Supporting Information Figs S1-S7 and Table S1
Fig. S1 Nucleotide and amino acid sequences of pepper CaDC1 cDNA. Amino acid
sequences are given below nucleotide sequences. The transcriptional start site is shown
in bold type and the termination codon is marked by an asterisk (*). The DC1 domain is
shaded in gray. Black Boxes indicate polyadenylation sequences.
1
Fig. S2 Alignment of pepper (Capsicum annuum) CaDC1 to other plant DC1 domaincontaining proteins. Comparison of the deduced amino acid sequences of pepper
(Capsicum annuum) CaDC1 with those of Nicotiana tabacum DC1 domain-containing
protein (accession no. BAF80452), Arabidopsis thaliana cysteine/histidine-rich C1
domain-containing protein (accession no. NP_181966), Populus trichocarpa predicted
protein (accession no. XP_002330857), Ricinus communis protein binding protein
(accession no. XP_002512181), and Vitis vinifera uncharacterized protein (accession no.
XP_002268131). Percent similarity to the CaDC1 protein is reported on the lower right.
Conserved cysteines and histidines are marked above the sequence.
2
Fig. S3 Subcellular localization of CaDC1-GFP and the CaDC1-NES-, nes-, NLS- or
nls-GFP fusion constructs transiently expressed in Nicotiana benthamiana leaves.
Fluorescing cells were visualized with a confocal microscope to determine patterns of
GFP fluorescence, together with the DAPI counterstaining to visualize nuclei. Bars, 50
μm
3
Fig. S4 Induction of the cell death response by CaDC1 transient expression in pepper
leaves. (a) Visible and UV light-illuminated phenotypes of leaves expressing empty
vector control (35S:00) or 35S:CaDC1 4 d after Agro-infiltration at various inoculum
concentrations (OD600). (b) Cell death levels measured in leaves transiently expressing
different constructs 4 d after Agro-infiltration (OD600 = 1.0). Cell death ratings were
done from 10 infiltration sites in the three independent experiments, based on the
following scales: 0, no cell death; 1, weak cell death (< 30%); 2, partial cell death (30–
70%); and 3, full cell death (70–100%). (c) Electrolyte leakage from leaves at different
time points after Agro-infiltration (OD600 = 1.0). (b, c) Values are presented as means ±
standard deviations from three independent experiments. Asterisks indicate
significant differences between empty vector control and CaDC1-expressing leaves
(Student's t test, P < 0.05).
4
Fig. S5 Effect of subcellular localization of CaDC1 and DC1 domain on induction of
the cell death response in pepper leaves. (a) Visible and UV light-illuminated
phenotypes of pepper leaves expressing 35S:00 (empty vector), 35S:CaDC1, 35S:DC1,
35S:CaDC1-NES and 35S:DC1-NES constructs 4 d after Agro-infiltration. (b)
Immunoblot analysis of c-Myc epitope-tagged CaDC1, DC1, CaDC1-NES, or DC1NES in transiently expressed leaves 24 and 48 h after Agro-infiltration. (c) Cell death
levels in leaves transiently expressing various constructs 4 d after Agro-infiltration. Cell
death ratings were done from 10 infiltration sites in the three independent experiments,
based on the following scales: 0, no cell death; 1, weak cell death (< 30%); 2, partial
cell death (30–70%); and 3, full cell death (70–100%). (d) Electrolyte leakage from
5
leaves at different time points after Agro-infiltration. (c, d) Values are presented as
means ± standard deviations from three independent experiments. Different letters
indicate significant differences, as analyzed by a Fisher's protected least significant
difference (LSD) test (P < 0.05).
6
Fig. S6 Effect of transient expression of 35S:CaDC1, 35S:CaDC1-NLS and
35S:CaDC1C constructs on induction of the cell death response in pepper leaves. (a)
Visible and UV light-illuminated phenotypes of pepper leaves transiently expressing
35S:00 (empty vector), 35S:CaDC1, 35S:CaDC1-NLS, 35S:CaDC1C and 35S:DC1
constructs 4 d after Agro-infiltration. (b) Immunoblot analysis of c-Myc epitope-tagged
CaDC1, DC1, CaDC1c, or CaDC1-NLS in pepper leaves 24, 48 and 72 h after Agro-
7
mediated transient expression. (c) Cell death levels in leaves transiently expressing
various constructs 4 d after Agro-infiltration. Cell death ratings were done from 10
infiltration sites using three independent experiments, based on the following scales: 0,
no cell death; 1, weak cell death (< 30%); 2, partial cell death (30–70%); and 3: full cell
death (70–100%). (d) Electrolyte leakage from leaves at different time points after
Agro-infiltration. (c, d) Values are presented as means ± standard deviations from three
independent experiments. Different letters indicate significant differences, as analyzed
by a Fisher's protected least significant difference (LSD) test (P < 0.05).
8
Fig. S7 Enhanced susceptibility of CaDC1-OX Arabidopsis transgenic lines to
Pseudomonas syringae pv. tomato (Pst) DC3000 and DC3000 (avrRpm1). (a) Disease
9
symptoms on leaves of wild-type (WT) and transgenic (# 2) leaves infected with Pst
DC3000 or DC3000 (avrRpm1) (106 cfu ml-1) 5 d after inoculation. (b) Bacterial growth
in leaves inoculated with Pst DC3000 or Pst DC3000 (avrRpm1) (105 cfu ml-1). (c)
Electrolyte leakage from leaf tissues inoculated with Pst DC3000 or Pst DC3000
(avrRpm1). Samples were taken at 0, 12 and 24 h after inoculation. Values are presented
as means ± standard deviations.
10
Table S1 Oligonucleotide sequences used for quantitative RT-PCR in this study
Accession
Gene
Sense primer
Antisense primer
no.
CaDC1
HM581976
CaPR1
AF053343
F:5'-AGCATTTTAGCCACCCCCAT-3'
R:5'-TTAACTGTCGCGCATTGCAG-3'
F:5'-CAGGATGCAACACTCTGGTGGR:5'-ATCAAAGGCCGGTTGGTC-3'
3'
F:5'R:5'CaDEF1
AF442388
CAAGGGAGTATGTGCTAGTGAGACTGCACAGCACTATCATTGCATAC-3'
3'
CaACT
GQ339766
F:5'-TTGGACTCTGGTGATGGTGTG-3'
R:5'-AACATGGTTGAGCCACCACTG-3'
AtPR1
AT2G14610
F:5'-GGAGCTACGCAGAACAACTA-3'
R:5'-AGTATGGCTTCTCGTTCACA-3'
AtPDF1.2
AT5G44420
F:5'-ATGGCTAAGTTTGCTTCC-3'
F:5'TTAACATGGGACGTAACAGATAC-3'
AtRD29a
AT5G52310
F:5'-GGAAGAGTCGGCTGTTTCAG-3'
R:5'-TGATGGAGAATTCGTGTCCA-3'
F:5'AtRbohD
AT5G47910
AACGGCCTCTTACTCTCTGCCAAGT- R:5'-TCATTTGCTTCTCCAACACG-3'
3'
AtACTIN2
AT3G18780
F:5'-AAGCTCTCCTTTGTTGCTGTT-3'
11
R:5'-GACTTCTGGGCATCTGAATCT-3'