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.