-411 008 (M.S.), India (A.P.G.)When Manduca sexta attacks Nicotiana attenuata, fatty acid-amino acid conjugates (FACs) in the larvae's oral secretions (OS) are introduced into feeding wounds. These FACs trigger a transcriptional response that is similar to the response induced by insect damage. Using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight, and liquid chromatography-tandem mass spectrometry, we characterized the proteins in phenolic extracts and in a nuclear fraction of leaves elicited by larval attack, and/or in leaves wounded and treated with OS, FAC-free OS, and synthetic FACs. Phenolic extracts yielded approximately 600 protein spots, many of which were altered by elicitation, whereas nuclear protein fractions yielded approximately 100 spots, most of which were unchanged by elicitation. Reproducible elicitor-induced changes in 90 spots were characterized. In general, proteins that increased were involved in primary metabolism, defense, and transcriptional and translational regulation; those that decreased were involved in photosynthesis. Like the transcriptional defense responses, proteomic changes were strongly elicited by the FACs in OS. A semiquantitative reverse transcription-PCR approach based on peptide sequences was used to compare transcript and protein accumulation patterns for 17 candidate proteins. In six cases the patterns of elicited transcript accumulation were consistent with those of elicited protein accumulation. Functional analysis of one of the identified proteins involved in photosynthesis, RuBPCase activase, was accomplished by virus-induced gene silencing. Plants with decreased levels of RuBPCase activase protein had reduced photosynthetic rates and RuBPCase activity, and less biomass, responses consistent with those of herbivore-attacked plants. We conclude that the response of the plant's proteome to herbivore elicitation is complex, and integrated transcriptome-proteome-metabolome analysis is required to fully understand this ubiquitous ecological interaction.The majority of studies examining the induced defense responses of plants after insect attack have focused on the dynamics of the specific genes, proteins, and metabolites that are thought to be responsible for changes in resistance elicited by attack. More recently, large-scale transcriptional analyses with microarrays have broadened the scope of the analysis and revealed coordinated changes in hundreds of transcripts, suggesting that large-scale shifts in metabolism accompany the activation of defense responses

The evolutionary plant–herbivore arms race sometimes gives rise to remarkably unique adaptation strategies. Here we report one such strategy in the lepidopteran herbivore Manduca sexta against its hostplant Nicotiana attenuata's major phytotoxins, 17-hydroxygeranyllinalool diterpene glycoside, lyciumoside IV and its malonylated forms. We show that alkalinity of larval regurgitant non-enzymatically demalonylates the malonylated forms to lyciumoside IV. Lyciumoside IV is then detoxified in the midgut by β-glucosidase 1-catalysed deglycosylation, which is unusual, as typically the deglycosylation of glycosylated phytochemicals by insects results in the opposite: toxin activation. Suppression of deglucosylation by silencing larval β-glucosidase 1 by plant-mediated RNAi causes moulting impairments and mortality. In the native habitat of N. attenuata, β-glucosidase 1 silencing also increases larval unpalatability to native predatory spiders, suggesting that the defensive co-option of lyciumoside IV may be ecologically advantageous. We infer that M. sexta detoxifies this allelochemical to avoid its deleterious effects, rather than co-opting it against predators.

Insect attack frequently down-regulates photosynthetic proteins. To understand how this influences the plant-insect interaction, we transformed Nicotiana attenuata to independently silence ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) activase (RCA) and RuBPCase and selected lines whose photosynthetic capacity was similarly reduced. Decreases in plant growth mirrored the decreases in photosynthesis, but the effects on herbivore performance differed. Both generalist (Spodoptera littoralis) and specialist (Manduca sexta) larvae grew larger on RCA-silenced plants, which was consistent with decreased levels of trypsin protease inhibitors and diterpene glycosides and increased levels of RuBPCase, the larvae's main dietary protein.RCA-silenced plants were impaired in their attack-elicited jasmonate (JA)-isoleucine (Ile)/leucine levels, but RuBPCasesilenced plants were not, a deficiency that could not be restored by supplementation with Ile or attributed to lower transcript levels of JAR4/6, the key enzyme for JA-Ile conjugation. From these results, we infer that JA-Ile/leucine signaling and the herbivore resistance traits elicited by JA-Ile are influenced by adenylate charge, or more generally, carbon availability in RCAbut not RuBPCase-silenced plants. Growth of generalist larvae on RuBPCase-silenced plants did not differ from growth on empty vector controls, but the specialist larvae grew faster on RuBPCase-silenced plants, which suggests that the specialist can better tolerate the protein deficiency resulting from RuBPCase silencing than the generalist can. We conclude that the plantherbivore interaction is more influenced by the particular mechanisms that reduce photosynthetic capacity after herbivore attack than by the magnitude of the decrease, which highlights the value of understanding defense mechanisms in evaluating growth-defense tradeoffs.An herbivore-attacked plant is known to reduce its photosynthetic capacity while increasing the production and accumulation of defense-related compounds (Walling, 2000;Hermsmeier et al., 2001;Kessler and Baldwin, 2002;Hahlbrock et al., 2003). In response to attack, plants must grow rapidly to compete and simultaneously maintain the defenses necessary to survive in environments with herbivores (Herms and Mattson, 1992). The dramatic up-regulation of defense metabolites likely requires metabolic adjustments in plant growth and reproduction (Halitschke et al., 2003;Reymond et al., 2004;Ralph et al., 2006). However, reductions in plant growth can also be understood as part of a plant's defense strategy, as reduced growth limits the availability of food and nutrition for the feeding insect (Hermsmeier et al., 2001;Hahlbrock et al., 2003). Allocating resources for resistance mechanisms is believed to reduce the availability of resources for growth; hence, resistance is thought to be costly in terms of plant growth and fitness (Heil and Baldwin, 2002). However, very little is known about how plants optimize their resource allocation when attacked by herbivores. Plan...

Insect damage to plants is known to up-regulate defense and down-regulate growth processes. While there are frequent reports about up-regulation of defense signaling and production of defense metabolites in response to herbivory, much less is understood about the mechanisms by which growth and carbon assimilation are down-regulated. Here we demonstrate that insect herbivory down-regulates the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in Arabidopsis (Arabidopsis thaliana), a pathway making primarily metabolites for use in photosynthesis. Simulated feeding by the generalist herbivore Spodoptera littoralis suppressed flux through the MEP pathway and decreased steady-state levels of the intermediate 1-deoxy-D-xylulose 5-phosphate (DXP). Simulated herbivory also increased reactive oxygen species content which caused the conversion of β-carotene to β-cyclocitral (βCC). This volatile oxidation product affected the MEP pathway by directly inhibiting DXP synthase (DXS), the rate-controlling enzyme of the MEP pathway in Arabidopsis and inducing plant resistance against S. littoralis. βCC inhibited both DXS transcript accumulation and DXS activity. Molecular models suggested that βCC binds to DXS at the binding site for the thymine pyrophosphate cofactor and blocks catalysis, which was confirmed by direct assays of βCC with the purified DXS protein in vitro. Another intermediate of the MEP pathway, 2-C-methyl-D-erythritol-2, 4-cyclodiphosphate, which is known to stimulate salicylate defense signaling, showed greater accumulation and enhanced export out of the plastid in response to simulated herbivory. Together, our work implicates βCC as a signal of herbivore damage in Arabidopsis that increases defense and decreases flux through the MEP pathway, a pathway involved in growth and carbon assimilation.