Subcellular Localization of OsERF83

To confirm the subcellular localization of OsERF83, we fused the coding regions of OsERF83 without the stop codon to the GFP. The cassette was driven by a 35S promoter and inserted into the pHBT vector (GenBank accession number EF090408) using the In-fusion system (Clonetech, CA, USA). 35S::NF-YA7-mCherry was used as the control for nuclear localization [23 (link)]. The final construct (35S::OsERF83-GFP) and the control vector (35S::NF-YA7-mCherry) were transfected into protoplasts (Oryza sativa cv. Dongjin) using a PEG-mediated protoplast transformation system [59 (link)]. The methods of rice protoplast preparation and transient gene expression were performed as described previously [60 (link)]. GFP and mCherry signals were observed 12 h after transfection using a confocal laser scanning microscope (Leica TCS SP8 STED, Wetzlar, Germany). Images were processed using Leica LAS AF Lite software. GFP was excited at 488 nm and the emitted light was detected between 512 and 560 nm. mCherry was excited at 587 nm and the emitted light was detected at 610 nm.