ポリリン酸ラベリング

PolyP labeling using E. coli PPBD was performed according to the method described by Saito et al. (2005) and Kuga et al. (2008) with some modifications.

For fluorescence microscopy

1. semithin sections of the resin-embedded roots were etched with 0.2% sodium ethoxide in ethanol for 5min.
2. Wash with 0.05% sodium ethoxide followed by 100% ethanol, 50% ethanol, and DW wash.
3. The sections were blocked with 1% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing low concentrations of salts (TBS-low salt; 25mM Tris-HCl pH 7.4, 13.7mM sodium chloride, and 0.27mM potassium chloride) for 10min.
4. Sections were first incubated in a mixture of 20μgml−1 PPBD, 10μgml−1 mouse anti-Xpress epitope antibody (#R910-25, Thermo Fisher Scientific, MA, United States), TBS-low salt, and 1% BSA for 2h at room temperature.
5. The sections were then washed with TBS-low salt buffer containing 0.05% Triton X-100, followed by TBS-low salt.
6. Samples were incubated with a goat anti-mouse IgG antibody conjugated with Alexa Fluor 488 (#A28175, Thermo Fisher Scientific) diluted to ratio 1:100 in TBS-low salt containing 1% BSA for 1h at room temperature.
7. The sections were washed with TBS-low salt containing 0.05% Triton X-100, followed by TBS-low salt and DW.
8. Alexa Fluor 488 fluorescence was excited with a band-pass filter BP470/40, and the emitted fluorescence was detected with BP525/50 using an epifluorescence microscope Axio Imager D1.

For TEM observation

1. ultrathin sections of the resin-embedded roots were etched with 0.2% sodium ethoxide in ethanol for 5min.
2. They were then washed with 0.02% sodium ethoxide, followed by 100% ethanol, 50% ethanol, and DW wash.
3. Specimens were blocked with 1% BSA in phosphate-buffered saline (PBS) containing low concentrations of salts (PBS-low salt; 10mM phosphate buffer pH 8.4, 13.7 mM sodium chloride, and 0.27 mM potassium chloride).
4. They were then incubated in a mixture of 20μgml−1 PPBD, 10μgml−1 mouse anti-Xpress epitope antibody, PBS-low salt, and 1% BSA
5. Wash with PBS-low salt buffer containing 0.05% Triton X-100, followed by PBS-low salt.
6. Samples were incubated with a goat anti-mouse IgG antibody conjugated with 6nm colloidal gold (#115-195-146, Jackson ImmunoResearch Laboratories, PA, United States) diluted 1:20 in PBS-low salt buffer containing 1% BSA for 1h at room temperature.
7. The samples were washed with PBS-low salt containing 0.05% Triton X-100, followed by PBS-low salt and DW wash.
8. Specimens were stained with 50% TI-Blue (Nisshin EM, Tokyo, Japan) for 10min, followed by lead citrate staining for 5min, and observed using a TEM (JEOL, Tokyo, Japan) at an accelerating voltage of 80kV.