Plants: Three tomato (Solanum lycopersicum) genotypes were used: 35S: prosystemin plants that overexpress JA (35S), spr2 plants that underexpress JA (spr2), and wild-type plants with normal JA levels (WT) (34, 35). The 35S, spr2, and WT plants are all derived from the same parent genotype of tomato cv Castlemart. Tomato seedlings were grown in individual 1.5 L pots filled with potting mix (peat moss, vermiculite, organic fertilizer, and perlite in a 10:10:10:1 ratio by volume) in a glasshouse under natural light at 25-28 °C and 50-70% relative humidity.
TYLCV-infected plants were obtained by agro-inoculation of seedlings at the three true-leaf stage with a TYLCV genome (GenBank accession number: AM282874) originally isolated from tomato plants in Shanghai, China (36, 37). Infected plants developed characteristic leaf-curl symptoms; infection was confirmed by PCR with the primer set TYLCV-61 and -473 (12).
Insects: Virus-free MED were originally collected in 2009 from Euphorbia pulcherrima growing near Beijing, China; they have since been maintained on virus-free S. lycopersicum within screen-mesh cages (0.6 × 0.6 × 0.6 m) in a greenhouse. Viruliferous MED were produced by confining 300 virus-free adults in cages with TYLCV-infected tomato plants. Viruliferous MED was confirmed by PCR with the primer set TYLCV-61 and -473 (12). Virus-free MED were obtained by confining 300 virus-free adults in cages with virus-free tomato plants. Both colonies were maintained for more than six generations in separate greenhouses under natural light at 25-28 °C and 50-70% RH. We confirmed that both MED colonies only contained MED by monitoring the mitochondrial cytochrome oxidase I (mtCOI) gene in 20 adults per generation (38).
Experiment I: Impact of JA levels on virus-free and viruliferous MED
We assessed the preference and performance of virus-free and viruliferous MED on plant genotypes differing in JA expression: JA-deficient spr2, control WT, and overexpressing 35S.
MED preference: We conducted paired-choice experiments assessing whether host preference of virus-free and viruliferous MED was affected by plant genotype. Plants at the 6-7 true leaf stage from each of the three tomato genotypes were placed in whitefly-proof screen cages (80 × 40 × 60 cm), with two plants of different genotypes per cage. The two plants were placed in opposite corners of the cage, and at least 100 (mean 100.7 + 0.35 [SE] MED per cage across all replicates) virus-free or viruliferous MED that were the same age and had been starved for 24 h were released in the center. After one day, we covered each plant with transparent plastic wrap to prevent whiteflies from relocating and counted the number of MED per plant. For all three plant genotypes, each treatment (= two-plant combination) was replicated (= single cage) nine times.
MED performance: We assessed nymphal survival (emerged adults/total eggs) and development time (days from egg to adult) by confining 20 adult MED (1:1 sex ratio) in a clip cage (30 mm in diameter; 20 mm in height) attached to the 3rd-6th true leaf from the top of a tomato seedling, one cage per plant, for 24 hours (19, 39). Each plant was used only once, and each treatment was replicated 30 times (30 virus-free and 30 viruliferous MED replicates) per genotype. After 24 hours, we removed the adults and used a stereomicroscope (Leica, M205C) to count egg production. On day 16, the first adult emerged; from that day onward, we collected emerging adults from each clip cage twice per day until all MED had matured. After all MED had matured, each whitefly-infested leaf was collected for quantification of TYLCV load using ELISA (40). Each treatment was replicated 30 times (30 virus-free and 30 viruliferous MED replicates) per genotype.
We assessed adult fecundity and longevity by transferring one newly-emerged virus-free or viruliferous female to a clip cage attached to the 3rd-6th true leaf from the top of a tomato seedling. Each plant was used only once, and each treatment was replicated 30 times (30 virus-free and 30 viruliferous MED replicates) per genotype. We took daily data on adult longevity and used a stereomicroscope to assess weekly egg production.
Experiment II: Impact of plant genotype and TYLCV infection on expression of JA-related genes and JA levels
We quantified JA levels in plants (6-7 true leaf stage) from each of the three tomato genotypes. After we attached individual clip cages to six leaves per plant, all six cages per plant received one of the three following treatments: control (no MED in any of the cages), virus-free MED (50 virus-free adult MED per cage), or viruliferous MED (50 viruliferous adult MED per cage). The number of MED per plant in this experiment (50) was identified using a pilot experiment as the minimum number of MED necessary to affect JA levels in a 24-h period. Clip cages and whiteflies were removed after 24 h and the six leaves per plant collected. Plants treated with viruliferous insects were confirmed to be infected by PCR with the primer set TYLCV-61 and -473 (12). JA levels in each leaf were quantified using a gas chromatography-mass spectrometry system (Agilent Technologies, Santa Clara, CA) (41) and TYLCV loads were quantified using ELISA (40). This protocol used 162 (three infestation treatments × three genotypes × nine replicates × two determination category for JA level and TYLCV titer) plants.
We used the same protocol, but with three plants per treatment, to measure the expression of four JA-related genes: lipoxygenase (LOX), 12-oxophytodienoate reductase 3 (OPR3), proteinase inhibitor II (PI II), and JA-amino acid synthetase 1 (JAR1). Both LOX and OPR3 are involved in JA biosynthesis, with LOX controlling the initial oxygenation of α-linolenic acid, a fatty acid substrate (42), and OPR3 catalyzing the reduction of the resulting 12-oxo-phytodienoic acid (OPDA; 43). Following biosynthesis, the presence of JA increases JAR1 expression as well as production of proteinase inhibitors via up regulation of the PI II gene (43). Actin (ACT) and ubiquitin 3 (UBI) (44) were used as reference genes (Table S1 in Supporting Information). Total RNA was extracted from 0.2 g of leaf tissue using an RNA extraction kit (Tiangen Biotech, Beijing, China), and 1.0 μg of RNA was used to synthesize the first-strand cDNA using the PrimeScript® RT reagent kit (Takara Bio, Tokyo, Japan) with gDNA Eraser (Perfect Real Time, TaKara, Shiga, Japan). The 25.0 μl reaction system contained 10.5 μl of ddH2O, 1.0 μl of cDNA, 12.5 μl of SYBR® Green PCR Master Mix (Tiangen Biotech, Beijing, China), and 0.5 μl of each primer. Relative RNA quantities were calculated using the comparative cycle threshold (2-ΔΔCt) method (45). Each treatment had 12 replicates (= 3 plants × 4 technical replicates) and used a minimum of four leaves per plant.
Experiment III: Impact of plant genotype on TYLCV titer and JA levels following infestation with viruliferous MED
A single clip cage containing five viruliferous MED was placed on a plant at the three-true-leaf stage. Twelve healthy plants from each of the three tomato genotypes were exposed. The clip cages and viruliferous whiteflies were removed after two days of exposure, and the plants kept individually in insect-proof cages. The number of MED per plant in this experiment (5) reflects prior work assessing the number of MED necessary to reliably transfer TYLCV to plants. After 5 d, the first true leaf from each of the three plants per genotype was collected for quantification of virus titer using TAS-ELISA (40); this procedure was repeated at day 10, 20 and 30. Each plant was only sampled a single time, and was discarded after the leaf was removed. This protocol used 36 (three genotypes × three replicates × four time points) plants.
JA levels in plants were sampled one day before infestation and one day after infestation with viruliferous MED. Three leaves were sampled per plant for both pre- and post-infestation analyses, and JA concentrations were determined using a GC-MS as described in Experiment II. Each plant was used only once. This protocol used 18 (three genotypes × three replicates × two sampling dates) plants.
Experiment IV: Impact of infestation by virus-free and viruliferous MED on volatile emissions from, and whitefly preference for, different plant genotypes
Plant volatile emissions: Plants from each of the three tomato genotypes (spr2, WT, and 35S) were exposed to one of three infestation treatments (clip cages with no whiteflies [=control], virus-free whiteflies, or viruliferous whiteflies) following the protocol detailed in experiment II. After two days, the clip cages and insects were removed from each plant and plant volatiles were collected using a slightly-modified version of the headspace collection system (6). Plant volatiles were collected for 6 h under continuous light, after which the whole plants were weighed (fresh weight, FW) to determine volatile quantity expressed per g FW. This protocol used 81 (three treatments × three genotypes × nine replicates) plants. TYLCV loads in leaves infested by virus-free and viruliferous whiteflies for each of the three plant genotypes was quantified using ELISA (40). This protocol used 81 (three treatments × three genotypes × nine replicates) plants.
We dissolved headspace samples in n-hexane, added 0.2 μg ml-1 of n-dodecane to the solvent as an internal standard, then subjected a 1 μl sample to the HP-5MS column (60 m long, 0.25 mm diameter and 0.25 μm film thickness, Agilent Technologies, Santa Clara, CA) of gas chromatography–mass spectrometry. The true standards of the detected volatiles were also injected in different concentrations ranging from 0.2 to 50 μg ml-1 hexane. Standard compounds were purchased from Beijing Huaerbo Technology Co., Ltd. The temperature profile was as follows: 50 oC for one min; 50 oC to 240 oC at 5 oC min-1; 240 oC for two min; 240 oC to 300 oC at 30 oC min-1; 300 oC for five min. The injection temperature was 270 oC, the source temperature was 200 oC, and the interface temperature was 280 oC. The column effluent was ionized by electron impact ionization (70 eV). Compounds were verified in the National Institute of Standards and Technology (NIST) database and mass spectra of the (co-) injected standards. Then compounds were quantified based on concentrations of true standards. All volatiles were analyzed separately.
MED preference: We conducted a series of paired-choice experiments as per the protocols described in experiment I. Briefly, plants from each of the three genotypes were exposed to one of two infestation treatments (clip cages with either virus-free or viruliferous whiteflies) following the protocol detailed above. After two days, clip cages were removed and plants of different genotypes were placed in opposite corners of a screen cage. Each treatment (= two plants of different genotypes) was replicated nine times for 27 (= three two-genotype combinations × nine plants per combination) replicates in each paired-choice experiment. We conducted the following four paired-choice experiments:
A. Virus-free MED choosing between plants of different genotypes that had both previously been fed upon by virus-free MED.
B. Viruliferous MED choosing between plants of different genotypes that had both previously been fed upon by virus-free MED.
C. Virus-free MED choosing between plants of different genotypes that had both previously been fed upon by viruliferous MED.
D. Viruliferous MED choosing between plants of different genotypes that had both previously been fed upon by viruliferous MED.
For the paired-choice experiments, we used t-tests to assess whether virus-free or viruliferous whiteflies exhibited a preference for one plant genotype over another. For the performance experiment, we used two-way ANOVAs to assess the effect of MED infection status (virus-free, viruliferous), plant genotype (JA-deficient spr2, normal WT, overexpressing 35S), and their interaction on whitefly life history parameters (development time, % survival to adulthood, longevity, and egg production) and whitefly preference. Data on survival from egg to adulthood was arcsine transformed prior to analysis to improve normality and homogeneity of variance.
Two-way ANOVAs were also used to compare the impact of MED infection status and plant genotype on endogenous JA levels and gene expression; gene expression data was square-root transformed prior to analysis where necessary.
We used repeated-measures ANOVA to assess whether plant genotypes differed in their virus titer following infestation with TYLCV, and whether plants differed in their pre- and post-infestation JA levels; for the time and genotype*time interactions, we report univariate unadjusted Epsilon F values. In cases where ANOVA revealed a significant main effect, Tukeys’ HSD (α = 0.05) was used to compare treatment means.
We analyzed the data on plant volatile emissions using one-way ANOVA to assess the effect of prior MED infestation (none, virus-free MED, viruliferous MED) on the concentrations of each volatile compound. In cases where there was a significant effect of MED infestation, Tukeys’ HSD (α = 0.05) was used to differentiate treatments. JMP 9.0.0 (SAS Institute, Durham NC) was used for all analyses.