Agroinoculation of TRV vectors

To generate pTRV2 containing the 3′ untranslated region of PhCESA3 (TRV2-PhCESA3), the gene sequence of 266 bp was PCR amplified using forward primers and reverse primers (Table S4), and the PCR products were inserted into the pTRV2 vector. Agrobacterium tumefaciens (strain GV3101) transformed with pTRV1 and pTRV2 derivatives were prepared as previously described^22,37. The Agrobacterium cells grown overnight were harvested and resuspended in inoculation buffer containing 10 mM MES, 200 mM acetosyringone, and 10 mM MgCl₂ to an OD₅₅₀ of 10. Following an additional 3 h of incubation at 28 °C, bacteria transformed with pTRV1 were mixed with bacteria containing the pTRV2 derivatives in a 1:1 ratio, and 200 to 400 μL of this mixture was injected into the stem or applied on the cut surface after removing the apical meristems of petunia plantlets. Approximately thirty plants were vaccinated with each vector. The inoculated plants were grown under greenhouse conditions (22–25 °C, 14 h light/10 h dark).