Feeding by the tobacco specialist Manduca sexta (Lepidoptera, Sphingidae) and application of larval oral secretions and regurgitant (R) to mechanical wounds are known to elicit: (a) a systemic release of mono-and sesquiterpenes, (b) a jasmonate burst, and (c) R-specific changes in transcript accumulation of putatively growth-and defense-related mRNAs in Nicotiana attenuata Torr. ex Wats. We identified several fatty acid-amino acid conjugates (FACs) in the R of M. sexta and the closely related species Manduca quinquemaculata which, when synthesized and applied to mechanical wounds at concentrations comparable with those found in R, elicited all three R-specific responses. Ion-exchange treatment of R, which removed all detectable FACs and free fatty acids (FAs), also removed all detectable activity. The biological activity of ion-exchanged R could be completely restored by the addition of synthetic FACs at R-equivalent concentrations, whereas the addition of FAs did not restore the biological activity of R. We conclude that the biological activity of R is not related to the supply of FAs to the octadecanoid cascade for endogenous jasmonate biosynthesis, but that FACs elicit the herbivore-specific responses by another mechanism and that the insect-produced modification of plant-derived FAs is necessary for the plant's recognition of this specialized herbivore.

Herbivore attack elicits costly defenses that are known to decrease plant fitness by using resources that are normally slated for growth and reproduction. Additionally, plants have evolved mechanisms for tolerating attack, which are not understood on a molecular level. Using 11 C-photosynthate labeling as well as sugar and enzyme measurements, we found rapid changes in sink-source relations in the annual Nicotiana attenuata after simulated herbivore attacks, which increased the allocation of sugars to roots. This herbivore-induced response is regulated by the ␤-subunit of an SnRK1 (SNF1-related kinase) protein kinase, GAL83, transcripts of which are rapidly down-regulated in source leaves after herbivore attack and, when silenced, increase assimilate transport to roots. This C diversion response is activated by herbivore-specific elicitors and is independent of jasmonate signaling, which regulates most of the plant's defense responses. Herbivore attack during early stages of development increases root reserves, which, in turn, delays senescence and prolongs flowering. That attacked GAL83-silenced plants use their enhanced root reserves to prolong reproduction demonstrates that SnRK1 alters resource allocation so that plants better tolerate herbivory. This tolerance mechanism complements the likely defensive value of diverting resources to a less vulnerable location within the plant.carbon-11 ͉ defense ͉ plant-herbivore interactions ͉ tolerance P lants have evolved a variety of mechanisms for reducing the negative impact of herbivore attack on fitness; these mechanisms include direct and indirect defenses and tolerance (1). Defenses are costly, expending energy and resources that could otherwise be used to grow and generate offspring. Inducible defenses allow plants to invest resources into defense only when needed. Although defenses limit the extent of damage, even well defended plants lose large amounts of tissue when attacked by herbivores that have adapted to their defenses. Then, plants would benefit from tolerance, which minimizes the fitness consequences of tissue loss to herbivores (2-4). Defense against, and tolerance of, herbivory are not mutually exclusive; most plantinsect interactions likely combine both (5, 6). In contrast to the rapid advances in our understanding of defense mechanisms, little is known about the traits that allow plants to tolerate herbivore damage.Tolerance, which is measured by comparing the fitness of a genotype in environments with and without attackers, remains uncharacterized at the molecular level (2, 7). At a physiological level, increases in photosynthetic rate, branching, and storage in belowground tissues are thought to be involved (8-10). These responses require the tuning of primary metabolism, for which mutant screens and other reverse genetic approaches with model plants have yet to yield molecular regulators. Host plants that have coevolved with adapted herbivores likely have elaborate defense and tolerance responses to minimize the fitness consequences of herbivory...

Plants respond to herbivore attack with a dramatic functional reorganization that involves the activation of direct and indirect defenses and tolerance, which in turn make large demands on primary metabolism. Here we provide the first characterization of the transcriptional reorganization that occurs after insect attack in a model plant-herbivore system: Nicotiana attenuata Torr. ex Wats.-Manduca sexta. We used mRNA differential display to characterize one-twentieth of the insect-responsive transcriptome of N. attenuata and verified differential expression for 27 cDNAs. Northern analyses were used to study the effects of folivory and exposure to airborne methyl jasmonate and for kinetic analyses throughout a 16-hlight/8-h-dark cycle. Sequence similarity searches allowed putative functions to be assigned to 15 transcripts. Genes were related to photosynthesis, electron transport, cytoskeleton, carbon and nitrogen metabolism, signaling, and a group responding to stress, wounding, or invasion of pathogens. Overall, transcripts involved in photosynthesis were strongly down-regulated, whereas those responding to stress, wounding, and pathogens and involved in shifting carbon and nitrogen to defense were strongly up-regulated. The majority of transcripts responded similarly to airborne methyl jasmonate and folivory, and had tissue-and diurnal-specific patterns of expression. Transcripts encoding Thr deaminase (TD) and a putative retrotransposon were absent in control plants, but were strongly induced after herbivory. Full-length sequences were obtained for TD and the pathogen-inducible ␣-dioxygenase, PIOX. Effects of abiotic and biotic stimuli were investigated for transcripts encoding TD, importin ␣, PIOX, and a GAL83-like kinase cofactor.

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The transcriptional changes in Nicotiana attenuata Torr. ex Wats. elicited by attack from Manduca sexta larvae were previously characterized by mRNA differential display (D. Hermsmeier, U. Schittko, I.T. Baldwin [2001] Plant Physiol 125: 683-700). Because herbivore attack causes wounding, we disentangled wound-induced changes from those elicited by M. sexta oral secretions and regurgitant (R) with a northern analysis of a subset of the differentially expressed transcripts encoding threonine deaminase, pathogen-induced oxygenase, a photosystem II light-harvesting protein, a retrotransposon homolog, and three unknown genes. R extensively modified wound-induced responses by suppressing wound-induced transcripts (type I) or amplifying the wound-induced response (type II) further down-regulating wound-suppressed transcripts (type IIa) or up-regulating wound-induced transcripts (type IIb). It is interesting that although all seven genes displayed their R-specific patterns in the treated tissues largely independently of the leaf or plant developmental stage, only the type I genes displayed strong systemic induction. Ethylene was not responsible for any of the specific patterns of expression. R collected from different tobacco feeding insects, M. sexta, Manduca quinquemaculata, and Heliothis virescens, as well as from different instars of M. sexta were equally active. The active components of M. sexta R were heat stable and active in minute amounts, comparable with real transfer rates during larval feeding. Specific expression patterns may indicate that the plant is adjusting its wound response to efficiently fend off M. sexta, but may also be advantageous to the larvae, especially when R suppress wound-induced plant responses.

As feeding by the tobacco specialist Manduca sexta L. is known to result in significantly higher jasmonate (JA) concentrations in Nicotiana plants than do mechanical simulations of the larval damage, we investigated whether M. sexta, which is known to rapidly consume large quantities of leaf material, can impair this "recognition" response by consuming the leaf tissue before it can respond with amplified JA levels. We report that oral secretions (OS) from M. sexta, but not from the cabbage specialist Pieris rapae, amplified the wound-induced JA response of Nicotiana attenuata Torr. Ex Wats., regardless of larval diet, instar and molting stage, and were still active after boiling and when diluted to 1/1000. The largest JA response occurred within 40 min in tissues adjacent to the OS application site. When 3 mm of leaf tissue immediately adjacent to the OS application site was excised within 40 s, the signal that elicits JA amplification was found to travel rapidly into the leaf, beyond the mandibular reach of the larvae. We conclude that M. sexta is not able to consume the evidence of feeding activity.

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Specialist herbivores are known to alter their host's wound-induced responses but the beneficiaries of these alterations are unknown. Nicotiana attenuata plants release a burst of ethylene specifically in response to feeding by Manduca sexta larvae, which is known to suppress wound- and methyl jasmonate (MeJA)-inducible nicotine accumulation. The ethylene burst may be a mechanism by which M. sexta larvae feed "stealthily" on their host plants or, alternatively, it may allow the plant to optimize its defense response against this specialist herbivore by reducing costs of induction. We examined the impact of the ethylene burst on defense-related fitness costs that are readily observed when plants are treated with MeJA and grown in competition with untreated plants. We elicited nicotine induction (with MeJA), the ethylene burst (with the ethylene releasing compound, ethephon) and inhibited the plant's ability to perceive ethylene (with applications of an antagonist of ethylene receptors, 1-methylcyclopropene, 1-MCP). By simultaneously applying MeJA and ethephon we mimicked the plant's hormonal response to larval attack. We hypothesized that if the ethylene burst benefited the plant, the fitness costs of MeJA induction should be reduced by ethephon and restored if the plants were additionally treated with 1-MCP. In a second experiment, we applied larval oral secretion (OS) to elicit endogenous hormone production and predicted that the 1-MCP treatment should reduce the fitness of OS-treated plants. Our measures of plant fitness, namely the rate of stalk elongation and lifetime capsule production, supported these predictions. We conclude that the ethylene burst elicited by this specialist herbivore can reduce MeJA-induced fitness costs and increase the competitive strength of OS-treated plants. Suppressed nicotine production is likely to contribute to, but is not sufficient to explain, the observed fitness outcomes. The intensity of intra-specific competition and herbivore attack will likely determine the adaptive value of the M. sexta-elicited ethylene response.