Constructs and stable transformation of L. japonicus

To create CRISPR/Cas9 constructs of CLE-RS1 or -RS2, targeting sites in the genes were designed using the CRISPR-P program (http://cbi.hzau.edu.cn/crispr/)⁶⁶. Oligonucleotide pairs (Supplementary Table ³) were annealed and cloned into a single guide RNA (sgRNA) cloning vector, pUC19_AtU6oligo, as previously described⁶⁷. Then, the sgRNA expression cassette prepared in pUC19_AtU6oligo was excised and replaced with OsU3:gYSA in pZH_gYSA_FFCas9, an all-in-one binary vector harboring a sgRNA, Cas9, and an HPT expression construct, as previously described⁶⁷. The recombinant plasmids were introduced into L. japonicus plants by A. tumefaciens-mediated stable transformation. Seeds were germinated on germination medium described above in a growth cabinet (24 °C dark for first 2 days, 24 °C 16 h light/8  h dark cycle for next 2 days). A. tumefaciens AGL1 strains harboring each construct were streaked on YEP plate with appropriate antibiotics for 2 days at 28 °C. Seedlings were placed in the A. tumefaciens suspension and then their hypocotyls were cut into about 3 mm pieces. The hypocotyl pieces were placed onto the top of pilled filter papers saturated with co-cultivation medium (1/10× Gamborg's B5 salt mixture, 1/10× Gamborg's vitamin solution, 0.2 μg ml⁻¹ BAP, 0.05 μg ml⁻¹ NAA, 5mM MES (pH 5.2), 20 μg ml⁻¹ acetosyringone, pH 5.5) and were incubated in a growth cabinet (21 °C dark) for 6 days. After that, the hypocotyl pieces were transferred to callus induction medium (1× Gamborg's B5 salt mixture, 1× Gamborg’s vitamin solution, 2% sucrose, 0.2 μg ml⁻¹ BAP, 0.05 μg ml⁻¹ NAA, 10 mM (NH₄)₂SO₄, 0.3% phytagel, 12.5 μg ml⁻¹ meropen, 15 μg ml⁻¹ Hygromycin B, pH 5.5) and were incubated in a growth cabinet (24 °C 16 h light/8 h dark cycle) for 2–3 weeks. The hypocotyl pieces were transferred to fresh callus induction medium every 5 days. When calluses became more than 1 mm in size, they were detached from the hypocotyls and transferred onto callus medium without hygromycin B, and were incubated for 3–7 weeks in a growth cabinet (24 °C 16 h light/8 h dark cycle) until leaf primordia became visible. The calluses were transferred onto new medium every 5 days. The calluses with leaf primordia then were transferred to shoot elongation medium (1× Gamborg's B5 salt mixture, 1× Gamborg’s vitamin solution, 2% sucrose, 0.2 μg ml⁻¹ BAP, 0.3% phytagel, 12.5 μg ml⁻¹ meropen, pH 5.5), and incubated until their shoot length became ~1 cm. Individual shoots were detached from calluses and transferred to root induction medium (1/2× Gamborg's B5 salt mixture, 1/2× Gamborg's vitamin solution, 1% sucrose, 0.5 μg ml⁻¹ NAA, 0.4% phytagel, 12.5 μg ml⁻¹ meropen, pH 5.5), and incubated for 10 days. Then, they were transferred to root induction medium without NAA and cultivated until their root length became ~2–3 cm. Thereafter, the transgenic plants were transplanted into vermiculite for further cultivation.