SUPPLEMENTAL DATA
Supplemental Figure 1. Sequence alignment of AtOS9, Yos9, mammalian OS-9s, and predicted plant Yos9/OS-9 homologs.
Yos9 (accession number: NP_010342), human Os-9 (NP_006803), and predicted Yos9/OS-9 homologs from Arabidopsis (AtOS9, NP_568525), Physcomitrella patens (PpOS9, XP_001756223), Oryza sativa (OsOS9, NP_001174928), Zea mays (ZmOS9, NP_001149187), and mouse (MmOS9, NP_001164497) were aligned using the ClustalW program. The aligned amino acid sequences were color shaded by the Boxshade program at the Mobyle portal ( Identical residues are shaded in red while similar residues are shaded in cyan. The blue box indicates the predicted signal peptide of AtOS9, blue dots show the positions of the predicted N-glycosylation sites, while triangles mark the ebs6-1 mutation and the three conserved amino acids that were mutated in several transgene constructs used in this study. The two green brackets delimit the predicted MRH domains while the red box indicates the HDEL ER retrieval sequence of Yos9.
Supplemental Figure 2: AtOS9 is coexpressed with known/predicted Arabidopsis ER chaperones.
Data were retrieved from ATTED-II [ (Obayashi et al., 2007)] after performing a simple search using the AtOS9 AGI code, At5g35080. Heavy lines indicate strong correlation [with a mutual rank (MR) value <5],thin linesmarknormally correlated genes (with an MR value between 5 and 30), while the orange line denotesconserved coexpressed gene pair.
Supplemental Figure 3. Expression of a genomic EBS2 gene nullifies the suppressive effect of the atos9-t mutation on the bri1-9 mutant.
(A) Images of 5-wk-old soil-grown plants of atos9-t bri1-9 and a gEBS2 atos9-t bri1-9 transgenic line that over-expresses a plant-specific CRT3.
(B) Immunoblot analysis of bri1-9. Equal amounts of total proteins extracted from 2-wk-old seedlings were treated with or without Endo H, separated by 7% SDS-PAGE, and analyzed by immunoblot with an anti-BRI1 antibody. Commassie blue staining of the Arabidopsis RbcS serves as a loading control. bri1-9C indicates the bri1-9 protein carrying complex type N-glycans while bri1-9H denotes the bri1-9 protein with high-mannose-type N-glycans.
Supplemental Figure 4. The ebs6-1 mutant carries amutant AtOS9 protein.
(A) Shown here is the region cropped from aligned sequences of Yos9/OS-9 and their homologs in Supplemental Figure 1, which contains the detected G191-E mutation in the ebs6-1 mutant.
(B) Immunoblot analysis of AtOS9. Equal amounts of total proteins extracted from 2-wk-old seedlings were separated by 10% SDS-PAGE and analyzed by immunoblot using an anti-AtOS9 antibody. Asterisk indicates a nonspecific band for loading control.
Supplemental Figure 5. The atos9-t ebs1-6 bri1-9 mutant remains insensitive to elf18.
(A and B) Images of 12-d-old seedlings grown on ½ MS medium supplemented without (A) or with (B) 100 nM elf18, a biologically active epitope derived from the bacterial translational elongation factor EF-Tu (Kunze et al., 2004). Five-day-old Arabidopsis seedlings grown on ½ MS medium were submerged with liquid ½ MS mediumsupplemented with or without 100 nM elf18 (Li et al., 2009)and were photographed 7 days later.
Supplemental Figure 6. The atos9-t mutation has no detectable effect on the stability of EBS5.
(A) Immunoblot analysis of the EBS5 abundance. Equal amounts of total proteins extracted from 2-wk-old seedlings were separated by 10% SDS-PAGE and analyzed by immunoblot with an affinity-purified anti-EBS5 antibody. Star indicates a cross-reacting band used for the loading control.
(B) Immnuoblot analysis of the EBS5 protein stability. Two-wk-old seedlings were transferred into liquid ½ MS medium containing 180 μM CHX. Equal amounts of seedlings were removed at indicated incubation times to extract total proteins in 2 X SDS sample buffer, which were subsequently analyzed by immunoblot with anaffinity-purified anti-EBS5 antibody. Coomassie blue staining of the Arabidopsis RbcS serves as a loading control.
Supplemental Figure 7. The simultaneous elimination of two Arabidopsis homologs of the yeast Hrd1 E3 ligase has no effect on the stability of AtOS9 or EBS5.
Two-wk-old seedlings of an athrd1a athrd1b bri1-9 triple mutant were transferred into liquid ½ MS medium containing 180 μM CHX. Equal amounts of seedlings were taken out at indicated incubation times to extract total proteins with 2 X SDS sample buffer, which were subsequently analyzed by immunoblot with ananti-AtOS9 (the 1st strip) or anti-EBS5 antibody (the 3rd strip). Coomassie blue staining of RbcS (the 2nd and 4th strips) serves as a loading control.
Supplemental Figure 8. The ERAD of bri1-9 requires both AtOS9 and EBS5.
(A and B) Image of 5-wk-old soil-grown plants of bri1-9, representative transgenic atos9-t bri1-9 lines carrying an empty vector of a p35:EBS5 transgene, representative transgenic ebs5-1 bri1-9 lines carrying an empty vector or a p35S:AtOS9 transgene.
Supplemental Figure 9. Both Yos9 and human OS-9 fail to rescue the atos9-t mutation.
Shown here (from left to right) are images of 5-wk-old soil-grown plants of representative transgenic atos9-t bri1-9 lines expressing p35S:AtOS9, p35S:HsOS-9, or p35S:Yos9 transgene.
Supplemental References:
Kunze, G., Zipfel, C., Robatzek, S., Niehaus, K., Boller, T., and Felix, G. (2004). The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16, 3496-3507.
Li, J., Zhao-Hui, C., Batoux, M., Nekrasov, V., Roux, M., Chinchilla, D., Zipfel, C., and Jones, J.D. (2009). Specific ER quality control components required for biogenesis of the plant innate immune receptor EFR. Proc Natl Acad Sci U S A 106, 15973-15978.
Obayashi, T., Kinoshita, K., Nakai, K., Shibaoka, M., Hayashi, S., Saeki, M., Shibata, D., Saito, K., and Ohta, H. (2007). ATTED-II: a database of co-expressed genes and cis elements for identifying co-regulated gene groups in Arabidopsis. Nucleic Acids Res 35, D863-869.
Supplemental Table 1.Oligonucleotides used in this study
Name / Sequence (5’-3’) / Usageebs6-
dCAPS / GGTATCACTCTCATGTATACACCCATG
CAGTGATTGAAGTGACCATTGCCC / genotyping of ebs6 mutation
NGA139 / AGAGCTACCAGATCCGATGG
GGTTTCGTTTCACTATCCAGG / mapping the EBS6 locus
CIW9 / CAGACGTATCAAATGACAAATG
GACTACTGCTCAAACTATTCGG / mapping the EBS6 locus
gAtOS9 / CGAGTCGACGAAGTCGTTGTCTAGTTGTCAC*
CGGATCCCAAGAATCAGCTATCATCTTAGG / amplifying an AtOS9 genomic fragment
cAtOS9 / CACCATGAGAATCACGCAGATCTTG
GAATCAGCTATCATCTTAGGTG / amplifying the AtOS9 cDNA by RT-PCR
HsOS9 / CACCATGGCGGCGGAAACGCTGC
GAAGTCAAATTCGTCCAGGTCC / amplifying a human OS-9 cDNA
Yos9 / CACCATGCAAGCTAAAATTATATATG
AAAGTTTATACTCCTCCTTGTTT / amplifying an Yos9 cDNA
cAtOS9-Y132A / GGGTTGGTGGTCTGCTGAGTTTTGTCATCAG†
CTGATGACAAAACTCAGCAGACCACCAACCC / to create Y132-A mutation in p35S:AtOS9 plasmid
cAtOS9-Q142E / CAGAAGTATGTGCGGGAGCTACACGTTGAGG
CCTCAACGTGTAGCTCCCGCACATACTTCTG / to create Q142-E mutation in p35S:AtOS9 plasmid
cAtOS9-E221N / GGTCACTTCAATCACTAACTTATCAACTTGC
GCAAGTTGATAAGTTAGTGATTGAAGTGACC / to create E221-N mutation in p35S:AtOS9 plasmid
AtOS9-C / CGGATCCGAAGTCGTTGTCTAGTTGTCAC
GATGTCGACCGGTTTTCAAGAATCAGCTATC / amplifying a partial AtOS9 cDNA
* the underlined sequences represent convenient restriction sites for the cloning purpose
† the blackened sequences indicate nucleotide mutations introduced in the primers.