Legends for Supporting Information
Figure S1. Amino acid sequences of the bHLH region and phylogenetic analysis of DPF
and DPF-like proteins. (a) Amino acid sequences of the bHLH region of the DPF protein
and rice homologous proteins. Black and gray backgrounds indicate identical and similar
amino acids, respectively. Basic HLH domains are indicated below the sequences. Arrows
in the basic region indicate the three residues, His (H), Glu (E) and Arg (R), comprising the
classic G-box binding region (Toledo-Ortiz et al., 2003). (b) Phylogenetic relationships
among the DPF and DPF-like proteins in plants. The corresponding sequence alignment is
shown in Figure S1c. Bootstrap values from 1,000 replications are indicated at each node.
Hv, Hordeum vulgare; Os, Oryza sativa; Sb, Sorghum bicolor; Ta, Triticum aestivum; Zm,
Zea mays. The bar corresponds to 0.1 amino acid substitutions per site. (c) Sequence
alignment of DPF (Os01g0196300), DPF-like proteins, and related proteins from various
plants. Black and gray backgrounds indicate identical and similar amino acids, respectively.
Figure S2. Typical gross morphology and leaf phenotype of DPF overexpressing plants in
the vegetative phase. Gross morphology (a) and phenotype of the 3rd youngest leaf blade
(b) of the descendants of DPF-OX-1 and -15 lines, and wild-type (WT) eight weeks after
sowing. Lesion mimics appeared in the DPF-OX-1 leaves. DPF-OX, DPF overexpressor;
WT, wild-type.
Figure S3. Lesion mimic phenotype of leaves overexpressing DPF in the ripening phase.
Flag leaves of the wild-type (WT), and descendants of DPF-OX-1, -15 and 16 lines. The
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image enclosed by a blue line is a close-up. The lesion mimic phenotype of flag leaves is
more severe than that of adult leaf blades in the vegetative phase (Figure S2).
Figure S4. Measurement of transcript levels of DPF and the accumulation of diterpenoid
phytoalexins in DPF-OX leaves. (a) Relative expression levels of DPF transcripts
normalized by RUBQ1. (b) Accumulation levels of MLs-A and B. (c) Accumulation levels
of PCs-A to E. The 3rd youngest leaf blades at the heading stage of each T1 plant were
sampled and used for the measurement. Three leaf discs were used for the measurement of
phytoalexins and the average is shown on the bar. Asterisks indicate that the lesion mimic
appeared in the sampled leaf. FW, fresh weight; n.d., not detected; WT, wild-type.
Figure S5. Accumulated levels of diterpenoid phytoalexins in DPF-OX husks. (a)
Accumulation levels of MLs-A and B. (b) Accumulation levels of PCs-A to E. Husks were
sampled from seeds of each harvested T1 plant and used for the measurement. Values are
means ± SD of four biological replicates. Asterisks show values that differ significantly
from the WT. *P<0.05; **P<0.01 (Student’s t-test). n.d., not detected; WT, wild-type.
Figure S6. Expression patterns of DPF-like1 & 2 in wild-type rice. (a) Expression patterns
in the leaf blade (LB) and roots in 10-day-old shoots. For LB, RNA was purified from the
youngest leaves. Values are means ± SD of three biological replicates. (b) The
CuCl2-inducible expression pattern after 24 h treatment.
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Figure S7. Expression patterns of DPF and several genes in MeJA or solvent (mock)
treated wild-type rice. (a) Expression pattern of DPF after 12 and 24 h treatments. (b)
Expression pattern after 24 h treatment. Values of the expression level of DPF are the same
as shown in (a). Values are means ± SD of three biological replicates. Asterisks show
values that differ significantly from the mock. *P<0.05; **P<0.01 (Student’s t-test).
Figure S8. Expression analyses of representative MEP pathway genes (DXS3, DXR and
HDR) and GGPS in the wild-type and two DPF-KD lines. RNA purified from 10-d-old
roots was used. Values are means ± SD of three biological replicates. Asterisks show values
that differ significantly from the WT. **P<0.01; ***P<0.001 (Student’s t-test).
Figure S9. Transient transactivation of promoters of DP biosynthetic genes by DPF. The
2-kb upstream regions of CPS2, KSL4 and CYP99A2 were used as the reporter plasmids.
pUCAP was used as a control plasmid. P35S, cauliflower mosaic virus 35S promoter; Tnos,
nopaline synthase terminator; hRluc, engineered Renilla luciferase gene (Promega).Values
are means ± SD of three biological replicates. Asterisks indicate that the activities with
DPF plasmid were significantly higher than that with the control plasmid (*P<0.05,
Student’s t-test).
Figure S10. Transient transactivation of the CPS2 promoter by DPF. (a) 400-bp upstream
region, 400-bp upstream region with mutated G-box element, 400-bp upstream region with
mutated G-box-like element, 400-bp upstream region with both elements mutated and the
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259-bp upstream region were used. G-box, 5-CACGTG-3; G-box like, 5-CACGTA-3;
N-box, 5-CACGAG-3; M, mutation; hRluc, engineered Renilla luciferase gene (Promega).
(b) 259-bp, 205-bp, 150-bp and 95-bp upstream regions were used.
Figure S11. Expression analyses of WRKY45 and DPF in wild-type and WRKY45-KD lines.
RNA was purified from 10-d-old roots. WT, wild-type; W45-KD, WRKY45-KD. Values are
means ± SD of four biological replicates. Asterisks show values that differ significantly
from the WT. **P<0.01 (Student’s t-test).
Figure S12. Distribution of N-boxes and G-boxes in the 2-kb upstream regions of DP
biosynthetic genes. The 5-untranslated region has not been reported; therefore, the 2-kb
upstream region from the translational start site is shown in KSL8. Black and white
triangles indicate the G-box and N-box, respectively. The asterisk indicates that because an
approximately 200-bp unreadable region exists around -700, there may be N-boxes in that
region.
Table S1. List of genes coexpressed with Os01g0196300 (DPF) by RiceFREND, a rice
gene coexpression database
Mutual rank is the index for coexpression.
Table S2. Primers used in this study
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Method S1. Plant materials
Method S2. Phylogenetic analysis
Method S3. Isolation and analysis of nucleic acids
Method S4. Plasmid construction and rice transformation
Method S5. CuCl2 , UV and methyl jasmonate treatments
Method S6. Transient expression assay
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