The experiment was carried out in eight open-top field chambers (OTCs). Four of the OTCs were continuously maintained at the current ambient level of CO₂ (about 400 ppm), and four were continuously maintained at an elevated level of CO₂ (about 750 ppm, the predicted level by the end of this century) (IPCC, 2013). Details of the automatic control system for CO₂ concentrations and OTCs are provided in Chen et al. (2005). Air temperature was measured three times daily (8:00, 14:00, and 18:00) throughout the experiment and did not differ significantly between the two sets of OTCs during the experiment.

Two near-isogenic tomato (Solanum lycopersicum) lines, the susceptible cultivar Moneymaker and the resistant cultivar Motelle (Mi-1.2), were used in our experiments. Motelle carries a 650-kb segment of S. peruvianum DNA that harbors the Mi-1.2 gene, which makes it genetically distinct from Moneymaker (Milligan et al., 1998). These lines were selected for study due to whitefly resistance (Nombela et al., 2000). Seeds of Moneymaker and Mi-1.2 (Motelle) plants were obtained from the National Engineer and Research Center for Vegetable, Academy of Agricultural and Forestry Sciences, Beijing, China. One week after germination, when the cotyledons were beginning to expand, the seedlings were transplanted singly into plastic pots (25 cm × 21 cm × 22 cm) containing sterilized loamy field soil (organic carbon 75 g/kg; available N 500 mg/kg; available P 200 mg/kg; available K 300 mg/kg). The pots were placed in ventilated insect-proof cages in octagonal OTCs until they grew to the 3- to 4-leaf stage. Pot placement was re-randomized within each OTC once each week. No chemical fertilizers and insecticides were used. Water was added to each pot every 2 days. Five groups of plants were used for the experiments described in the following sections (Supplementary Figure S1).

Six undamaged 8-week-old plants of each genotype in each OTC (=24 plants per treatment and 96 plants in total) were selected for measurement of photosynthetic rate and plant growth traits. The net photosynthetic rate was determined according to Guo et al. (2012) with some modification. The net photosynthetic rate of each plant was measured with a Li-Cor 6400 gas exchange system (Li-Cor Inc., Lincoln, NE, USA). The fourth mature leaf from the base of the stem was selected for measurement. All measurements were done between 9:00 and 12:00 am. The CO₂ concentration of the incoming air was adjusted to 400 μmol mol^-1 CO₂ or 750 μmol mol^-1. Relative humidity corresponded to ambient conditions. Before gas exchange was measured, photosynthetic active radiation for the leaf in the measuring cuvette was increased in steps to 1200 μmol m^-2 s^-1. When the CO₂ assimilation rate was stable for at least 2 min, measurements were recorded. After that, the plants were harvested for measurement of biomass, stem diameter, and height.

The plant–virus interactions could be affected by both plant physiology and vector transmission ability, thus, in current study, we determined the effects of elevated CO₂ on the disease incidence and index of TYLCV by either agroinoculation or transmitted by whitefly. For agroinoculation of TYLCV, 25 8-week-old plants of each genotype in each OTC (25 plants × 4 OTC × 2 genotypes × 2 CO₂ levels and 400 plants in total) were selected and agroinoculated as described previously (Huang et al., 2012). The TYLCV infection of tomato plants was achieved using Agrobacterium tumefaciens-mediated infectious inoculation (Zhang et al., 2009; Al Abdallat et al., 2010), and the infectious 2 clone (pBINPLUS-SH2-1.4A) of TYLCV- Israel [China: Shangai2] was constructed into A. tumefaciens strain EHA105 as described previously (Zhang et al., 2009). The infectious clone of TYLCV was provided by Professor Xueping Zhou (State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China). The culture of TYLCV clone was grown in LB culture medium with kanamycin (50 μg/ml) and rifampicin (50 μg/ml) at 28°C (250 rpm) for 24 h (OD₆₀₀ = 1.5). The bacteria culture was centrifuged for 10 min at 2500 g and resuspended with 50 ml buffer (10 mM MgC1₂, 10 mM 2-(N-morpholino) ethanesulfonic acid, 200 μM acetosyringone) after which 0.2 ml of the culture was injected three times into the phloem (about 1 mm in depth) of the tomato stem at the three to four leaf stage to achieve inoculation; a sterile syringe (1 ml) with a beveled needle (0.5 mm × 20 mm) was used for injection. Inoculated plants were grown in ventilated cages in the OTCs. The incidence of TYLCV infection (percentage of plants with disease symptoms) and the disease index were determined 6 weeks after agroinoculation. Disease index values were determined as follows (Curvers et al., 2010):

where Ni represents the number of plants in disease symptom ranking i, Ri represents the disease symptom rank (i = 0–4), N represents the total number of plants investigated, and Rh represents the highest disease symptom rank. Disease symptoms were ranked mainly according to Friedmann et al. (1998): 0 = no visible symptoms: inoculated plants show the same growth and development as non-inoculated plants; 1 = very slight yellowing of apical leaf margins; 2 = some yellowing and minor curling of leaf ends; 3 = widespread leaf yellowing, curling, and cupping, with some reduction in size, but plants continue to develop; 4 = severe plant stunting and yellowing, and pronounced cupping and curling of leaves; plants stop growing.

Bemisia tabaci of the B biotype (Middle East Asia Minor 1, aka MEAM 1), which were kindly provided by Professor Youjun Zhang of the Institute of Vegetable and Flower, Chinese Academy of Agricultural Sciences, were reared on cabbage (non-host of TYLCV) grown in insect-proof wooden cages as previously described (Jiu et al., 2007). Viruliferous whiteflies were caged on the TYLCV-infected tomato plants in a separate greenhouse. Whiteflies from the viruliferous colony were confirmed to be infected with TYLCV prior to infestation by PCR analysis (Zhang et al., 2009). For transmission of TYLCV to tomato plants by B. tabaci, 60 8-week-old plants of each genotype in each OTC were randomly selected, and each of 20 plants was infested by 5, 15, or 25 viruliferous B. tabaci for 48 h (20 plants × 4 OTC × 2 genotypes × 2 CO₂ levels × 3 whiteflies densities and 960 plants in total). The virus incidence and disease index of the tomato plants were determined 6 weeks after B. tabaci infestation.

To determine the effect of TYLCV infection on B. tabaci numbers and fecundity on tomato, 16 5-week-old plants of each genotype in each OTC were randomly selected. Eight plants were agroinoculated with TYLCV, and the other eight were not. Three weeks later, we checked the TYLCV copy numbers of the new emerged leaf by qPCR and confirmed that they are all successfully infected by TYLCV. Then, 4 8-week plants from each tomato genotype and TYLCV treatment per OTC (4 plants × 4 OTC × 2 genotypes × 2 CO₂ levels × 2 TYLCV treatment and 128 plants in total) were selected. Five newly emerged females and five newly emerged males were released onto each plant; each plant was kept in a separate whitefly proof, ventilated cage (120 mesh). After 28 days, the numbers of each developmental stage of B. tabaci were determined for each of the four replicates in each OTC.

To determine the effect of TYLCV infection of tomato on B. tabaci fecundity, 4 8-week plants from each tomato genotype and TYLCV treatment per OTC (4 plants × 4 OTC × 2 genotypes × 2 CO₂ levels × 2 TYLCV treatment and 128 plants in total) were randomly selected, one mated females were introduced into each plant with a whitefly proof, ventilated cage. The females were then transferred daily to fresh leaves until they died, and the number of eggs deposited by each female was determined.

Forty-eight 4-week-old tomato plants were agroinoculated with the virus. Once the plants exhibited obvious symptoms 4 weeks later and were confirmed as TYLCV infected by detecting the TYLCV copies with RT-PCR in the systemic leaves according to Zhang et al. (2009), we started to inoculate whiteflies. To determine the effects of plant genotype and CO₂ level on transmission of TYLCV by B. tabaci, 100 adult whiteflies were caged on the second true leaf (numbered from the apex down) of each TYLCV-infected tomato plants to obtain enough viruliferous whiteflies. After a 48-h acquisition access period, 20 viruliferous whiteflies were then caged on the second true leaf of each of four 5-week-old tomato plants (at the four-leaf stage) at three time points of each genotype in each OTC (4 plants × 4 OTC × 2 genotypes × 2 CO₂ levels × 3 three time points and 192 plants in total) (Rubinstein and Czosnek, 1997). The whiteflies were removed after 8, 24, and 48 h inoculation access period. Infection was assessed 4 weeks later based on the appearance of TYLCV symptoms and on the number of copies of TYLCV in the leaf tissue, which was determined according to Zhang et al. (2009).

To determine the effects of plant genotype and CO₂ level on the acquisition of TYLCV by B. tabaci, four six-leaf stage virus-infected tomato plants (9-week-old) of each genotype at each time point in each OTC were selected (4 plants × 4 OTC × 2 genotypes × 2 CO₂ levels × 3 three time points and 192 plants in total); the plants had been agroinoculated about 4 weeks earlier. Before releasing whiteflies, we confirmed as TYLCV infected by detecting the TYLCV copies with PCR in the systemic leaves according to Zhang et al. (2009). Fifty adult B. tabaci were caged on the second true leaf (numbered from the apex down). After acquisition access periods of 2, 8, and 24 h, ten B. tabaci were removed from each cage, and the TYLCV copy number in each group of ten B. tabaci was determined.

For measurement of the contents of the phytohormones JA and SA and the activities of the defensive enzymes phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) in tomato plants as affected by TYLCV and CO₂ level, four 5-week-old plants of each genotype in each OTC were agroinoculated with TYLCV; another four plants of each genotype in each OTC were not inoculated and served as controls. Four weeks later, 500 mg of leaves were collected from each plant. The leaf samples were immediately stored in liquid N until analyzed.

For measurement of expression of JA- and SA-dependent defense genes, 16 5-week-old plants of each genotype in each OTC were agroinoculated with TYLCV, and another 16 plants of each genotype in each OTC were not inoculated and served as controls. After 0, 2, 8, and 24 h, the leaves of four plants (±inoculation) of each genotype in each OTC were harvested. The leaf samples were immediately stored in liquid N until analyzed.

The contents of endogenous JA and SA in the plant leaves were measured as described by Sun et al. (2013). The activities of PAL and LOX were measured according to Guo et al. (2012).

For real-time quantitative PCR, each treatment sample had four technical replicates for each of the biological replications. The RNeasy Mini Kit (Qiagen, Valencia, CA, USA) was used to isolate total RNAs from tomato leaves (0.05 g from samples stored at -70°C), and about 2 μg quantities of the RNAs were used to generate the cDNAs with the QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA). The mRNA amounts of four target genes were quantified by real-time quantitative PCR: proteinase inhibitor (PI-1), lipoxygenase (LOX2), phenylalanine ammonia lyase (PAL5), and pathogenesis-related protein (PR1a). Specific primers for each gene were designed from the tomato EST sequences using PRIMER5 software (Supplementary Table S1). The PCR reactions were performed in a 20-μL total reaction volume including 10 μL of 2x SYBRs Premix EX TaqTM (Qiagen) master mix, 5 mM of each gene-specific primer, and 1 μL of cDNA template. PCR reactions were carried out on an Mx 3000P detection system (Stratagene, USA) as follows: 5 min at 95°C; then 40 cycles of 10 s at 95°C and 20 s at 62°C; and finally one cycle of 30 s at 95°C, 30 s at 55°C, and 30 s at 95°C. A standard curve was derived from the serial dilutions to quantify the copy numbers of target mRNAs. The relative level of each target gene was standardized by comparing the copy numbers of target mRNAs with the copy number of β-actin (Actin7) (the housekeeping gene; Zhai et al., 2013), which remains constant under different treatment conditions. The β-actin mRNAs of the control were examined in every plate of PCR to eliminate systematic error.

All data were checked for normality and equality of residual error variances and were appropriately transformed (log or square-root) if needed to satisfy the assumptions of analysis of variance. A split-split plot design was used to analyze the univariate responses of the phytohormone contents, enzyme activities, and gene expression in plants (ANOVA, PASW Statistics 18.0, SPSS Inc., Chicago, IL, USA). In the following ANOVA model, CO₂ and block (a pair of OTCs with ambient and elevated CO₂) were the main effects, tomato genotype was the subplot effect, and TYLCV infection level was the sub-subplot effect:

where C is the CO₂ treatment (i = 2), B is the block (j = 4), G is the tomato genotype (k = 2), and H is the virus infection treatment (l = 2). e_m(ijkl) represents the error because of the smaller scale differences between samples and variability within blocks (ANOVA, SAS Institute). Effects were considered significant if P < 0.05. Because the effect of block and the interactive effects of block and other factors were not significant (P > 0.45), the effect of block and its interaction with other factors are not presented to simplify the presentation. Tukey’s multiple range tests were used to separate means when ANOVAs were significant. For analysis of the plant growth traits (biomass, stem diameter, plant height, and photosynthetic rate), TYLCV incidence and index, and the ability of B. tabaci to acquire and transmit TYLCV under two CO₂ levels, a split-plot design was also applied, with CO₂ and block as the main effects and tomato genotype as the subplot effect.

Under ambient CO₂, growth and photosynthesis did not significantly differ between Moneymaker and Mi-1.2 plants except for the height (Figure 1; Supplementary Table S2). Elevated CO₂ increased biomass by 38.2%, height by 28.6%, and photosynthetic rate by 75.1% for Moneymaker plants, and increased biomass by 15.5%, height by 33.3%, and photosynthetic rate by 62.3 % for Mi-1.2 plants. Mi-1.2 plants had a lower biomass, a lower photosynthetic rate, and a greater height than Moneymaker plants under elevated CO₂ (Figures 1A,C,D).

For the plants that were agroinoculated with TYLCV, elevated CO₂ significantly decreased TYLCV disease incidence and index values for Moneymaker plants but increased those values for Mi-1.2 plants (Figure 2; Supplementary Table S3). For plants that were inoculated with TYLCV by B. tabaci, TYLCV incidence and index values increased as the number of B. tabaci added increased (Figure 3; Supplementary Table S4). Elevated CO₂ decreased the TYLCV incidence and disease index values for Moneymaker plants but increased those values for Mi-1.2 plants when infested by the same number of viruliferous B. tabaci (Figure 3).

Elevated CO₂ did not significantly affect the abundance or fecundity of B. tabaci on either healthy or virus-infected plants regardless of plant genotype (Figure 4; Supplementary Table S5). Fecundity was lower on healthy Mi-1.2 plants than on healthy Moneymaker plants under ambient CO₂. Under elevated CO₂, in contrast, neither B. tabaci fecundity nor abundance significantly differed between the two plant genotypes. B. tabaci abundance and fecundity were lower on TYLCV-infected plants than on healthy plants regardless of CO₂ level or plant genotype (Figure 4).

After whiteflies had fed on the TYLCV-infected plants for 2, 24, or 48 h, the number of TYLCV-DNA copies per B. tabaci was significantly lower under elevated CO₂ than under ambient CO₂ in the case of Moneymaker plants but the opposite was true in the case of Mi-1.2 plants (Figures 5A,B). Under ambient CO₂, B. tabaci contained fewer TYLCV-DNA copies when reared on TYLCV-infected Mi-1.2 plants than on TYLCV-infected Moneymaker plants (Figures 5A,B). Under elevated CO₂, B. tabaci contained a higher number of TYLCV-DNA copies when reared on TYLCV-infected Mi-1.2 plants than on TYLCV-infected Moneymaker plants (Figures 5A,B; Supplementary Table S6), which is consistent with the TYLCV disease incidence and index of both genotypes before whitefly acquired TYLCV from plants (Supplementary Figure S2).

After viruliferous B. tabaci had fed on plants for 24 h, numbers of TYLCV-DNA copies in Moneymaker plants were unaffected by CO₂ level but were higher in Mi-1.2 plants under elevated CO₂ than under ambient CO₂ (Figures 5C,D). After a 48 h transmission access period, Mi-1.2 plants contained fewer TYLCV-DNA copies than Moneymaker plants under ambient CO₂ but contained higher numbers of TYLCV-DNA copies under elevated CO₂ (Figures 5C,D).

In Moneymaker plants that were not infected by TYLCV, elevated CO₂ increased SA content and PAL activity but decreased JA content and LOX activity (Figure 6; Supplementary Table S7). Elevated CO₂ increased SA content and decreased JA content of Mi-1.2 plants (Figures 6A,B). After agroinoculation of TYLCV infection for 48 h, elevated CO₂ increased the SA and JA contents and PAL and LOX activities of Moneymaker plants. In contrast, elevated CO₂ decreased SA and PAL activity but increased JA content and LOX activity of Mi-1.2 plants (Figure 6). Under ambient CO₂, SA content and PAL activity were lower in infected Moneymaker plants than in infected Mi-1.2 plants. Under elevated CO₂, however, SA content and PAL activity were lower in the Mi-1.2 plants than in Moneymaker plants regardless of TYLCV infection.

From 8 to 48 h post-infection with TYLCV artificially, elevated CO₂ increased the expression of genes encoding PAL5 and PR1a involved in the SA-signaling pathway of Moneymaker plants but decreased their expression in Mi-1.2 plants (Figures 7A,B; Supplementary Table S8). The expression of genes encoding LOX2 and PI1-1 in the JA-signaling pathway, however, was not greatly affected by elevated CO₂ (Figures 7C,D; Supplementary Table S8). TYLCV infection tended to up-regulate the expression of genes encoding PAL5 and PR1a but to down-regulate the expression of LOX2 and PI1-1 regardless of plant genotype (Figure 7). Compared with Moneymaker plants, Mi-1.2 plants had a higher expression of genes encoding PAL5 and PR1a under ambient CO₂ but the reverse was true under elevated CO₂ (Figure 7). The expression pattern of genes involved in the SA- signaling pathway across the treatments suggested that the SA-signaling pathway is an important part of plant response to TYLCV infection.