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The western corn rootworm (WCR) decimates maize crops worldwide. One potential way to control this pest is treatment with entomopathogenic nematodes (EPNs) that harbor bacterial symbionts that are pathogenic to insects. However, WCR larvae sequester benzoxazinoid secondary metabolites that are produced by maize and use them to increase their resistance to the nematodes and their symbionts. Here we report that experimental evolution and selection for bacterial symbionts that are resistant to benzoxazinoids improve the ability of a nematode–symbiont pair to kill WCR larvae. We isolated five Photorhabdus symbionts from different nematodes and increased their benzoxazinoid resistance through experimental evolution. Benzoxazinoid resistance evolved through multiple mechanisms, including a mutation in the aquaporin-like channel gene aqpZ. We reintroduced benzoxazinoid-resistant Photorhabdus strains into their original EPN hosts and identified one nematode–symbiont pair that was able to kill benzoxazinoid-sequestering WCR larvae more efficiently. Our results suggest that modification of bacterial symbionts might provide a generalizable strategy to improve biocontrol of agricultural pests.Nematode bacterial symbionts were evolved in vitro to resist insect-sequestered plant toxins to improve the insect-killing efficacy of their nematode host.

Plant domestication often alters plant traits, including chemical and physical defenses against herbivores. In squash, domestication leads to reduced levels of cucurbitacins and leaf trichomes, influencing interactions with insects. However, the impact of domestication on inducible defenses in squash remains poorly understood. Here, we investigated the chemical and physical defensive traits of wild and domesticated squash (Cucurbita argyrosperma), and compared their responses to belowground and aboveground infestation by the root-feeding larvae and the leaf-chewing adults of the banded cucumber beetle Diabrotica balteata (Coleoptera: Chrysomelidae). Wild populations contained cucurbitacins in roots and cotyledons but not in leaves, whereas domesticated varieties lacked cucurbitacins in all tissues. Belowground infestation by D. balteata larvae did not increase cucurbitacin levels in the roots but triggered the expression of cucurbitacin biosynthetic genes, irrespective of domestication status, although the response varied among different varieties. Conversely, whereas wild squash had more leaf trichomes than domesticated varieties, the induction of leaf trichomes in response to herbivory was greater in domesticated plants. Leaf herbivory varied among varieties but there was a trend of higher leaf damage on wild squash than domesticated varieties. Overall, squash plants responded to both belowground and aboveground herbivory by activating chemical defense-associated gene expression in roots and upregulating their physical defense in leaves, respectively. While domestication suppressed both chemical and physical defenses, our findings suggest that it may enhance inducible defense mechanisms by increasing trichome induction in response to herbivory.

The outcome of plant-mediated interactions among herbivores from several feeding guilds has been studied intensively. However, our understanding on the effects of nematode root herbivory on leaf miner oviposition behavior and performance remain limited. In this study, we evaluated whether Meloidogyne incognita root herbivory affects Tuta absoluta oviposition preference on Solanum lycopersicum plants and the development of the resulting offspring. To investigate the M. incognita-herbivory induced plant systemic responses that might explain the observed biological effects, we measured photosynthetic rates, leaf trypsin protease inhibitor activities, and analyzed the profile of volatiles emitted by the leaves of root-infested and non-infested plants. We found that T. absoluta females avoided laying eggs on the leaves of root-infested plants, and that root infestation negatively affected the pupation process of T. absoluta. These effects were accompanied by a strong suppression of leaf volatile emissions, a decrease in photosynthetic rates, and an increase in the activity of leaf trypsin protease inhibitors. Our study reveals that root attack by nematodes can shape leaf physiology, and thereby increases plant resistance.

Plant invertases are sucrolytic enzymes that are essential for the regulation of carbohydrate metabolism and source–sink relationships. While their activity has been well documented during abiotic and biotic stresses, the role of proteinaceous invertase inhibitors in regulating these changes is unknown. Here, we identify a putative Nicotiana attenuata cell wall invertase inhibitor (NaCWII) which is strongly up-regulated in a jasmonate (JA)-dependent manner following simulated attack by the specialist herbivore Manduca sexta. To understand the role of NaCWII in planta, we silenced its expression by RNA interference and measured changes in primary and secondary metabolism and plant growth following simulated herbivory. NaCWII-silenced plants displayed a stronger depletion of carbohydrates and a reduced capacity to increase secondary metabolite pools relative to their empty vector control counterparts. This coincided with the attenuation of herbivore-induced CWI inhibition and growth suppression characteristic of wild-type plants. Together our findings suggest that NaCWII may act as a regulatory switch located downstream of JA accumulation which fine-tunes the plant's balance between growth and defense metabolism under herbivore attack. Although carbohydrates are not typically viewed as key factors in plant growth and defense, our study shows that interfering with their catabolism strongly influences plant responses to herbivory.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Insect herbivores use different cues to locate host plants. The importance of CO 2 in this context is not well understood. We manipulated CO 2 perception in western corn rootworm (WCR) larvae through RNAi and studied how CO 2 perception impacts their interaction with their host plant. The expression of a carbon dioxide receptor, DvvGr2 , is specifically required for dose-dependent larval responses to CO 2 . Silencing CO 2 perception or scrubbing plant-associated CO 2 has no effect on the ability of WCR larvae to locate host plants at short distances (<9 cm), but impairs host location at greater distances. WCR larvae preferentially orient and prefer plants that grow in well-fertilized soils compared to plants that grow in nutrient-poor soils, a behaviour that has direct consequences for larval growth and depends on the ability of the larvae to perceive root-emitted CO 2 . This study unravels how CO 2 can mediate plant–herbivore interactions by serving as a distance-dependent host location cue. Living deep in the ground and surrounded by darkness, soil insects must rely on the chemicals released by plants to find the roots they feed on. Carbon dioxide, for example, is a by-product of plant respiration, which, above ground, is thought to attract moths to flowers and flies to apples; underground, however, its role is still unclear. This gaseous compound can travel through soil and potentially act as a compass for root-eating insects. Yet, it is also produced by decaying plants or animals, which are not edible. It is therefore possible that insects use this signal as a long-range cue to orient themselves, but then switch to another chemical when closer to their target to narrow in on an actual food source. To test this idea, Arce et al. investigated whether carbon dioxide guides the larvae of Western corn rootworm to maize roots. First, the rootworm genes responsible for sensing carbon dioxide were identified and switched off, making the larvae unable to detect this gas. When the genetically engineered rootworms were further than 9cm from maize roots, they were less able to locate that food source; closer to the roots, however, the insects could orient themselves towards the plant. This suggests that the insects use carbon dioxide at long distances but rely on another chemicals to narrow down their search at close range. To confirm this finding, Arce et al. tried absorbing the carbon dioxide using soda lime, leading to similar effects: carbon dioxide sensitive insects stopped detecting the roots at long but not short distances. Additional experiments then revealed that the compound could help insects find the best roots to feed on. Indeed, eating plants that grow on rich terrain – for instance, fertilized soils – helps insects to grow bigger and faster. These roots also release more carbon dioxide, in turn attracting rootworms more frequently. In the United States and Eastern Europe, Western corn rootworms inflict major damage to crops, highlighting the need to understand and manage the link between fertilization regimes, carbon dioxide release and how these pests find their food.

Plant responses to herbivore attack are regulated by phytohormonal networks. To date, the role of the auxin indole-3-acetic acid (IAA) in this context is not well understood. We quantified and manipulated the spatiotemporal patterns of IAA accumulation in herbivore-attacked Nicotiana attenuata plants to unravel its role in the regulation of plant secondary metabolism. We found that IAA is strongly, rapidly, and specifically induced by herbivore attack. IAA is elicited by herbivore oral secretions and fatty acid conjugate elicitors and is accompanied by a rapid transcriptional increase of auxin biosynthetic YUCCA-like genes. IAA accumulation starts 30 to 60 s after local induction and peaks within 5 min after induction, thereby preceding the jasmonate (JA) burst. IAA accumulation does not require JA signaling and spreads rapidly from the wound site to systemic tissues. Complementation and transport inhibition experiments reveal that IAA is required for the herbivore-specific, JA-dependent accumulation of anthocyanins and phenolamides in the stems. In contrast, IAA does not affect the accumulation of nicotine or 7-hydroxygeranyllinalool diterpene glycosides in the same tissue. Taken together, our results uncover IAA as a rapid and specific signal that regulates a subset of systemic, JA-dependent secondary metabolites in herbivore-attacked plants.

Plants defend themselves against herbivores through the production of toxic and deterrent metabolites. Adapted herbivores can tolerate and sometimes sequester these metabolites, allowing them to feed on defended plants and become toxic to their own enemies. Can herbivore natural enemies overcome sequestered plant defense metabolites to prey on adapted herbivores? To address this question, we studied how entomopathogenic nematodes cope with benzoxazinoid defense metabolites that are produced by grasses and sequestered by a specialist maize herbivore, the western corn rootworm. We find that nematodes from US maize fields in regions in which the western corn rootworm was present over the last 50 y are behaviorally and metabolically resistant to sequestered benzoxazinoids and more infective toward the western corn rootworm than nematodes from other parts of the world. Exposure of a benzoxazinoid-susceptible nematode strain to the western corn rootworm for 5 generations results in higher behavioral and metabolic resistance and benzoxazinoid-dependent infectivity toward the western corn rootworm. Thus, herbivores that are exposed to a plant defense sequestering herbivore can evolve both behavioral and metabolic resistance to plant defense metabolites, and these traits are associated with higher infectivity toward a defense sequestering herbivore. We conclude that plant defense metabolites that are transferred through adapted herbivores may result in the evolution of resistance in herbivore natural enemies. Our study also identifies plant defense resistance as a potential target for the improvement of biological control agents.

Jasmonates regulate plant secondary metabolism and herbivore resistance. How they influence primary metabolites and how this may affect herbivore growth and performance are not well understood. We profiled sugars and starch of jasmonate biosynthesis-deficient and jasmonate-insensitive Nicotiana attenuata plants and manipulated leaf carbohydrates through genetic engineering and in vitro complementation to assess how jasmonate-dependent sugar accumulation affects the growth of Manduca sexta caterpillars. We found that jasmonates reduce the constitutive and herbivore-induced concentration of glucose and fructose in the leaves across different developmental stages. Diurnal, jasmonate-dependent inhibition of invertase activity was identified as a likely mechanism for this phenomenon. Contrary to our expectation, both in planta and in vitro approaches showed that the lower sugar concentrations led to increased M. sexta growth. As a consequence, jasmonate-dependent depletion of sugars rendered N. attenuata plants more susceptible to M. sexta attack. In conclusion, jasmonates are important regulators of leaf carbohydrate accumulation and this determines herbivore growth. Jasmonate-dependent resistance is reduced rather than enhanced through the suppression of glucose and fructose concentrations, which may contribute to the evolution of divergent resistance strategies of plants in nature.

Natural enemies of herbivores are expected to adapt to the defence strategies of their preys or hosts. Such adaptations may also include their capacity to cope with plant metabolites that herbivores sequester as a defence. In this study, we evaluated the ability of Mexican entomopathogenic nematodes (EPN) to resist benzoxazinoids that are sequestered from maize roots by the western corn rootworm (WCR, Diabrotica virgifera virgifera; Coleoptera: Chrysomelidae), an important maize pest in America and Europe. From maize fields throughout Mexico, we retrieved 40 EPN isolates belonging to five different species, with a majority identified as Heterorhabditis bacteriophora . In the laboratory, all nematodes readily infected non-sequestering larvae of the banded cucumber beetle ( D. balteata ), while infectivity varied strongly for WCR larvae. While some H. bacteriophora isolates seemed negatively affected by benzoxazinoids, most showed to be resistant. Thus, EPN from Mexican maize fields can cope with these plant defence metabolites, but the results also indicate that WCR larvae possess other mechanisms that help to resist EPN. This work contributes to a better understanding of the capacity of herbivore natural enemies to resist plant defence metabolites. Furthermore, it identifies several benzoxazinoid-resistant EPN isolates that may be used to control this important maize pest.