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Supporting Information Notes S1 and Figs S1–S3
Jasmonate and ethylene signaling mediate whitefly-induced interference with
indirect plant defense in Arabidopsis thaliana
Peng-Jun Zhang1,2, Colette Broekgaarden3, Si-Jun Zheng1, Tjeerd A. L. Snoeren1,
Joop J. A. van Loon1, Rieta Gols1, Marcel Dicke1
1
Laboratory of Entomology, Wageningen University, PO. Box 8031, 6700 EH
Wageningen, the Netherlands; 2Institute of Plant Protection and Microbiology,
Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; 3Wageningen
UR Plant Breeding, Wageningen University and Research Centre, PO Box 386, 6700
AJ Wageningen, the Netherlands
Notes S1 Additional details of plant treatments, qRT-PCR, and statistical analysis
Materials and Methods
Plant Treatments
6-8 week-old Arabidopsis thaliana plants from the three accessions Col-0, dde2-2,
and ein2-1, were subjected to the following treatments:
1) Caterpillar treatment: Five second-instar larvae were transferred to the plant, and
allowed to feed on the plant for 48 h. After this period, the amount of feeding damage
was measured by counting the number of removed mm2 using mm paper.
2) Whitefly treatment: Fifty adult whiteflies were introduced onto the plant in an
approximately 1:1 male-to-female sex ratio. The whiteflies were allowed to feed and
oviposit on the plant for 48 h. Thereafter, the number of eggs laid by whitefly females
was recorded.
3) Caterpillar plus whitefly treatment: Five second-instar larvae were transferred to a
plant that was placed into a cage (21.0 cm high, 13.5 cm diam). Immediately after
introducing the caterpillars, 50 adult whiteflies were introduced into the cage. The
caterpillars and whiteflies were allowed to feed on the plant for 48 h. Thereafter, the
leaf damage caused by larvae and the number of eggs laid by whiteflies was recorded,
respectively.
Nine or ten replicate plants were used in each treatment. The plants were maintained
individually in small plastic cylinders (21.0 cm high, 13.5 cm diam.) and covered with
a meshed lid.
Quantitative Real-Time PCR. To validate the results from the microarray
experiments, the expression values of four selected genes were determined using
quantitative RT-PCR (qRT-PCR) on the same RNA pools. One microgram of total
RNA was treated with DNaseI (Invitrogen) according to the manufacturer’s
instructions and subsequently converted into cDNA using the iScripts cDNA synthesis
kit (Bio-Rad). Gene-specific primers for At4g31720 and At1g22410 were obtained
from literature (Czechowski et al., 2004; Ajjawi et al., 2010). All other gene-specific
primers were designed with the Beacon Designer software (Premier Biosoft
International, USA) set to an annealing temperature of 56°C. The primer sequences
are shown in Table S3. Primers were tested for their efficiency and gene specificity by
performing dilution series and melt curve analyses respectively. Quantitative RT-PCR
analysis was performed with a Rotor-Gene 6000 machine with a 72-well rotor
(Corbett Research, Australia) using SYBR Green to monitor dsDNA synthesis. Each
amplification reaction contained 12.5 μl ABsolute
TM
QPCR SYBR® Green Mix
(ABgene®, UK), 10 ng cDNA, and 400 nM of each gene-specific primer in a final
volume of 25 μl. All reactions were performed in duplicate and average values were
used in the analyses. The PCR program was used as described by Zheng et al. (2007).
Threshold cycle (Ct) values were normalized for differences in cDNA synthesis by
subtracting
the
Ct
value
of
the
constitutively expressed
gene
GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) (GenBank accession M64116) from the
Ct value of the gene of interest. Normalized gene expression was then calculated as
2-∆Ct and subsequently log2 transformed.
Statistics
The leaf damage caused by P. xylostella was compared between dde2-2 and Col-0,
and between ein2-1 and Col-0, respectively. Leaf damage of the plants was analyzed
by a two-factor ANOVA (two plant genotypes  two levels of whitefly treatment).
Similarly, the numbers of eggs laid by B. tabaci were compared between dde2-2 and
Col-0, and between ein2-1 and Col-0, respectively. Egg counts were analyzed by a
two-factor ANOVA (two plant genotypes  two levels of caterpillar treatment).
Means were compared pairwise using one-way ANOVA. To meet assumptions of
normality and homoscedasticity, egg counts were log-transformed before they were
subjected to the analysis.
Results and Discussion
Infestation intensity of P. xylostella. In the dde2-2 vs. Col-0 comparison, neither
plant genotype nor whitefly presence affected leaf damage caused by P. xylostella
(plant: F1,32 = 1.41, P = 0.24; whitefly: F1,32 = 1.1, P = 0.30); also the interaction of
plant  whitefly was not statistically significant (F1,32 = 0.27, P = 0.61; Fig. S1A).
Similarly, in the ein2-1 vs. Col-0 comparison, neither plant genotype nor whitefly
presence affected the leaf damage caused by P. xylostella (plant: F1,36 = 2.27, P = 0.14;
whitefly: F1,36 = 0.01, P = 0.94); also, the interaction of plant  whitefly was not
statistically significant (F1,36 = 0.50, P = 0.49; Fig. S1B). These data indicate that both
reduced JA or ET response and whitefly infestation did not interfere with the
infestation intensity of P. xylostella.
Infestation intensity of B. tabaci. To determine the effect of caterpillar feeding on
the feeding by B. tabaci, we assessed oviposition rate of B. tabaci as a proxy of
feeding rate under different treatments. In the dde2-2 vs. Col-0 comparison, neither
plant genotype nor caterpillar presence affected the number of eggs laid by B. tabaci
(plant: F1,32 = 2.04, P = 0.16; whitefly: F1,32 = 0.06, P = 0.80); also the interaction
plant  caterpillar was not statistically significant (F1,32 = 0.02, P = 0.90; Fig. S2A).
Similarly, in the ein2-1 vs. Col-0 comparison, neither plant genotype nor caterpillar
presence affected the number of eggs laid by B. tabaci (plant: F1,36 = 2.41, P = 0.13;
whitefly: F1,36 = 0.43, P = 0.52); also the interaction of plant  caterpillar was not
statistically significant (F1,36 = 1.35, P = 0.25; Fig. S2B). These data indicate that both
reduced JA or ET response and caterpillar infestation did not interfere with the
infestation intensity of B. tabaci.
Comparison of microarray and qRT-PCR analysis of four genes. To validate the
microarray data, we selected four genes that showed significantly different expression
levels between plants infested by P. xylostella and plants infested by P. xylostella and
B. tabaci, to be analyzed with qRT-PCR. The qRT-PCR and microarray analyses
showed similar expression patterns among the four treatments (Fig. S3), indicating the
reliability of the microarray data.
References
Ajjawi I, Lu Y, Savage LJ, Bell SM, Last RL. 2010. Large-scale reverse genetics in
Arabidopsis: Case studies from the chloroplast 2010 project. Plant Physiology
152: 529-540.
Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK. 2004. Real-time
RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented
sensitivity reveals novel root- and shoot-specific genes. The Plant Journal 38:
366-379.
Zheng SJ, Van Dijk JP, Bruinsma M, Dicke M. 2007 Sensitivity and speed of
induced defense of cabbage (Brassica oleracea L.): Dynamics of BoLOX
expression patterns during insect and pathogen attack. Molecular Plant-Microbe
Interactions 20: 1332-1345.
DBM+WF
DBM
Leaf damage (mm2)
60
A
60
n.s.
50
40
n.s.
B
n.s.
50
n.s.
40
30
30
20
20
10
10
0
0
Col-0
dde2-2
Col-0
ein2-1
Fig. S1 Amount of leaf damage (mm2) caused by P. xylostella on Arabidopsis. (A)
Effect of B. tabaci infestation on the amount of leaf damage caused by P. xylostella on
Col-0 vs. dde2-2 plants; (B) Effect of B. tabaci infestation on the amount of leaf
damage caused by P. xylostella on Col-0 vs. ein2-1 plants. Values are shown as the
mean ( SE) of nine biological replicates. “n.s.” represents non-significant differences
from control plants as determined by one-way ANOVA. [DBM = P. xylostella;
DBM+WF = P. xylostella and B. tabaci].
Number of eggs laid by WF
WF
140
DBM+WF
80
A
120
100
80
n.s.
B
70
60
n.s.
50
n.s.
n.s.
40
60
30
40
20
20
10
0
Col-0
dde2-2
0
Col-0
ein2-1
Fig. S2 The mean number of eggs laid by B. tabaci females on Arabidopsis. (A)
Effect of P. xylostella infestation on the mean number of eggs laid by fifty adult B.
tabaci on Col-0 vs. dde2-2 plants; (B) Effect of P. xylostella infestation on the mean
number of eggs laid by fifty adult B. tabaci on Col-0 vs. ein2-1 plants. Values are
shown as the mean ( SE) of ten biological replicates. “n.s.” represents
non-significant differences from control plants as determined by one-way ANOVA.
[WF = B. tabaci; DBM+WF = P. xylostella and B. tabaci].
Microarray
8.0
qRT-PCR
At4g31720
0.04
At4g31720
0.03
7.5
0.02
7.0
0.01
0
6.5
Relative transcript levels
8.5
0.03
At5g50850
At5g50850
8.0
0.02
7.5
0.01
7.0
0
6.5
9.0
At1g22410
0.03
At1g22410
8.5
0.02
8.0
7.5
0.01
7.0
0
6.5
12.5
0.04
At4g25080
11.5
At4g25080
0.03
10.5
9.5
0.02
8.5
0.01
7.5
0
6.5
Control
WF
DBM
DBM+WF
Control
WF
DBM
DBM+WF
Fig. S3 Comparison of microarray and qRT-PCR analysis of four genes. The relative
transcript levels of the selected four genes (At4g31720, At5g50850, At1g22410,
At4g25080) in plants after infestation by B. tabaci (WF), P. xylostella (DBM), or both
(WF+DBM), and control plants (Control). The transcript levels of these four genes
have been normalized to the amount of GAPDH transcripts in each sample. On top of
all bars the standard error of the mean is indicated.