Plasmid construction and plant transformation
The coding sequence of each gene was first cloned into pDONR221 vector by BP reaction following the manufacture’s instruction (Life Technologies, cat. #11789-020). These entry vectors were recombined into destination vectors by LR reactions (Life Technologies, cat. #11791-020). For Crispr-cas9 constructs, two guide RNAs, target 1 and 2, or Target 3 and 4 were designed following the instruction in the website www.skl.scau.edu.cnand included in primers for PCR amplification (Xie et al., 2017). DNA fragments amplified using the plasmid pCBC-DT1T2.2 as a template were ligated into pG3K-U6SC vector using Golden Gate Assembly method, and all constructs were transformed into Agrobacterium strains LBA4404 harboring the helper plasmids pVS1-VIR2 (Zhang et al., 2019). Transgenic Arabidopsis seedlings with overexpression constructs were generated throughAgrobaterium -mediated floral dipping method (Clough & Bent, 1998). Transgenic tomato plants using cultivar ‘Heinz 1706’ were generated throughAgrobaterium -mediated transformation as previously described (H. J. Sun, Uchii, Watanabe, & Ezura, 2006).