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.