Fd0524

The 5′ and 3′ flanking regions (863 bp and 800 bp respectively) of the PpATG3 gene were amplified using 2 primer pairs P1/P2 and P3/P4, respectively. Two fragments were digested with KpnI/HindIII and XbaI/BamHI respectively (Thermo Scientific FastDigest, FD0524, FD0504, FD0684 and FD0054), and cloned by sequential into the pTN182 vector upstream and downstream of the nptII gene driven by a Pm35S promoter (geneticin resistance gene cassettes) (http://moss.nibb.ac.jp/). Construct was linearized with KpnI and BamHI prior to transformation. Transformation was completed as described previously [48 (link)]. The primers for genotyping the PpATG3 gene knockout were used can be found in Additional file 10. PpUbiquitin [56 (link)] and PpAdePRT [57 (link)] was used to assess DNA and cDNA template quality, respectively.

Full length coding sequence (lacking the stop codon) of PpATG3 was PCR amplified from P. patens cDNA template using the primers PpATG3-F and PpATG3-R (Additional file 10). The resulting 921-bp PCR fragment was cloned using KpnI and XbaI restriction enzymes (Thermo Scientific FastDigest, FD0524 and FD0684) into the vector pM999 [55 (link)] by sticky end ligation. The pM999 contains the eGFP coding sequence driven by a 35S promoter. The chimeric gene p35S:PpATG3-eGFP was constructed by fusing the PpATG3 cDNA sequence to the 5′ end frame of eGFP. After this, the resulting plasmid was introduced into the protoplast of P. patens by PEG-mediated transformation [48 (link)]. 48 h after culture at 25 °C, protoplasts were observed by a confocal microscopy for GFP fluorescence.