Drastic Genome Reduction in an Herbivore's Pectinolytic Symbiont.

Pectin, an integral component of the plant cell wall, is a recalcitrant substrate against enzymatic challenges by most animals. In characterizing the source of a leaf beetle's (Cassida rubiginosa) pectin-degrading phenotype, we demonstrate its dependency on an extracellular bacterium housed in specialized organs connected to the foregut. Despite possessing the smallest genome (0.27 Mb) of any organism not subsisting within a host cell, the symbiont nonetheless retained a functional pectinolytic metabolism targeting the polysaccharide's two most abundant classes: homogalacturonan and rhamnogalacturonan I. Comparative transcriptomics revealed pectinase expression to be enriched in the symbiotic organs, consistent with enzymatic buildup in these structures following immunostaining with pectinase-targeting antibodies. Symbiont elimination results in a drastically reduced host survivorship and a diminished capacity to degrade pectin. Collectively, our findings highlight symbiosis as a strategy for an herbivore to metabolize one of nature's most complex polysaccharides and a universal component of plant tissues.

Injury Activates Ca2+/Calmodulin-Dependent Phosphorylation of JAV1-JAZ8-WRKY51 Complex for Jasmonate Biosynthesis.

Insect herbivory causes severe damage to plants and threatens the world's food production. During evolutionary adaptation, plants have evolved sophisticated mechanisms to rapidly accumulate a key defense hormone, jasmonate (JA), that triggers plant defense against herbivory. However, little is known about how plants initially activate JA biosynthesis at encounter with herbivory. Here, we uncover that a novel JAV1-JAZ8-WRKY51 (JJW) complex controls JA biosynthesis to defend against insect attack. In healthy plants, the JJW complex represses JA biosynthesis to restrain JA at a low basal level to ensure proper plant growth. When plants are injured by insect attack, injury rapidly triggers calcium influxes to activate calmodulin-dependent phosphorylation of JAV1, which disintegrates JJW complex and activates JA biosynthesis, giving rise to the rapid burst of JA for plant defense. Our findings offer new insights into the highly sophisticated defense systems evolved by plants to defend against herbivory.

Tissue-specific plant toxins and adaptation in a specialist root herbivore.

In coevolution between plants and insects, reciprocal selection often leads to phenotype matching between chemical defense and herbivore offense. Nonetheless, it is not well understood whether distinct plant parts are differentially defended and how herbivores adapted to those parts cope with tissue-specific defense. Milkweed plants produce a diversity of cardenolide toxins and specialist herbivores have substitutions in their target enzyme (Na+/K+-ATPase), each playing a central role in milkweed-insect coevolution. The four-eyed milkweed beetle (Tetraopes tetrophthalmus) is an abundant toxin-sequestering herbivore that feeds exclusively on milkweed roots as larvae and less so on milkweed leaves as adults. Accordingly, we tested the tolerance of this beetle's Na+/K+-ATPase to cardenolide extracts from roots versus leaves of its main host (Asclepias syriaca), along with sequestered cardenolides from beetle tissues. We additionally purified and tested the inhibitory activity of dominant cardenolides from roots (syrioside) and leaves (glycosylated aspecioside). Tetraopes' enzyme was threefold more tolerant of root extracts and syrioside than leaf cardenolides. Nonetheless, beetle-sequestered cardenolides were more potent than those in roots, suggesting selective uptake or dependence on compartmentalization of toxins away from the beetle's enzymatic target. Because Tetraopes has two functionally validated amino acid substitutions in its Na+/K+-ATPase compared to the ancestral form in other insects, we compared its cardenolide tolerance to that of wild-type Drosophila and CRISPR-edited Drosophila with Tetraopes' Na+/K+-ATPase genotype. Those two amino acid substitutions accounted for >50% of Tetraopes' enhanced enzymatic tolerance of cardenolides. Thus, milkweed's tissue-specific expression of root toxins is matched by physiological adaptations in its specialist root herbivore.

Transgenerational phenotypic responses to herbicide stress are more rapid than to shade and simulated herbivory in Arabidopsis.

Weeds in agricultural settings continually adapt to stresses from ecological and anthropogenic sources, in some cases leading to resistant populations. However, consequences of repeated sub-lethal exposure of these stressors on fitness and stress "memory" over generations remain poorly understood. We measured plant performance over a transgenerational experiment with Arabidopsis thaliana where plants were exposed to sub-lethal stress induced by the herbicides glyphosate or trifloxysulfuron, stresses from clipping or shading in either one (G1) or four successive generations (G1-G4), and control plants that never received stress. We found that fourth-generation (G4) plants that had been subjected to three generations of glyphosate or trifloxysulfuron stress produced higher post-stress biomass, seed weight, and rosette area as compared to that produced by plants that experienced stress only in the first generation (G1). By the same measure, clipping and shade were more influential on floral development time (shade) and seed weight (clipping) but did not show responsive phenotypes for vegetative metrics after multiple generations. Overall, we found that plants exhibited more rapid transgenerational vegetative "stress memory" to herbicides while reproductive plasticity was stressor dependent and similar between clipping/shade and anthropogenic stressors. Our study suggests that maternal plant stress memory aids next-generation plants to respond and survive better under the same stressors.

Insect herbivory within modern forests is greater than fossil localities.

Fossilized leaves provide the longest running record of hyperdiverse plant-insect herbivore associations. Reconstructions of these relationships over deep time indicate strong links between environmental conditions, herbivore diversity, and feeding damage on leaves. However, herbivory has not been compared between the past and the modern era, which is characterized by intense anthropogenic environmental change. Here, we present estimates for damage frequencies and diversities on fossil leaves from the Late Cretaceous (66.8 Ma) through the Pleistocene (2.06 Ma) and compare these estimates with Recent (post-1955) leaves collected via paleobotanical methods from modern ecosystems: Harvard Forest, United States; the Smithsonian Environmental Research Center, United States; and La Selva, Costa Rica. Total damage frequency, measured as the percentage of leaves with any herbivore damage, within modern ecosystems is greater than any fossil locality within this record. This pattern is driven by increased frequencies across nearly all functional feeding groups within the Recent. Diversities of total, specialized, and mining damage types are elevated within the Recent compared with fossil floras. Our results demonstrate that plants in the modern era are experiencing unprecedented levels of insect damage, despite widespread insect declines. Human influence, such as the rate of global climate warming, influencing insect feeding and timing of life cycle processes along with urbanization and the introduction of invasive plant and insect species may drive elevated herbivory. This research suggests that the strength of human influence on plant-insect interactions is not controlled by climate change alone but rather, the way in which humans interact with terrestrial landscape.

A de novo long-read genome assembly of the sacred datura plant (Datura wrightii) reveals a role of tandem gene duplications in the evolution of herbivore-defense response.

The sacred datura plant (Solanales: Solanaceae: Datura wrightii) has been used to study plant-herbivore interactions for decades. The wealth of information that has resulted leads it to have potential as a model system for studying the ecological and evolutionary genomics of these interactions. We present a de novo Datura wrightii genome assembled using PacBio HiFi long-reads. Our assembly is highly complete and contiguous (N50 = 179Mb, BUSCO Complete = 97.6%). We successfully detected a previously documented ancient whole genome duplication using our assembly and have classified the gene duplication history that generated its coding sequence content. We use it as the basis for a genome-guided differential expression analysis to identify the induced responses of this plant to one of its specialized herbivores (Coleoptera: Chrysomelidae: Lema daturaphila). We find over 3000 differentially expressed genes associated with herbivory and that elevated expression levels of over 200 genes last for several days. We also combined our analyses to determine the role that different gene duplication categories have played in the evolution of Datura-herbivore interactions. We find that tandem duplications have expanded multiple functional groups of herbivore responsive genes with defensive functions, including UGT-glycosyltranserases, oxidoreductase enzymes, and peptidase inhibitors. Overall, our results expand our knowledge of herbivore-induced plant transcriptional responses and the evolutionary history of the underlying herbivore-response genes.

Aspen recovery in northern Yellowstone: A comment on Brice et al. (2021).

Aspen sapling recruitment increased as browsing by elk decreased, following the 1995-96 reintroduction of wolves in Yellowstone National Park. We address claims by Brice et al. (2021) that previous studies exaggerated recent aspen recovery. We conclude that their results actually supported previous work showing a trophic cascade benefiting aspen.

Does spatial variation in insect herbivory match variations in plant quality? A meta-analysis.

Variation in herbivore pressure has often been predicted from patterns in plant traits considered as antiherbivore defences. Here, we tested whether spatial variation in field insect herbivory is associated with the variation in plant quality by conducting a meta-analysis of 223 correlation coefficients between herbivory levels and the expression of selected plant traits. We found no overall correlation between herbivory and either concentrations of plant secondary metabolites or values of physical leaf traits. This result was due to both the large number of low correlations and the opposing directions of high correlations in individual studies. Field herbivory demonstrated a significant association only with nitrogen: herbivore pressure increased with an increase in nitrogen concentration in plant tissues. Thus, our meta-analysis does not support either theoretical prediction, i.e., that plants possess high antiherbivore defences in localities with high herbivore pressure or that herbivory is low in localities where plant defences are high. We conclude that information about putative plant defences is insufficient to predict plant losses to insects in field conditions and that the only bottom-up factor shaping spatial variation in insect herbivory is plant nutritive value. Our findings stress the need to improve a theory linking plant putative defences and herbivory.

De Novo Transcriptome Assembly of Cedar (Cedrela odorata L.) and Differential Gene Expression Involved in Herbivore Resistance.

Timber trees are targets of herbivorous attacks. The identification of genes associated with pest resistance can be accomplished through differential expression analysis using transcriptomes. We reported the de novo assembly of cedar (Cedrela odorata L.) transcriptome and the differential expression of genes involved in herbivore resistance. The assembly and annotation of the transcriptome were obtained using RNAseq from healthy cedar plants and those infested with Chrysobothris yucatanensis. A total of 325.6 million reads were obtained, and 127,031 (97.47%) sequences were successfully assembled. A total of 220 herbivory-related genes were detected, of which 170 genes were annotated using GO terms, and 161 genes with 245 functions were identified-165, 75, and 5 were molecular functions, biological processes, and cellular components, respectively. To protect against herbivorous infestation, trees produce toxins and volatile compounds which are modulated by signaling pathways and gene expression related to molecular functions and biological processes. The limited number of genes identified as cellular components suggests that there are minimal alterations in cellular structure in response to borer attack. The chitin recognition protein, jasmonate ZIM-domain (JAZ) motifs, and response regulator receiver domain were found to be overexpressed, whereas the terpene synthase, cytochrome P450, and protein kinase domain gene families were underexpressed. This is the first report of a cedar transcriptome focusing on genes that are overexpressed in healthy plants and underexpressed in infested plants. This method may be a viable option for identifying genes associated with herbivore resistance.

Secret Weapon of Insects: The Oral Secretion Cocktail and Its Modulation of Host Immunity.

Plants and insects have co-existed for almost 400 million years and their interactions can be beneficial or harmful, thus reflecting their intricate co-evolutionary dynamics. Many herbivorous arthropods cause tremendous crop loss, impacting the agro-economy worldwide. Plants possess an arsenal of chemical defenses that comprise diverse secondary metabolites that help protect against harmful herbivorous arthropods. In response, the strategies that herbivores use to cope with plant defenses can be behavioral, or molecular and/or biochemical of which salivary secretions are a key determinant. Insect salivary secretions/oral secretions (OSs) play a crucial role in plant immunity as they contain several biologically active elicitors and effector proteins that modulate plants' defense responses. Using this oral secretion cocktail, insects overcome plant natural defenses to allow successful feeding. However, a lack of knowledge of the nature of the signals present in oral secretion cocktails has resulted in reduced mechanistic knowledge of their cellular perception. In this review, we discuss the latest knowledge on herbivore oral secretion derived elicitors and effectors and various mechanisms involved in plant defense modulation. Identification of novel herbivore-released molecules and their plant targets should pave the way for understanding the intricate strategies employed by both herbivorous arthropods and plants in their interactions.

Interactive Effects of Temperature, Aridity, and Plant Stoichiometry on Insect Herbivory: Past and Present.

AbstractThe influence of climate on deep-time plant-insect interactions is becoming increasingly well known, with temperature, CO2 increases (and associated stoichiometric changes in plants), and aridity likely playing a critical role. In our modern climate, all three factors are shifting at an unprecedented rate, with uncertain consequences for biodiversity. To investigate effects of temperature, stoichiometry (specifically that of nitrogen), and aridity on insect herbivory, we explored insect herbivory in three modern floral assemblages and in 39 fossil floras, especially focusing on eight floras around a past hyperthermal event (the Paleocene-Eocene Thermal Maximum) from Bighorn Basin (BB). We find that higher temperatures were associated with increased herbivory in the past, especially among BB sites. In these BB sites, non-N2-fixing plants experienced a lower richness but higher frequency of herbivory damage than N2-fixing plants. Herbivory frequency but not richness was greater in BB sites compared with contemporaneous, nearby, but less arid sites from Hanna Basin. Compared with deep-time environments, herbivory frequency and richness are higher in modern sites, suggesting that current accelerated warming uniquely impacts plant-insect interactions. Overall, our work addresses multiple aspects of climate change using fossil data while also contextualizing the impact of modern anthropogenic change on Earth's most diverse interactions.

Trichomes and unique gene expression confer insect herbivory resistance in Vitis labrusca grapevines.

BACKGROUND: Grapevine (Vitis) is one of the world's most valuable fruit crops, but insect herbivory can decrease yields. Understanding insect herbivory resistance is critical to mitigating these losses. Vitis labrusca, a wild North American grapevine species, has been leveraged in breeding programs to generate hybrid grapevines with enhanced abiotic and biotic stress resistance, rendering it a valuable genetic resource for sustainable viticulture. This study assessed the resistance of V. labrusca acc. 'GREM4' and Vitis vinifera cv. 'PN40024' grapevines to Popillia japonica (Japanese beetle) herbivory and identified morphological and genetic adaptations underlying this putative resistance. RESULTS: 'GREM4' displayed greater resistance to beetle herbivory compared to 'PN40024' in both choice and no-choice herbivory assays spanning periods of 30 min to 19 h. 'GREM4' had significantly higher average leaf trichome densities than 'PN40024' and beetles preferred to feed on the side of leaves with fewer trichomes. When leaves from each species that specifically did not differ in trichome densities were fed on by beetles, significantly less leaf area was damaged in 'GREM4' (3.29mm2) compared to 'PN40024' (9.80mm2), suggesting additional factors beyond trichomes contributed to insect herbivory resistance in 'GREM4'. Comparative transcriptomic analyses revealed 'GREM4' exhibited greater constitutive (0 h) expression of defense response and secondary metabolite biosynthesis genes compared to 'PN40024', indicative of heightened constitutive defenses. Upon herbivory, 'GREM4' displayed a greater number of differentially expressed genes (690) compared to 'PN40024' (502), suggesting a broader response. Genes up-regulated in 'GREM4' were enriched in terpene biosynthesis, flavonoid biosynthesis, phytohormone signaling, and disease defense-related functions, likely contributing to heighted insect herbivory defense, while genes differentially expressed in 'PN40024' under herbivory were enriched in xyloglucan, cell wall formation, and calcium ion binding. The majority of genes implicated in insect herbivory defense were orthologs with specific expression patterns in 'GREM4' and 'PN40024', but some paralogous and genome-specific genes also likely contributed to conferring resistance. CONCLUSIONS: Our findings suggest that 'GREM4' insect herbivory resistance was attributed to a combination of factors, including trichomes and unique constitutive and inducible expression of genes implicated in terpene, flavonoid, and phenylpropanoid biosynthesis, as well as pathogen defense.

Artificial light at night and warming impact grazing rates and gonad index of the sea urchin Centrostephanus rodgersii.

Artificial light at night (ALAN) is a growing threat to coastal habitats, and is likely to exacerbate the impacts of other stressors. Kelp forests are dominant habitats on temperate reefs but are declining due to ocean warming and overgrazing. We tested the independent and interactive effects of ALAN (dark versus ALAN) and warming (ambient versus warm) on grazing rates and gonad index of the sea urchin Centrostephanus rodgersii. Within these treatments, urchins were fed either 'fresh' kelp or 'treated' kelp. Treated kelp (Ecklonia radiata) was exposed to the same light and temperature combinations as urchins. We assessed photosynthetic yield, carbon and nitrogen content and C : N ratio of treated kelp to help identify potential drivers behind any effects on urchins. Grazing increased with warming and ALAN for urchins fed fresh kelp, and increased with warming for urchins fed treated kelp. Gonad index was higher in ALAN/ambient and dark/warm treatments compared to dark/ambient treatments for urchins fed fresh kelp. Kelp carbon content was higher in ALAN/ambient treatments than ALAN/warm treatments at one time point. This indicates ocean warming and ALAN may increase urchin grazing pressure on rocky reefs, an important finding for management strategies.

Alarm calls of sagebrush converge when herbivory is high.

Herbivory is a major threat to virtually all plants, so adaptations to avoid herbivory will generally be selected. One potential adaptation is the ability to 'listen in' on the volatile cues emitted by plants that are experiencing herbivory and to then respond by ramping up defences. The nature of these volatile cues is poorly understood. Sagebrush (Artemisia tridentata) plants that were exposed to cues of experimentally damaged neighbours experienced less herbivory; this induction was most effective if emitter and receiver plants had similar volatile emission profiles, termed chemotypes. Previously, we observed that sagebrush populations that were in locations with high herbivory exhibited little diversity of volatiles compared to populations with low herbivory. Several hypotheses could produce this correlation. High risk of herbivory could have selected for individuals that converged on a common 'alarm cue' that all individuals would respond to. In this case, individuals of locally rare chemotypes that were less able to eavesdrop would experience more damage than common chemotypes when herbivores were abundant. Alternatively, low chemotypic diversity could allow higher levels of damage to plants. In this case, rare chemotypes would experience less damage than common chemotypes. We examined the chemotypes of sagebrush individuals from multiple sites and found that rare chemotypes experienced more damage than common chemotypes when herbivores were abundant. This pattern was seen among sites and among years with different densities of herbivores. This result is consistent with the hypothesis that herbivory selects for individuals that are effective communicators and shapes the communication system.

Neutrality in plant-herbivore interactions.

Understanding the distribution of herbivore damage among leaves and individual plants is a central goal of plant-herbivore biology. Commonly observed unequal patterns of herbivore damage have conventionally been attributed to the heterogeneity in plant quality or herbivore behaviour or distribution. Meanwhile, the potential role of stochastic processes in structuring plant-herbivore interactions has been overlooked. Here, we show that based on simple first principle expectations from metabolic theory, random sampling of different sizes of herbivores from a regional pool is sufficient to explain patterns of variation in herbivore damage. This is despite making the neutral assumption that herbivory is caused by randomly feeding herbivores on identical and passive plants. We then compared its predictions against 765 datasets of herbivory on 496 species across 116° of latitude from the Herbivory Variability Network. Using only one free parameter, the estimated attack rate, our neutral model approximates the observed frequency distribution of herbivore damage among plants and especially among leaves very well. Our results suggest that neutral stochastic processes play a large and underappreciated role in natural variation in herbivory and may explain the low predictability of herbivory patterns. We argue that such prominence warrants its consideration as a powerful force in plant-herbivore interactions.

Butterfly eggs prime anti-herbivore defense in an annual but not perennial Arabidopsis species.

MAIN CONCLUSION: Unlike Arabidopsis thaliana, defenses of Arabidopsis lyrata against Pieris brassicae larval feeding are not primable by P. brassicae eggs. Thus, egg primability of plant anti-herbivore defenses is not phylogenetically conserved in the genus Arabidopsis. While plant anti-herbivore defenses of the annual species Arabidopsis thaliana were shown to be primable by Pieris brassicae eggs, the primability of the phylogenetically closely related perennial Arabidopsis lyrata has not yet been investigated. Previous studies revealed that closely related wild Brassicaceae plant species, the annual Brassica nigra and the perennial B. oleracea, exhibit an egg-primable defense trait, even though they have different life spans. Here, we tested whether P. brassicae eggs prime anti-herbivore defenses of the perennial A. lyrata. We exposed A. lyrata to P. brassicae eggs and larval feeding and assessed their primability by (i) determining the biomass of P. brassicae larvae after feeding on plants with and without prior P. brassicae egg deposition and (ii) investigating the plant transcriptomic response after egg deposition and/or larval feeding. For comparison, these studies were also conducted with A. thaliana. Consistent with previous findings, A. thaliana's response to prior P. brassicae egg deposition negatively affected conspecific larvae feeding upon A. thaliana. However, this was not observed in A. lyrata. Arabidopsis thaliana responded to P. brassicae eggs with strong transcriptional reprogramming, whereas A. lyrata responses to eggs were negligible. In response to larval feeding, A. lyrata exhibited a greater transcriptome change compared to A. thaliana. Among the strongly feeding-induced A. lyrata genes were those that are egg-primed in feeding-induced A. thaliana, i.e., CAX3, PR1, PR5, and PDF1.4. These results suggest that A. lyrata has evolved a robust feeding response that is independent from prior egg exposure.

Reassessing the alternative ecosystem states proposition in the African savanna-forest domain.

Ecologists are being challenged to predict how ecosystems will respond to climate changes. According to the Multi-Colored World (MCW) hypothesis, climate impacts may not manifest because consumers such as fire and herbivory can override the influence of climate on ecosystem state. One MCW interpretation is that climate determinism fails because alternative ecosystem states (AES) are possible at some locations in climate space. We evaluated theoretical and empirical evidence for the proposition that forest and savanna are AES in Africa. We found that maps which infer where AES zones are located were contradictory. Moreover, data from longitudinal and experimental studies provide inconclusive evidence for AES. That is, although the forest-savanna AES proposition is theoretically sound, the existing evidence is not yet convincing. We conclude by making the case that the AES proposition has such fundamental consequences for designing management actions to mitigate and adapt to climate change in the savanna-forest domain that it needs a more robust evidence base before it is used to prescribe management actions.

Middle Jurassic insect mines on gymnosperms provide missing links to early mining evolution.

We investigated the mining mode of insect feeding, involving larval consumption of a plant's internal tissues, from the Middle Jurassic (165 million years ago) Daohugou locality of Northeastern China. Documentation of mining from the Jurassic Period is virtually unknown, and results from this time interval would address mining evolution during the temporal gap of mine-seed plant diversifications from the previous Late Triassic to the subsequent Early Cretaceous. Plant fossils were examined with standard microscopic procedures for herbivory and used the standard functional feeding group-damage-type system of categorizing damage. All fossil mines were photographed and databased. We examined 2014 plant specimens, of which 27 occurrences on 14 specimens resulted in eight, new, mine damage types (DTs) present on six genera of bennettitalean, ginkgoalean, and pinalean gymnosperms. Three conclusions emerge from this study. First, these mid-Mesozoic mines are morphologically conservative and track plant host anatomical structure rather than plant phylogeny. Second, likely insect fabricators of these mines were three basal lineages of polyphagan beetles, four basal lineages of monotrysian moths, and a basal lineage tenthredinoid sawflies. Third, the nutrition hypothesis, indicating that miners had greater access to nutritious, inner tissues of new plant lineages, best explains mine evolution during the mid-Mesozoic.

Cardiac glycosides protect wormseed wallflower (Erysimum cheiranthoides) against some, but not all, glucosinolate-adapted herbivores.

The chemical arms race between plants and insects is foundational to the generation and maintenance of biological diversity. We asked how the evolution of a novel defensive compound in an already well-defended plant lineage impacts interactions with diverse herbivores. Erysimum cheiranthoides (Brassicaceae), which produces both ancestral glucosinolates and novel cardiac glycosides, served as a model. We analyzed gene expression to identify cardiac glycoside biosynthetic enzymes in E. cheiranthoides and characterized these enzymes via heterologous expression and CRISPR/Cas9 knockout. Using E. cheiranthoides cardiac glycoside-deficient lines, we conducted insect experiments in both the laboratory and field. EcCYP87A126 initiates cardiac glycoside biosynthesis via sterol side-chain cleavage, and EcCYP716A418 has a role in cardiac glycoside hydroxylation. In EcCYP87A126 knockout lines, cardiac glycoside production was eliminated. Laboratory experiments with these lines revealed that cardiac glycosides were highly effective defenses against two species of glucosinolate-tolerant specialist herbivores, but did not protect against all crucifer-feeding specialist herbivores in the field. Cardiac glycosides had lesser to no effect on two broad generalist herbivores. These results begin elucidation of the E. cheiranthoides cardiac glycoside biosynthetic pathway and demonstrate in vivo that cardiac glycoside production allows Erysimum to escape from some, but not all, specialist herbivores.

Legacy effects of premature defoliation in response to an extreme drought event modulate phytochemical profiles with subtle consequences for leaf herbivory in European beech.

Extreme droughts can have long-lasting effects on forest community dynamics and species interactions. Yet, our understanding of how drought legacy modulates ecological relationships is just unfolding. We tested the hypothesis that leaf chemistry and herbivory show long-term responses to premature defoliation caused by an extreme drought event in European beech (Fagus sylvatica L.). For two consecutive years after the extreme European summer drought in 2018, we collected leaves from the upper and lower canopy of adjacently growing drought-stressed and unstressed trees. Leaf chemistry was analyzed and leaf damage by different herbivore-feeding guilds was quantified. We found that drought had lasting impacts on leaf nutrients and on specialized metabolomic profiles. However, drought did not affect the primary metabolome. Drought-related phytochemical changes affected damage of leaf-chewing herbivores whereas damage caused by other herbivore-feeding guilds was largely unaffected. Drought legacy effects on phytochemistry and herbivory were often weaker than between-year or between-canopy strata variability. Our findings suggest that a single extreme drought event bears the potential to long-lastingly affect tree-herbivore interactions. Drought legacy effects likely become more important in modulating tree-herbivore interactions since drought frequency and severity are projected to globally increase in the coming decades.