Yeast complementation assay
Mg2+ transport ability of OsMTP1 protein was tested by complementation of a yeast CM66 mutant, which lacks both Mg2+ transporters of ALR1 and ALR2 gene, and CM52 (Bi et al., 2009). Full-length coding sequence of the OsMTP1 andmOsMTP1 (mutated OsMTP1 ) were PCR-amplified and ligated into BamHI/SalI-digested pRS425-ADH1 p plasmid. The plasmid was transformed into Escherichia coli and the ADH1p::OsMTP1and ADH1p::mOsMTP1 plasmids were transformed into CM66 and CM52 yeast cell lines on standard synthetic media lacking leucine (SD-LEU) supplemented with 10 mM MgCl2. The transformed yeast cell lines were cultured in liquid SD-LEU and adjusted to 1.0 OD600, diluted in a 10-fold series, with sterilized water. Ten microliter of the yeast cells were spotted on solid SD-LEU media containing 0, 0.1, or 1 of MgCl2. For the liquid media assay, growth rates of each transformant was monitored for 66 h using a SPECTROstarNano.
The Na+ transport ability of OsMTP1 protein was examined using wild-type yeast strain FM391 (MATa his1Δ leu2Δ0 met15Δ0 ura3Δ0 , Research Genetics). The plasmids harboringADH1p::OsMTP1 , ADH1p::mOsMTP1 , and empty vector were transformed into the FM391 and selected on SD-LEU media. The transformants were cultured in complete media (YPD) and spotted on solid YPD media containing 0, 0.5, or 1 M of NaCl and 1M of NaCl with 0.1 or 1 mM MgCl2. The liquid media assay was conducted for 48 h under same conditions described above.