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SUPPORTING INFORMATION
Identification of a cis-element that mediates multiple pathways of the ER
stress response in rice
Shimpei Hayashi, Hideyuki Takahashi, Yuhya Wakasa, Taiji Kawakatsu, and
Fumio Takaiwa
Figure S1. Inducibility of gene expression by OsbZIP60deltaC.
Figure S2. Inducibility of OsBiP2–5 expression by OsbZIP50s-GR.
Figure S3. ChIP-PCR analysis of some ER stress-responsive promoters.
Figure S4. Binding specificities of OsbZIP60 to pUPRE-II variants.
Figure S5. Binding affinity between bZIP domains of OsbZIP50 and OsbZIP60.
Figure S6. Alignment of OsbZIP50 binding sequences.
Table S1. Oligonucleotides used in this study.
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FIGURE LEGENDS
Figure S1. Inducibility of gene expression by OsbZIP60deltaC.
Inducibility of OsbZIP50-dependent genes in transgenic plants expressing
OsbZIP60deltaC under control of the dexamethazone (DEX)-inducible promoter.
(a) Structure of T-DNA used for the generation of transgenic plants. 35S Pro,
CaMV 35S promoter; GAL4-VP16-GR, a chimeric protein consisting of the DNA
binding domain of the GAL4 transcription factor, the VP16 transcriptional
activation domain, and the glucocorticoid receptor (GR); UAS, upstream
activation sequence; OsbZIP60dC, OsbZIP60deltaC. In transgenic plants,
GAL4-VP16-GR proteins were constitutively produced. DEX treatment allowed
the nuclear localisation of the chimeric proteins and thereby activated the
UAS-containing promoter. (b) Expression of OsbZIP50-dependent genes after
treatment with or without DEX. First-strand cDNA derived from equal amounts of
total RNA were analyzed by PCR with specific primer sets. Similar results were
observed in more than two independent experiments. OsbZIP60dC (trans),
OsbZIP60 transcripts derived from the transgene.
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Figure S2. Inducibility of BiP2–5 expression by OsbZIP50s-GR.
mRNA levels of OsBiP paralogue genes in transgenic plants that expressed
OsbZIP50s-GR after treatment for 4 h with both CHX and DEX. Quantitative
RT-PCR analysis of seven biological replicates.
Figure S3. ChIP-PCR analysis of some ER stress-responsive promoters.
Quantitative PCR analysis of DNA fragments precipitated with anti-OsbZIP50
antibodies. Short regions upstream of the transcription initiation site of genes
were analyzed. DNA sample before immunoprecipitation was used as INPUT.
Similar results were observed in more than two independent experiments.
Figure S4. Binding specificities of OsbZIP60 to pUPRE-II variants.
(a) Competition between labelled DNA fragments containing ERSE and
non-labelled DNA fragments (1 and 2), as revealed by EMSA. Arrow indicates
complexes containing GST-OsbZIP60 and labelled DNA fragments. (b, c)
Competition between the labelled fragment D in Figure 3 and non-labelled DNA
fragments are shown by EMSA. Closed arrowhead indicates complexes of
GST-OsbZIP60 with labelled DNA fragment D. The experiment in (c) was
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performed under the same conditions as Figure 4, except for the use of
OsbZIP60 instead of OsbZIP50. Upper panel in (c) is part of Figure 4.
Figure S5. Binding affinity between bZIP domains of OsbZIP50 and
OsbZIP60.
Yeast strain YRG-2 was transformed with the vectors pGBKT7 and pGADT7
(Clontech), which harbour the ORF encoding the bZIP domain of OsbZIP50 or
OsbZIP60. Transformants were grown on synthetic complete medium, as
indicated.
Figure S6. Alignment of OsbZIP50 binding sequences.
(a) Competition between labelled fragment D in Figure 3 and non-labelled DNA
fragments, as revealed by EMSA. Arrowhead indicates complexes containing
GST-OsbZIP50 and labelled DNA fragment D. WRKY45, Os05g0322900; BiP2,
Os03g0710500; BiP3, Os05g0367800; BiP5, Os08g0197700. (b, c) Alignment
of OsbZIP50 binding sequences. DNA sequences that showed binding ability to
OsbZIP50 in EMSA were aligned, and a sequence logo was created using the
WebLogo program (Crooks et al., 2004). Lower panel in (c), which is part of
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Figure 3d, shows the effects of transversion mutations in the positions indicated
in the sequence logo on the binding affinity of fragment D to OsbZIP50.