Nicotiana attenuata LECTIN RECEPTOR KINASE1 suppresses the insect-mediated inhibition of induced defense responses during Manduca sexta herbivory

Nicotiana attenuata has the capacity to respond specifically to herbivory by its natural herbivore, Manduca sexta, through the perception of elicitors in larval oral secretions. We demonstrate that Lectin receptor kinase 1 (LecRK1) functions during M. sexta herbivory to suppress the insect-mediated inhibition of jasmonic acid (JA)-induced defense responses. Gene function analysis performed by reducing LecRK1 expression in N. attenuata by both virus-induced gene silencing and inverted repeated RNA interference (ir-lecRK1 plants) revealed that LecRK1 was essential to mount a full defense response against M. sexta folivory; larvae growing on ir-lecRK1 plants were 40 to 100% larger than those growing on wild-type plants. The insect-induced accumulation of nicotine, diterpene-glucosides, and trypsin protease inhibitors, as well as the expression of Thr deaminase, was severalfold reduced in ir-lecRK1 plants compared with the wild type. The accumulation of JA and JA-Ile was unaffected during herbivory in ir-lecRK1 plants; however, salicylic acid (SA) accumulation was increased by twofold. The expression of nahG in ir-lecRK1 plants prevented the increased accumulation of SA and restored the defense response against M. sexta herbivory. The results suggest that LecRK1 inhibits the accumulation of SA during herbivory, although other mechanisms may also be affected.     

BAK1 regulates the accumulation of jasmonic acid and the levels of trypsin proteinase inhibitors in Nicotiana attenuata's responses to herbivory

BAK1 is a co-receptor of brassinosteroid (BR) receptor BRI1, and plays a well-characterized role in BR signalling. BAK1 also physically interacts with the flagellin receptor FLS2 and regulates pathogen resistance. The role of BAK1 in mediating Nicotiana attenuata's resistance responses to its specialist herbivore, Manduca sexta, was examined here. A virus-induced gene-silencing system was used to generate empty vector (EV) and NaBAK1-silenced plants. The wounding- and herbivory-induced responses were examined on EV and NaBAK1-silenced plants by wounding plants or simulating herbivory by treating wounds with larval oral secretions (OS). After wounding or OS elicitation, NaBAK1-silenced plants showed attenuated jasmonic acid (JA) and JA-isoleucine bursts, phytohormone responses important in mediating plant defences against herbivores. However, these decreased JA and JA-Ile levels did not result from compromised MAPK activity or elevated SA levels. After simulated herbivory, NaBAK1-silenced plants had EV levels of defensive secondary metabolites, namely, trypsin proteinase inhibitors (TPIs), and similar levels of resistance to Manduca sexta larvae. Additional experiments demonstrated that decreased JA levels in NaBAK1-VIGS plants, rather than the enzymatic activity of JAR proteins or Ile levels, were responsible for the reduced JA-Ile levels observed in these plants. Methyl jasmonate application elicited higher levels of TPI activity in NaBAK1-silenced plants than in EV plants, suggesting that silencing NaBAK1 enhances the accumulation of TPIs induced by a given level of JA. Thus NaBAK1 is involved in modulating herbivory-induced JA accumulation and how JA levels are transduced into TPI levels in N. attenuata.     

Silencing NOA1 elevates herbivory-induced jasmonic acid accumulation and compromises most of the carbon-based defense metabolites in Nicotiana attenuata(F)

Nitric oxide-associated protein 1 (NOA1) is involved in various abiotic stress responses and is required for plant resistance to pathogen infections. However, the role of NOA1 in plant-herbivore interactions has not been explored. We created NOA1-silenced Nicotiana attenuata plants (irNaNOA1). Compared with wild-type (WT) plants, irNaNOA1 plants had highly decreased photosynthesis rates. We further examined various traits important for plant defense against its specialist herbivore Manduca sexta by treating WT and irNaNOA1 plants with mechanical wounding and M. sexta oral secretions (OS). NOA1-silenced plants showed elevated levels of herbivory-induced jasmonic acid (JA), but decreased JA-isoleucine conjugate (JA-Ile) levels. The decreased JA-Ile levels did not result from compromised JAR (jasmonic acid resistant) activity in irNOA1 plants. Moreover, nitrogen-rich defensive compounds, nicotine and trypsin proteinase inhibitors, did not differ between WT and irNaNOA1 plants. In contrast, concentrations of most carbon-based defensive compounds were lower in these plants than in WT plants, although the levels of chlorogenic acid were not changed. Therefore, silencing NOA1 alters the allocation of carbon resources within the phenylpropanoid pathway. These data suggest the involvement of NOA1 in N. attenuata's defense against M. sexta attack, and highlight its role in photosynthesis, and biosynthesis of jasmonates and secondary metabolites.     

Jasmonate perception regulates jasmonate biosynthesis and JA-Ile metabolism: the case of COI1 in Nicotiana attenuata

CORONATINE INSENSITIVE 1 (COI1) is a well-known key player in processes downstream of jasmonic acid (JA) biosynthesis: silencing COI1 in Nicotiana attenuata (ir-coi1) makes plants insensitive to JA, prevents the up-regulation of JA-mediated defenses and decreases the plant's resistance to herbivores and pathogens. In agreement with previous studies, we observed that regulation of several JA biosynthesis genes elicited by Manduca sexta oral secretions (OS) is COI1 dependent. In response to wounding and application of OS ir-coi1 plants accumulate 75% less JA compared with wild-type plants (WT), resembling JA levels found in plants silenced in the key enzyme in JA biosynthesis LIPOXYGENASE 3 (as-lox). However, while OS-elicited as-lox plants also accumulated lower levels of the JA-conjugate JA-isoleucine (JA-Ile) than did WT plants, JA-Ile accumulation in ir-coi1 was higher, prolonged and peaked with a delay of 30 min. In vivo substrate feeding experiments of N. attenuata demonstrate that the increased and prolonged JA-Ile accumulation pattern in ir-coi1 is not the result of altered substrate availability, i.e. of JA and/or Ile, but is due to an approximately 6-fold decrease in JA-Ile turnover. These results provide the first evidence for a second, novel regulatory feedback function of COI1 in enhancing JA-Ile turnover. Hence, in addition to its control over JA biosynthesis, COI1 might fine-tune the dynamics of the jasmonate response after induction by herbivore elicitors.     

Ectopic expression of AtJMT in Nicotiana attenuata: creating a metabolic sink has tissue-specific consequences for the jasmonate metabolic network and silences downstream gene expression

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink that affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA, and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatiotemporal characteristics of the jasmonate metabolic network.     

Stability of plant defense proteins in the gut of insect herbivores

Plant defense against insect herbivores is mediated in part by enzymes that impair digestive processes in the insect gut. Little is known about the evolutionary origins of these enzymes, their distribution in the plant kingdom, or the mechanisms by which they act in the protease-rich environment of the animal digestive tract. One example of such an enzyme is threonine (Thr) deaminase (TD), which in tomato (Solanum lycopersicum) serves a dual role in isoleucine (Ile) biosynthesis in planta and Thr degradation in the insect midgut. Here, we report that tomato uses different TD isozymes to perform these functions. Whereas the constitutively expressed TD1 has a housekeeping role in Ile biosynthesis, expression of TD2 in leaves is activated by the jasmonate signaling pathway in response to herbivore attack. Ingestion of tomato foliage by specialist (Manduca sexta) and generalist (Trichoplusia ni) insect herbivores triggered proteolytic removal of TD2's C-terminal regulatory domain, resulting in an enzyme that degrades Thr without being inhibited through feedback by Ile. This processed form (pTD2) of TD2 accumulated to high levels in the insect midgut and feces (frass). Purified pTD2 exhibited biochemical properties that are consistent with a postingestive role in defense. Shotgun proteomic analysis of frass from tomato-reared M. sexta identified pTD2 as one of the most abundant proteins in the excrement. Among the other tomato proteins identified were several jasmonate-inducible proteins that have a known or proposed role in anti-insect defense. Subtilisin-like proteases and other pathogenesis-related proteins, as well as proteins of unknown function, were also cataloged. We conclude that proteomic analysis of frass from insect herbivores provides a robust experimental approach to identify hyperstable plant proteins that serve important roles in defense.     

Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata

Inhibition of jasmonic acid (JA) signaling has been shown to decrease herbivore resistance, but the responsible mechanisms are largely unknown because insect resistance is poorly understood in most model plant systems. We characterize three members of the lipoxygenase (LOX) gene family in the native tobacco plant Nicotiana attenuata and manipulate, by antisense expression, a specific, wound- and herbivory-induced isoform (LOX3) involved in JA biosynthesis. In three independent lines, antisense expression reduced wound-induced JA accumulation but not the release of green leaf volatiles (GLVs). The impaired JA signaling reduced two herbivore-induced direct defenses, nicotine and trypsin protease inhibitors (TPI), as well as the potent indirect defense, the release of volatile terpenes that attract generalist predators to feeding herbivores. All these defenses could be fully restored by methyl-JA (MeJA) treatment, with the exception of the increase in TPI activity, which was partially restored, suggesting the involvement of additional signals. The impaired ability to produce chemical defenses resulted in lower resistance to Manduca sexta attack, which could also be restored by MeJA treatment. Expression analysis using a cDNA microarray, specifically designed to analyze M. sexta-induced gene expression in N. attenuata, revealed a pivotal role for LOX3-produced oxylipins in upregulating defense genes (protease inhibitor, PI; xyloglucan endotransglucosylase/hydrolase, XTH; threonine deaminase, TD; hydroperoxide lyase, HPL), suppressing both downregulated growth genes (RUBISCO and photosystem II, PSII) and upregulated oxylipin genes (alpha-dioxygenase, alpha-DOX). By genetically manipulating signaling in a plant with a well-characterized ecology, we demonstrate that the complex phenotypic changes that mediate herbivore resistance are controlled by a specific part of the oxylipin cascade.     

Nicotiana attenuata α-DIOXYGENASE1 through its production of 2-hydroxylinolenic acid is required for intact plant defense expression against attack from Manduca sexta larvae

Nicotiana attenuata α-DIOXYGENASE1 (α-DOX1) is an oxylipin-forming gene elicited during herbivory by fatty acid amino acid conjugates (FACs) contained in oral secretions of Manduca sexta. To understand the roles of Naα-DOX1 and its major product, 2-hydroxylinolenic acid (2-hydroxylinolenic acid), in N.?attenuata's anti-herbivore defenses, we used a transgenic line specifically silenced in Naα-DOX1 expression (ir-α-dox1) and monitored 2-HOT production in M.?sexta-damaged tissues and its role in influencing the production of direct defense compounds and resistance to this insect. Attack by M.?sexta larvae amplified 2-HOT formation at the feeding sites; a reaction probably facilitated by Naα-DOX1's high pH optimum which allows 2-HOT formation to occur in the more alkaline conditions at the feeding sites or potentially in the insect mouth parts after the leaf tissue is ingested. Manduca sexta larvae performed better on ir-α-dox1 plants than on wild-type (WT) plants as a result of attenuated herbivory-specific JA and 2-HOT bursts as well as JA-inducible well-established defenses (nicotine, caffeoylputrescine and trypsin proteinase inhibitors). Repeated applications of 2-HOT to wounds before insect feeding partly amplified JA-controlled defenses and restored the resistance of ir-α-dox1 plants. We conclude that 2-HOT, produced by attack-activated α-DOX1 activity, participates in defense activation during insect feeding.     

Pithy protection: Nicotiana attenuata's jasmonic acid-mediated defenses are required to resist stem-boring weevil larvae

Folivory is the best studied plant-herbivore interaction, but it is unclear whether the signaling and resistance traits important for the defense of leaves are also important for other plant parts. Larvae of the tobacco stem weevil, Trichobaris mucorea, burrow into stems of Nicotiana attenuata and feed on the pith. Transgenic N. attenuata lines silenced in signaling and foliar defense traits were evaluated in a 2-year field study for resistance against attack by naturally occurring T. mucorea larva. Plants silenced in early jasmonic acid (JA) biosynthesis (antisense [as]-lipoxygenase3 [lox3]; inverted repeat [ir]-allene oxide cyclase), JA perception (as-coronatine insensitive1), proteinase inhibitors (ir-pi), and nicotine (ir-putrescine methyl-transferase) direct defenses and lignin (ir-cad) biosynthesis were infested more frequently than wild-type plants. Plants unable to emit C(6) aldehydes (as-hpl) had lower infestation rates, while plants silenced in late steps in JA biosynthesis (ir-acyl-coenzyme A oxidase, ir-opr) and silenced in diterpene glycoside production (ir-geranylgeranyl pyrophosphate synthase) did not differ from wild type. Pith choice assays revealed that ir-putrescine methyl-transferase, ir-coronatine insensitive1, and ir-lox3 pith, which all had diminished nicotine levels, were preferred by larvae compared to wild-type pith. The lack of preference for ir-lox2 and ir-cad piths, suggest that oviposition attraction and vascular defense, rather than pith palatability accounts for the higher attack rates observed for these plants. We conclude that traits that influence a plant's apparency, stem hardness, and pith direct defenses all contribute to resistance against this herbivore whose attack can be devastating to N. attenuata's fitness.