Salivary signals of European corn borer induce indirect defenses in tomato

Abstract

Plants can recognize the insect elicitors and activate its defense mechanisms. European Corn Borer (ECB; Ostrinia nubilalis) saliva, produced from the labial salivary glands and released through the spinneret, is responsible for inducing direct defenses in host plants. Glucose oxidase (GOX) present in the ECB saliva induced direct defenses in tomato. By contrast, GOX activity in ECB saliva was insufficient to trigger defenses in maize, suggesting that host-specific salivary elicitors are responsible for inducing direct defenses in host plants. Our current study further examined whether ECB saliva can trigger indirect defenses in tomato. Relative expression levels of TERPENE SYNTHASE5 (TPS5) and HYDROPEROXIDE LYASE (HPL), marker for indirect defenses in host plants, were monitored. Quantitative real-time PCR analysis revealed that ECB saliva can induce the expression of TPS5 and HPL, suggesting that salivary signals can induce indirect defenses in addition to the direct defenses. Further experiments are required to identify different ECB elicitors that are responsible for inducing direct and indirect defenses in host plants.

Caterpillar oral secretions and saliva are discrete secretions that play a crucial role in inducing/suppressing plant defenses. For example, the action of Spodoptera littoralis (Egyptian cotton worm) and Pieris brassicae (Large white butterfly) oral secretions (OS) suppressed the defense responses in Arabidopsis leading to an enhanced larval weight.¹ In contrast, components present in the OS (e.g., volicitin, caeliferins, etc.) activate indirect plant defenses by releasing plant volatiles and recruiting predators of the attacking herbivore.^2,3 In addition, caterpillar saliva plays a key role in inducing/suppressing plant defenses.^4-7 The glucose oxidase (GOX) and ATP hydrolyzing enzymes present in the saliva of Helicoverpa zea (Tomato fruit worm) can suppress defense responses in tobacco and tomato, respectively.^4,5 In contrast, GOX in H. zea saliva trigger defenses in tomato.⁶ Very recently, we have shown that host-specific salivary signals induce defenses in different host plants.⁷ Salivary components of Ostrinia nubilalis (European Corn Borer; ECB) (Lepidoptera: Crambidae) significantly induced the defense-related transcript proteinase inhibitor, Proteinase Inhibitor 2 (PIN2) and Maize Protease Inhibitor (MPI) in tomato and maize, respectively. Furthermore, ECB saliva induced the expression of LIPOXYGENASE (LOX) and 12-OXO-PHYTODIENOIC ACID (OPR) genes that are involved in the jasmonic acid (JA) biosynthesis/signaling pathway. GOX present in the saliva of ECB induced defenses in tomato, however, it failed to trigger defenses in maize, suggesting that ECB saliva contains multiple salivary signals that could elicit defense responses in different host plants.⁷

ECB saliva triggers the induction of indirect defenses in tomato

The release of blend of volatile organic compounds (VOC) from plants can attract natural enemies of the attacking herbivores. Caterpillar saliva not only modulates the direct defenses in plants, but also alters the production of volatile, indirect defenses, and/or the genes encoding for their synthesis.^8-10 To determine if ECB saliva affects indirect gene expression in tomato, the spinneret, the structure that secretes saliva, was cauterized/ablated to prevent salivation.⁷ This allowed us to compare the gene expression in tomato that were infested with ablated (no saliva) or unablated (saliva secreted) ECB larvae for 24 h. Using qRT-PCR we monitored the expression of TERPENE SYNTHASE5 (TPS5) and HYDROPEROXIDE LYASE (HPL), genes which regulate biosynthesis of terpene and green leafy volatiles (GLVs) in tomato, after ECB infestation. As shown in Figure 1A, ECB feeding with intact spinneret significantly upregulated the expression of TPS5 compared with ECB with ablated spinneret. Plants that were uninfested with ECB were used as the control. Previously, it was shown that the monoterpenes (TPS5) was significantly higher in the leaves of tomato as opposed to stem.¹¹ This data further suggests that plants can indeed perceive the insects on foliage and induce indirect defense mechanisms. Similarly, ECB feeding with intact spinneret clearly induced the expression of HPL relative to ECB with ablated spinneret (Fig. 1B). Studies have shown that downstream products of HPL cascades, i.e., GLVs, can act as critical signaling compounds in providing defense against insects.^12,13 Our data suggests that ECB feeding significantly induces the expression of HPL which potentially produce the downstream blends of VOC, thereby attracting the predators/parasitoids of ECB.

Figure 1. European corn borer (ECB) saliva induces indirect defenses in tomato. (A) Relative expression of TERPENE SYNTHASE5 (TPS5) and (B) HYDROPEROXIDE LYASE (HPL) in tomato leaves, 24 h after feeding with ECB intact and ablated spinneret. Tomato plants that were not infested with ECB were used as the control. (C) Relative expression of TPS5 and (D) HPL in tomato leaves, 24 h after wounding and application of 20 μl of PBS and ECB saliva. Unwounded plants were used as the control. Different letters above the bars indicate values that are significantly different from each other (ANOVA, P < 0.05, LSD procedure). Error bars represent ± SE (n = 4).

Since the saliva of ECB is released through the spinneret, we collected saliva directly from ECB.⁷ Application of pure saliva to the wounded sites of tomato leaves significantly induced the expression of TPS5 and HPL compared with wounded tomato leaves that received phosphate buffered saline (PBS) (Fig. 1C and 1D). Unwounded plants were used as the control. These results are supportive of our suggestion that ECB saliva plays a critical role in inducing the indirect defenses in tomato. Further experiments are required to monitor the volatile profiles of tomato plants in response to ECB feeding. This will further help us to characterize whether a few compounds or a complex blend of volatiles are responsible for inducing tomato indirect defenses in response to ECB herbivory. The isoprene-derived terpenoids and fatty acid-derived GLV are best studied classes of HIPVs (Herbivore-Induced Plant Volatiles). However, these two classes of compounds are released at different times after herbivore attack. Terpenoids are released at a delayed time whereas GLV are released immediately after insect herbivory.^14-16 Although our data suggests that ECB saliva induces the expression of both TPS5 and HPL after 24 h (Fig. 1), it is highly plausible that ECB infestation can produce different GLV blend immediately after the insect attack and at a delayed time. Time-course volatile profiling of tomato plants after ECB infestation will help us to set apart the different compounds that are responsible for inducing indirect defenses in tomato. Nonetheless, our data clearly suggests that ECB saliva, in addition to inducing direct defenses,⁷ also activates indirect defenses in tomato.