Xba I
35S P.
tAPX or GUS intron
tAPX or GUS
A
Xba I
NOS T.
pGWB80/tAPX (or GUS)
Xba I
Spe I
B
G10-90 P.
XVE
T3A
Lex A P.
ccdB
T3A
pMDC7
C
XVE
T3A
Lex A P.
tAPX or GUS intron
tAPX or GUS
G10-90 P.
T3A
pMDC7/tAPX (or GUS) RNAi
Maruta et al., Supplemental Fig. S1.
Plasmid construction for estrogen inducible RNAi. To construct the plasmid for
estrogen-inducible silencing of tAPX, DNA fragment containing the 3’-untranslational
region of tAPX was cloned into the donor vector, pDONR201, and then recloned into
the destination vector, pGWB80. An open reading frame of the beta-Glucuronidase
(GUS) gene was also cloned into pGWB80 as a control. PCR and in vitro BP and LR
recombination reactions were carried out according to the manufacturer’s instructions
(Invitrogen). The specific primers with attB1 and attB2 sequences were as follows:
attB1-tAPX3’ (5’-AAAAAGCAGGCTGAGGACAGTCATGGACAGTG-3’), attB2-tAPX3’
(5’-AGAAAGCTGGGTTCACCACGTAATTATGTATGTAGGT-3’),
attB1-GUS
(5’AAAAAGCAGGCTATGTTACGTCCTGTAGAAAC-3’),
and
attB2-GUS
(5’AGAAAGCTGGGTTCATTGTTTGCCTCCCTGCT-3’).
DNA
sequencing
was
performed using the dideoxy chain terminator method with an automatic DNA
sequencer
(ABI
PRISMTM
310,
Applied
Biosystems,
http://www.appliedbiosystems.com/). The plasmids obtained, pGWB80/tAPX and
pGWB80/GUS (A), were digested with a restriction enzyme Xba I, and then blunted
by a Mung Bean Nuclease (Takara). The estrogen-inducible expression vector,
pMDC7 (B), was also digested with restriction enzymes, Xba I and Spe I, and blunted.
The regions of an RNAi construct having an inverted repeat of tAPX and GUS were
ligated to the blunted pMDC7.
A
B
XVE
tAPX
tAPX
RNAitrigger
Actin8
Actin8
C
0
12
16
20
24 (h)
tAPX
RNAitrigger
Actin8
Maruta et al., Supplemental Fig. S2.
Association between expression of tAPX and dsRNA in the IS-tAPX-19 plants
during treatment with estrogen. (A) IS-GUS-2, IS-tAPX-2, and IS-tAPX-19 plants
(T2 generation) were grown under normal light for 17 days. The transcript levels of
tAPX and XVE were determined by sem-quantitative RT-PCR. (B) Seventeen-day-old
IS-GUS-2, IS-tAPX-2, and IS-tAPX-19 plants were sprayed with a 100 µM estrogen.
At 24 h after the estrogen treatment, the expression of tAPX and RNAi-triggers was
determined by semi-quantitative RT-PCR. The expression of RNAi-triggers was
indicated using a primer set for the intron region of RNAi-triggers (Supplemental Fig.
S1C). Primer sequences were as follows; intron-F (5’-GGTGAGATCTTACTTCTCCT3’), intron-R (5’-CGAAACTATTTCGCCGAATC-3’). (C) Seventeen-day-old IS-tAPX-19
plants were sprayed with a 100 µM estrogen. A time-course analysis of the expression
of tAPX and RNAi-triggers was performed.
Relative mRNA level
3
3
2
1
*
0
8
3
ACS7
*
*
*
5
*
0
PDIL1-1
*
2
*
1
0
ANAC074
*
4
2
hydrolase
10
UP
6
*
0
3
*
2
*
1
0
F-box
*
IS-GUS-2-17
2
1
Relative mRNA level
Relative mRNA level
10
*
4
15
Relative mRNA level
Relative mRNA level
7
6
5
4
3
2
1
0
AtbZIP65
Relative mRNA level
Relative mRNA level
5
IS-tAPX-19-23
*
wild type
KO-tAPX
0
Maruta et al., Supplemental Fig. S3.
The transcript levels of RTS genes in the estrogen-treated IS-tAPX-19-23 or the
KO-tAPX plants. (A) Seventeen-day-old IS-GUS-2-17 and IS-tAPX-19-23 plants
were sprayed with a 100 µM estrogen, and kept under normal light. At 48 h after the
estrogen treatment, the transcript levels of RTS genes (AtbZIP65, ACS7, UP, F-box,
hydrolase, PDIL1-1, and ANAC074) were measured by q-PCR. Error bars indicate SD
(n = 3). Significant differences: *P < 0.05 vs. the value for IS-GUS-2-17 plants. (B)
The transcript levels of RTS genes in 17-day-old wild-type and KO-tAPX plants were
measured by q-PCR. Error bars indicate SD (n = 3). Significant differences: *P < 0.05
vs. the value for wild-type plants.
Relative
mRNA level
6
At3g08970
At2g29490
At2g29470
At2g29460
At1g17170
At2g43510
At1g10585
At3g53230
At4g37370
At3g49620
At4g37990
At2g41380
At4g22530
At3g54150
At1g26420
At1g26380
At3g26830
At3g28210
At1g57630
At4g01870
At3g11340
At2g43820
At1g22400
At1g19020
At1g05340
At2g21640
At3g09350
At1g13340
At4g39670
At1g62300
At5g13080
At2g32190
4
2
0
Relative
mRNA level
6
4
2
0
Relative
mRNA level
6
4
2
0
Relative
mRNA level
6
4
2
0
IS-GUS-2-17
IS-tAPX-19-23
Maruta et al., Supplemental Fig. S4.
The transcript levels of general oxidative stress response markers in the
estrogen-treated IS-tAPX-19-23 plants. Seventeen-day-old IS-GUS-2-17 and IStAPX-19-23 plants were sprayed with a 100 µM estrogen, and kept under normal light.
At 48 h after the estrogen treatment, the transcript levels of “general oxidative stress
response markers” were measured by q-PCR. Error bars indicate SD (n = 3).
IS-GUS-2-17
IS-tAPX-19-23
Mock
Estrogen
Treatment
Maruta et al., Supplemental Fig. S5.
Effect of lack of tAPX on cold acclimation under LL. Seventeen-day-old IS-GUS2-17 and IS-tAPX-19-23 plants were sprayed with a 100 µM estrogen solution or
water (mock), and transferred to cold stress conditions (10 µmol photons m-2 s-1,
4°C) for 2 weeks. The treatment with estrogen was performed every 3 days in order
to maintain the tAPX silencing. At 14 days after cold stress, the IS-GUS-2-17 and IStAPX-19-23 plants were photographed. The same results were obtained in three
independent experiments.
A
CBF1/
DREB1B
CBF2
DREB1C
COR6.6
COR15B
COR414TM1
COR414TM2
Relative mRNA level
1.5
1.0
*
*
0.5
0
wild type
wild type
KO-tAPX
C
0.6
0.4
Fv/Fm
B
KO-tAPX
0.2
0
Maruta et al., Supplemental Fig. S6.
Effect of lack of tAPX on cold acclimation. (A) The wild-type and KO-tAPX plants
were grown under light for 17 days. The transcript levels of RTS genes
(CBF1/DREB1B, CBF2/DREB1C, COR6.6, COR15B, COR414-TM1, and COR414TM2) were measured by q-PCR. Error bars indicate SD (n = 3). Significant
differences: *P < 0.05 vs. the value for wild-type plants. (B) Seventeen-day-old wildtype and KO-tAPX plants were transferred to cold stress conditions (100 µmol
photons m-2 s-1, 4°C). At 2 weeks after cold stress, the wild-type and KO-tAPX plants
were photographed. (C) Fv/Fm values in the leaves of wild-type and KO-tAPX at 2
weeks after cold stress were measured using a Closed FluorCam 800MF. Error bars
indicate SD (n = 3).
ICS2
Relative mRNA level
1.5
TIR domain TolB-related
RLP7
protein
protein
RLP23
RLP34
1.0
0.5
*
*
*
*
*
0
RLP39
RLP41
NIMIN-3
AtNUDX6
LCR68
Relative mRNA level
1.5
LCR70
*
1.0
*
0.5
*
*
*
0
wild type
KO-tAPX
Maruta et al., Supplemental Fig. S7.
Effect of lack of tAPX on the transcript levels of RTS genes involved in disease
resistance. The wild-type and KO-tAPX plants were grown under normal light for 17
days. The transcript levels of RTS genes (ICS2, TolB, TIR, RLP7, RLP23, RLP34,
RLP39, RLP41, NIMIN3, NUDX6, LCR68, and LCR70) were measured by q-PCR.
Error bars indicate SD (n = 3). Significant differences: *P < 0.05 vs. the value for wildtype plants.
1.0
4
0.8
0.6
0.4
0.2
0
Total SA (µg g-1 FW)
Free SA (µg g-1 FW)
wild type
3
KO-tAPX
2
1
0
Maruta et al., Supplemental Fig. S8.
Effect of lack of tAPX on the levels of SA. The wild-type and KO-tAPX plants were
grown under normal light for 17 days. The levels of free and total SA in the wild-type
and KO-tAPX plants were measured as described under ‘‘Experimental Procedures.’’
A
IS-GUS-2-17
IS-tAPX-19-23
1.0
0.8
Fv/Fm
Mock
0.6
0.4
IS-GUS-2-17
Estrogen
Treatment
0.2
0
IS-tAPX-19-23
0
24
48
72 (h)
Maruta et al., Supplemental Fig. S9.
Effect of tAPX silencing on HL sensitivity. Seventeen-day-old IS-GUS-2-17 and IStAPX-19-23 plants were sprayed with a 100 µM estrogen solution or water (mock),
and kept under NL. At 48 h after the treatments, IS-GUS-2-17 and IS-tAPX-19-23
plants were exposed to HL (1000 µmol photons m-2 s-1). (A) At 72 h after HL, the ISGUS-2-17 and IS-tAPX-19-23 plants were photographed. The same results were
obtained in three independent experiments. (B) Fv/Fm values in the leaves of IS-GUS2-17 and IS-tAPX-19-23 were measured using a Closed FluorCam 800MF. Error bars
indicate SD (n = 5).