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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.