Jasmonate-dependent induction of polyphenol oxidase activity in tomato foliage is important for defense against Spodoptera exigua but not against Manduca sexta

Abstract

Background: Jasmonates are involved in plant defense, participating in the timely induction of defense responses against insect herbivores from different feeding guilds and with different degrees of host specialization. It is less clear to what extent the induction of plant defense is controlled by different members of the jasmonate family and how specificity of the response is achieved. Using transgenic plants blocked in jasmonic acid (JA) biosynthesis, we previously showed that JA is required for the formation of glandular trichomes and trichome-borne metabolites as constitutive defense traits in tomato, affecting oviposition and feeding behavior of the specialist Manduca sexta. In contrast, JA was not required for the local induction of defense gene expression after wounding. In JA-deficient plants, the JA precursor oxophytodienoic acid (OPDA) substituted as a regulator of defense gene expression maintaining considerable resistance against M. sexta larvae. In this study, we investigate the contribution of JA and OPDA to defense against the generalist herbivore Spodoptera exigua.

Results: S. exigua preferred JA-deficient over wild-type tomato plants as a host for both oviposition and feeding. Feeding preference for JA-deficient plants was caused by constitutively reduced levels of repellent terpenes. Growth and development of the larvae, on the other hand, were controlled by additional JA-dependent defense traits, including the JA-mediated induction of foliar polyphenol oxidase (PPO) activity. PPO induction was more pronounced after S. exigua herbivory as compared to mechanical wounding or M. sexta feeding. The difference was attributed to an elicitor exclusively present in S. exigua oral secretions.

Conclusions: The behavior of M. sexta and S. exigua during oviposition and feeding is controlled by constitutive JA/JA-Ile-dependent defense traits involving mono- and sesquiterpenes in both species, and cis-3-hexenal as an additional chemical cue for M. sexta. The requirement of jasmonates for resistance of tomato plants against caterpillar feeding differs for the two species. While the OPDA-mediated induction of local defense is sufficient to restrict growth and development of M. sexta larvae in absence of JA/JA-Ile, defense against S. exigua relied on additional JA/JA-Ile dependent factors, including the induction of foliar polyphenol oxidase activity in response to S. exigua oral secretions.

Figure 1

Jasmonate levels and defense-related phenotypes of transgenic tomato plants silenced for OPR3 expression by RNAi. The figure summarizes the main findings of study [40] addressing the effect of JA/JA-Ile deficiency of OPR3-silenced plants on constitutive and induced defenses against the specialist herbivore M. sexta (green, red and yellow arrows indicating up- or down-regulation and no change in OPR3-RNAi as compared to control plants, respectively). OPR3-RNAi plants contain less JA/JA-Ile as compared to the wild type, and there is no wound-induced increase in JA or JA-Ile (left panel). As a result of JA/JA-Ile deficiency, trichome density and terpene content are reduced, while cis-3-hexenal concentration is increased in OPR3-RNAi as compared to wild-type plants (right panel, top). OPR3-RNAi plants are preferred by gravid M. sexta females for oviposition, and by the larvae for feeding (right panels, center). The development of M. sexta larvae is indistinguishable on OPR3-RNAi and wild-type plants (right panel, bottom). Resistance against larval feeding is thus maintained in the absence of JA/JA-Ile and was attributed to the local induction of defense gene expression by OPDA.

Figure 2

S. exigua prefers JA-deficient over wild-type plants for oviposition and feeding. (A) Oviposition preference was analyzed in dual-choice assays as the number of egg deposits on OPR3-RNAi (green bars) as compared to wild-type plants (WT₁, UC82B; blue bars). Data are shown for three independent OPR3-RNAi lines individually on the left (lines A15, A52, and P3), and as the mean of the three lines +/− SD on the right (paired t-test: **P = 0.007). (B) Feeding preference was analyzed in dual-choice assays using three independent OPR3-RNAi lines (green) and the jai1 mutant (yellow) with the corresponding wild types (WT₁, UC82B; WT₂, Castlemart; blue). Each experiment consisted of three mutant and wild-type leaf discs offered to three larvae for feeding. Preference is shown as percent consumed leaf area after four hours. Data represent the mean +/− SD of at least 20 replicates (n = 28, 27, 37, and 20 for J55, J18, A52, and the jai1 mutant). Asterisks indicate significant preference (Wilcoxon signed rank test: ***P < 0.001).

Figure 3

Feeding preference of S. exigua larvae is determined by terpene content. (A) Dual-choice test for feeding preference comparing trichome-cured wild-type (blue) and OPR3-RNAi leaves (green) were performed as in Figure 2B. The consumed leaf area is shown in percent as the mean +/− SD of 58 experiments. Differences between the means are not significant (Wilcoxon signed rank test: P = 0.895). (B) Dual-choice tests comparing artificial diet to which cis-3-hexenal (n = 44) or a blend of mono- and sesquiterpenes (n = 86) were added in concentrations reflecting the content of wild-type (blue) or OPR3-RNAi trichomes (green). Diet consumption after 20 hrs is shown in percent as the mean +/− SD. Asterisks indicate significant preference (Wilcoxon signed rank test: ***P < 0.001).

Figure 4

S. exigua larvae perform better on jai1 mutants than on wild type. The experiment involved 300 and 150 four-day-old larvae on wild-type and jai1 plants, respectively. (A) Percent survival of S. exigua larvae on wild type (WT₂, Castlemart, blue) and the jai1 mutant (yellow). (B) Larval development on wild-type (blue) and jai1 (yellow) host plants. Larval mass is given in mg as the mean +/− SD. Asterisks indicate significant differences (Wilcoxon signed rank test: *** P < 0.001). (C) S. exigua larvae at the end of the experiment, prior to pupation (scale bar = 1 cm).

Figure 5

S. exigua larvae perform better on OPR3-RNAi plants than on wild type. The experiment involved 246 and 175 four-day-old larvae on wild-type and OPR3-RNAi plants, respectively. (A) Percent survival of S. exigua larvae on wild type (WT₁, UC82B, blue) and 3 independent OPR3-RNAi lines (J55, J18, A52; green). (B) Larval development on wild-type (blue) and OPR3-RNAi (green) host plants. Larval mass is given in mg as the mean +/− SD. Asterisks indicate significant differences (Wilcoxon signed rank test: *** P < 0.001). (C) S. exigua larvae at the end of the experiment, prior to pupation (scale bar = 1 cm). (D) One representative of wild-type and OPR3-RNAi host plants at the end of the experiment.

Figure 6

Performance of S. exigua larvae on trichome-cured OPR3-RNAi and wild-type plants (broken lines) as compared to untreated controls (solid lines). (A) Larval development on wild-type (blue) and OPR3-RNAi (green) host plants. Larval mass is given in mg as the mean +/− SD. (B) Percent survival of S. exigua larvae on wild-type (blue) and OPR3-RNAi (green) host plants. 300 and 150 larvae were used on untreated and trichome-cured wild type, while 200 and 150 larvae were used on untreated and trichome-cured OPR3-RNAi plants, respectively.

Figure 7

Induction of PPO activity by S. exigua and M. sexta feeding. PPO activity was assayed in wild-type (blue) and OPR3-RNAi plants (green) before (C), 48 and 72 hours after insect feeding. (A) PPO induction by S. exigua. (B) PPO induction by M. sexta. Data were normalized to PPO levels in unwounded wild-type controls and represent the mean +/− SD of 2 to 3 independent experiments each with four leaf samples. Significant differences between wild-type and OPR3-RNAi plants are indicated (t-test; ** P < 0.01, *** P < 0.001).

Figure 8

Induction of PPO activity by mechanical wounding and insect oral secretions. PPO activity was assayed in wild-type leaves 72 hours after mechanical wounding (W) or wounding with addition of insect (M. sexta or S. exigua) oral secretions (W + OS). OS were diluted 1:1 in water and applied in their native state (OS_n) or after heat denaturation (OS_d). Data are shown for one of three independent experiments, representing the mean +/− SD of four biological replicates each including pooled leaf material from three plants. Different letters indicate significant differences in PPO fold-induction normalized to unwounded controls (C; One-Way-ANOVA (F_5,18 = 12.534, P < 0.001) and post-hoc Holm-Sidak for multiple comparisons at P < 0.05).