Thermocycle 3000

Competent cells of Agrobacterium tumefaciens strain EHA105 (Hood et al., 1993) were transformed by electroporation with the p9‐C‐HS‐D4 binary vector and used to transform plantlets of Malus × domestica, cultivars ‘Gala’ and ‘Golden Delicious’, as described by Joshi et al. (2011). Transformations were performed in duplicate (V1 and V3 for ‘Gala’; V2 and V4 for ‘Golden Delicious’; Table 1) using from 300 to 1000 leaf explants (Table 1). Regenerated plants (obtained after 6–7 months from co‐culture with Agrobacterium) were screened to detect the presence of the T‐DNA cassette (Table 1). For each plant, genomic DNA was extracted from 2 leaves using the Illustra™ Nucleon DNA Extraction Kit PHYTOPURE™ (GE Healthcare), quantified on the NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific), diluted to 50 ng/μL and used for PCRs using the thermocycle‐3000 (Biometra), the GoTaq^® Green Master Mix 2X (Promega, Fitchburg, MA) and primers Cas9, VirG and MdTOPO6 (0.4 μm) listed in Table S1.

To produce the binary vector used for apple and A. thaliana transformations (Supplementary Tables S1, S2), genomic DNA was extracted from apple leaf tissue using the Illustra^TM Nucleon DNA Extraction Kit PHYTOPURE^TM (GE Healthcare). Extracted DNA was quantified on the NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific) and then used in a PCR aimed at amplifying 2 kb of intergenic genomic DNA sequence upstream of the transcription start site of MdmiR285N gene. PCR was performed on 40 ng of starting DNA using the thermocycle-3000 (Biometra), the Phusion® High-Fidelity DNA Polymerase (Thermo Fisher Scientific) and the pair of primers attB-MdmiR285N_Prom reported in Supplementary Table S4. The PCR product was directly cloned into a pENTR/D TOPO vector (Invitrogen), and subsequently the MdmiR285N promoter region was recombined by LR reaction (Invitrogen) into the GATEWAY^TM binary vector pKGWFS7⁶⁷ in-frame with the downstream GFP-GUS gene fusion system.