Subcellular localization
For transient expression analysis in Arabidopsis protoplasts, the
full-length coding sequence of each BnPHT5;1b gene was amplified
from ‘Westar 10’ genome and then cloned into the PM999 vector driven by
the CaMV35S promoter using the
In-Fusion HD Cloning kit (Takara Bio). Transformation of Arabidopsis
mesophyll protoplasts was performed with the method of Yoo et al.
(2007). After 12-16 hrs incubation, the transformed protoplasts were
treated with pure water to release vacuoles for GFP signal imaging (TCS
SP8, Leica). For subcellular localization in tobacco (Nicotiana
benthamiana ), full-length coding sequences of each BnPHT5;1bgene was cloned into the PMDC83 vector driven by the CaMV35S promoter.
The Agrobacterium GV3101 strain containing 35S:BnPHT5;1b :GFP was
co-injected into 4-week-old tobacco leaves with a mCherry labeled
tonoplast marker (AtγTIP :mCherry) and plasma membrane marker
(AtPIP2;: mCherry), respectively. After 2 days, the fluorescence
signals were examined using laser confocal fluorescence microscopy
(STELLARIS, Leica).