Cyclitol metabolism is a central feature of Burkholderia leaf symbionts.

The symbioses between plants of the Rubiaceae and Primulaceae families with Burkholderia bacteria represent unique and intimate plant-bacterial relationships. Many of these interactions have been identified through PCR-dependent typing methods, but there is little information available about their functional and ecological roles. We assembled 17 new endophyte genomes representing endophytes from 13 plant species, including those of two previously unknown associations. Genomes of leaf endophytes belonging to Burkholderia s.l. show extensive signs of genome reduction, albeit to varying degrees. Except for one endophyte, none of the bacterial symbionts could be isolated on standard microbiological media. Despite their taxonomic diversity, all endophyte genomes contained gene clusters linked to the production of specialized metabolites, including genes linked to cyclitol sugar analog metabolism and in one instance non-ribosomal peptide synthesis. These genes and gene clusters are unique within Burkholderia s.l. and are likely horizontally acquired. We propose that the acquisition of secondary metabolite gene clusters through horizontal gene transfer is a prerequisite for the evolution of a stable association between these endophytes and their hosts.

Mosquito metabolomics reveal that dengue virus replication requires phospholipid reconfiguration via the remodeling cycle.

Dengue virus (DENV) subdues cell membranes for its cellular cycle by reconfiguring phospholipids in humans and mosquitoes. Here, we determined how and why DENV reconfigures phospholipids in the mosquito vector. By inhibiting and activating the de novo phospholipid biosynthesis, we demonstrated the antiviral impact of de novo-produced phospholipids. In line with the virus hijacking lipids for its benefit, metabolomics analyses indicated that DENV actively inhibited the de novo phospholipid pathway and instead triggered phospholipid remodeling. We demonstrated the early induction of remodeling during infection by using isotope tracing in mosquito cells. We then confirmed in mosquitoes the antiviral impact of de novo phospholipids by supplementing infectious blood meals with a de novo phospholipid precursor. Eventually, we determined that phospholipid reconfiguration was required for viral genome replication but not for the other steps of the virus cellular cycle. Overall, we now propose that DENV reconfigures phospholipids through the remodeling cycle to modify the endomembrane and facilitate formation of the replication complex. Furthermore, our study identified de novo phospholipid precursor as a blood determinant of DENV human-to-mosquito transmission.

Wild Wheat Rhizosphere-Associated Plant Growth-Promoting Bacteria Exudates: Effect on Root Development in Modern Wheat and Composition.

Diazotrophic bacteria isolated from the rhizosphere of a wild wheat ancestor, grown from its refuge area in the Fertile Crescent, were found to be efficient Plant Growth-Promoting Rhizobacteria (PGPR), upon interaction with an elite wheat cultivar. In nitrogen-starved plants, they increased the amount of nitrogen in the seed crop (per plant) by about twofold. A bacterial growth medium was developed to investigate the effects of bacterial exudates on root development in the elite cultivar, and to analyze the exo-metabolomes and exo-proteomes. Altered root development was observed, with distinct responses depending on the strain, for instance, with respect to root hair development. A first conclusion from these results is that the ability of wheat to establish effective beneficial interactions with PGPRs does not appear to have undergone systematic deep reprogramming during domestication. Exo-metabolome analysis revealed a complex set of secondary metabolites, including nutrient ion chelators, cyclopeptides that could act as phytohormone mimetics, and quorum sensing molecules having inter-kingdom signaling properties. The exo-proteome-comprised strain-specific enzymes, and structural proteins belonging to outer-membrane vesicles, are likely to sequester metabolites in their lumen. Thus, the methodological processes we have developed to collect and analyze bacterial exudates have revealed that PGPRs constitutively exude a highly complex set of metabolites; this is likely to allow numerous mechanisms to simultaneously contribute to plant growth promotion, and thereby to also broaden the spectra of plant genotypes (species and accessions/cultivars) with which beneficial interactions can occur.

Dengue virus reduces AGPAT1 expression to alter phospholipids and enhance infection in Aedes aegypti.

More than half of the world population is at risk of dengue virus (DENV) infection because of the global distribution of its mosquito vectors. DENV is an envelope virus that relies on host lipid membranes for its life-cycle. Here, we characterized how DENV hijacks the mosquito lipidome to identify targets for novel transmission-blocking interventions. To describe metabolic changes throughout the mosquito DENV cycle, we deployed a Liquid chromatography-high resolution mass spectrometry (LC-HRMS) workflow including spectral similarity annotation in cells, midguts and whole mosquitoes at different times post infection. We revealed a major aminophospholipid reconfiguration with an overall early increase, followed by a reduction later in the cycle. We phylogenetically characterized acylglycerolphosphate acyltransferase (AGPAT) enzyme isoforms to identify those that catalyze a rate-limiting step in phospholipid biogenesis, the acylation of lysophosphatidate to phosphatidate. We showed that DENV infection decreased AGPAT1, but did not alter AGPAT2 expression in cells, midguts and mosquitoes. Depletion of either AGPAT1 or AGPAT2 increased aminophospholipids and partially recapitulated DENV-induced reconfiguration before infection in vitro. However, only AGPAT1 depletion promoted infection by maintaining high aminophospholipid concentrations. In mosquitoes, AGPAT1 depletion also partially recapitulated DENV-induced aminophospholipid increase before infection and enhanced infection by maintaining high aminophospholipid concentrations. These results indicate that DENV inhibition of AGPAT1 expression promotes infection by increasing aminophospholipids, as observed in the mosquito's early DENV cycle. Furthermore, in AGPAT1-depleted mosquitoes, we showed that enhanced infection was associated with increased consumption/redirection of aminophospholipids. Our study suggests that DENV regulates aminophospholipids, especially phosphatidylcholine and phosphatidylethanolamine, by inhibiting AGPAT1 expression to increase aminophospholipid availability for virus multiplication.

MS-CleanR: A Feature-Filtering Workflow for Untargeted LC-MS Based Metabolomics.

Untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS) is currently the gold-standard technique to determine the full chemical diversity in biological samples. However, this approach still has many limitations; notably, the difficulty of accurately estimating the number of unique metabolites profiled among the thousands of MS ion signals arising from chromatograms. Here, we describe a new workflow, MS-CleanR, based on the MS-DIAL/MS-FINDER suite, which tackles feature degeneracy and improves annotation rates. We show that implementation of MS-CleanR reduces the number of signals by nearly 80% while retaining 95% of unique metabolite features. Moreover, the annotation results from MS-FINDER can be ranked according to the database chosen by the user, which enhance identification accuracy. Application of MS-CleanR to the analysis of Arabidopsis thaliana grown in three different conditions fostered class separation resulting from multivariate data analysis and led to annotation of 75% of the final features. The full workflow was applied to metabolomic profiles from three strains of the leguminous plant Medicago truncatula that have different susceptibilities to the oomycete pathogen Aphanomyces euteiches. A group of glycosylated triterpenoids overrepresented in resistant lines were identified as candidate compounds conferring pathogen resistance. MS-CleanR is implemented through a Shiny interface for intuitive use by end-users (available at https://github.com/eMetaboHUB/MS-CleanR).

Comparison of the Phytochemical Composition of Serenoa repens Extracts by a Multiplexed Metabolomic Approach.

Phytochemical extracts are highly complex chemical mixtures. In the context of an increasing demand for phytopharmaceuticals, assessment of the phytochemical equivalence of extraction procedures is of utmost importance. Compared to routine analytical methods, comprehensive metabolite profiling has pushed forward the concept of phytochemical equivalence. In this study, an untargeted metabolomic approach was used to cross-compare four marketed extracts from Serenoa repens obtained with three different extraction processes: ethanolic, hexanic and sCO2 (supercritical carbon dioxide). Our approach involved a biphasic extraction of native compounds followed by liquid chromatography coupled to a high-resolution mass spectrometry based metabolomic workflow. Our results showed significant differences in the contents of major and minor compounds according to the extraction solvent used. The analyses showed that ethanolic extracts were supplemented in phosphoglycerides and polyphenols, hexanic extracts had higher amounts of free fatty acids and minor compounds, and sCO2 samples contained more glycerides. The discriminant model in this study could predict the extraction solvent used in commercial samples and highlighted the specific biomarkers of each process. This metabolomic survey allowed the authors to assess the phytochemical content of extracts and finished products of S. repens and unequivocally established that sCO2, hexanic and ethanolic extracts are not chemically equivalent and are therefore unlikely to be pharmacologically equivalent.

Antileishmanial Compounds Isolated from Psidium Guajava L. Using a Metabolomic Approach.

With an estimated annual incidence of one million cases, leishmaniasis is one of the top five vector-borne diseases. Currently available medical treatments involve side effects, including toxicity, non-specific targeting, and resistance development. Thus, new antileishmanial chemical entities are of the utmost interest to fight against this disease. The aim of this study was to obtain potential antileishmanial natural products from Psidium guajava leaves using a metabolomic workflow. Several crude extracts from P. guajava leaves harvested from different locations in the Lao People's Democratic Republic (Lao PDR) were profiled by liquid chromatography coupled to high-resolution mass spectrometry, and subsequently evaluated for their antileishmanial activities. The putative active compounds were highlighted by multivariate correlation analysis between the antileishmanial response and chromatographic profiles of P. guajava mixtures. The results showed that the pooled apolar fractions from P. guajava were the most active (IC50 = 1.96 ± 0.47 µg/mL). Multivariate data analysis of the apolar fractions highlighted a family of triterpenoid compounds, including jacoumaric acid (IC50 = 1.318 ± 0.59 µg/mL) and corosolic acid (IC50 = 1.01 ± 0.06 µg/mL). Our approach allowed the identification of antileishmanial compounds from the crude extracts in only a small number of steps and can be easily adapted for use in the discovery workflows of several other natural products.

In vivo validation of anti-malarial activity of crude extracts of Terminalia macroptera, a Malian medicinal plant.

BACKGROUND: Plasmodium falciparum malaria is still one of the most deadly pathology worldwide. Efficient treatment is jeopardized by parasite resistance to artemisinin and its derivatives, and by poor access to treatment in endemic regions. Anti-malarial traditional remedies still offer new tracks for identifying promising antiplasmodial molecules, and a way to ensure that all people have access to care. The present study aims to validate the traditional use of Terminalia macroptera, a Malian plant used in traditional medicine. METHODS: Terminalia macroptera was collected in Mali. Leaves (TML) and roots ethanolic extracts (TMR) were prepared and tested at 2000 mg/kg for in vivo acute toxicity in Albino Swiss mice. Antiplasmodial activity of the extracts was assessed against a chloroquine resistant strain P. falciparum (FcB1) in vitro. In vivo, anti-malarial efficacy was assessed by a 4-day suppressive test at 100 mg/kg in two malaria murine models of uncomplicated malaria (Plasmodium chabaudi chabaudi infection) and cerebral malaria (Plasmodium berghei strain ANKA infection). Constituents of TMR were characterized by ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry. Top ranked compounds were putatively identified using plant databases and in silico fragmentation pattern. RESULTS: Lethal dose of TML and TMR were greater than 2000 mg/kg in Albino Swiss mice. According to the OECD's Globally Harmonized System of Classification, both extracts are non-toxic orally. Antiplasmodial activity of T. macroptera extracts was confirmed in vitro against P. falciparum FcB1 strain with IC50 values of 1.2 and 1.6 µg/mL for TML and TMR, respectively. In vivo, oral administration of TML and TMR induced significant reduction of parasitaemia (37.2 and 46.4% respectively) in P. chabaudi chabaudi infected mice at the 7th day of infection compared to untreated mice. In the cerebral malaria experimental model, mice treated with TMR and TML presented respectively 50 and 66.7% survival rates at day 9 post-infection when all untreated mice died. Eleven major compounds were found in TMR. Among them, several molecules already known could be responsible for the antiplasmodial activity of the roots extract of T. macroptera. CONCLUSIONS: This study confirms both safety and anti-malarial activity of T. macroptera, thus validating its traditional use.

Transcriptome diversity among rice root types during asymbiosis and interaction with arbuscular mycorrhizal fungi.

Root systems consist of different root types (RTs) with distinct developmental and functional characteristics. RTs may be individually reprogrammed in response to their microenvironment to maximize adaptive plasticity. Molecular understanding of such specific remodeling--although crucial for crop improvement--is limited. Here, RT-specific transcriptomes of adult rice crown, large and fine lateral roots were assessed, revealing molecular evidence for functional diversity among individual RTs. Of the three rice RTs, crown roots displayed a significant enrichment of transcripts associated with phytohormones and secondary cell wall (SCW) metabolism, whereas lateral RTs showed a greater accumulation of transcripts related to mineral transport. In nature, arbuscular mycorrhizal (AM) symbiosis represents the default state of most root systems and is known to modify root system architecture. Rice RTs become heterogeneously colonized by AM fungi, with large laterals preferentially entering into the association. However, RT-specific transcriptional responses to AM symbiosis were quantitatively most pronounced for crown roots despite their modest physical engagement in the interaction. Furthermore, colonized crown roots adopted an expression profile more related to mycorrhizal large lateral than to noncolonized crown roots, suggesting a fundamental reprogramming of crown root character. Among these changes, a significant reduction in SCW transcripts was observed that was correlated with an alteration of SCW composition as determined by mass spectrometry. The combined change in SCW, hormone- and transport-related transcript profiles across the RTs indicates a previously overlooked switch of functional relationships among RTs during AM symbiosis, with a potential impact on root system architecture and functioning.

Adaptation of a microbead assay for the easy evaluation of traditional anti-sickling medicines: application to DREPANOSTAT and FACA.

CONTEXT: Sickle cell disease is a common inherited blood disorder affecting millions of people worldwide. Due to lack of progress in drug discovery for a suitable treatment, sufferers often turn to traditional medicines that take advantage of the plant extracts activity used by traditional healers. OBJECTIVE: This study optimizes an anti-sickling screening test to identify preparations capable of reverting sickle cells back to the morphology of normal red blood cells. We focused on the miniaturization and practicability of the assay, so that it can be adapted to the laboratory conditions commonly found in less developed countries. MATERIALS AND METHODS: We tested two traditional anti-sickling herbal medicines, FACA® and DREPANOSTAT®, composed of Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler (Rutaceae) and Calotropis procera (Aiton) Dryand. (Apocynaceae) at screening concentrations of hydroethanol extracts from 0.2 to 1 mg/mL. Potential bioactive molecules present in the extracts were profiled using Ultra High Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (UHPLC-HRMS/MS) method, identified through HRMS, MS/MS spectra and in silico fragmentation tools. RESULTS: Hydroethanol extracts of FACA® and DREPANOSTAT® showed low anti-sickling activity, inhibiting less than 10% of the sickling process. The UHPLC-HRMS/MS profiles identified 28 compounds (18 in FACA® and 15 in DREPANOSTAT®, including common compounds) among which l-phenylalanine is already described as potential anti-sickling agent. When used as positive control, 7 mg/mL phenylalanine reduced the sickled RBC to 52%. DISCUSSION AND CONCLUSIONS: This assay has been optimized for the easy screening of plant extracts or extracted compounds from bioassay guided fractionation, valuable to laboratories from less developed countries.

A Physiological and Behavioral Mechanism for Leaf Herbivore-Induced Systemic Root Resistance.

Indirect plant-mediated interactions between herbivores are important drivers of community composition in terrestrial ecosystems. Among the most striking examples are the strong indirect interactions between spatially separated leaf- and root-feeding insects sharing a host plant. Although leaf feeders generally reduce the performance of root herbivores, little is known about the underlying systemic changes in root physiology and the associated behavioral responses of the root feeders. We investigated the consequences of maize (Zea mays) leaf infestation by Spodoptera littoralis caterpillars for the root-feeding larvae of the beetle Diabrotica virgifera virgifera, a major pest of maize. D. virgifera strongly avoided leaf-infested plants by recognizing systemic changes in soluble root components. The avoidance response occurred within 12 h and was induced by real and mimicked herbivory, but not wounding alone. Roots of leaf-infested plants showed altered patterns in soluble free and soluble conjugated phenolic acids. Biochemical inhibition and genetic manipulation of phenolic acid biosynthesis led to a complete disappearance of the avoidance response of D. virgifera. Furthermore, bioactivity-guided fractionation revealed a direct link between the avoidance response of D. virgifera and changes in soluble conjugated phenolic acids in the roots of leaf-attacked plants. Our study provides a physiological mechanism for a behavioral pattern that explains the negative effect of leaf attack on a root-feeding insect. Furthermore, it opens up the possibility to control D. virgifera in the field by genetically mimicking leaf herbivore-induced changes in root phenylpropanoid patterns.

Cucurbitacins from the Leaves of Citrullus colocynthis (L.) Schrad.

Two new tetracyclic cucurbitane-type triterpene glycosides were isolated from an ethyl acetate extract of Citrullus colocynthis leaves together with four known cucurbitacins. Their structures were established on the basis of their spectroscopic data (mainly NMR and mass spectrometry). Evaluation of the in vitro cytotoxic activity of the isolated compounds against two human colon cancer cell lines (HT29 and Caco-2) and one normal rat intestine epithelial cell line (IEC6), revealed that one of the isolated compounds presented interesting specific cytotoxic activity towards colorectal cell lines.

Study of leaf metabolome modifications induced by UV-C radiations in representative Vitis, Cissus and Cannabis species by LC-MS based metabolomics and antioxidant assays.

UV-C radiation is known to induce metabolic modifications in plants, particularly to secondary metabolite biosynthesis. To assess these modifications from a global and untargeted perspective, the effects of the UV-C radiation of the leaves of three different model plant species, Cissus antarctica Vent. (Vitaceae), Vitis vinifera L. (Vitaceae) and Cannabis sativa L. (Cannabaceae), were evaluated by an LC-HRMS-based metabolomic approach. The approach enabled the detection of significant metabolite modifications in the three species studied. For all species, clear modifications of phenylpropanoid metabolism were detected that led to an increased level of stilbene derivatives. Interestingly, resveratrol and piceid levels were strongly induced by the UV-C treatment of C. antarctica leaves. In contrast, both flavonoids and stilbene polymers were upregulated in UV-C-treated Vitis leaves. In Cannabis, important changes in cinnamic acid amides and stilbene-related compounds were also detected. Overall, our results highlighted phytoalexin induction upon UV-C radiation. To evaluate whether UV-C stress radiation could enhance the biosynthesis of bioactive compounds, the antioxidant activity of extracts from control and UV-C-treated leaves was measured. The results showed increased antioxidant activity in UV-C-treated V. vinifera extracts.

Root-associated Streptomyces produce galbonolides to modulate plant immunity and promote rhizosphere colonization.

The rhizosphere, which serves as the primary interface between plant roots and the soil, constitutes an ecological niche for a huge diversity of microbial communities. Currently, there is little knowledge on the nature and the function of the different metabolites released by rhizospheric microbes to facilitate colonization of this highly competitive environment. Here, we demonstrate how the production of galbonolides, a group of polyene macrolides that inhibit plant and fungal inositol phosphorylceramide synthase (IPCS), empowers the rhizospheric Streptomyces strain AgN23, to thrive in the rhizosphere by triggering the plant's defence mechanisms. Metabolomic analysis of AgN23-inoculated Arabidopsis roots revealed a strong induction in the production of an indole alkaloid, camalexin, which is a major phytoalexin in Arabidopsis. By using a plant mutant compromized in camalexin synthesis, we show that camalexin production is necessary for the successful colonization of the rhizosphere by AgN23. Conversely, hindering galbonolides biosynthesis in AgN23 knock-out mutant resulted in loss of inhibition of IPCS, a deficiency in plant defence activation, notably the production of camalexin, and a strongly reduced development of the mutant bacteria in the rhizosphere. Together, our results identified galbonolides as important metabolites mediating rhizosphere colonization by Streptomyces.

Metabolomics reveals herbivore-induced metabolites of resistance and susceptibility in maize leaves and roots.

Plants respond to herbivory by reprogramming their metabolism. Most research in this context has focused on locally induced compounds that function as toxins or feeding deterrents. We developed an ultra-high-pressure liquid chromatography time-of-flight mass spectrometry (UHPLC-TOF-MS)-based metabolomics approach to evaluate local and systemic herbivore-induced changes in maize leaves, sap, roots and root exudates without any prior assumptions about their function. Thirty-two differentially regulated compounds were identified from Spodoptera littoralis-infested maize seedlings and isolated for structure assignment by microflow nuclear magnetic resonance (CapNMR). Nine compounds were quantified by a high throughput direct nano-infusion tandem mass spectrometry/mass spectrometry (MS/MS) method. Leaf infestation led to a marked local increase of 1,3-benzoxazin-4-ones, phospholipids, N-hydroxycinnamoyltyramines, azealic acid and tryptophan. Only few changes were found in the root metabolome, but 1,3-benzoxazin-4-ones increased in the vascular sap and root exudates. The role of N-hydroxycinnamoyltyramines in plant-herbivore interactions is unknown, and we therefore tested the effect of the dominating p-coumaroyltyramine on S. littoralis. Unexpectedly, p-coumaroyltyramine was metabolized by the larvae and increased larval growth, possibly by providing additional nitrogen to the insect. Taken together, this study illustrates that herbivore attack leads to the induction of metabolites that can have contrasting effects on herbivore resistance in the leaves and roots.

Natural aristolactams and aporphine alkaloids as inhibitors of CDK1/cyclin B and DYRK1A.

In an effort to find potent inhibitors of the protein kinases DYRK1A and CDK1/Cyclin B, a systematic in vitro evaluation of 2,500 plant extracts from New Caledonia and French Guyana was performed. Some extracts were found to strongly inhibit the activity of these kinases. Four aristolactams and one lignan were purified from the ethyl acetate extracts of Oxandra asbeckii and Goniothalamus dumontetii, and eleven aporphine alkaloids were isolated from the alkaloid extracts of Siparuna pachyantha, S. decipiens, S. guianensis and S. poeppigii. Among these compounds, velutinam, aristolactam AIIIA and medioresinol showed submicromolar IC50 values on DYRK1A.

Metabotyping of Andean pseudocereals and characterization of emerging mycotoxins.

Pseudocereals are best known for three crops derived from the Andes: quinoa (Chenopodium quinoa), canihua (C. pallidicaule), and kiwicha (Amaranthus caudatus). Their grains are recognized for their nutritional benefits; however, there is a higher level of polyphenism. Meanwhile, the chemical food safety of pseudocereals remains poorly documented. Here, we applied untargeted and targeted metabolomics approaches by LC-MS to achieve both: i) a comprehensive chemical mapping of pseudocereal samples collected in the Andes; and ii) a quantification of their contents in emerging mycotoxins. An inventory of the fungal community was also realized to better know the fungi present in these grains. Metabotyping permitted to add new insights into the chemotaxonomy of pseudocereals, confirming the previously established phylotranscriptomic clades. Sixteen samples from Peru (out of 27) and one from France (out of one) were contaminated with Beauvericin, an emerging mycotoxin. Several mycotoxigenic fungi were detected, including Aspergillus sp., Penicillium sp., and Alternaria sp.

Deciphering anti-infectious compounds from Peruvian medicinal Cordoncillos extract library through multiplexed assays and chemical profiling.

High prevalence of parasitic or bacterial infectious diseases in some world areas is due to multiple reasons, including a lack of an appropriate health policy, challenging logistics and poverty. The support to research and development of new medicines to fight infectious diseases is one of the sustainable development goals promoted by World Health Organization (WHO). In this sense, the traditional medicinal knowledge substantiated by ethnopharmacology is a valuable starting point for drug discovery. This work aims at the scientific validation of the traditional use of Piper species ("Cordoncillos") as firsthand anti-infectious medicines. For this purpose, we adapted a computational statistical model to correlate the LCMS chemical profiles of 54 extracts from 19 Piper species to their corresponding anti-infectious assay results based on 37 microbial or parasites strains. We mainly identified two groups of bioactive compounds (called features as they are considered at the analytical level and are not formally isolated). Group 1 is composed of 11 features being highly correlated to an inhibiting activity on 21 bacteria (principally Gram-positive strains), one fungus (C. albicans), and one parasite (Trypanosoma brucei gambiense). The group 2 is composed of 9 features having a clear selectivity on Leishmania (all strains, both axenic and intramacrophagic). Bioactive features in group 1 were identified principally in the extracts of Piper strigosum and P. xanthostachyum. In group 2, bioactive features were distributed in the extracts of 14 Piper species. This multiplexed approach provided a broad picture of the metabolome as well as a map of compounds putatively associated to bioactivity. To our knowledge, the implementation of this type of metabolomics tools aimed at identifying bioactive compounds has not been used so far.

The mycoparasite Pythium oligandrum induces legume pathogen resistance and shapes rhizosphere microbiota without impacting mutualistic interactions.

Pythium oligandrum is a soil-borne oomycete associated with rhizosphere and root tissues. Its ability to enhance plant growth, stimulate plant immunity and parasitize fungal and oomycete preys has led to the development of agricultural biocontrol products. Meanwhile, the effect of P. oligandrum on mutualistic interactions and more generally on root microbial communities has not been investigated. Here, we developed a biological system comprising P. oligandrum interacting with two legume plants, Medicago truncatula and Pisum sativum. P. oligandrum activity was investigated at the transcriptomics level through an RNAseq approach, metabolomics and finally metagenomics to investigate the impact of P. oligandrum on root microbiota. We found that P. oligandrum promotes plant growth in these two species and protects them against infection by the oomycete Aphanomyces euteiches, a devastating legume root pathogen. In addition, P. oligandrum up-regulated more than 1000 genes in M. truncatula roots including genes involved in plant defense and notably in the biosynthesis of antimicrobial compounds and validated the enhanced production of M. truncatula phytoalexins, medicarpin and formononetin. Despite this activation of plant immunity, we found that root colonization by P. oligandrum did not impaired symbiotic interactions, promoting the formation of large and multilobed symbiotic nodules with Ensifer meliloti and did not negatively affect the formation of arbuscular mycorrhizal symbiosis. Finally, metagenomic analyses showed the oomycete modifies the composition of fungal and bacterial communities. Together, our results provide novel insights regarding the involvement of P. oligandrum in the functioning of plant root microbiota.

Induction and detoxification of maize 1,4-benzoxazin-3-ones by insect herbivores.

In monocotyledonous plants, 1,4-benzoxazin-3-ones, also referred to as benzoxazinoids or hydroxamic acids, are one of the most important chemical barriers against herbivores. However, knowledge about their behavior after attack, mode of action and potential detoxification by specialized insects remains limited. We chose an innovative analytical approach to understand the role of maize 1,4-benzoxazin-3-ones in plant-insect interactions. By combining unbiased metabolomics screening and simultaneous measurements of living and digested plant tissue, we created a quantitative dynamic map of 1,4-benzoxazin-3-ones at the plant-insect interface. Hypotheses derived from this map were tested by specifically developed in vitro assays using purified 1,4-benzoxazin-3-ones and active extracts from mutant plants lacking 1,4-benzoxazin-3-ones. Our data show that maize plants possess a two-step defensive system that effectively fends off both the generalist Spodoptera littoralis and the specialist Spodoptera frugiperda. In the first step, upon insect attack, large quantities of 2-ß-d-glucopyranosyloxy-4,7-dimethoxy-1,4-benzoxazin-3-one (HDMBOA-Glc) are formed. In the second step, after tissue disruption by the herbivores, highly unstable 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one (HDMBOA) is released by plant-derived ß-glucosidases. HDMBOA acts as a strong deterrent to both S. littoralis and S. frugiperda. Although constitutively produced 1,4-benzoxazin-3-ones such as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) are detoxified via glycosylation by the insects, no conjugation of HDMBOA in the insect gut was found, which may explain why even the specialist S. frugiperda has not evolved immunity against this plant defense. Taken together, our results show the benefit of using a plant-insect interface approach to elucidate plant defensive processes and unravel a potent resistance mechanism in maize.