Pre-symbiotic response of the compatible host spruce and low-compatibility host pine to the ectomycorrhizal fungus Tricholoma vaccinum.

Mutualistic ectomycorrhizal symbiosis requires the exchange of signals even before direct contact of the partners. Volatiles, and specifically volatile terpenoids, can be detected at a distance and may trigger downstream signaling and reprogramming of metabolic responses. The late-stage ectomycorrhizal fungus Tricholoma vaccinum shows high host specificity with its main host spruce, Picea abies, while rarely associations can be found with pine, Pinus sylvestris. Hence, a comparison of the host and the low-compatibility host's responses can untangle differences in early signaling during mycorrhiza formation. We investigated sesquiterpenes and identified different patterns of phytohormone responses with spruce and pine. To test the specific role of volatiles, trees were exposed to the complete volatilome of the fungus versus volatiles present when terpene synthases were inhibited by rosuvastatin. The pleiotropic response in spruce included three non-identified products, a pyridine derivative as well as two diterpenes. In pine, other terpenoids responded to the fungal signal. Using exposure to the fungal volatilome with or without terpene synthesis inhibited, we could find a molecular explanation for the longer time needed to establish the low-compatibility interaction.

Metal-dependent enzyme symmetry guides the biosynthetic flux of terpene precursors.

Terpenoids account for more than 60% of all natural products, and their carbon skeletons originate from common isoprenoid units of different lengths such as geranyl pyrophosphate and farnesyl pyrophosphate. Here we characterize a metal-dependent, bifunctional isoprenyl diphosphate synthase from the leaf beetle Phaedon cochleariae by structural and functional analyses. Inter- and intramolecular cooperative effects in the homodimer strongly depend on the provided metal ions and regulate the biosynthetic flux of terpene precursors to either biological defence or physiological development. Strikingly, a unique chain length determination domain adapts to form geranyl or farnesyl pyrophosphate by altering enzyme symmetry and ligand affinity between both subunits. In addition, we identify an allosteric geranyl-pyrophosphate-specific binding site that shares similarity with end-product inhibition in human farnesyl pyrophosphate synthase. Our combined findings elucidate a deeply intertwined reaction mechanism in the P. cochleariae isoprenyl diphosphate synthase that integrates substrate, product and metal-ion concentrations to harness its dynamic potential.

8-HQA adjusts the number and diversity of bacteria in the gut microbiome of Spodoptera littoralis.

Quinolinic carboxylic acids are known for their metal ion chelating properties in insects, plants and bacteria. The larval stages of the lepidopteran pest, Spodoptera littoralis, produce 8-hydroxyquinoline-2-carboxylic acid (8-HQA) in high concentrations from tryptophan in the diet. At the same time, the larval midgut is known to harbor a bacterial population. The motivation behind the work was to investigate whether 8-HQA is controlling the bacterial community in the gut by regulating the concentration of metal ions. Knocking out the gene for kynurenine 3-monooxygenase (KMO) in the insect using CRISPR/Cas9 eliminated production of 8-HQA and significantly increased bacterial numbers and diversity in the larval midgut. Adding 8-HQA to the diet of knockout larvae caused a dose-dependent reduction of bacterial numbers with minimal effects on diversity. Enterococcus mundtii dominates the community in all treatments, probably due to its highly efficient iron uptake system and production of the colicin, mundticin. Thus host factors and bacterial properties interact to determine patterns of diversity and abundance in the insect midgut.

Egg toxic compounds in the animal kingdom. A comprehensive review.

Covering: 1951 to 2022Packed with nutrients and unable to escape, eggs are the most vulnerable stage of an animal's life cycle. Consequently, many species have evolved chemical defenses and teamed up their eggs with a vast array of toxic molecules for defense against predators, parasites, or pathogens. However, studies on egg toxins are rather scarce and the available information is scattered. The aim of this review is to provide an overview of animal egg toxins and to analyze the trends and patterns with respect to the chemistry and biosynthesis of these toxins. We analyzed their ecology, distribution, sources, occurrence, structure, function, relative toxicity, and mechanistic aspects and include a brief section on the aposematic coloration of toxic eggs. We propose criteria for a multiparametric classification that accounts for the complexity of analyzing the full set of toxins of animal eggs. Around 100 properly identified egg toxins are found in 188 species, distributed in 5 phyla: cnidarians (2) platyhelminths (2), mollusks (9), arthropods (125), and chordates (50). Their scattered pattern among animals suggests that species have evolved this strategy independently on numerous occasions. Alkaloids are the most abundant and widespread, among the 13 types of egg toxins recognized. Egg toxins are derived directly from the environment or are endogenously synthesized, and most of them are transferred by females inside the eggs. Their toxicity ranges from ρmol kg-1 to mmol kg-1, and for some species, experiments support their role in predation deterrence. There is still a huge gap in information to complete the whole picture of this field and the number of toxic eggs seems largely underestimated.

Ectomycorrhizal Influence on the Dynamics of Sesquiterpene Release by Tricholoma vaccinum.

Tricholoma vaccinum is an ectomycorrhizal basidiomycete with high host specificity. The slow-growing fungus is able to produce twenty sesquiterpenes, including α-barbatene, sativene, isocaryophyllene, α-cuprenene, ß-cedrene, ß-copaene, 4-epi-α-acoradiene, and chamigrene in axenic culture. For the three major compounds, Δ6-protoilludene, ß-barbatene, and an unidentified oxygenated sesquiterpene (m/z 218.18), changed production during co-cultivation with the ectomycorrhizal partner tree, Picea abies, could be shown with distinct dynamics. During the mycorrhizal growth of T. vaccinum-P. abies, Δ6-protoilludene and the oxygenated sesquiterpene appeared at similar times, which warranted further studies of potential biosynthesis genes. In silico analyses identified a putative protoilludene synthesis gene, pie1, as being up-regulated in the mycorrhizal stage, in addition to the previously identified, co-regulated geosmin synthase, ges1. We therefore hypothesize that the sesquiterpene synthase pie1 has an important role during mycorrhization, through Δ6-protoilludene and/or its accompanied oxygenated sesquiterpene production.

New Guaianolide Sesquiterpene Lactones and Other Constituents from Pyrethrum pulchrum.

Pyrethrum pulchrum is a rare Mongolian plant species that has been traditionally used as an ingredient in various remedies. Bioactivity-guided fractionation performed on the methanol extract of its aerial parts led to the isolation of 2 previously undescribed guaianolide-type sesquiterpene lactones, namely 1ß,10ß-epoxy-8α-hydroxyguaia-3,11(13)-dien-6,12-olide (1: ) and 1,8,10-trihydroxyguaia-3,11(13)-dien-6,12-olide (2: ), along with the isolation or chromatographic identification of 11 compounds, arglabin (3: ), 3ß-hydroxycostunolide (4: ), isocostic acid (5: ), (E)-9-(2-thienyl)-6-nonen-8-yn-3-ol (6: ), (Z)-9-(2-thienyl)-6-nonen-8-yn-3-ol (7: ), N 1,N 5,N 10,N 14-tetra-p-coumaroyl spermine (8: ), chlorogenic acid (9: ), 3,5-di-O-caffeoylquinic acid (10: ), 3,5-di-O-caffeoylquinic acid methyl ester (11: ), 3,4-di-O-caffeoylquinic acid (12: ), and tryptophan (13: ). Their structures were assigned based on spectroscopic and spectrometric data. The antimicrobial, antiproliferative and cytotoxic activities of selected compounds were evaluated. The new compounds showed weak to moderate antimicrobial activity. Arglabin (3: ), the major sesquiterpene lactone found in the methanol extract of P. pulchrum, exhibited the highest activity against human cancer lines, while compound 1: also possesses significant antiproliferative activity against leukemia cells.

Involvement of CYP347W1 in neurotoxin 3-nitropropionic acid-based chemical defense in mustard leaf beetle Phaedon cochleariae.

Chrysomelina beetlesstore 3-nitropropionic acid in form of a pretoxin, isoxazolin-5-one glucoside-conjugated ester, to protect themselves against predators. Here we identified a cytochrome P450 monooxygenase, CYP347W1, to be involved in the production of the 3-nitropropionic acid moiety of the isoxazolin-5-one glucoside ester. Knocking down CYP347W1 led to a significant depletion in the concentration of the isoxazolin-5-one glucoside ester and an increase in the concentration of the isoxazolin-5-one glucoside in the larval hemolymph. Enzyme assays with the heterologously expressed CYP347W1 showed free ß-alanine was not the direct substrate. Homology modeling indicated that ß-alanine-CoA ester can fit into CYP347W1's active site. Furthermore, we proved that Phaedon cochleariae eggs are not able to de novo synthesize 3-NPA, although both isoxazolin-5-one glucoside and its 3-NPA-conjugated ester are present in the eggs. These results provide direct evidence for the involvement of CYP347W1 in the biosynthesis of a P. cochleariae chemical defense compound.

Chromane Derivatives from Underground Parts of Iris tenuifolia and Their In Vitro Antimicrobial, Cytotoxicity and Antiproliferative Evaluation.

Phytochemical investigation of the ethanol extract of underground parts of Iris tenuifolia Pall. afforded five new compounds; an unusual macrolide termed moniristenulide (1), 5-methoxy-6,7-methylenedioxy-4-O-2'-cycloflavan (2), 5,7,2',3'-tetrahydroxyflavanone (3), 5-hydroxy-6,7-dimethoxyisoflavone-2'-O-ß-d-glucopyranoside (9), 5,2',3'-dihydroxy-6,7-dimethoxyisoflavone (10), along with seven known compounds (4-8, 11-12). The structures of all purified compounds were established by analysis of 1D and 2D NMR spectroscopy and HR-ESI-MS. The antimicrobial activity of the compounds 1-3, 5, 9, and 10 was investigated using the agar diffusion method against fungi, Gram-positive and Gram-negative bacteria. In consequence, new compound 3 was found to possess the highest antibacterial activity against Enterococcus faecalis VRE and Mycobacterium vaccae. Cell proliferation and cytotoxicity tests were also applied on all isolated compounds and plant crude extract in vitro with the result of potent inhibitory effect against leukemia cells. In particular, the newly discovered isoflavone 10 was active against both of the leukemia cells K-562 and THP-1 while 4-6 of the flavanone type compounds were active against only THP-1.

A partially sex-reversed giant kelp sheds light into the mechanisms of sexual differentiation in a UV sexual system.

In UV sexual systems, sex is determined during the haploid phase of the life cycle and males have a V chromosome whereas females have a U chromosome. Previous work in the brown alga Ectocarpus revealed that the V chromosome has a dominant role in male sex determination and suggested that the female developmental programme may occur by 'default'. Here, we describe the identification of a genetically male giant kelp strain presenting phenotypic features typical of a female, despite lacking the U-specific region. The conversion to the female developmental programme is however incomplete, because gametes of this feminized male are unable to produce the sperm-attracting pheromone lamoxirene. We identify the transcriptomic patterns underlying the male and female specific developmental programmes, and show that the phenotypic feminization is associated with both feminization and de-masculinization of gene expression patterns. Importantly, the feminization phenotype was associated with dramatic downregulation of two V-specific genes including a candidate male-determining gene. Our results reveal the transcriptional changes associated with sexual differentiation in a UV system, and contribute to disentangling the role of sex-linked and autosomal gene expression in the initiation of sex-specific developmental programmes. Overall, the data presented here imply that the U-specific region is not required to initiate the female developmental programme, but is critical to produce fully functional eggs, arguing against the idea that female is the 'default' sex in this species.

Function of sesquiterpenes from Schizophyllum commune in interspecific interactions.

Wood is a habitat for a variety of organisms, including saprophytic fungi and bacteria, playing an important role in wood decomposition. Wood inhabiting fungi release a diversity of volatiles used as signaling compounds to attract or repel other organisms. Here, we show that volatiles of Schizophyllum commune are active against wood-decay fungi and bacteria found in its mycosphere. We identified sesquiterpenes as the biologically active compounds, that inhibit fungal growth and modify bacterial motility. The low number of cultivable wood inhabiting bacteria prompted us to analyze the microbial community in the mycosphere of S. commune using a culture-independent approach. Most bacteria belong to Actinobacteria and Proteobacteria, including Pseudomonadaceae, Sphingomonadaceae, Erwiniaceae, Yersiniaceae and Mariprofundacea as the dominating families. In the fungal community, the phyla of ascomycetes and basidiomycetes were well represented. We propose that fungal volatiles might have an important function in the wood mycosphere and could meditate interactions between microorganisms across domains and within the fungal kingdom.

Transcriptomics Reveal the Survival Strategies of Enterococcus mundtii in the Gut of Spodoptera littoralis.

The complex interaction between a higher organism and its resident gut flora is a subject of immense interest in the field of symbiosis. Many insects harbor a complex community of microorganisms in their gut. Larvae of Spodoptera littoralis, a lepidopteran pest, house a bacterial community that varies both spatially (along the length of the gut) and temporally (during the insect's life cycle). To monitor the rapid adaptation of microbes to conditions in the gut, a GFP-tagged reporter strain of E. mundtii, a major player in the gut community, was constructed. After early-instar S. littoralis larvae were fed with the tagged microbes, these were recovered from the larval fore- and hindgut by flow cytometry. The fluorescent reporter confirmed the persistence of E. mundtii in the gut. RNA-sequencing of the sorted bacteria highlighted various strategies of the symbiont's survival, including upregulated pathways for tolerating alkaline stress, forming biofilms and two-component signaling systems for quorum sensing, and resisting oxidative stress. Although these symbionts depend on the host for amino acid and fatty acids, differential regulation among various metabolic pathways points to an enriched lysine synthesis pathway of E. mundtii in the hindgut of the larvae.

The fate of chlorophyll in phytophagous insects goes beyond nutrition.

Chlorophyll (Chl) is a natural compound that is found in all autotrophic plants. Since phytophagous insects ingest the photosynthetically active material with the plant leaves, the question arises if and how herbivores deal with Chl and its degradation products. Here we review findings on Chl degradation in phytophagous insects and highlight the role of these ubiquitous plant metabolites for plant-feeding insects. Due to the anaerobic gut of many insects, the degradation is limited to the removal of the peripheral substituents, while the tetrapyrrole core remains intact. Proteins, such as red fluorescent protein, P252 (a novel 252-kDa protein), and chlorophyllide binding protein have been reported to occur in the insect gut and might be indirectly connected to Chl degradation. Besides of an nutritional value, e.g., by taking up Mg2+ ions or by sequestration of carbon from the phytol side chain, the Chl degradation products may serve the insect, after binding to certain proteins, as antimicrobial, antifungal, and antiviral factors. The protein complexes may also confer protection against reactive oxygen species. The antibiotic potential of proteins and degradation products does not only benefit phytophagous insects but also human being in medical application of cancer treatment for instance. This review highlights these aspects from a molecular, biochemical, and ecological point of view.

Phytohormones and volatile organic compounds, like geosmin, in the ectomycorrhiza of Tricholoma vaccinum and Norway spruce (Picea abies).

The ectomycorrhizospheric habitat contains a diverse pool of organisms, including the host plant, mycorrhizal fungi, and other rhizospheric microorganisms. Different signaling molecules may influence the ectomycorrhizal symbiosis. Here, we investigated the potential of the basidiomycete Tricholoma vaccinum to produce communication molecules for the interaction with its coniferous host, Norway spruce (Picea abies). We focused on the production of volatile organic compounds and phytohormones in axenic T. vaccinum cultures, identified the potential biosynthesis genes, and investigated their expression by RNA-Seq analyses. T. vaccinum released volatiles not usually associated with fungi, like limonene and ß-barbatene, and geosmin. Using stable isotope labeling, the biosynthesis of geosmin was elucidated. The geosmin biosynthesis gene ges1 of T. vaccinum was identified, and up-regulation was scored during mycorrhiza, while a different regulation was seen with mycorrhizosphere bacteria. The fungus also released the volatile phytohormone ethylene and excreted salicylic and abscisic acid as well as jasmonates into the medium. The tree excreted the auxin, indole-3-acetic acid, and its biosynthesis intermediate, indole-3-acetamide, as well as salicylic acid with its root exudates. These compounds could be shown for the first time in exudates as well as in soil of a natural ectomycorrhizospheric habitat. The effects of phytohormones present in the mycorrhizosphere on hyphal branching of T. vaccinum were assessed. Salicylic and abscisic acid changed hyphal branching in a concentration-dependent manner. Since extensive branching is important for mycorrhiza establishment, a well-balanced level of mycorrhizospheric phytohormones is necessary. The regulation thus can be expected to contribute to an interkingdom language.

Cellular Base of Mint Allelopathy: Menthone Affects Plant Microtubules.

Plants can use volatiles for remote suppression of competitors. Mints produce essential oils, which are known to affect the growth of other plants. We used a comparative approach to identify allelopathic compounds from different Mints (genus Mentha, but also including Cat Mint, Nepeta cataria, and Corean Mint, Agastache rugosa, belonging to sisters clades within the Mentheae) using the standard cress germination assay as readout. To understand the mechanism behind this allelopathic effect, we investigated the response of tobacco BY-2 cell lines, expressing GFP-tagged markers for microtubules and actin filaments to these essential oils. Based on the comparison between bioactivity and chemical components, we identified menthone as prime candidate for the allelopathic effect, and confirmed this bioactivity targeted to microtubules experimentally in both, plant cells (tobaccoBY-2), and seedlings (Arabidopsis thaliana). We could show that menthone disrupted microtubules and induced mortality linked with a rapid permeabilization (less than 15 min) of the plasma membrane. This mortality was elevated in a tubulin marker line, where microtubules are mildly stabilized. Our study paves the way for the development of novel bioherbicides that would be environmentally friendly.

Pyrrolic and Dipyrrolic Chlorophyll Degradation Products in Plants and Herbivores.

The degradation of chlorophyll, the omnipresent green pigment, has been investigated intensively over the last 30 years resulting in many elucidated tetrapyrrolic degradation products. With a comparison to the degradation of the structurally similar heme, we hereby propose a novel additional chlorophyll degradation mechanism to mono- and dipyrrolic products. This is the first proof of the occurrence of a family of mono- and dipyrrols in leaves that are previously only known as heme degradation products. This product family is also found in spit and feces of herbivores with specific metabolomic patterns reflecting the origin of the samples. Based on chromatographic and mass spectrometric evidence as well as on mechanistic considerations we also suggest several tentative new degradation products. One of them, dihydro BOX A, was fully confirmed as a novel natural product by synthesis and comparison of its spectroscopic data.

SpitWorm, a Herbivorous Robot: Mechanical Leaf Wounding with Simultaneous Application of Salivary Components.

Induction of jasmonate-mediated plant defense against insect herbivory is initiated by a combination of both mechanical wounding and chemical factors. In order to study both effects independently on plant defense induction, SpitWorm, a computer-controlled device which mimics the damage pattern of feeding insect larvae on leaves and, in addition, can apply oral secretions (OS) or other solutions to the 'biting site' during 'feeding,' was developed and evaluated. The amount of OS left by a Spodoptera littoralis larva during feeding on Phaseolus lunatus (lima bean) leaves was estimated by combining larval foregut volume, biting rate, and quantification of a fluorescent dye injected into the larvae's foregut prior to feeding. For providing OS amounts by SpitWorm equivalent to larval feeding, dilution and delivery rate were optimized. The effectiveness of SpitWorm was tested by comparing volatile organic compounds (VOC) emissions of P. lunatus leaves treated with either SpitWorm, MecWorm, or S. littoralis larvae. Identification and quantification of emitted VOCs revealed that SpitWorm induced a volatile bouquet that is qualitatively and quantitatively similar to herbivory. Additionally, RT-qPCR of four jasmonic acid responsive genes showed that SpitWorm, in contrast to MecWorm, induces the same regulation pattern as insect feeding. Thus, SpitWorm mimics insect herbivory almost identically to real larvae feeding.

Omega hydroxylated JA-Ile is an endogenous bioactive jasmonate that signals through the canonical jasmonate signaling pathway.

Jasmonates are fatty acid derivatives that control several plant processes including growth, development and defense. Despite the chemical diversity of jasmonates, only jasmonoyl-L-isoleucine (JA-Ile) has been clearly characterized as the endogenous ligand of the jasmonate co-receptors (COI1-JAZs) in higher plants. Currently, it is accepted that ω-hydroxylation of JA-Ile leads to inactivation of the molecule. This study shows that ω-hydroxylated JA-Ile (12-OH-JA-Ile) retains bioactivity and signals through the canonical JA-pathway. The results suggest that 12-OH-JA-Ile differentially activates a subset of JA-Ile co-receptors that may control and/or modulate particular jasmonate dependent responses. It is proposed that after a strong immune response mediated by JA-Ile, the ω-hydroxylated form modulates JA-Ile activated processes thereby improving plant resilience.

A cytochrome P450 from the mustard leaf beetles hydroxylates geraniol, a key step in iridoid biosynthesis.

Larvae of the leaf beetle Phaedon cochleariae synthesize the iridoid chysomelidial via the mevalonate pathway to repel predators. The normal terpenoid biosynthesis is integrated into the dedicated defensive pathway by the ω-hydroxylation of geraniol to (2E,6E)-2,6-dimethylocta-2,6-diene-1,8-diol (ω-OH-geraniol). Here we identify and characterize the P450 monooxygenase CYP6BH5 as the geraniol hydroxylase using integrated transcriptomics, proteomics and RNA interference (RNAi). In the fat body, 73 cytochrome P450s were identified, and CYP6BH5 was among those that were expressed specifically in fat body. Double stranded RNA mediated knockdown of CYP6BH5 led to a significant reduction of ω-hydroxygeraniol glucoside in the hemolymph and, later, of the chrysomelidial in the defensive secretion. Heterologously expressed CYP6BH5 converted geraniol to ω-OH-geraniol. In addition to geraniol, CYP6BH5 also catalyzes hydroxylation of other monoterpenols, such as nerol and citronellol to the corresponding α,ω-dihydroxy compounds.

The Ca2+ Channel CNGC19 Regulates Arabidopsis Defense Against Spodoptera Herbivory.

Cellular calcium elevation is an important signal used by plants for recognition and signaling of environmental stress. Perception of the generalist insect, Spodoptera litura, by Arabidopsis (Arabidopsis thaliana) activates cytosolic Ca2+ elevation, which triggers downstream defense. However, not all the Ca2+ channels generating the signal have been identified, nor are their modes of action known. We report on a rapidly activated, leaf vasculature- and plasma membrane-localized, CYCLIC NUCLEOTIDE GATED CHANNEL19 (CNGC19), which activates herbivory-induced Ca2+ flux and plant defense. Loss of CNGC19 function results in decreased herbivory defense. The cngc19 mutant shows aberrant and attenuated intravascular Ca2+ fluxes. CNGC19 is a Ca2+-permeable channel, as hyperpolarization of CNGC19-expressing Xenopus oocytes in the presence of both cyclic adenosine monophosphate and Ca2+ results in Ca2+ influx. Breakdown of Ca2+-based defense in cngc19 mutants leads to a decrease in herbivory-induced jasmonoyl-l-isoleucine biosynthesis and expression of JA responsive genes. The cngc19 mutants are deficient in aliphatic glucosinolate accumulation and hyperaccumulate its precursor, methionine. CNGC19 modulates aliphatic glucosinolate biosynthesis in tandem with BRANCHED-CHAIN AMINO ACID TRANSAMINASE4, which is involved in the chain elongation pathway of Met-derived glucosinolates. Furthermore, CNGC19 interacts with herbivory-induced CALMODULIN2 in planta. Together, our work reveals a key mechanistic role for the Ca2+ channel CNGC19 in the recognition of herbivory and the activation of defense signaling.