Molecular cloning and subcellular localization analyses
To identify the T insertion mutation in OsMTP1 gene, genomic DNA was extracted using DNeasy Plant Mini Kit (Qiagen, USA) and target region was amplified using Q5 DNA polymerase (NEB, USA) with appropriate primers (Table S1). For cloning the full-length coding sequence of theOsMTP1 gene, total RNA was extracted from 1-week-old seedlings of the sitl1 and WT using RNeasy Plant Mini Kit (Qiagen). First-strand cDNA was synthesized using the 1st strand cDNA synthesis kit (Takara-Bio, Otsu, Japan) with oligo dT and Q5 DNA polymerase was used to amplify the full-length OsMTP1 gene. The PCR-amplified product was purified and cloned into the directional TOPO® vector (Invitrogen). The plasmids were extracted and verified by Sanger DNA sequencing.
For the subcellular localization, the OsMTP1 gene was cloned into the binary vector ImpGWB405 (H. Li & Durbin, 2009) containing the C-terminal sGFP using the Gateway™ LR Clonase™ II Enzyme Mix (Invitrogen). For transient expression, 10 µg of each35S::OsMTP1-sGFP , 35S::sGFP (Nakagawa et al., 2007), andpm-rk (CD3-1007) (Lim et al., 2018) was transfected into rice protoplasts using a 40% PEG solution as described (Nelson, Cai, & Nebenfuhr, 2007). Transfected protoplasts were incubated overnight in the dark and then subcellular localization was observed using a confocal microscope.