S156AT168AY175A (AAA) were purified as GST-fusion proteins and incubated with GSTfused

Transcription

1 Supplemental Materials Supplemental Figure S1 (a) Phenotype of the wild type and grik1-2 grik2-1 plants after 8 days in darkness. (b) Recovery of the wild type and grik1-2 grik2-1 after 60h-submergence treatment. Pictures were taken after 4 days of recovery. (c) Phenotype of the wild type and grik1-2 grik2-1 under various sugar supplements. Pictures show seedlings growth on MS plates supplemented with sucrose 1% (control), and with additional 3% glucose, sucrose or sorbitol. Scale bar: 1 cm. The bar graph plots the rates of green seedlings of Col-0 (black bar) and grik1-2 grik2-1 (gray bar) (mean ± SE, n=4). * P<0.001 in t-test. (d) Seedlings of the wild type (Col-0), grik2-1, grik1-2, and grik1-2 grik2-1 mutants grown on MS agar plates were transferred to fresh with or without mannitol. The line graph depicts primary root length of seedlings 12d after transfer to MS agar plates with indicated concentration of mannitol (mean ± S.E., n=20). Supplemental Figure S2 Control experiments for the in vitro kinase assays. (a) In vitro kinase assay as in Figure 3 but without GRIK1 (arrowhead) or SOS2KN (arrow). (b) Coomassie staining of GST-GRIK1 loaded with 10-fold more protein than that in (a) to show its purity. (c) The SOS2 mutant variants S156A (SA), T168A (TA), Y175A (YA) and S156AT168AY175A (AAA) were purified as GST-fusion proteins and incubated with GSTfused GRIK1. Proteins were separated by SDS-PAGE followed by Coomassie staining (left) and autoradiograph (right). (d) In vitro kinase assay as in Figure 6 with or without GRIK1, SOS2-308 (arrowhead) or SOS1-CT (arrows). Note that the small amount of GRIK1 was enough to activate SOS2, even though GRIK1 bands were not observed. Proteins were separated by SDS-PAGE followed 1

2 by Coomassie staining (left) and autoradiograph (right). Supplemental Figure S3 Transgenic lines with similar expression levels of SOS2-S159A (SA), T168A (TA), and Y175A (YA) in the sos2-2 background do not perform as the transgenic lines with wild-type SOS2 in the sos2-2 under salt stress condition. (a) Seedlings grown on MS agar medium were transferred to fresh MS agar plates with 100 mm NaCl. Photographs were taken 12 d after transfer. (b) Northern blot for SOS2 transcript was performed with total RNA extracted from wild type (WT), sos2-2 transgenic plants expressing various mutant forms of SOS2, and sos2-2. RNA was extracted 12h after transfer to MS agar plates with 100 mm NaCl. rrna (ethidium bromide stained) was used as a loading control. Supplemental Figure S4 Decimal dilutions of overnight cultures of the yeast strain YPDahl55 (sak1δ::kanmx elm1δ::kanmx tos3δ::trp1) lacking all three upstream regulatory kinases of SNF1 and expressing the Arabidopsis GRIK1 protein or the dead-kinase mutant K137R were spotted in YP media plates with 2% glucose or a mixture of 2% glycerol and 3% ethanol, and incubated for 2 days. Restoration of growth in non-fermentable carbon sources demonstrated genetic complementation of SAK1, ELM1 and TOS3 absence by the plant GRIK1 kinase. Supplemental Table 1 Primer sequence 2

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8 Supplemental Table 1 Primer sequence GRIK1 cloning point mutation TDNA line identification to check expression forward for pgex4t1 forward for p414gpd reverse pgex4t1forward SOS2 S159A reverse SOS2 T168A reverse SOS2 Y175A reverse GRIK1 K137R reverse pgex4t1 reverse SOS2 S159A forward SOS2 T168A forward SOS2 Y175A forward GRIK1 K137R forward GRIK1-2 LP GRIK1-2 RP GRIK2-1 LP GRIK1-1 RP GRIK1 forward GRIK1 reverse GRIK2 forward GRIK1 reverse tubulin forward tubulin reverse 5'-TAGAATTCATGTTTTGTGATAGTTTTGCATTTGCCC-3' 5'-AAGGATCCACCATGTTTTGTGATAGTTTTGCATTTGC-3' 5'-TACTCGAGTCAGCTATGGTTTTGATCTTCTTCTTCT-3' 5'-GTGGTGGCGACCATCCTCCAA-3' 5'-GCAATGCAGCTAGCCCGAAATCCGAAACCTTCAG-3' 5'-GTTCCACACGTGGCACGCAGAAGTTCTACTCCTTCC-3' 5'-TGGAGCTACAGCGTTCGGAGTTCCACATGTGGTA-3' 5'-AAAAGCCCGGATAGCATAATGCTTGTCATCG-3' 5'-TGTGTCAGAGGTTTTCACCGTC-3' 5'-GGATTTCGGGCTAGCTGCATTGCCTCAGGAAGGAGT-3' 5'-TCTGCGTGCCACGTGTGGAACTCCGAACTATGTAGC-3' 5'-AACTCCGAACGCTGTAGCTCCAGAGGTACTTAGTG-3' 5'-TATGCTATCCGGGCTTTTCACAAGTCACATT-3' 5'-TGGAGTTTCGACATGTTGCGAT-3' 5'-TGGTGATCCAATTTCCAATGAGC-3' 5'-TTTTGGAGCATCCTAACATCG-3' 5'-TGAACCATTGGAGGCTAAAAGA-3' 5'-GGTCTCCTTTGCAAAGATCCA-3' 5'-AAGAATTCAAGAGTCTTCTCAATACATGACCG-3' 5'-GGTCTCCTTTGCAAAGATCCG-3' 5'-AAGAATTCATGACCCCAAGCGAGGTC-3' 5'-TCGTGGATCACAGCAATACA-3' 5'-CCTCCTGCACTTCCACTTCG-3' BiFC GRIK1 forward 5 -CCGCGTGGATCCATGTTTTGTGATAGTTTTG -3 GRIK1 reverse 5 -CGGCGCCCGGGGCTATGGTTTTGATCTTC -3 1

9 RT-PCR T-DNA end forward GRIK1 1 st strand reverse GRIK1 4 th exon reverse 5'-CATTAAAAACGTCCGCAATGTG-3' 5'-TCTCCCGGTACTAGTAACCAG-3' 5'-GAAGAACATCACCCATAGCAG-3' 2