Xbai and hindiii

Ratiometric pHluorin is a pH-sensitive GFP variant^{71 (link),72 (link)}, which carries a cryptic intron^{72 (link)}, leading to aberrant mRNA processing and the absence of protein expression. Silent mutations were introduced into the pHluorin coding sequence, using a primer pair (Supplementary Table 3), to eliminate the cryptic intron sequence and restore pHluorin expression in A. thaliana. The resulting amino acid sequence is 100% identical to the original pHluorin. The aha9-4 line is resistant to kanamycin, most likely due to a kanamycin resistant marker being associated with the T-DNA inserted into AHA9. Therefore, the pLat52::pHluorin sequence was shuttled into pMDC99^{70 (link)}, which in plants is selected for by hygromycin B. Briefly, pLat52::pHluorin plasmid and pMDC99 were mixed and digested using XbaI and HindIII (NEB), effectively excising pLat52::pHluorin and the Gateway Cassette. The digestion product was ligated with T4 DNA ligase (NEB) and transformed into Escherichia coli strain TOP10. Positive clones containing the resulting pMP5249 vector were selected on LB plates supplemented with 50 mg ml^–1 kanamycin (Supplementary Fig. 13 for vector map).

End products from the in vitro end-joining assays, with 5.7 kb substrate with non-cohesive ends, were purified by phenol-chloroform extraction and ethanol precipitation. End-joined regions were amplified by polymerase chain reaction using HotStarTaq DNA polymerase (Qiagen) and primers (5′-ATGCAAGCTTCCTTTATTACCCAGAAGTCAG ATGC-3′ and 5′-ATGCTCTAGAGTAAACTCGCCCAGAAGCTAGG-3′; substrate sequences are underlined and restriction endonuclease sites are highlighted in bold). PCR was carried out in a 50-μl reaction mixture for 35 cycles (denaturation, 94 °C; annealing, 62 °C; extension, 72 °C) and amplified end-joined products were purified by ethanol precipitation, digested with XbaI and HindIII (New England BioLabs), and cloned into the pUC18 vector using HB101 competent cells. DNA from at least 30 clones for each sample from three independent experiments was sequenced using the M13F primer.

S. tuberosum var. ‘Desirée’ were grown in Magenta GA7 boxes with MS Reg media [48 (link)]. RNA was extracted from ~1-month-old tissue using TRI Reagent, as per the manufacturer’s instructions (Molecular Research Center, Cincinnati, OH, USA). RNA was cleaned with a Zymo Research RNA Clean and Concentrator kit (Irvine, CA, USA). cDNA was synthesized according to protocol using a Thermo Fisher Scientific SuperScript III First-Strand Synthesis System (Waltham, MA, USA). The FtsZ1 coding sequence was amplified using primers 1 and 2 (all primers used in this study can be found in Supplementary Table S3). The FtsZ1 amplicon and pUC19 were digested with XbaI and HindIII (NEB, Ipswich, MA, USA). One microliter of calf intestinal alkaline phosphatase was added to the pUC19 digestion for dephosphorylation, and the amplicon was ligated into the vector. The vector was Sanger sequenced with M13 forward and reverse primers.