Jasmonic acid is a key regulator of spider mite-induced volatile terpenoid and methyl salicylate emission in tomato

Abstract

The tomato (Lycopersicon esculentum) mutant def-1, which is deficient in induced jasmonic acid (JA) accumulation upon wounding or herbivory, was used to study the role of JA in the direct and indirect defense responses to phytophagous mites (Tetranychus urticae). In contrast to earlier reports, spider mites laid as many eggs and caused as much damage on def-1 as on wild-type plants, even though def-1 lacked induction of proteinase inhibitor activity. However, the hatching-rate of eggs on def-1 was significantly higher, suggesting that JA-dependent direct defenses enhanced egg mortality or increased the time needed for embryonic development. As to gene expression, def-1 had lower levels of JA-related transcripts but higher levels of salicylic acid (SA) related transcripts after 1 d of spider mite infestation. Furthermore, the indirect defense response was absent in def-1, since the five typical spider mite-induced tomato-volatiles (methyl salicylate [MeSA], 4,8,12-trimethyltrideca-1,3,7,11-tetraene [TMTT], linalool, trans-nerolidol, and trans-beta-ocimene) were not induced and the predatory mite Phytoseiulus persimilis did not discriminate between infested and uninfested def-1 tomatoes as it did with wild-type tomatoes. Similarly, the expression of the MeSA biosynthetic gene salicylic acid methyltransferase (SAMT) was induced by spider mites in wild type but not in def-1. Exogenous application of JA to def-1 induced the accumulation of SAMT and putative geranylgeranyl diphosphate synthase transcripts and restored MeSA- and TMTT-emission upon herbivory. JA is therefore necessary to induce the enzymatic conversion of SA into MeSA. We conclude that JA is essential for establishing the spider mite-induced indirect defense response in tomato.

Figure 1.

Performance of spider mites on wild-type (white bars) and def-1 (black bars) plants. Leaf area damaged by spider mites (A) was determined from five sets of plants infested with four adult female spider mites. There was no significant (NS) difference between the damage inflicted on wild-type leaves and def-1 leaves (ANOVA with df = 1; F = 0.22; P = 0.65). Spider mite fecundity (B) was determined from 12 sets of plants infested with seven adult female spider mites on one leaf per plant. Eggs were counted daily. The figure shows the average total egg production per mite in 5 d. There was no significant (NS) difference between the number of eggs produced on wild-type and def-1 (ANOVA with df = 1; F = 2.0; P = 0.17). The numbers of juveniles that emerged per egg (C) was calculated for days 5, 6, and 7. Egg hatching was not observed before day 5. The hatching rate on def-1 was significantly (S) higher than on the wild type with P = 0.014 (repeated measures ANOVA with df = 1; F = 7.4).

Figure 2.

Proteinase inhibitor activity in tomato leaves. The figure shows the percentage of inhibition of chymotrypsin activity in wild-type (white bars) and def-1 (black bars) leaves. Samples of infested and control plants were taken after 1 and 4 d of spider mite infestation. Bars annotated with the same letter were not significantly different (P > 0.05) after ANOVA and the Fisher post hoc test.

Figure 3.

Spider mite-induced gene expression in wild type and def-1. RNA gel-blot analysis of genes related to secondary metabolism (PAL), SA signaling (PR-P6 and PR-P23) and lipid signaling (DGK) in wild type and def-1 during 5 d of spider mite-infestation. For both plants the day 0 control (C) is shown, which is representative for all other days of the control plants. To check for equal loading, blots were hybridized with a probe for polyubiquitin (UBQ; TC115895). The bar graphs represent quantification (means and SEs of duplicated experiments) of the hybridizing bands, which were normalized for ubiquitin. The results were evaluated by means of ANOVA. Expression between wild type and def-1 was significantly different for P6 (P = 0.007), P23 (P = 0.013), DGK (P < 0.0001), and PAL (P < 0.0001).

Figure 4.

Olfactory response of Phytoseiulus persimilis to tomato plants infested with Tetranychus urticae and uninfested control plants. A, Percentages of predatory mites that chose for odors of infested plants (black bars) or for odors of uninfested plants (gray bars) after 3 or 4 d (tests with wild type and def-1 were conducted independently). Results were analyzed with a replicated test for goodness of fit. An asterisk (^*) denotes P = 0.001. None of the other experiments tested significantly different (0.84 > P < 0.98). B, Total number of predatory mites per experiment that did not make a choice within 5 min (white bars).

Figure 5.

MeSA and TMTT emission by JA-treated def-1 plants. The data shown represents the amounts of MeSA (A) and TMTT (B) emitted per gram fresh weight (FW) of spider mite-infested def-1 pretreated with 3 mL water (def control), 3 mL of 0.05, 0.25, or 0.5 mm JA and spider mite-infested wild type (WT control) during 4 d. Data was evaluated using ANOVA followed by Dunnett's post hoc test for comparing a control mean to other group means (separate ANOVAs on MeSA and TMTT, P < 0.04). A single asterisk (^*) denotes P < 0.05 and double asterisks (**) denote P < 0.01 after Dunnett's test (df = 8). Vertical bars indicate the means and ses. NS denotes not significant different from def control. The infested wild-type data were obtained from an independent experiment (Kant et al., 2004) and are shown for comparison.

Figure 6.

Spider mite-induced expression of SAMT in def-1 and wild type. RNA gel-blot analysis of the gene encoding SAMT during spider mite infestation. For all plants the control for day 2 (C) is shown, which is representative for all other days of the control plants. Ethidium bromide-stained gels are shown to indicate equal loading. The bar graph represents quantification (means and SEs of duplicated experiments) of the hybridizing bands.

Figure 7.

Spider mite-induced expression of SAMT, GGPPS, and WIPI genes in JA-treated wild type and def-1. Intact infested wild-type (WT) and def-1 plants had been pretreated with 0.25 mm JA (+) or water (−). RT-PCR analysis is shown for SAMT, GGPPS, WIPI, and UBQ. The bar graphs represent quantification (means and ses of duplicated experiments) of the hybridizing bands that were normalized for ubiquitin.