Fig. 5: CRISPR-Cas9-mediated gene editing of GhSWEET genes reduces Xcm-induced water-soaked lesion development.
From: Activation of three targets by a TAL effector confers susceptibility to bacterial blight of cotton
A The CRISPR-GhSWEET14a/b gene-edited cotton line harbors early frameshift deletions in both GhSWEET14a and GhSWEET14b. The target site was amplified by PCR from genomic DNAs of the edited line for amplicon sequencing. Nucleotide deletions are denoted by red dashes with amino acid sequences displayed above and below, frameshift mutations indicated by red letters and shaded boxes for gRNA target sequences and PAM sites with numbers relative to the translational start codon as 1. B Water-soaked symptom development is reduced in CRISPR-GhSWEET14a/b cotton upon Xcm TX4 infection. Cotton plants were syringe-inoculated with Xcm TX4 or HM2.2 at OD600 = 0.05 with photos shown at 5 dpi. C The CRISPR-pGhSWEET10D line contains a 7-nucleotide deletion in the GhSWEET10D promoter at the Avrb6 EBE, determined by PCR amplificon sequencing. Nucleotide deletions are denoted by red dashes with sequencing chromatogram shown below, shaded boxes for the gRNA target sequence and PAM site and the underlined sequence for Avrb6 EBE. D Xcm H1005-mediated upregulation of GhSWEET10D is reduced in the CRISPR-pGhSWEET10D line. RT-qPCR analysis was done as in Fig. 3A, B. Data are shown as mean ± s.d. (n = 3) from three independent repeats. Asterisks indicate a significant difference (*p < 0.05) compared to the control line using a two-tailed Student’s t-test. E Lesion development induced by Xcm H1005 is reduced in the CRISPR-pGhSWEET10D line. Twenty-one-day-old cotton plants were vacuum-infiltrated with Xcm H1005 at OD600 = 0.0001 followed by counting water-soaked lesions on the 3rd true leaf at 10 dpi. Data are shown as mean ± s.e. (n = 6) from six independent repeats. Asterisks indicate a significant difference (*p < 0.05) compared to the control line using a two-tailed Student’s t-test. F The CRISPR-pGhSWEET10D line exhibits reduced water-soaked symptoms following Xcm H1005 infection. Inoculations were done as in Fig. 1C. Photos shown at 4 dpi. The experiments in E, F were repeated three times with a similar result. Source data are provided as a Source Data file.
