Construction of Recombinant TRV-based Vector

The TRV2-GFP empty vector with EGFP-tagged coat protein (CP) fragment (Figure 7) exhibited similar efficiency in infection and gene silencing to TRV2 vector [51 (link)]; then, it was linearized using restriction enzymes (Xba I and Sac I) at 37 °C for 30 min in this study. The linearized vector was then linked with the isolated PlHB31 fragment using a Vazyme One Step Cloning Kit (Takara, Shiga, Japan), and the product was identified by agarose gel electrophoresis and purified using a Mini BEST Agarose Gel Extraction kit (Takara, Shiga, Japan). The purified recombinant plasmid was transformed into 100 μL of Escherichia. coli DH5α competent cells (Bioteke, Beijing, China) and then incubated in Luria–Bertani (LB) liquid medium for 1 h (37 °C, 200 rpm); thereafter, competent cells were selected on LBK plates containing 50 mg/L kanamycin after 12–16 h of incubation (37 °C, 200 rpm, in darkness). Monocolonies were inoculated into fresh LB medium, and plasmids were extracted for further DNA sequence alignment to verify the correct insertion of the fragment.