2.3 Vector construction and tobacco transformation
For RNAi vector construction, the different CDS fragments ofNtMYB12a and MtMYB12b genes were amplified with gene specific primers containing attB adaptor, the correct fragments were then cloned into pHellsgate 2 vectors via BP reaction. The complete open reading frame of NtMYB12a gene without the stop codon was amplified and digested with the endonucleases Kpn I and Spe I (Supplementary Figure S6a), and then cloned into the pCAMBIA1300 vector to obtain the fusion NtMYB12a-GFP, under the control of the CaMV35S promoter (Supplementary Figure S6b). Similarly, the NtMYB12a CDS sequence was also amplified and digested with the endonucleases XhoI and ApaI, and then cloned into pGreen-35S-GR (GLUCOCORTICOID RECEPTOR) to obtain an in frame fusion of NtMYB12a-GR (Chen et al., 2014). For Cas9/sgRNA vector construction, 20 nucleotides located in the first exon ofNtMYB12a gene was selected and used for sgRNA generation via overlapping PCR method as described previously (Xie et al., 2017). The sgRNA was then subcloned into the pORE-Cas9 binary vector. The predicted promoter fragment (2 kb) of NtMYB12a gene was amplified and digested with Hind III and BamH I, and then cloned into pBI121 to construct theProNtMYB12a: GUS vector. All the primers used in the vector constructions are listed in Supplementary Table S3.
The recombinant plasmids were transformed into Agrobacterium tumefaciens strain GV3101, which was used for the transgenic manipulation via leaf disks method (Palmgren, Mattson, & Okkels, 1993). The positive transgenic plants were selected with hygromycin or basta, confirmed by DNA and RNA analyses (Supplementary Figure S6c), and then self-pollinated twice to generate T2 transgenic progeny. To detect the mutations generated by Cas9/sgRNA, an about 250 bp fragment of NtMYB12a gene containing the target sites was amplified and used for Hi-TOM sequencing analysis as described in previous study (Q. Liu et al., 2019). The individual plants with >90% mutations were chosen for the subsequent studies (Supplementary Figure S6d).