35S:miR-168a and 35S:rSlAGO1 vector construction and tomato transformation
Pre-SlmiR-168a was prepared using gene-specific primers. The sequence-confirmed polymerase chain reaction (PCR) fragment was cloned into pCAMBIA3301/Luc, which harboured two 35S Cauliflower mosaic virus promoters, the marker gene for kanamycin resistance, phosphinothricin, and luciferase. The bacterial colonies were screened for positive recombinant plasmids by colony PCR, restriction enzyme digestion, and sequence analysis. Binary vectors containing the expected insert were subsequently transferred into Agrobacterium tumefaciens GV3101 cells by electroporation. The competent cells harbouring the vector were transformed into JZ18 tomatoes using a tomato genetic transformation system. The obtained T1 transformants and their corresponding T2 families were assessed for the expression of the target gene by quantitative real-time PCR (qRT-PCR) and evaluation of the presence of the kanamycin marker gene. All primers used in this study are listed in Supplementary Table S1. To generate rSlAGO1 (theSlmiR-168a -resistant construct), mutations in theSlmiR-168a target site of SlAGO1 were inserted using two-step PCR mutagenesis. The 35S:SlAGO1 transformants were obtained using the same method as described above.