14-3-3 Proteins and Other Candidates form Protein-Protein Interactions with the Cytosolic C-terminal End of SOS1 Affecting Its Transport Activity.

The plasma membrane transporter SOS1 (SALT-OVERLY SENSITIVE1) is vital for plant survival under salt stress. SOS1 activity is tightly regulated, but little is known about the underlying mechanism. SOS1 contains a cytosolic, autoinhibitory C-terminal tail (abbreviated as SOS1 C-term), which is targeted by the protein kinase SOS2 to trigger its transport activity. Here, to identify additional binding proteins that regulate SOS1 activity, we synthesized the SOS1 C-term domain and used it as bait to probe Arabidopsis thaliana cell extracts. Several 14-3-3 proteins, which function in plant salt tolerance, specifically bound to and interacted with the SOS1 C-term. Compared to wild-type plants, when exposed to salt stress, Arabidopsis plants overexpressing SOS1 C-term showed improved salt tolerance, significantly reduced Na+ accumulation in leaves, reduced induction of the salt-responsive gene WRKY25, decreased soluble sugar, starch, and proline levels, less impaired inflorescence formation and increased biomass. It appears that overexpressing SOS1 C-term leads to the sequestration of inhibitory 14-3-3 proteins, allowing SOS1 to be more readily activated and leading to increased salt tolerance. We propose that the SOS1 C-term binds to previously unknown proteins such as 14-3-3 isoforms, thereby regulating salt tolerance. This finding uncovers another regulatory layer of the plant salt tolerance program.

. Putative interaction partners of the recombinant AtSOS1-Cterminus protein under control conditions identified by mass-spectrometry. AT5G65430.3 14-3-3-like protein GF14 kappa, General Regulatory Factor 8, involved in brassinosteroid mediated signalling pathway, located in chloroplast, cytoplasm, nucleus, plant-type cell wall, plasma membrane, functions in protein phosphorylated amino acid binding AT1G02500 SAM1 (S-adenosylmethionine synthase 1); Catalyzes the formation of S-adenosylmethionine from methionine and ATP. This protein is involved in a subpathway that synthesizes S-adenosyl-L-methionine from L-methionine. This subpathway is part of the pathway S-adenosyl-L-methionine biosynthesis, which is itself part of Amino-acid biosynthesis AT5G16050 14-3-3-like protein GF14 upsilon, General Regulatory Factor 5, involved in the response to cadmium ion, located in Golgi apparatus, cell wall, chloroplast, stroma, cytoplasm, cytosol, mitochondrion, nuclear envelope, plasma membrane, involved in brassinosteroid mediated signaling pathway, functions in protein phosphorylated amino acid binding AT3G02520. 1 14-3-3-like protein GF14 nu, General Regulatory Factor 7, involved in brassinosteroid mediated signaling pathway, functions in protein phosphorylated amino acid binding ATMG01190 ATPA (ATP synthase subunit alpha, mitochondrial); Functions in ATP hydrolysis coupled proton transport AT2G16950 TRN1 (Transportin-1); Functions in intracellular protein transport, nuclear protein import as nuclear transport receptor

Gene number Predicted protein function
AT2G36880 METK3 (S-adenosylmethionine synthase 3); Protein is involved in a subpathway that synthesizes S-adenosyl-L-methionine from L-methionine. This subpathway is part of the pathway S-adenosyl-L-methionine biosynthesis, which is itself part of Amino-acid biosynthesis AT5G35360 CAC2 (Biotin carboxylase, chloroplastic); Protein is involved in a subpathway that synthesizes malonyl-CoA from acetyl-CoA AT1G12840 VHA-C (V-type proton ATPase subunit C); Subunit of the peripheral V1 complex of vacuolar ATPase. Subunit C is necessary for the assembly of the catalytic sector of the enzyme and is likely to have a specific function in its catalytic activity.    weeks. Plants were grown for an additional 2 weeks before analyses were initiated. Growth in hydroponics was carried out in a nutrient medium as described in [1]. Experiments were performed with 5 week old plants, watered either with or without NaCl. Plant material from shoot and root tissue was used for ion and metabolite quantification and mRNA isolation.
For hydroponic experiments plants were cultivated in sterile liquid culture medium according to [2]. Liquid culture medium was replaced after 7 days with ±100 mM NaCl. Seeds used for sterile culture were surface sterilized in 5% sodium hypochloride and subsequently incubated for 2 d in the dark at 4 °C for stratification.
The seedlings were harvested after 10 days and washed twice in deinonized water to remove external medium.
Generally, plant tissues were collected and immediately frozen in liquid nitrogen until use. Solute extraction from Arabidopsis tissues and spectroscopic quantification of sugars and starch were performed as described earlier [3].

Generation of p35s::SOS1 c-term StrepII plants
To create C-term overexpressor mutants the SOS1 amino acid sequence 446 to 1146 was cloned as a Strep-tagII-SOS1 C-terminus fusion construct and introduced into the plant genome via the floral dip method [4]. The Strep-tagII (WSHPQFEK) was introduced with the aim to quantify the recombinant SOS1-C-terminus protein via immuno-detection. It was added by the usage of the following oligonucleotides: SOS1C_Strep_for (5'-TTTGAATTCATGTGGAGCCACCCACAGTTCGAAAAGTTTGTTCTA CGC-3') and SOS1_XbaI_rev (5'-

TTTTCTAGATCATAGATCGTTCCTGAAAACGATTTTACT-3').
We excised the mutated sequence by using EcoRI and XbaI and inserted it into the correspondingly prepared original pHannibal vector [5]. Then, the Strep-tagII-SOS1 C-terminus construct was cut by NotI and inserted into the correspondingly prepared original pART27 vector [6]. The fidelity of all constructs was confirmed by complete sequencing. Transformation of the pART27 vector harboring the p35s::SOS1 c-term StrepII construct was performed using Agrobacterium tumefaciens, strain GV3101. Positive transformants were isolated by Kanamycin selection.

Gene expression analysis
mRNA from Arabidopsis was extracted from frozen leaf and root material using the NucleoSpin® RNA Plant Kit (Machery-Nagel, Düren, Germany). Contaminating DNA was removed by DNase digestion. The  [7] and either normalized to the phosphatase subunit pp2a (At1g13320) or to starting mRNA levels. Gene-specific oligonucleotides used for qRT-PCR are listed in Suppl. Table S3.

Interaction studies on AtSOS1-C-terminus using Bimolecular Fluorescence Complementation (BiFC)
We verified of the interaction between the SOS1-C-terminus and the identified binding partners in vivo via the Bimolecular Fluorescence Complementation (BiFC) method. For this purpose we generated YFP N and YFP C fusion constructs using the GATEWAY™ specific destination vectors pUBC-cYFP-Dest, pUBC-nYFP-Dest, pUBN-cYFP-Dest and pUBN-nYFP-Dest [8]. The cDNA of the gene of interest was amplified by PCR using gene-specific primers harboring the attB1 and attB2 sites, then cloned via BP reaction into pDONRZEO (Invitrogen, Darmstadt, Germany) and via LR reaction into the specific destination vector (for primers used see Suppl. Table S3). Purified vector DNAs were introduced into Agrobacterium strain GV3101::PM90 which was subsequently used to transform epidermal cells of Nicotiana benthamiana. After an expression period of 3-5 days epidermal cells were analyzed with a Leica TCS SP5II confocal laser scanning microscope system.

Metabolite/ion extraction and quantifications
For isolation of sugars, proline and cations, plant material was ground under liquid N2. Subsequently, one ml of deionized water was added to 100 mg tissue, the preparation was thoroughly mixed and kept for 15 min at Nucledur 100-5 C18-ec column (Macherey-Nagel, Düren, Germany). A gradient comprising 100 mM sodium acetate and acetonitrile (0-15%) was used to separate the amino acids. The AQC amino acids were detected by fluorescence with excitation at 250 nm and emission at 395 nm.

Heterologous synthesis of AtSOS1 C-terminus (aa978-1146) and At14-3-3 ω
To generate a N-terminal GST-tag fusion construct, the AtSOS1 C-terminus(aa978-1146) coding sequence was amplified and inserted into a pDEST™15 plasmid (ThermoFisher Scientific). The pDEST™15 construct containing AtSOS1 C-terminus (aa978-1146) was transformed into BL21-SI cells. The transformed bacterial cells were grown at 30°C in Luria-Bertani medium without NaCl. At an OD600 of 0.6 initiation of expression was induced and cells were harvested 18 h post-induction by centrifugation in an Eppendorf centrifuge (6000rpm, 15 min, 4°C), resuspended in buffer medium (10 mM Tris-HCl, pH 7.4) and immediately frozen in liquid nitrogen. To avoid proteolytic degradation phenylmethane sulphonyl-fluoride (PMSF, 1 mM) and protease inhibitor cocktail (Roche, Penzberg, Germany) was added. Cell lysis was performed using a cell disrupter (Emulsiflex) at 5000-10000PSI. After centrifugation (10000xg, 15 min at 4°C), cell debris was removed and the supernatant was collected for the following purification of the soluble protein.
To generate an N-terminal (10xHis)-tag fusion construct, the At14-3-3 ω coding sequence was amplified and inserted into a pET16b plasmid (Novagen). The pET16b construct containing At14-3-3 ω was transformed into Cell lysis was supported by sonication. Via centrifugation (12500 rpm, 20 min, 4°C) cell debris were removed and the supernatant was collected for the following purification of the soluble protein.

Pull-down assay
The GST-tagged AtSOS1 C-terminus(aa978-1146) peptide was transferred from elution buffer (50 mM Tris-HCl, pH 9.0, 7.5 mM reduced glutathione) to binding buffer medium (0.1 M MOPS, pH 7.5) by the use of an Amicon® Ultra Centrifugal filter (MerckMillipore, http://www.merckmillipore.com) and subsequently coupled to Affi-Gel10 matrix (Bio-Rad, http://www.bio-rad.com). Leaves of 5-week-old Arabidopsis plants were grounded in PBS medium, and the resulting extract was filtered prior to centrifugation. The supernatant was flushed over a chromatography column containing Affi-Gel10 affinity media to which the peptide had been coupled. The