On the Origin of SERKs: Bioinformatics Analysis of the Somatic Embryogenesis Receptor Kinases  Marije aan den Toorn, Catherine Albrecht, Sacco de Vries  Molecular Plant  Volume 8, Issue 5, Pages 762-782 (May 2015) DOI: 10.1016/j.molp.2015.03.015 Copyright © 2015 The Author Terms and Conditions

Figure 1 Neighbor-Joining Tree of the SERK Protein Family. Neighbor-joining (NJ) tree based on a multiple sequence alignment of the sequence of 71 proteins in Table 1. These include 67 putative SERK proteins and four A. thaliana LRR-II non-SERK proteins (included as an outgroup). Bootstrap values (in percentages) from 1000 replicates are shown next to the branches. Branches with a replicate percentage below 50% are collapsed. The evolutionary distances were computed using the Dayhoff matrix-based method. Four distinct groups can be inferred from this NJ tree; LRRII are proteins that group together with the A. thaliana non-SERK proteins and are thus not true SERKs. SERK Dicot S3/4 proteins are sequences solely from dicotyledons that group together with A. thaliana SERK3, SERK4, and SERK5, while the SERK Dicot S 1/2 proteins are dicot SERKs that group with A. thaliana S1/2. All SERK sequences from non-vascular plants group together (Non-Vascular) as do the sequences from monocotyledons (Monocot). Molecular Plant 2015 8, 762-782DOI: (10.1016/j.molp.2015.03.015) Copyright © 2015 The Author Terms and Conditions

Figure 2 Multiple Sequence Alignment of SPP and C-Terminal Tail Domains. (A) Detail of the SERK multiple sequence alignment (MSA) focused on the SPP domain. The dicot SERK3/4 protein sequences are clearly distinct from the other SERK protein sequences by missing an otherwise conserved cysteine pair preceding the SPP domain, and having a shorter, more heterogeneous SPP domain. (B) Detail of the SERK MSA focused on the C-terminal tail domain. Although highly homologous among the monocot, non-vascular, and dicot SERK1/2 protein sequences, the dicot SERK3/4 show more divergence in this domain, and especially a higher occurrence of tyrosine residues across the domain. Molecular Plant 2015 8, 762-782DOI: (10.1016/j.molp.2015.03.015) Copyright © 2015 The Author Terms and Conditions

Figure 2 Multiple Sequence Alignment of SPP and C-Terminal Tail Domains. (A) Detail of the SERK multiple sequence alignment (MSA) focused on the SPP domain. The dicot SERK3/4 protein sequences are clearly distinct from the other SERK protein sequences by missing an otherwise conserved cysteine pair preceding the SPP domain, and having a shorter, more heterogeneous SPP domain. (B) Detail of the SERK MSA focused on the C-terminal tail domain. Although highly homologous among the monocot, non-vascular, and dicot SERK1/2 protein sequences, the dicot SERK3/4 show more divergence in this domain, and especially a higher occurrence of tyrosine residues across the domain. Molecular Plant 2015 8, 762-782DOI: (10.1016/j.molp.2015.03.015) Copyright © 2015 The Author Terms and Conditions

Figure 3 Conservation in the SERK Ectodomain. Conservation score for each amino acid as determined using the ConSurf algorithm (Ashkenazy et al., 2010) was plotted onto the reported structure of the SERK1 ectodomain (PDB: 4LSC; Santiago et al., 2013). Higher scores, plotted as red onto the structure, represent residues of higher conservation, whereas lower scores, indicated by cyan, depict more variable residues. Input was the MSA of 57 SERK protein sequences. (A) Convex or solvent-exposed side of the SERK LRR. (B) Concave side of the SERK LRR, which has interactions with the main ligand receptors. (C and D) Hydrophobicity plots (hydrophobic regions are depicted by orange, hydrophilic regions by blue) of the convex (C) and concave (D) sides. Molecular Plant 2015 8, 762-782DOI: (10.1016/j.molp.2015.03.015) Copyright © 2015 The Author Terms and Conditions

Figure 4 Conservation in the SERK Kinase Domain. The ConSurf algorithm (Ashkenazy et al., 2010) was used to plot the conservation score per amino acid onto the crystal structure of AtSERK3 kinase domain (PDB: 3TL8; Cheng et al., 2011), using the MSA of 57 SERK kinase sequences as input. Most of the kinase domain is conserved between the family members; all non-conserved residues (depicted in cyan space-fill) are found on one side of the kinase domain, away from the catalytic cleft of the kinase. Molecular Plant 2015 8, 762-782DOI: (10.1016/j.molp.2015.03.015) Copyright © 2015 The Author Terms and Conditions

Figure 5 In Contrast to SERK1 and SERK3, SERK2 Does Not Interact with BRI1. Co-immunoprecipitation of SERK-GFP and BRI1 proteins. Transgenic Arabidopsis seedlings expressing SERK1, SERK2, and SERK3 driven by their own promoter and tagged with GFP were treated with brassinazole (BZR, 2.5 μM for 3 days), propiconazole (PPC, 1 μM for 3 days), or epibrassinosteroid (BL, 1 μM for 10 min). Total protein (input) was subjected to immunoprecipitation with anti-GFP beads followed by immunoblot analysis with anti-GFP antibodies to detect SERK-GFP or anti-BRI1 antibodies to detect BRI1. Molecular weight of detected proteins is indicated on the left in kilodaltons. These experiments were repeated three times with similar results. Molecular Plant 2015 8, 762-782DOI: (10.1016/j.molp.2015.03.015) Copyright © 2015 The Author Terms and Conditions

Figure 6 Schematic Representation of the Chimeric Constructs. Molecular Plant 2015 8, 762-782DOI: (10.1016/j.molp.2015.03.015) Copyright © 2015 The Author Terms and Conditions

Figure 7 SERK1 or SERK2 Extracellular Domain Is Required to Restore Male Sporogenesis. (A) Inflorescence of a serk1 serk2 double mutant, expressing the SERK2 gene driven by the SERK3 promoter (PSERK3:SERK2-YFP), and the S2ES3K and S3ES2K chimeras under the SERK3 promoter. Plants expressing PSERK3:SERK2-YFP and PSERK3:S2ES3K-YFP show normal seedpods; plants expressing PSERK3:S3ES2K-YFP are sterile. (B) The PSERK3:SERK2-YFP, PSERK3:S2ES3K-YFP, and PSERK3:S3ES2K-YFP proteins are visualized in the anther tissue by confocal microscopy. T, tapetum. (C) Western blot showing the expression of the chimeric proteins. Molecular Plant 2015 8, 762-782DOI: (10.1016/j.molp.2015.03.015) Copyright © 2015 The Author Terms and Conditions

Figure 8 BR-Phenotype Complementation by Chimeric SERK-GFP Proteins. (A) BR-induced root growth inhibition of complemented serk1 serk3 mutant with chimeric SERK proteins tagged with GFP. Root growth of 8-day-old seedlings grown on medium containing an increasing amount of epibrassinolide: 2.5, 5, and 10 nM. Two representative independent lines for each chimeric construct are presented, #1 and #2. Bars represent SE (n = 20). These experiments were repeated three times with similar results. (B) Leaf phenotype of complemented serk1 serk3 mutant with chimeric SERK-GFP proteins. The leaf growth was assessed by measuring the ratio leaf length per leaf width (n = 10). Two representative independent lines for each chimeric construct are presented, #1 and #2. These experiments were repeated twice with similar results. (C) Western blot analysis showing the expression of the transgene in the lines used for the root inhibition assay and leaf phenotype. Molecular weight of detected proteins is indicated on the left in kilodaltons. (D) Complex formation between SERK2 and BRI1 is restored in the chimeric proteins S2ES3K and S3ES2K. Transgenic PBRI1:BRI1-GFP Arabidopsis seedlings co-expressing S1ES3K, S3ES1K, S2ES3K, or S3ES2K driven by the SERK3 promoter and tagged with mCherry were used for the experiment. Total protein (input) was subjected to immunoprecipitation with anti-RFP immunoaffinity beads, followed by immunoblot analysis with anti-RFP antibodies to detect SERK-RFP and anti-GFP antibodies to detect BRI1-GFP. Molecular weight of detected proteins is indicated on the left in kilodaltons. Molecular Plant 2015 8, 762-782DOI: (10.1016/j.molp.2015.03.015) Copyright © 2015 The Author Terms and Conditions

Figure 9 None of the Chimeric Proteins Restores PAMP Signaling. (A) PAMP-induced seedling growth inhibition of complemented serk1 serk3 mutant with chimeric SERK proteins. Seedling growth inhibition in response to increasing concentrations of flg22 in Col[0], serk3, serk1serk3, and complemented serk1serk3 mutants with different swap domain SERK proteins. Seedlings were weighed 10 days after treatment. Results are given as absolute (left) or relative growth (right) measured as fresh weight. Results are mean ± SE (n = 10). All experiments were repeated at least three times with similar results. (B) The chimeric proteins are not impaired in their ability to interact with FLS2. Transgenic Arabidopsis seedlings expressing SERK3, S1ES3K, and S3ES1K driven by SERK3 promoter and tagged with GFP were treated with flagellin (flg22, 1 μM for 10 min). Total protein (input) was subjected to immunoprecipitation with anti-GFP immunoaffinity beads, followed by immunoblot analysis with anti-GFP antibodies to detect SERK-GFP and anti-FLS2 antibodies to detect FLS2. Molecular weight of detected proteins is indicated on the left in kilodaltons. Molecular Plant 2015 8, 762-782DOI: (10.1016/j.molp.2015.03.015) Copyright © 2015 The Author Terms and Conditions