4.2. Transient Expression Assay in N. benthamiana

Plantlets of N. benthamiana were grown in a growth chamber at 26 °C with a 16 h photoperiod. The transformation protocol of N. benthamiana was modified from the procedure described in Shan et al. [25]. Agrobacterium tumefaciens strain EHA105 harboring the final ABE binary construct was cultured on Lysogeny broth (LB) medium supplemented with 50 mg L⁻¹ Kanamycin at 28 °C. The EHA105 cells were then pelleted and resuspended in MES buffer in the dark for 4 h at room temperature (RT) before infiltration. Leaves of 3–4 weeks N. benthamiana plants were infiltrated with Agrobacterium tumefaciens EHA105 strains carrying the final binary vectors with OD600 = 0.4. The leaves were harvested three days post infiltration, and the genomic DNA was extracted for base-editing identification according to the instruction of the DNAsecure plant kit (TIANGEN, Beijing, China).