Yeast strains and growth assay
StPIP1;1 and StPIP1;3 were cloned from ‘Topas’ root cDNA using primers listed in Supplementary Table S2. PCR products were cloned in the USER-yeast expression vector pYeDP60u (Hamann & Moller, 2007) and pRS426-pTPIu using an uracil excision-based improved high-throughput USER cloning technique (Nour-Eldin, Hansen, Norholm, Jensen, & Halkier, 2006). A S. cerevisiae deletion mutant strain (Δmep1-3(-ura/-leu) (Anna Maria Marini’s lab collection)) was co-transformed with either the two empty vectors (EV) pRS426-pTPIu and pYeDP60u (negative control transformant), or hAQP8 (in pYeDP60u) and an EV (pRS425-pTPIu) (positive control transformant). The two PIP isoforms,StPIP1;1 or StPIP1;3 in pYeDP60u, were co-transformed with maize ZmPIP2;5 in pRS425-pTPIu to localize the individual PIPs to the PM for transport assays. Co-transformation of StPIP1;1 orStPIP1;3 in pRS426-pTPI:N-terminal-green fluorescent protein (GFP) with ZmPIP2;5 in pRS425-pTPIu was performed for transport assays and for confocal microscopy (G. P. Bienert, Heinen, Berny, & Chaumont, 2014). Transformed yeast was selected on synthetic medium containing 2% glucose, 50 mM succinic acid/Tris base, pH 6.5, 0.7% yeast nitrogen base without amino acids (Difco) and 0.2% proline. For the ammonium transport assay, yeast cells were diluted to different OD600 values (1, 0.01, and 0.0001) and spotted on synthetic medium containing 2% galactose, 50 mM succinic acid/Tris base, pH 6.5, 0.7% yeast nitrogen base without amino acids (Difco) and different concentrations of ammonium (2, 4, 5, 10, 20 mM ammonium, supplied as ammonium sulfate) or 0.2% proline as positive control. Growth was documented after 8-10 days at 30 °C.