRAS9 line #18–2 were fused to pCAMBIA1381::GUS. Effector construct pHB-pthA5 and reporter vector T0 proCsGRAS9 line #18–2 Type III (−86 bp)::GUS were used in GUS assays (C). Asterisks indicate statistically significant differences (****p < 0.0001) using Student’s t-test. Truncated promoter fragments of the 628-bp Type I (−62 bp) promoter from T0 proCsGRAS9 line #23 were fused to pCAMBIA1381::GUS. Effector constructs pHB-pthA5 or pHB-pthA6 and reporter vector T0 proCsGRAS9 line #23 Type I (−62 bp)::GUS were used for GUS assays (D). Asterisks indicate statistically significant differences by one-way ANOVA with Tukey’s test (**p < 0.01, ***p < 0.001). E Leaves of WT and T0 proCsGRAS9-edited C. sinensis cv. 'Anliu' lines (#9, #18–2, and #23) were infiltrated with Xcc suspensions (OD₆₀₀ = 1.0) of Xcc049E/EV, Xcc049E/pthA4, and mixed suspensions of Xcc049E/pthA4 + pthA5 and Xcc049E/pthA4 + pthA6 in a 1:1 ratio. Citrus canker symptoms were evaluated 21 days after inoculation. F Bacterial growth of Xcc strains in the leaves of WT and proCsGRAS9 mutants of 'Anliu' sweet orange was monitored 17 days post-inoculation. Error bars represent standard deviations of three biological replicates. Different letters indicate statistically significant differences by two-way ANOVA with Tukey’s test (p < 0.05). G-H Relative expression of CsGRAS9 (G) and CsLOB1 (H) in leaves of WT and T0 proCsGRAS9-edited C. sinensis cv. 'Anliu' lines (#9, #18–2, and #23) measured 48 h post-inoculation (OD₆₀₀ = 1.0). CsEf1a was used as a constitutive standard. Different letters indicate statistically significant differences by two-way ANOVA with Tukey’s test (p < 0.05)