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	Fig. 1. Homologous interaction between OsHKT1;3 and OsCNIH1. Homologous interaction between OsHKT1;3 and OsCNIH1 confirmed with the mbSUS in yeast with selective medium in the absence (IS0) or in the presence of methionine (0.5mM; IS500). Corroboration of the interaction between OsHKT1;3 and OsCNIH1 was demonstrated by activation of LacZ and revealed with X-Gal as a substrate. Representative results of three different assays are shown.
	Fig. 2. OsCNIH1 sequence analysis. (A) A Kyte–Doolittle hydropathy plot shows three potential transmembrane domains (I, II, and III; values >0) in OsCNIH1. (B) Sequence alignment of OsCNIH1 with OsCNIH2, AtCNIH1, AtCNIH2, AtCNIH3 AtCNIH4, AtCNIH5, ScErv14, and cornichon homologues from Drosophila (Drosophila melanogaster), worm (Caenorhabditis elegans), zebra fish (Danio rerio), and human isoform 4 (Homo sapiens). Accession numbers are Os06g04500, Os12g32180, At4g12090, At1g12340, At1g12390, At1g62880, At3g12180, YGL054C Erv14, NP_477068, CAB01516, NP_001028278, and NP_001264129.1, respectively. Black bars indicate putative transmembrane domains; arrows indicate conserved residues involved in binding to COPII in yeast (I96, F97, and L100); the grey bar denotes an acidic domain. (C) Pairwise comparison between the cornichon proteins listed in (B) shows the percentage identity (upper right values) and number of identical residues (bottom left values). (This figure is available in colour at JXB online.)
	Fig. 3. BiFC confirms the interaction of OsCNIH1 with OsHKT1;3 and demonstrates the likely oligomerization of OsCNIH1 in transiently transfected tobacco leaves. (A, E) Reciprocal interaction between YFC–OsCNIH1/YFN–OsHKT1;3 and YFN–OsCNIH1/YFC–OsHKT1;3 confirms the interaction of the two proteins in the ER. (B) Co-expression of YFC–OsCNIH1 and YFN–-OsCNIH1 indicates the possible oligomerization of cornichon in the ER. Absence of a fluorescence complementation signal indicates that: OsCNIH1 does not interact with AtPIP2A (C, H); OsHKT1;3 does not form oligomers (D); and the transporter and the aquaporin do not interact (F, G). (I) Oligomerization of AtPIP2A. Scale bar=25 μm.
	Fig. 4. Intracellular co-localization of OsCNIH1 with OsHKT1;3 in plants. (A) Expression of OsCNIH1–mCherry (left) and OsHKT1;3–EYFP (centre), and co-localization of the two proteins (right) in tobacco leaves. (B) Expression of OsCNIH1–mCherry (left) and the ER marker AtWAK2–Citrine (centre), and co-localization of the two proteins (right). (C) Expression of OsHKT1;3–EYFP (centre) and the Golgi marker GmMan1–mCherry (left), and co-localization of the two proteins (right). (D) Scatter plots of pixel distribution of the magenta (y-axis) and green (y-axis) channels employing the Costes algorithm for images shown in (A, left), (B, centre), and (C, right). Scale bar=25 μm.
	Fig. 5. OsCNIH1 is also located at the Golgi and ERES. (A) Expression of OsCNIH1–mCherry (left) and the Golgi marker GmMan1–Citrine (centre), and the co-localization of the two proteins (right). (B) Expression of OsCNIH1–mCherry (left) and the ERES/COPII marker AtSec24–YFP (centre), and overlapping of the two images showing the co-localization of the two proteins (right). Arrows indicate the punctate sites of co-localization (white signal). Scale bar=25 μm.
	Fig. 6. OsHKT1;3 does not localize to the ERES or the plasma membrane and forms aggregates upon exposure to brefeldin A. (A) Expression of OsHKT1;3–mCherry (left) and the ERES/COPII marker AtSec24–EYFP (centre), and overlapping of the two images (right). (B) Co-localization analysis of OsHKT1;3–EYFP (left) with the plasma membrane marker AtPIP2A–mCherry (centre), and overlapping of the two images (right). The intracellular localization of OsHKT1;3–EYFP, seen as fluorescent puncta distributed throughout the cell (C, left), was modified after incubation of the epidermis with brefeldin A at 25 μM for 15min, resulting in the formation of aggregated bodies (C, right). Scale bar=25 μm.
	Fig. 7. Transport properties of OsHKT1;3. (A) Original traces of currents activated by voltage pulses between –200 mV and 50 mV in 20 mV steps from a control water-injected oocyte exposed to 30mM NaCl (left), or expressing OsHKT1;3 exposed to the bath solution either without (centre) or with 30mM NaCl (right). (B) I–V plot from currents recorded in an oocyte expressing OsHKT1;3 and exposed to different concentrations of NaCl (mM); E r (arrows). (C) I–V plot from currents recorded from a control oocyte and exposed to the same NaCl solutions as in (B). (D) Plot showing the linear relationship between E r and extracellular Na+ concentrations from oocytes expressing OsHKT1;3 in the absence (circles) or presence of 1mM KCl (squares). Lines are least square linear regressions fits with a slope of 54.2 mV per decade. (E) Sodium transport kinetics were voltage dependent. Lines are fits to the Michaelis–Menten equation at the corresponding voltages with r 2 ≥0.9. (F) The affinity (K m) of OsHKT1;3 for Na+ was voltage dependent. The line is a fit to Equation 1. Data are from more than five oocytes from 3–4 different frogs and correspond to the mean ±SD.
	Fig. 8. Co-expression of OsHKT1;3 and OsCNIH1 in Xenopus oocytes caused retention of the transporter in the ER, preventing the activation of Na+ currents. (A) Sodium inward currents activated by voltage ramps from an oocyte expressing OsHKT1;3 (left). Co-expression of OsHKT1;3 and OsCNIH1 (cRNA ratio injected 1:1) in a Xenopus oocyte (right). (B) Expression of OsHKT1;3–EGFP was observed as puncta at the plasma membrane of a Xenopus oocyte (left); upon co-expression with OsCNIH1, fluorescence was observed exclusively in a reticulated structure (centre). Autofluorescence is from a water-injected oocyte (right). Scale bar=250 μm. (This figure is available in colour at JXB online.)
	Fig. 9. OsCNIH1 restores the intracellular expression of OsHKT1;3 in the yeast mutant BY4741Δerv14 and sensitivity to NaCl. (A) Fluorescence and DIC images of living BY4741 and BY4741Δerv14 cells expressing OsHKT1;3–GFP observed by confocal fluorescence microscopy and co-expressing either OsCNIH1 or ERV14p. Scale bar=5 μm. (B) Drop-test assay on yeast strains BYT4741 (top) and BYT45Δerv14 (bottom) grown in YNB solid medium with different Na+ concentrations and transformed with pGRU1 and pDR-F1, OsHKT1;3 and pDR-F1, OsHKT1;3 and OsCNIH1, or OsHKT1;3 and ERV14. Representative results of at least three different experiments are shown.

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