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.