Metabolism of plant-derived toxins from its insect host increases the success of the entomopathogenic fungus Beauveria bassiana.

Beauveria bassiana is a soil fungus that parasitizes a large number of arthropod species, including numerous crop pests, causing white muscardine disease and is therefore used as a biological insecticide. However, some insects, such as the cabbage aphid (Brevicoryne brassicae), defend themselves chemically by sequestering dietary pro-toxins (glucosinolates) from their Brassicales host plants. Glucosinolates are accumulated by cabbage aphids and activated to form toxic isothiocyanates when under attack. While isothiocyanate formation protects aphids against most attackers, B. bassiana is still able to infect the cabbage aphid under natural conditions. We therefore investigated how this fungus is able to circumvent the chemical defense system of the cabbage aphid. Here, we describe how B. bassiana infection activates the cabbage aphid defense system, but the resulting toxins are metabolized by B. bassiana via the mercapturic acid pathway, of which the first step is catalyzed by glutathione-S-transferases of low substrate specificity. This detoxification pathway enhances B. bassiana growth when isothiocyanates are present in natural concentrations, and so appears to be an important factor in fungal parasitization of these chemically defended aphids.

Adaptations of Pseudoxylaria towards a comb-associated lifestyle in fungus-farming termite colonies.

Characterizing ancient clades of fungal symbionts is necessary for understanding the evolutionary process underlying symbiosis development. In this study, we investigated a distinct subgeneric taxon of Xylaria (Xylariaceae), named Pseudoxylaria, whose members have solely been isolated from the fungus garden of farming termites. Pseudoxylaria are inconspicuously present in active fungus gardens of termite colonies and only emerge in the form of vegetative stromata, when the fungus comb is no longer attended ("sit and wait" strategy). Insights into the genomic and metabolic consequences of their association, however, have remained sparse. Capitalizing on viable Pseudoxylaria cultures from different termite colonies, we obtained genomes of seven and transcriptomes of two Pseudoxylaria isolates. Using a whole-genome-based comparison with free-living members of the genus Xylaria, we document that the association has been accompanied by significant reductions in genome size, protein-coding gene content, and reduced functional capacities related to oxidative lignin degradation, oxidative stress responses and secondary metabolite production. Functional studies based on growth assays and fungus-fungus co-cultivations, coupled with isotope fractionation analysis, showed that Pseudoxylaria only moderately antagonizes growth of the termite food fungus Termitomyces, and instead extracts nutrients from the food fungus biomass for its own growth. We also uncovered that Pseudoxylaria is still capable of producing structurally unique metabolites, which was exemplified by the isolation of two novel metabolites, and that the natural product repertoire correlated with antimicrobial and insect antifeedant activity.

Insights into the Metabolomic Capacity of Podaxis and Isolation of Podaxisterols A-D, Ergosterol Derivatives Carrying Nitrosyl Cyanide-Derived Modifications.

Cultures of a termite-associated and a free-living member of the fungal genus Podaxis, revived from spores maintained in century-old herbarium collections, were analyzed for their insecticidal and antimicrobial effects. Their secondary metabolomes were explored to uncover possible adaptive mechanisms of termite association, and dereplication of LC-HRMS/MS data sets led to the isolation of podaxisterols A-D (1-4), modified ergosterol derivatives that result from a Diels-Alder reaction with endogenous nitrosyl cyanide. Chemical structures were determined based on HRMS/MS and NMR analyses as well as X-ray crystallography. The putative origin of the endogenous fungal nitrosyl cyanide and ergosterol derivatives is discussed based on results obtained from stable isotope experiments and in silico analysis. Our "omics"-driven analysis of this underexplored yet worldwide distributed fungal genus builds a foundation for studies on a potential metabolic adaptations to diverse lifestyles.

Alternative transcript splicing regulates UDP-glucosyltransferase-catalyzed detoxification of DIMBOA in the fall armyworm (Spodoptera frugiperda).

Herbivorous insects often possess the ability to detoxify chemical defenses from their host plants. The fall armyworm (Spodoptera frugiperda), which feeds principally on maize, detoxifies the maize benzoxazinoid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by stereoselective re-glucosylation using a UDP-glucosyltransferase, SfUGT33F28. SfUGT33F28 activity is induced by feeding on a DIMBOA-containing diet, but how this induction is regulated is unknown. In the present work, we describe the alternative splicing of the SfUGT33F28 transcript. Variant transcripts are differentially expressed in response to DIMBOA, and this transcriptional response is mediated by an insect aryl hydrocarbon receptor. These variants have large deletions leading to the production of truncated proteins that have no intrinsic UGT activity with DIMBOA but interact with the full-length enzyme to raise or lower its activity. Therefore, the formation of SfUGT33F28 splice variants induces DIMBOA-conjugating UGT activity when DIMBOA is present in the insect diet and represses activity in the absence of this plant defense compound.

Chemical Modification of Biomarkers through Accelerated Degradation: Implications for Ancient Plant Identification in Archaeo-Organic Residues.

Biochemical and biomolecular archaeology is increasingly used to elucidate the consumption, use, origin, and trade of plants in the past. However, it can be challenging to use biomarkers to identify the taxonomic origin of archaeological plants due to limited knowledge of molecular survival and degradation for many key plant compounds in archaeological contexts. To gain a fundamental understanding of the chemical alterations associated with chemical degradation processes in ancient samples, we conducted accelerated degradation experiments with essential oil derived from cedar (Cedrus atlantica) exposed to materials commonly found in the archaeological record. Using GC-MS and multivariate analysis, we detected a total of 102 compounds across 19 treatments that were classified into three groups. The first group comprised compounds that were abundant in fresh cedar oil but would be unlikely to remain in ancient residues due to rapid degradation. The second group consisted of compounds that remained relatively stable or increased over time, which could be potential biomarkers for identifying cedar in archaeological residues. Compounds in the third group were absent in fresh cedar oil but were formed during specific experiments that could be indicative for certain storage conditions. These results show that caution is warranted for applying biomolecular profiles of fresh plants to ancient samples and that carefully designed accelerated degradation experiments can, at least in part, overcome this limitation.

Identification of a Sulfatase that Detoxifies Glucosinolates in the Phloem-Feeding Insect Bemisia tabaci and Prefers Indolic Glucosinolates.

Cruciferous plants in the order Brassicales defend themselves from herbivory using glucosinolates: sulfur-containing pro-toxic metabolites that are activated by hydrolysis to form compounds, such as isothiocyanates, which are toxic to insects and other organisms. Some herbivores are known to circumvent glucosinolate activation with glucosinolate sulfatases (GSSs), enzymes that convert glucosinolates into inactive desulfoglucosinolates. This strategy is a major glucosinolate detoxification pathway in a phloem-feeding insect, the silverleaf whitefly Bemisia tabaci, a serious agricultural pest of cruciferous vegetables. In this study, we identified and characterized an enzyme responsible for glucosinolate desulfation in the globally distributed B. tabaci species MEAM1. In in vitro assays, this sulfatase showed a clear preference for indolic glucosinolates compared with aliphatic glucosinolates, consistent with the greater representation of desulfated indolic glucosinolates in honeydew. B. tabaci might use this detoxification strategy specifically against indolic glucosinolates since plants may preferentially deploy indolic glucosinolates against phloem-feeding insects. In vivo silencing of the expression of the B. tabaci GSS gene via RNA interference led to lower levels of desulfoglucosinolates in honeydew. Our findings expand the knowledge on the biochemistry of glucosinolate detoxification in phloem-feeding insects and suggest how detoxification pathways might facilitate plant colonization in a generalist herbivore.

The Fall Armyworm Spodoptera frugiperda Utilizes Specific UDP-Glycosyltransferases to Inactivate Maize Defensive Benzoxazinoids.

The relationship between plants and insects is continuously evolving, and many insects rely on biochemical strategies to mitigate the effects of toxic chemicals in their food plants, allowing them to feed on well-defended plants. Spodoptera frugiperda, the fall armyworm (FAW), accepts a number of plants as hosts, and has particular success on plants of the Poaceae family such as maize, despite their benzoxazinoid (BXD) defenses. BXDs stored as inert glucosides are converted into toxic aglucones by plant glucosidases upon herbivory. DIMBOA, the main BXD aglucone released by maize leaves, can be stereoselectively re-glucosylated by UDP-glycosyltransferases (UGTs) in the insect gut, rendering it non-toxic. Here, we identify UGTs involved in BXD detoxification by FAW larvae and examine how RNAi-mediated manipulation of the larval glucosylation capacity toward the major maize BXD, DIMBOA, affects larval growth. Our findings highlight the involvement of members of two major UGT families, UGT33 and UGT40, in the glycosylation of BXDs. Most of the BXD excretion in the frass occurs in the form of glucosylated products. Furthermore, the DIMBOA-associated activity was enriched in the gut tissue, with a single conserved UGT33 enzyme (SfUGT33F28) being dedicated to DIMBOA re-glucosylation in the FAW gut. The knock-down of its encoding gene reduces larval performance in a strain-specific manner. This study thus reveals that a single UGT enzyme is responsible for detoxification of the major maize-defensive BXD in this pest insect.

Plant Defensive ß-Glucosidases Resist Digestion and Sustain Activity in the Gut of a Lepidopteran Herbivore.

Two-component activated chemical defenses are a major part of many plants' strategies to disrupt herbivory. The activation step is often the ß-glucosidase-catalyzed removal of a glucose moiety from a pro-toxin, leading to an unstable and toxic aglycone. While some ß-glucosidases have been well studied, several aspects of their roles in vivo, such as their precise sites of enzymatic activity during and after ingestion, and the importance of particular isoforms in plant defense are still not fully understood. Here, plant defensive ß-glucosidases from maize, white mustard and almonds were shown to resist digestion by larvae of the generalist lepidopteran Spodoptera littoralis, and the majority of the ingested activities toward both general and plant pro-toxic substrates was recovered in the frass. Among other proteins potentially involved in defense, we identified specific plant ß-glucosidases and a maize ß-glucosidase aggregating factor in frass from plant-fed insects using proteomic methods. We therefore found that, while S. littoralis larvae efficiently degraded bulk food protein during digestion, ß-glucosidases were among a small number of plant defensive proteins that resist insect digestive proteolysis. These enzymes remain intact in the gut lumen and frass and can therefore further catalyze the activation of plant defenses after ingestion, especially in pH-neutral regions of the digestive system. As most of the ingested enzymatic activity persists in the frass, and only particular ß-glucosidases were detected via proteomic analyses, our data support the involvement of specific isoforms (maize ZmGlu1 and S. alba MA1 myrosinase) in defense in vivo.

ZEITLUPE in the Roots of Wild Tobacco Regulates Jasmonate-Mediated Nicotine Biosynthesis and Resistance to a Generalist Herbivore.

The jasmonate (JA) phytohormone signaling system is an important mediator of plant defense against herbivores. Plants deficient in JA signaling are more susceptible to herbivory as a result of deficiencies in defensive trait expression. Recent studies have implicated the circadian clock in regulating JA-mediated defenses, but the molecular mechanisms linking the clock to JA signaling are unclear. Here, we report that wild tobacco (Nicotiana attenuata) plants rendered deficient in the clock component ZEITLUPE (ZTL) by RNA interference have attenuated resistance to the generalist herbivore Spodoptera littoralis This effect can be attributed in part to reduced concentrations of nicotine, an abundant JA-regulated toxin produced in N. attenuata roots and transported to shoots. RNA interference targeting ZTL dramatically affects the root circadian clock and reduces the expression of nicotine biosynthetic genes. Protein-protein interaction experiments demonstrate that ZTL regulates JA signaling by directly interacting with JASMONATE ZIM domain (JAZ) proteins in a CORONATINE-INSENSITIVE1- and jasmonoyl-isoleucine conjugate-independent manner, thereby regulating a JAZ-MYC2 module that is required for nicotine biosynthesis. Our study reveals new functions for ZTL and proposes a mechanism by which a clock component directly influences JA signaling to regulate plant defense against herbivory.

Glucosinolate Desulfation by the Phloem-Feeding Insect Bemisia tabaci.

Glucosinolates are plant secondary defense metabolites confined nearly exclusively to the order Brassicales. Upon tissue rupture, glucosinolates are hydrolyzed to various bioactive breakdown products by the endogenous plant enzyme myrosinase. As the feeding of chewing insect herbivores is associated with plant tissue damage, these insects have developed several independent strategies for coping with the glucosinolate-myrosinase defense system. On the other hand, our knowledge of how phloem-feeding insects interact with the glucosinolate-myrosinase system is much more limited. In fact, phloem feeders might avoid contact with myrosinase altogether so their susceptibility to intoxication by glucosinolate hydrolysis products is unclear. Previous studies utilizing Arabidopsis thaliana plants accumulating high levels of aliphatic- or indolic-glucosinolates indicated that both glucosinolate groups have moderate negative effects on the reproductive performance of Bemisia tabaci, a generalist phloem-feeding insect. To get a deeper understanding of the interaction between B. tabaci and glucosinolate-defended plants, adults were allowed to feed on artificial diet containing intact glucosinolates or on Brussels sprout and A. thaliana plants, and their honeydew was analyzed for the presence of possible metabolites. We found that B. tabaci is capable of cleaving off the sulfate group of intact glucosinolates, producing desulfoglucosinolates that cannot be activated by myrosinases, a mechanism described to date only in several chewing insect herbivores. The presence of desulfated glucosinolates in the honeydew of a generalist phloem-feeder may indicate the necessity to detoxify glucosinolates, likely due to some level of cellular damage during feeding, which results in glucosinolate activation, or as a mechanism to circumvent the non-enzymatic breakdown of indolic glucosinolates.

A mode of action of glucosinolate-derived isothiocyanates: Detoxification depletes glutathione and cysteine levels with ramifications on protein metabolism in Spodoptera littoralis.

Glucosinolates are activated plant defenses common in the order Brassicales that release isothiocyanates (ITCs) and other hydrolysis products upon tissue damage. The reactive ITCs are toxic to insects resulting in reduced growth, delayed development and occasionally mortality. Generalist lepidopteran larvae often detoxify ingested ITCs via conjugation to glutathione (GSH) and survive on low glucosinolate diets, but it is not known how this process influences other aspects of metabolism. We investigated the impact of the aliphatic 4-methylsulfinylbutyl-ITC (4msob-ITC, sulforaphane) on the metabolism of Spodoptera littoralis larvae, which suffer a significant growth decline on 4msob-ITC-containing diets while excreting ITC-glutathione conjugates and their derivatives in the frass. The most striking effects were a decrease of GSH in midgut tissue and hemolymph due to losses by conjugation to ITC during detoxification, and a decline of the GSH biosynthetic precursor cysteine. Protein content was likewise reduced by ITC treatment suggesting that protein is actively catabolized in an attempt to supply cysteine for GSH biosynthesis. The negative growth and protein effects were relieved by dietary supplementation with cystine. Other consequences of protein breakdown included deamination of amino acids with increased excretion of uric acid and elevated lipid content. Thus metabolic detoxification of ITCs provokes a cascade of negative effects on insects that result in reduced fitness.

The role of glucosinolates and the jasmonic acid pathway in resistance of Arabidopsis thaliana against molluscan herbivores.

Although slugs and snails play important roles in terrestrial ecosystems and cause considerable damage on a variety of crop plants, knowledge about the mechanisms of plant immunity to molluscs is limited. We found slugs to be natural herbivores of Arabidopsis thaliana and therefore investigated possible resistance mechanisms of this species against several molluscan herbivores. Treating wounded leaves with the mucus residue ('slime trail') of the Spanish slug Arion lusitanicus increased wound-induced jasmonate levels, suggesting the presence of defence elicitors in the mucus. Plants deficient in jasmonate biosynthesis and signalling suffered more damage by molluscan herbivores in the laboratory and in the field, demonstrating that JA-mediated defences protect A. thaliana against slugs and snails. Furthermore, experiments using A. thaliana mutants with altered levels of specific glucosinolate classes revealed the importance of aliphatic glucosinolates in defending leaves and reproductive structures against molluscs. The presence in mollusc faeces of known and novel metabolites arising from glutathione conjugation with glucosinolate hydrolysis products suggests that molluscan herbivores actively detoxify glucosinolates. Higher levels of aliphatic glucosinolates were found in plants during the night compared to the day, which correlated well with the nocturnal activity rhythms of slugs and snails. Our data highlight the function of well-known antiherbivore defence pathways in resistance against slugs and snails and suggest an important role for the diurnal regulation of defence metabolites against nocturnal molluscan herbivores.

Metabolism of glucosinolate-derived isothiocyanates to glutathione conjugates in generalist lepidopteran herbivores.

The defensive properties of the glucosinolate-myrosinase system in plants of the order Brassicales have been attributed to the formation of toxic isothiocyanates generated upon tissue damage. Lepidopteran herbivores specialised on brassicaceous plants have been shown to possess biochemical mechanisms preventing the formation of isothiocyanates. Yet, no such mechanisms are known for generalist lepidopterans which also occasionally but successfully feed on plants of the Brassicales. After feeding on Arabidopsis thaliana plants, faeces of Spodoptera littoralis larvae contained glutathione conjugate derivatives (cysteinylglycine- and cysteinyl-isothiocyanate-conjugates) of the plant's major glucosinolate hydrolysis product, 4-methylsulfinylbutyl isothiocyanate. When caterpillars fed on leaves of A. thaliana containing [¹4C]4-methylsulfinylbutyl glucosinolate, more than half of the ingested radioactivity was excreted as the unmetabolised corresponding isothiocyanate, and only 11% as glutathione conjugate derivatives. However, these conjugates were demonstrated to be the major metabolites of isothiocyanates in S. littoralis, and their abundance was shown to correlate with the amount of isothiocyanates ingested. Analysis of larval faeces from several species of generalist lepidopterans (Spodoptera exigua, S. littoralis, Mamestra brassicae, Trichoplusia ni and Helicoverpa armigera) fed on different Brassicaceae revealed that glutathione conjugates arise from a variety of aliphatic and aromatic isothiocyanates derived from dietary glucosinolates.