Volume 3, Issue 4, Pages 740-750 (July 2010) Requirement of a Homolog of Glucosidase II β-Subunit for EFR-Mediated Defense Signaling in Arabidopsis thaliana  von Numers Nina , Survila Mantas , Aalto Markku , Batoux Martine , Heino Pekka , Palva E. Tapio , Li Jing   Molecular Plant  Volume 3, Issue 4, Pages 740-750 (July 2010) DOI: 10.1093/mp/ssq017 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 A Scheme Depicting Glucose Trimming by Glucosidases I and II. Asparagine-linked oligosaccharides are transferred to nascent polypeptides in the lumen of the rough ER by oligosaccharyltransferase using a dolichol pyrophosphate-linked oligosaccharide as the donor substrate. The first step in the trimming of N-glycosylated proteins is to generate mature oligosaccharide chains of glycoproteins. This process includes the cleavage of all three glucose residues by ER-resident glucosidases. Glucosidase I (GCSI) removes the terminal α 1,2-linked glucose followed by the removal of the two inner α 1,3-linked glucose residues by GCS II. Molecular Plant 2010 3, 740-750DOI: (10.1093/mp/ssq017) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 Comparative Analysis of Primary Polypeptide Sequences. (A) The alignment of the full-length human protein kinase C substrate 80K-H (PKCSH) and its homolog in Arabidopsis. The programs CLUSTALW (http://align.genome.jp/) and BOXSHADE (www.ch.embnet.org/software/BOX_form.html) were used for aligning the two protein sequences. Similar or identical amino acids are shaded with gray and black, respectively. The asterisk indicates the predicted cleavage site of the signal peptide. The first two boxed regions at the N-terminus are corresponding to a putative EF-hand motif and a C-type lectin domain, respectively. The boxed region at the C-terminus indicates a mannose-6-phosphate receptor domain, where asterisks represent the residues implicated in mannose binding. The poorly matched stretch is underlined. (B) The alignment of plant-specific stretches in the putative calmodulin-binding protein (At5g56360). The cut-off E-value 6e-05 was used for retrieving protein sequences. The accession numbers in GenBank (www.ncbi.nlm.nih.gov/Genbank/) and the species harboring At5g56360 homologs are XP_002283762 (Vitis vinifera), CAO22122 (Vitis vinifera), XP_002304848 (Populus trichocarpa), XP_002299093 (Populus trichocarpa), XP_002523398 (Ricinus communis), NP_001146004 (Zea mays), NP_001146236 (Zea mays), NP_001042732 (Oryza sativa), and XP_002525454 (Ricinus communis). Molecular Plant 2010 3, 740-750DOI: (10.1093/mp/ssq017) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Data-Mining on the Transcriptional Changes of AtGSCIIβ (At5g56360) and the Related Arabidopsis Genes. The image was generated with the research tools of the Genevestigator V3 program (www.genevestigator.ethz.ch), which collected 4070 high-quality 22K Affymetrix arrays covering 267 different stimuli in wild-type Arabidopsis. Molecular Plant 2010 3, 740-750DOI: (10.1093/mp/ssq017) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 Molecular Identification of Three AtGCSIIβ T-DNA Insertion Lines. (A) A schematic picture of the T-DNA insertions in the AtGCSIIβ locus. Green, red, and blue boxes indicate UTRs, exons, and introns, respectively. The numbers 1, 529, 2933, 3024, and 4365 represent the translation start site, the position of T-DNA insertion confirmed by sequencing PCR fragments with the primer pairs from the AtGCSIIβ gene and T-DNA left border, and the stop codon of AtGCSIIβ, respectively. (B) Homozygous lines were selected by PCR. Equal amounts of DNA templates from wild-type plants (WT) and the T-DNA tagged lines were mixed to confirm the appearance of the insert with the AtGCSIIβ-specific primer pairs. PCR fragments of expected size were amplified from DNA templates of WT and the DNA mixture of WT and mutants, but not from mutants only. (C) RT–PCR analysis of AtGCSIIβ transcripts in WT and mutants. The levels of RPL4 were assayed as controls. Molecular Plant 2010 3, 740-750DOI: (10.1093/mp/ssq017) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 T-DNA Insertion Mutants in atgcsIIβ Show Differential Sensitivity to elf18 in Growth Assays. (A) Wild-type (WT) and atgcsIIβ seedlings grown for 7 d in the presence of 50 nM elf18. (B) Inhibitory effect of elf18 on seedling growth. Results are mean values ± SD (n = 8). Letters ‘a’ and ‘b’ indicate significant differences at the level of p < 0.05 (t-test) between atgcsIIβ mutants and WT or efr-1 plants, respectively. Experiments were repeated three times, with similar results. Molecular Plant 2010 3, 740-750DOI: (10.1093/mp/ssq017) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 6 Elf18-Responsiveness and Bacterial Infection in atgcsIIβ Mutants in Comparison with Wild-Type (WT).(A) Oxidative burst measured in relative light units (RLU) in leaf discs. Results are shown as the mean values ± SD (n = 12).(B) MAP kinase activation. In vitro kinase reactions were performed with myelin basic protein as a substrate in the presence of [γ-32P]ATP. Coomassie blue staining is shown to demonstrate equal loading of proteins in each lane.(C) RT–qPCR analysis of elf18-induction of marker genes WRKY33 and CYP81F2. The data represent the mean values ± SD (n = 4).(D) Callose deposition. Leaf samples were collected 20 h after infiltration with 100 nM elf18 and stained with aniline blue for visualization of callose. Results are mean values ± SD (n = 18).(E) Bacterial susceptibility assays in WT and atgcsIIβ mutants. Plants were infected by spraying with suspensions of Pst DC3000 (OD600 = 0.02). In planta-grown bacteria were quantified with leaf discs after 72-h inoculation. Asterisks indicate significant difference (P < 0.05) from WT control as determined by Student's t-test. Results are means ± SE (n = 8). All experiments were repeated at least twice, with similar results. Molecular Plant 2010 3, 740-750DOI: (10.1093/mp/ssq017) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 7 Secretion of CPY in Yeast. The gtb1Δ mutant carrying the Arabidopsis transgene AtGCSIIβ is indicated by gtb1Δ/AtGCSIIβ. Wild-type strain (WT) and prc1Δ mutant were used as a positive and negative controls for detecting both precursor forms (pro-CPY) and enzymatically active forms of CPY, respectively. Equal amounts of total protein extracts from each strain were used for radio-labeling and immunoprecipitation (data not shown). Molecular Plant 2010 3, 740-750DOI: (10.1093/mp/ssq017) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions