In fusion enzyme

Bik-1 mRNA containing its 5’UTR and open reading frame (but lacking the Bik-1 LNA target site) was amplified from cDNAs via PCR and cloned into pT3TS-nCas9n vector (addgene #46757, a gift from Ariel Bazzini). Bik-1 mRNA was then synthesized using mMESSAGE mMACHINE^™ T3 Transcription Kit (AM1348). The mRNA was purified using RNeasy kit from Qiagen. The Bik-1 mRNA (153 pg) combined with Bik-1 LNA (22 pg) or Bik-1 LNA alone were co-injected into embryos at one-cell stage as the rescue experiment. Injected embryos then collected at 12 hpf for in situ hybridization.
We used In-Fusion enzyme (Takara Bio, #638947) to create a single nucleotide substitution on the 69th amino acid (Leucine to Stop) of Gag. The Bik-1 mRNA with the mutation (Rescue-Stop) was synthesized and purified as above. The Bik-1 mRNA with the mutation (153 pg) combined with Bik-1 LNA (22 pg) or Bik-1 LNA alone were co-injected into embryos at one-cell stage as in the original rescue experiment. Injected embryos were then collected at 12 hpf for in situ hybridization.

An overexpression vector containing PsnNAC090 was constructed using a homologous fusion method. The homologous arm containing a Spe1 digestion site was introduced at both ends of PsnNAC090 and reconstituted with a pBI121 vector using infusion enzyme (bought from Takara). The recombinant vectors were transferred into GV3101 Agrobacterium tumefaciens. Positive strains were cultured with OD600 of 0.6 in LB liquid medium containing 50 mg/L rifampicin and 50 mg/L kanamycin. The leaves of three-week-old tobacco plants were cut to 1.0 × 1.0 cm and soaked in the bacterial solution for 10 min. Then, the leaf pieces were cultured in screening medium containing 50 mg/L kanamycin and 200 mg/L ceftriaxone sodium until resistant young shoots sprouted. Then, the resistant seedlings were obtained by culturing resistant shoots in MS medium containing 50 mg/L kanamycin and 200 mg/L ceftriaxone sodium. DNA was extracted from the leaves of resistant seedlings for molecular identification using specific primers of pBI121-GFP vectors (Supplementary Table S2 and Figure S1). Three molecularly positive tobacco lines were randomly selected to collect their T₁ seeds. Then, the T₁ seeds were sterilized and screened in MS medium containing 100 mg/L kanamycin and 200 mg/L ceftriaxone sodium, and T₂ and T₃ seeds were obtained in the same way.

To generate 35S:GFP-ATM/Col-0, the full-length ATM CDS sequence was amplified and then inserted into pEGAD vector [33 (link)]. To construct ProATM:GUS/Col-0, a 3 kb genomic promoter sequence was amplified and inserted into pBI101 vector [34 (link)]. The amplified fragments were inserted into respective vectors by using the in-fusion enzyme (TAKARA, Shiga, Japan). All constructs were transformed into Agrobacterium tumefaciens cells (strain GV3101) which was used to transform Col-0 plants by the floral dip method [35 (link)]. All primer sequences used here are listed in Supplementary Table S1.