Fig. 4
Verification of the key functional elements of the Xa7 gene. (a) The key sequences in the promoter targeted by genome editing. EBEAvrXa7 are displayed in blue. The sgRNA targeting sequences are underlined. W6–4 and W7–4 are T1 homozygous transgenic lines obtained using Target1 site-specific editing, W8–7 and W9–6 are T2 homozygous transgenic lines obtained using Target2 site-specific editing. (b) Transcription of Xa7 in the homozygous promoter mutant lines in response to Xoo strain PXO86. IRBB7C, W6–4C, W7–4C, W8–7C, and W9–6C stand for the lines that were inoculated with Xoo strain PXO86 at day0. IRBB7T, W6–4T, W7–4T, W8–7T, and W9–6T stand for the same lines inoculated with Xoo strain PXO86 at 3 days after inoculation. The average expression level of Xa7 in IRBB7 at 0 d (IRBB7C) was set as “1”. The expression levels of other lines were used to compare with that of IRBB7C. “**” indicates a significant difference between IRBB7C and other plants at P < 0.01. (c) Disease phenotype of the Xa7 homozygous promoter mutants after inoculation with the Xoo strain PXO86. Scale bar stands for 5 cm. (d) Xa7 coding region for genome editing. The sgRNA targeting sequences are underlined. ATG code initiation site is marked in red, base insertions are marked in blue lowercase letters, while base deletions are marked as “-”. W12–1, W12–6, W13–4, and W15–3 are T1 homozygous transgenic lines obtained using Target3 and Target4 site-specific editing. (e) Transcription of Xa7 in the homozygous CDS mutant lines in response to Xoo strain PXO86. IRBB7C, W12–1C, W12–6C, W13–4C, and W15–3C stand for the lines that were inoculated with Xoo strain PXO86 at day0. IRBB7T, W12–1 T, W12–6 T, W13–4 T, and W15–3 T stand for the same lines inoculated with Xoo strain PXO86 at 3 days after inoculation. The average expression level of Xa7 in IRBB7 at 0 d (IRBB7C) was set as “1”. The expression levels of other lines were used to compare with that of IRBB7C. “**” stand for a significant difference between IRBB7C and other plants at P < 0.01. (f) Disease phenotype of Xa7 homozygous CDS mutant lines after inoculation with the Xoo strain PXO86. Scale bar stands for 5 cm. Each data in (b) and (e) represents the mean ± standard deviation (n = 3 replicates). Student’s t-test analysis indicates a significant difference (compared with control, ** P < 0.01)