Pi transport activity assay in yeast
To generate vectors for yeast expression, the VPT1 and BnPHT5;1b coding
regions were amplified from Col-0 and ‘Westar 10’ and cloned into the
PRS426–ADH1 vector by the In-Fusion
HD Cloning kit (PT5162-1, Takara Bio). The PRS426–ADH1-PHO84 and
PRS426–ADH1- PHO91 vectors were provided (Xu et al. 2019). These
constructs and empty vector were transformed independently into the
yeast strain YP100. Yeast was grown in SD/-Trp-Ura media with 0.67%
yeast nitrogen base, 2% galactose, 0.2% -Trp-Ura amino acids, 1.5%
agar at pH 5.6 and incubated at 30 °C for 3 days. Colonies were picked
for further growth in SD/- Trp-Ura liquid media until they reached an
OD600 of 0.1. The strains were collected and washed twice with sterile
water and then diluted 10-, 100-, 1000-fold with ddH2O.
Yeasts containing 5 µL diluted solution was spotted on SD (YNB-Pi)
/-Trp-Ura media plates supplemented with 2% glucose, 30 mM
KH2PO4 at 30 °C for survival test.