Differential timing of spider mite-induced direct and indirect defenses in tomato plants

Abstract

Through a combined metabolomics and transcriptomics approach we analyzed the events that took place during the first 5 d of infesting intact tomato (Lycopersicon esculentum) plants with spider mites (Tetranychus urticae). Although the spider mites had caused little visible damage to the leaves after 1 d, they had already induced direct defense responses. For example, proteinase inhibitor activity had doubled and the transcription of genes involved in jasmonate-, salicylate-, and ethylene-regulated defenses had been activated. On day four, proteinase inhibitor activity and particularly transcript levels of salicylate-regulated genes were still maintained. In addition, genes involved in phospholipid metabolism were up-regulated on day one and those in the secondary metabolism on day four. Although transcriptional up-regulation of the enzymes involved in the biosynthesis of monoterpenes and diterpenes already occurred on day one, a significant increase in the emission of volatile terpenoids was delayed until day four. This increase in volatile production coincided with the increased olfactory preference of predatory mites (Phytoseiulus persimilis) for infested plants. Our results indicate that tomato activates its indirect defenses (volatile production) to complement the direct defense response against spider mites.

Figure 1.

Spider mite performance and damage to tomato leaves. A, Represents the average damaged leaf area in millimeters squared over a period of 5 d inflicted by 1, 5, 15, and 50 adult female spider mites. The vertical bars represent the ses. B, Represents the number of eggs (black line) and juveniles (dotted line) produced by adult females (2-d-old at day 1 of the experiment). The vertical bars represent the se. C, Spider mites caused chlorotic lesions when feeding on tomato leaf tissue. The photo shows the accumulated damage on day five inflicted by 0, 1, 5, 15, and 50 adult female spider mites.

Figure 2.

Spider mite-induced gene expression. Three leaves of 3-week-old tomato plants were each infested with 15 spider mites and RNA was isolated after 1, 2, 3, and 4 d. RNA gel-blots (Verdonk et al., 2003) were hybridized with pathogenesis related protein P6 (TC 115911), diacylglycerol kinase (DGK; BE434771), or polyubiquitin (UBQ) as loading control. The ethidium bromide-stained gel (EtBr) is shown to confirm equal loading. Control plants were grown under identical conditions without spider mites.

Figure 3.

Proteinase inhibitor activity is induced by spider mites. Three leaves of 3-week-old tomato plants were each infested with 15 spider mites in duplicate experiments. After 1 and 4 d, infested leaves were collected and pooled per plant. Leaves were also collected and pooled from uninfested control plants. Proteinase inhibitor activity (percent inhibition of 10 μg chymotrypsin activity/500 μg protein) in extracts of infested (black bars) and uninfested leaves (white bars) was determined following the protocol of Stout et al. (1994). The vertical bars indicate the means and sds of each treatment group. An asterisk (*) denotes a significant difference at P < 0.05, double asterisks (**) at P < 0.01 (Student's t test).

Figure 4.

Volatile compounds emitted by 3-week-old tomato plants infested by spider mites (black lines) and by uninfested control plants (dotted lines). In each replicate experiment we sampled the volatiles of 3 control plants and 3 treatment plants at 24 h intervals during 5 d. The graphs show the average relative production during 5 d for: (A) methyl salicylate; (B) trans-β-ocimene; (C) trans-nerolidol; (D) linalool; (E) β-farnesene; and (F) cis-nerolidol, calculated from 9 independent replicate experiments. The vertical bars represent the ses.

Figure 5.

Olfactory response of P. persimilis to tomato plants infested with T. urticae and uninfested control plants. The figure shows the fraction of predatory mites that chose for the odors of infested plants (black bars), for uninfested plants (gray bars), or that did not make a choice within 5 min (white bars). Two replicate choice tests (n = 24 and n = 34) on day 3 and 2 replicates (n = 60 and n = 50) on day 4 are presented. An asterisk (*) denotes significant deviation from H₀ (predatory mites chose infested plants and uninfested plants in a ratio of 1:1) according to a replicated test for goodness-of-fit at α = 0.01.