1. HKT1 form Arabidopsis relative extremophile Thellungiella work as Na/K co-transporter

2. Figure 1 A ScTRK1 665 SPTWWGFWTAMSAFNDLGLTLTPNSMMSFNKAVYPLIVMIWFIIIGNTGFPILLRCIIWILHKIT 729 SP + +TA+S +D G T +M+ F K L ++I + +G+T FP L IW + KI+ TsHKT2 192 SPLTFSVFTAVSTLSDCGFVPTNENMIIFRKNSGLLWLLIPQVFMGDTLFPCFLVLAIWGLHKIT 256 TpHKT2 190 SPLTFSIFTTVSTLSDCGFVPTNENMVIFRKNSGLLWLLIPQVFMGDTLFPCFLISLIWGLDKIT 254 TpHKT1 198 SPLTFSIFTTVSTFGNCGFVPTNENMAIFRKNSGLLWLLIPQVFMGNTLFPCFLFLLIWGLDKIT 262 AlHKT1 191 SPLTFSVFTTVSTFANCGFVPTNENMIIFRKNSGLIWLLIPQVLMGNTLFPCFLALLIWGLYKIT 255 AtHKT1 196 SPLTFSVFTTVSTFANCGFVPTNENMIIFRKNSGLIWLLIPQVLMGNTLFPCFLVLLIWGLYKIT 260 TsHKT1 198 SPLTFSVFTAVSTFGNCGFVPTNENMIIFRKNSGLLWLLIPQALMGNTLFPCFLLFLVSGLDKIT 262 ******:**:***:.:********** ********:******.:**:******* : ** *** P B M2 B

3. Figure 1 B Subfamily 1 Subfamily 2

4. Figure 1. Sequence comparison and phylogenetic analysis of HKT homologs in Thellungiella salsuginea. A, Comparison of HKT homologs from Arabidopsis and Thellungiella species. Amino acid sequences in the second pore loop region (P B ) and the adjacent transmembrane domain (M2 B ) are aligned by clustalw (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Yeast TRK1 (ScTRK1) sequence is included for comparison.http://www.ebi.ac.uk/Tools/msa/clustalw2/ The conserved glycine residues in the PB region (Mäser et al., 2002) are indicated by an arrow. The aspartic acid residues specific for TsHKT2 and TpHKT2 are highlighted with boxes. B, Unrooted minimum-evolution phylogenetic tree of protein sequences of HKT homologs with 10,000 bootstrap replicates. Accession numbers and species for all sequences are listed in Supplemental Table S31. Yeast TRK protein sequences (ScTRK1 and ScTRK2) were included as an outgroup. Subfamilies are as defined by Platten et al. (2006). The tree was generated using MEGA5 (http://www.megasoftware.net/). The scale bar shows 0.2 substitutions per site.http://www.megasoftware.net/

5. pMAS BAR MAS3 / 35S GUS OCS3 / AscI SwaI BamHI SpeI 561bps TsHKT cDNA fragment (131-692) LB RB C HKT ACT2 77-181-2 WT TsHKT-RNAi D A (hr) Thellungiella tssos1-4 ACT2/8 HKT 0 8 24 150mM NaCl (hr) Arabidopsis atsos1-1 0 8 24 100mM NaCl B Figure 2

6. Figure 2. Expression of HKT homologs and construction of T. salsuginea TsHKT-RNAi transgenic plants. A and B, HKT expression patterns are analysed with semi-quantitative RT-PCR in T. salsuginea (A) and A. thaliana (B). Two week-old T. salsuginea and 10 day-old A. Thaliana seedlings were treated with 150mM and 100mM NaCl, respectively, for indicated times. Compared are T. salsuginea Shandong (Ts WT) and A. thaliana Col-gl1 (At WT) and the lines with compromised SOS1 expression (tssos1-4 and atsos1-1, respectively). Actin (ACT2/8) is used as a reference transcript. C, Diagrammatic representation of the TsHKT-RNAi construct. D, TsHKT expression was inhibited in T. Salsuginea lines transformed with the TsHKT-RNAi construct. Shown are two representative TsHKT-RNAi transgenic lines (77-1 and 81-2 81-2 and 77-1) in comparison to a vector control (WT).

7. +NaCl D WT TsHKT-RNAi 77-181-2 F Figure 3 A +NaCl 10 mm B -NaCl 10 mm E

8. Figure 3. Salt-sensitive phenotypes of T. salsuginea TsHKT-RNAi lines A-C, T. salsuginea plants harboring either the vector control (WT) or the TsHKT-RNAi construct (lines 77-1 and 81-2) were treated with no salt (A) or 300mM NaCl (B) as described in Materials and Methods. Representative leaf sizes were compared in the lower panel. After the salt treatment, the fresh weights were compared between the WT and TsHKT-RNAi lines (C), with error bars representing standard errors from three independent repeats (n=30). D Leaf size of A and B. E, Root growth of WT and TsHKT-RNAi seedlings was compared under salt stress. Ten-days-old seedlings grown on ½ MS were transferred to K + deficient media (Materials and Methods) supplemented with 100 mM NaCl. Pictures were taken after additional 10 days of vertical growth (E). A magnification of the root hair zone is shown (F).

9. B Figure 4 75mM Na Control OX.AtHKT1 OX.TsHKT2 athkt1-3 WT OX.AtHKT1 OX.TsHKT2 OX.AtHKT1 sos3-1 OX.TsHKT2 athkt1-3 Col-gl1 athkt1-3 A 10 mm 250 mM NaCl CONTROL 250 mM NaCl +1 mM KCl AXT3K (Δena1-4Δnha1 Δnhx1) C

10. Figure 4. Comparison of salt stress responses of A. thaliana and yeast strains ectopically expressing TsHKT2 and AtHKT1. A-C, Four-day old Arabidopsis seedlings of over-expressing TsHKT2 (TsHKT2-OX) or AtHKT1 (AtHKT1-OX), on the wild type (Col-gl1) or an AtHKT1 knockout mutant (athkt1-3) background, were transferred to salt media (Supplemental Table). Pictures were taken after vertical growth for 7 days (A). A magnified picture is presented (C), to show the shoot phenotype. D, TsHKT2 and AtHKT1 were ectopically expressed in the Saccharomyces cerevisiae strain, AXT3K (Δena1-4Δnha1 Δnhx1), which lacks the Na + -efflux system. Cells transformed with a vector control and AtKAT1 were included, as negative and positive controls, respectively. Yeast cells were plated on selective media containing 0 and 250 mM NaCl, with and without 1 mM KCl.

11. A C 10 mm TsHKT-RNAi WT 81-2 77-1 B 10 mm Figure 5 10 mm 18 mm Root length (mm) WT 81-2 77-1 TsHKT1-RNAi Figure 5. Compromised growth of T. salsuginea TsHKT-RNAi lines under K + -limiting conditions. A and B, T. salsuginea seedlings Harboring either the vector control (Ts WT) or TsHKT-RNAi construct (lines 77-1 and 81-2) were grown on 1/2 MS for 10 days and transferred to either K + deficient media (A) or the same media supplemented with 10mM KCl (B). The K + deficient media was modified from MS media, containing no KNO 3 and KH 2 PO 4 replaced with (NH 4 ) 2 HPO 4 (Materials and Methods and Supplemental Table 3). Pictures were taken after the transferred seedlings incubated in vertical positions for 15 days. C, The root growth in B is quantified, with error bars representing the standard errors of three repeats (n=30).

12. Figure 6 WT OX.TsHKT2OX.AtHKT1 Col-gl1 WT OX.TsHKT2 OX.AtHKT1 Col-gl1 WT OX.TsHKT2 OX.AtHKT1 Col-gl1 B Control 1 mM KCl300 mM NaCl + 1 mM KCl CY162 (Mat a, ura3–52,his3D200, his44–15, trkD1,trkD2::pcK64) C OX.AtHKT1 OX.TsHKT2 OX.AtHKT1 sos3-1 OX.TsHKT2 athkt1-3 TsHKT2 ACT2 Col-gl1 athkt1-3 A WT 10 mm

13. Figure 6. Distinct responses to K + -limiting conditions of A. thaliana and yeast strains ectopically expressing TsHKT2 and AtHKT1. A and B, Four-day old Arabidopsis seedlings of over-expressing TsHKT2 (TsHKT2-OX) or AtHKT1 (AtHKT1-OX), on the wild type (Col-gl1) or an AtHKT1 knockout mutant (athkt1-3) background, were transferred to K + deficient media (Supplemental Table 3) supplemented with 20mM NaCl. Pictures were taken after 7 days incubation in vertical positions (A) and the root growth quantified (B), with error bars representing the standard errors of three repeats (n=30) Semi-quantitative RT-PCR is used to select transgenic lines with comparable levels of ectopic HKT expression (A, the upper part). The root growth of the transgenic lines selected from A is compared with the WT, in the K + deficient media (Supplemental Table 3) supplemented with 1mM Na + and 0, 1 or 5mM K + ions C, Growth of the yeast strain CY162 (Mat a, ura3–52,his3D200, his44–15, trkD1,trkD2::pcK64) cells harboring the vector control (pYES2), TsHKT2, AtHKT1 and AtKAT1 is compared on media containing either 1mM K +, 300 mM Na + or both of them combined.

14. Supplementary Figure 1 WT 81-2 77-1 TsHKT-RNAi WT 81-2 77-1 TsHKT-RNAi K + /Na + ratio whole plant K + (mg-g -1 DW whole plant) K + (mg-g -1 DW roots) K + (mg-g -1 DW shoots) AB C D

15. Supplemental Figure S1. Potassium contents in TsHKT-RNAi lines (lines 77-1 and 81-2) compared to the vector control (WT). Four week-old hydroponically grown plants were irrigated with 250 NaCl for 24hr. Na + and K + contents were measured by inductively coupled plasma optical emission spectroscopy (ICP-OES). A, K + contents and B, K + /Na + ratio of whole plants. C, K+ contents in the root and D, in the shoot tissues.

16. Supplementary Figure 2 TpHKT1 MERVVAKLAKLRSQLAKFRSTFFLYFFYFISFSFLGFLALKITKPRTTPRPHDLDLFFTS 60 TsHKT2 MERVVDKLAKIFSQHAKSLPLFFLYFFYFLFFSFLGFLALKISKPRTTSRPHDLDLFFTS 60 TpHKT2 MERVVAKLARSRSP-------FFLYFLYFLSFSFVGFLALKISKPRTSSRPHDLDLFFTS 53 AlHKT1 MDRVAAKIAKIRSQLMKSSSLFLLYFIYFLLFSFLGFLALKITKPRTTSRPHDLDLFFTS 60 AtHKT1 MDRVVAKIAKIRSQLTKLRSLFFLYFIYFLFFSFLGFLALKITKPRTTSRPHDFDLFFTS 60 TsHKT1 MERVGAKLAKFRLQLAKNPSVLCLYFVYFLSFSFLGFLALKISKPRTTSRPHDLDLFFTS 60 *:*: *:*: : **:.**: **.:*******:****:.****:*:**** M1 A P A ↓(TRK and class II/III HKTs have G here) TpHKT1 VSAITASSMSTVDMEVFSNTQLIIITILMFLGGDIFTSFLDLYFSHFTNFVFPHNK-IRH 119 TsHKT2 VSAITVSSMSTIDMEVFSNTQLIIITILMFLGGEIFTSFVNLYFSHFINF-----K-IKH 114 TpHKT2 VSAITVSSMSTVDMEVFSNTQLIIITILMLLGGEIFTSFLHLYFTHYTNFVFPHNK-LRH 112 AlHKT1 VSAITVSSMSTVDMEVFSNTQLIFLTILMFLAGEIFTSFLNLYFQHFTNFVFPHNK-IRH 119 AtHKT1 VSAITVSSMSTVDMEVFSNTQLIFLTILMFLGGEIFTSFLNLYVSYFTKFVFPHNK-IRH 119 TsHKT1 VSAITVSSMSTIDMEVFSNTHLIFLTILMLLGGEVFTSFLTLYFSHFTKFVLPHNKNIRH 120 *****.*****:********:**::****:*.*::****: **. :: :* * : * P A M2 A TpHKT1 LMGSFSLKRPTEDRLSDIENVTTDHQKRPRQINERASKCLYSVVLGYHLVTNLAGSVLLL 179 TsHKT2 LVGSFNFDRPINDPGSDLENVT-NHVKLSSQINERASKCLYSVVLGYLFVTNIAGSTLLL 173 TpHKT2 LVCSLSLDTSIDDRGRDLENVT-DNFKGPSQIIERASKCLYSVVLGYHLVTNLAGSMLLL 171 AlHKT1 LVGSFNSD------RCDLETVT-DHREGLSKIEERASKCLYSVVLSYHLVSNLVGSVLLL 172 AtHKT1 ILGSYNSDSSIED-RCDVETVT-DYREGLIKIDERASKCLYSVVLSYHLVTNLVGSVLLL 177 TsHKT1 LMGSFDLDSPIEDRRIDLENVT-DHRVDPSQINERASKCLYSVVLGYHLVTNIAGSVLVL 179 : *.. *:*.** : :* ***********:.* :*:.:.** *:* M1 B

17. ↓ TpHKT1 VYVSFVQTAKDVLSSKKISPLTFSIFTTVSTFGNCGFVPTNENMAIFRKNSGLLWLLIPQ 239 TsHKT2 LYVNFVKTARDVLSSKKISPLTFSVFTAVSTLSDCGFVPTNENMIIFRKNSGLLWLLIPQ 233 TpHKT2 LYVSFVKTAKDVLSSKAISPLTFSIFTTVSTLSDCGFVPTNENMVIFRKNSGLLWLLIPQ 231 AlHKT1 VYVNFVKTARDVLSSKDISPLTFSVFTTVSTFANCGFVPTNENMIIFRKNSGLIWLLIPQ 232 AtHKT1 VYVNFVKTARDVLSSKEISPLTFSVFTTVSTFANCGFVPTNENMIIFRKNSGLIWLLIPQ 237 TsHKT1 VYVNFVKTAGDVLSSKEISPLTFSVFTAVSTFGNCGFVPTNENMIIFRKNSGLLWLLIPQ 239 :**.**:** ****** *******:**:***: :********** ********:****** P B M2 B TpHKT1 VFMGNTLFPCFLFLLIWGLDKITKREEFGYILKHRKTMGCSHLLSLRLCVLLGLTVLGFV 299 TsHKT2 VFMGDTLFPCFLVLAIWGLHKITNREELGYILKNHKKMGYSHLLSVRLCVLLALTVLGLV 293 TpHKT2 VFMGDTLFPCFLISLIWGLDKITKREEYGYILKNHKKMGYSHLLSIRLCGLLCLTVLGLV 291 AlHKT1 VLMGNTLFPCFLALLIWGLYKITKRDEFGYILKNQKKMGYSHLLSVRLCVLLGVTVLGFL 292 AtHKT1 VLMGNTLFPCFLVLLIWGLYKITKRDEYGYILKNHNKMGYSHLLSVRLCVLLGVTVLGFL 297 TsHKT1 ALMGNTLFPCFLLFLVSGLDKITKYDEFGYILNNYKKMGYYHLLSVRRCVLLGLTVLGFL 299.:**:******* : ** ***: :* ****:: :.** ****:* * ** :****:: M2 B M1c ↓ TpHKT1 MIQFVVFCTFEWNSESLKHMNPYEKLVGSLFQVVNSRQTGENIVDLSTLSPAILVLFIVM 359 TsHKT2 MIQFLLFCTFEWNSESLEGMNSYEKLVGSLFQVVNSRHTGETVVDLSTLSPAILVLFILM 353 TpHKT2 MIQLLLFCTFEWNSESLEGMSSYEKLVGSLFQVVNSRHTGETIVDLSTLSPAILVLFIIM 351 AlHKT1 IIQLLFFCAFEWTSESLEGMSSYEKLVGSLFQVVNSRHTGETIVDLSTLSPAILVLFILM 352 AtHKT1 IIQLLFFCAFEWTSESLEGMSSYEKLVGSLFQVVNSRHTGETIVDLSTLSPAILVLFILM 357 TsHKT1 TIQLIFFCVYEWSSESLEGMNWYEKFVGSLFQVVNSRHTGETILDVSTLSPTILILFILM 359 **::.**.:**.* **: *. ***:* *********:***.::*.:****:**:***:* M1c P C M2 c Supplementary Figure 2 (conti’d)

18. TpHKT1 MYLPQYTLFMPLTEDKN--KKEGEDLSRNENKEKKSGLFVSQLSFLAICVFFISITESQN 417 TsHKT2 MYLPPYTLFMPLTVEKN--KKEGEHDSGDEIKGKKNGFYVSQLTFLAICIFLISTTESQK 411 TpHKT2 MYLPPYTLFMPLAEEGN--KKEGECDSKNQKKRKMNRFFVSQLTFLAICIFLISITESQK 409 AlHKT1 MYLPSYTLFMPLTEQKTIEKEG-DDDSGNGKKVTKSGLFVSQLSFLTICIFLISITERQK 411 AtHKT1 MYLPPYTLFMPLTEQKTIEKEGGDDDSENGKKVKKSGLIVSQLSFLTICIFLISITERQN 417 TsHKT1 MYLPPYTLFMPFTEET---NEKGEDDSENGKKRKKSGLFVSQLSFLVVCIVLISISEREK 416 **** ******:: : :. :. : *.. : ****:**.:*:.:** :* :: M2 c M1 D ↓ ↓ TpHKT1 LRRDPLNFNILNITFEVISAFGNVGFTTGYSCERRLDISNGSCKDTSYGFVGRWSPNGKF 477 TsHKT2 LRRDPLNFNILNITFEVISAYGNVGFTTGYSCERRLDISDGSCKDASYGFAGRWSPVGKF 471 TpHKT2 LRRDPLNFSILNITLEVFSAYGNVGFTTGYNCERRLDTSDNSCKDASYGFAGRWSPTGKF 469 AlHKT1 LRRDPLNFNVLNITLEVISAYGNVGFTTGYSCERRLDISDGSCKDAGYGFAGRWSPVGKF 471 AtHKT1 LQRDPINFNVLNITLEVISAYGNVGFTTGYSCERRVDISDGGCKDASYGFAGRWSPMGKF 477 TsHKT1 LRRDPLNFNVLNITLEVISAYGNVGFTTGYSCKRRLNVSDGGCEDASYGFVGRWSPAGKV 476 *:***:**.:****:**:**:*********.*:**:: *:..*::.***.***** **. P D M2 D ↓ ↓ ↓ TpHKT1 ILIIVMFYGRFKTFTAKSGRAWILYP---- 503 TsHKT2 ILIIVMFYGKFKQFSAKSGRAWILYPSSS- 500 TpHKT2 ILIVVMFYGKFKQFTAKSGRAWILYPSSSY 499 AlHKT1 VLIIVMFYGRFKQFTAKSGRAWILYPSSS- 500 AtHKT1 VLIIVMFYGRFKQFTAKSGRAWILYPSSS- 506 TsHKT1 ILILVMFYGRFKHFTPKSGRAWILYPSSF- 505 :**:*****:** *:.********** M2 D Supplementary Figure 2 (conti’d)

19. Supplemental Figure 2. Alignment of HKT protein sequences of crucifer species. The source of the sequences is described in Supplemental Table S1. The alignment was generated with clustalw (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The 4 pore loophttp://www.ebi.ac.uk/Tools/msa/clustalw2/ regions (P A to P D ) and the transmembrane domains (M1 A /M2 A to M1 D /M2 D ) are labelled with underlines. The conserved glycine residues in the pore loop regions (Mäser et al., 2002) and the positively charged residues in the M2 D domain (Kato et al., 2007) are indicated with arrows. The aspartic acid residues uniquely found in TsHKT2 and TpHKT2 are marked with red boxes.

20. TsHKT2 TsHKT1 ACT2 0H 24H 150 mM salt Supplementary Figure 3 Supplemental Figure S3. Induction of TsHKT2 by salt. RNA samples were prepared from two-week old Thellungiella salsuginea plants Treated with 150mM NaCl. Semi-quantitative RT-PCR was performed using primers specific to the TsHKT1 and TsHKT2 sequences (Supplemental Table S 2). The amplicon size was identical (350bp) for both TsHKT genes.

21. Supplementary Figure 4 athkt1-1 WT athkt1-3 athkt1-4 WT 81-2 77-1 TsHKT-RNAi 18 mm 10 mm 18 mm 10 mm athkt1-4 athkt1-3 athkt1-1 WT Root length (mm) C B A

22. Further study 1.Localization confirmation of both HKTs 2.TsHKT1 Protein stability upon salt stress.