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Plants produce an extraordinary array of natural products (specialized metabolites). Notably, these structurally complex molecules are not evenly distributed throughout plant tissues but are instead synthesized and stored in specific cell types. Elucidating both the biosynthesis and function of natural products would be greatly facilitated by tracking the location of these metabolites at the cell-level resolution. However, detection, identification, and quantification of metabolites in single cells, particularly from plants, have remained challenging. Here, we show that we can definitively identify and quantify the concentrations of 16 molecules from four classes of natural products in individual cells of leaf, root, and petal of the medicinal plant Catharanthus roseus using a plate-based single-cell mass spectrometry method. We show that identical natural products show substantially different patterns of cell-type localization in different tissues. Moreover, we show that natural products are often found in a wide range of concentrations across a population of cells, with some natural products at concentrations of over 100 mM per cell. This single-cell mass spectrometry method provides a highly resolved picture of plant natural product biosynthesis partitioning at a cell-specific resolution.
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Wounding occurs across multicellular organisms. Wounds can affect host mobility and reproduction, with ecological consequences for competitive interactions and predator-prey dynamics. Wounds are also entry points for pathogens. An immune response is activated upon injury, resulting in the deposition of the brown-black pigment melanin in insects. Despite the abundance of immunity studies in the laboratory and the potential ecological and evolutionary implications of wounding, the prevalence of wounding in wild-collected insects is rarely systematically explored. We investigated the prevalence and potential causes of wounds in wild-collected Drosophilidae flies. We found that 31% of Drosophila melanogaster were wounded or damaged. The abdomen was the most frequently wounded body part, and females were more likely to have melanized patches on the ventral abdomen, compared with males. Encapsulated parasitoid egg frequency was approximately 10%, and just under 1% of Drosophilidae species had attached mites, which also caused wounds. Wounding is prevalent in D. melanogaster, likely exerting selection pressure on host immunity for two reasons: on a rapid and efficient wound repair and on responding efficiently to opportunistic infections. Wounding is thus expected to be an important driver of immune system evolution and to affect individual fitness and population dynamics.
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The bloom and bust patterns of microalgae in aquatic systems contribute massively to global biogeochemical cycles. The decline of algal blooms is mainly caused by nutrient limitation resulting in cell death, the arrest of cell division and the aging of surviving cells. Nutrient intake can re-initiate proliferation, but the processes involved are poorly understood. Here we characterize how the bloom-forming diatom Coscinodiscus radiatus recovers from starvation after nutrient influx. Rejuvenation is mediated by extracellular vesicles that shuttle reactive oxygen species, oxylipins and other harmful metabolites out of the old cells, thereby re-enabling their proliferation. By administering nutrient pulses to aged cells and metabolomic monitoring of the response, we show that regulated pathways are centred around the methionine cycle in C. radiatus. Co-incubation experiments show that bacteria mediate aging processes and trigger vesicle production using chemical signalling. This work opens new perspectives on cellular aging and rejuvenation in complex microbial communities.
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The representation of odors in the locust antennal lobe with its >2,000 glomeruli has long remained a perplexing puzzle. We employed the CRISPR-Cas9 system to generate transgenic locusts expressing the genetically encoded calcium indicator GCaMP in olfactory sensory neurons. Using two-photon functional imaging, we mapped the spatial activation patterns representing a wide range of ecologically relevant odors across all six developmental stages. Our findings reveal a functionally ring-shaped organization of the antennal lobe composed of specific glomerular clusters. This configuration establishes an odor-specific chemotopic representation by encoding different chemical classes and ecologically distinct odors in the form of glomerular rings. The ring-shaped glomerular arrangement, which we confirm by selective targeting of OR70a-expressing sensory neurons, occurs throughout development, and the odor-coding pattern within the glomerular population is consistent across developmental stages. Mechanistically, this unconventional spatial olfactory code reflects the locust-specific and multiplexed glomerular innervation pattern of the antennal lobe.
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Antenas de Artrópodos , Odorantes , Neuronas Receptoras Olfatorias , Animales , Neuronas Receptoras Olfatorias/metabolismo , Antenas de Artrópodos/fisiología , Olfato/fisiología , Saltamontes/fisiología , Animales Modificados Genéticamente , Sistemas CRISPR-Cas/genética , Vías Olfatorias/fisiología , Receptores Odorantes/metabolismo , Receptores Odorantes/genética , Locusta migratoria/fisiología , Calcio/metabolismoRESUMEN
Plants assimilate inorganic nitrogen (N) to glutamine. Glutamine is the most abundant amino acid in most plant species, the N-supplying precursor of all N-containing compounds in the cell and the first organic nitrogen molecule formed from inorganic nitrogen taken up by the roots. In addition to its role in plant nutrition, glutamine most likely also has a function as a signaling molecule in the regulation of nitrogen metabolism. We investigated whether glutamine influences the high-affinity transporter system for nitrate uptake. Therefore, we analyzed the expression of the nitrate transporter NRT2.4, which is inducible by N deficiency, in Arabidopsis thaliana grown under different nitrogen starvation scenarios, comparing nitrate or glutamine as the sole nitrogen source. Using the reporter line ProNRT2.4:GFP and two independent knockout lines, nrt2.4-1 and nrt2.4-2, we analyzed gene expression and amino acid profiles. We showed that the regulation of NRT2.4 expression depends on available nitrogen in general, for example on glutamine as a nitrogen source, and not specifically on nitrate. In contrast to high nitrate concentrations, amino acid profiles changed to an accumulation of amino acids containing more than one nitrogen during growth in high glutamine concentrations, indicating a switch to nitrogen storage metabolism. Furthermore, we demonstrated that the nrt2.4-2 line shows unexpected effects on NRT2.5 gene expression and the amino acids profile in shoots under high glutamine supply conditions compared to Arabidopsis wild type and nrt2.4-1, suggesting non-NRT2.4-related metabolic consequences in this knockout line.
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Non-mycorrhizal but beneficial fungi often mitigate (a)biotic stress-related traits in host plants. The underlying molecular mechanisms are mostly still unknown, as in the interaction between the endophytic growth-promoting soil fungus Mortierella hyalina and Arabidopsis thaliana. Here, abiotic stress in the form of nitrogen (N) deficiency was used to investigate the effects of the fungus on colonized plants. In particular, the hypothesis was investigated that fungal infection could influence N deficiency via an interaction with the high-affinity nitrate transporter NRT2.4, which is induced by N deficiency. For this purpose, Arabidopsis wild-type nrt2.4 knock-out and NRT2.4 reporter lines were grown on media with different nitrate concentrations with or without M. hyalina colonization. We used chemical analysis methods to determine the amino acids and phytohormones. Experimental evidence suggests that the fungus does not modulate NRT2.4 expression under N starvation. Instead, M. hyalina alleviates N starvation in other ways: The fungus supplies nitrogen (15N) to the N-starved plant. The presence of the fungus restores the plants' amino acid homeostasis, which was out of balance due to N deficiency, and causes a strong accumulation of branched-chain amino acids. We conclude that the plant does not need to invest in defense and resources for growth are maintained, which in turn benefits the fungus, suggesting that this interaction should be considered a mutualistic symbiosis.
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Proteínas de Arabidopsis , Arabidopsis , Mortierella , Proteínas de Arabidopsis/genética , Nitrógeno/metabolismo , Mortierella/metabolismo , Nitratos/metabolismo , Aminoácidos/metabolismo , Homeostasis , Regulación de la Expresión Génica de las Plantas , Proteínas de Transporte de Anión/metabolismo , Raíces de Plantas/metabolismoRESUMEN
Strychnine is a natural product that, through isolation, structural elucidation and synthetic efforts, shaped the field of organic chemistry. Currently, strychnine is used as a pesticide to control rodents1 because of its potent neurotoxicity2,3. The polycyclic architecture of strychnine has inspired chemists to develop new synthetic transformations and strategies to access this molecular scaffold4, yet it is still unknown how plants create this complex structure. Here we report the biosynthetic pathway of strychnine, along with the related molecules brucine and diaboline. Moreover, we successfully recapitulate strychnine, brucine and diaboline biosynthesis in Nicotiana benthamiana from an upstream intermediate, thus demonstrating that this complex, pharmacologically active class of compounds can now be harnessed through metabolic engineering approaches.
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Vías Biosintéticas , Ingeniería Metabólica , Estricnina , Vías Biosintéticas/genética , Estricnina/análogos & derivados , Estricnina/biosíntesis , Estricnina/química , Nicotiana/química , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Many herbivorous insects exploit defense compounds produced by their host plants for protection against predators. Ingested plant defense compounds are absorbed via the gut epithelium and stored in the body, a physiological process that is currently not well understood. Here, we investigated the absorption of plant defense compounds from the gut in the horseradish flea beetle, Phyllotreta armoraciae, a specialist herbivore known to selectively sequester glucosinolates from its brassicaceous host plants. Feeding experiments using a mixture of glucosinolates and other glucosides not found in the host plants showed a rapid and selective uptake of glucosinolates in adult beetles. In addition, we provide evidence that this uptake mainly takes place in the foregut, whereas the endodermal midgut is the normal region of absorption. Absorption via the foregut epithelium is surprising as the apical membrane is covered by a chitinous intima. However, we could show that this cuticular layer differs in its structure and overall thickness between P. armoraciae and a non-sequestering leaf beetle. In P. armoraciae, we observed a thinner cuticle with a less dense chitinous matrix, which might facilitate glucosinolate absorption. Our results show that a selective and rapid uptake of glucosinolates from the anterior region of the gut contributes to the selective sequestration of glucosinolates in P. armoraciae.
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Symbiosis is a dominant form of life that has been observed numerous times in marine ecosystems. For example, macroalgae coexist with bacteria that produce factors that promote algal growth and morphogenesis. The green macroalga Ulva mutabilis (Chlorophyta) develops into a callus-like phenotype in the absence of its essential bacterial symbionts Roseovarius sp. MS2 and Maribacter sp. MS6. Spatially resolved studies are required to understand symbiont interactions at the microscale level. Therefore, we used mass spectrometry profiling and imaging techniques with high spatial resolution and sensitivity to gain a new perspective on the mutualistic interactions between bacteria and macroalgae. Using atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionisation high-resolution mass spectrometry (AP-SMALDI-HRMS), low-molecular-weight polar compounds were identified by comparative metabolomics in the chemosphere of Ulva. Choline (2-hydroxy-N,N,N-trimethylethan-1-aminium) was only determined in the alga grown under axenic conditions, whereas ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was found in bacterial presence. Ectoine was used as a metabolic marker for localisation studies of Roseovarius sp. within the tripartite community because it was produced exclusively by these bacteria. By combining confocal laser scanning microscopy (cLSM) and AP-SMALDI-HRMS, we proved that Roseovarius sp. MS2 settled mainly in the rhizoidal zone (holdfast) of U. mutabilis. Our findings provide the fundament to decipher bacterial symbioses with multicellular hosts in aquatic ecosystems in an ecologically relevant context. As a versatile tool for microbiome research, the combined AP-SMALDI and cLSM imaging analysis with a resolution to level of a single bacterial cell can be easily applied to other microbial consortia and their hosts. The novelty of this contribution is the use of an in situ setup designed to avoid all types of external contamination and interferences while resolving spatial distributions of metabolites and identifying specific symbiotic bacteria.
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Numerous hematophagous insects are attracted to ammonia, a volatile released in human sweat and breath.1-3 Low levels of ammonia also attract non-biting insects such as the genetic model organism Drosophila melanogaster and several species of agricultural pests.4,5 Two families of ligand-gated ion channels function as olfactory receptors in insects,6-10 and studies have linked ammonia sensitivity to a particular olfactory receptor in Drosophila.5,11,12 Given the widespread importance of ammonia to insect behavior, it is surprising that the genomes of most insects lack an ortholog of this gene.6 Here, we show that canonical olfactory receptors are not necessary for responses to ammonia in Drosophila. Instead, we demonstrate that a member of the ancient electrogenic ammonium transporter family, Amt, is likely a new type of olfactory receptor. We report two hitherto unidentified olfactory neuron populations that mediate neuronal and behavioral responses to ammonia in Drosophila. Their endogenous ammonia responses are lost in Amt mutant flies, and ectopic expression of either Drosophila or Anopheles Amt confers ammonia sensitivity. These results suggest that Amt is the first transporter known to function as an olfactory receptor in animals and that its function may be conserved across insect species.
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Compuestos de Amonio , Proteínas de Drosophila , Drosophila melanogaster , Neuronas Receptoras Olfatorias , Receptores Odorantes , Amoníaco , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Receptores Odorantes/genéticaRESUMEN
Insect odorant receptors (ORs) detect volatile chemical cues with high sensitivity. These ORs operate as ligand-gated ion channels and are formed by heptahelical OrX and Orco (co-receptor) proteins. A highly conserved calmodulin (CaM) binding site (CBS) 336SAIKYWVER344 within the second intracellular loop of Drosophila melanogaster Orco constitutes a target for regulating OR performance. Here we asked how a point mutation K339N in this CBS affects the olfactory performance of Drosophila melanogaster. We first asked how this mutation would affect the odor responses of olfactory sensory neurons (OSNs). Using Ca2+ imaging in an ex-vivo antenna preparation, we activated all OR (OrX/Orco) expressing neurons using the synthetic agonist VUAA1. In a next attempt, we restricted the OR spectrum to Or22a expressing neurons (Or22a/Orco) and stimulated these OSNs with the ligand ethyl hexanoate. In both approaches, we found that flies carrying the K339N point mutation in Orco display a reduced olfactory response. We also found that the mutation abolishes the capability of OSNs to sensitize by repeated weak odor stimuli. Next, we asked whether OrcoK339N might affect the odor localization performance. Using a wind tunnel bioassay, we found that odor localization in flies carrying the OrcoK339N mutation was severely diminished.
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Calmodulina/metabolismo , Proteínas de Drosophila/genética , Receptores Odorantes/metabolismo , Animales , Sitios de Unión , Calmodulina/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Masculino , Odorantes , Percepción Olfatoria/fisiología , Neuronas Receptoras Olfatorias/metabolismo , Polimorfismo de Nucleótido Simple/genética , Unión Proteica , Receptores Odorantes/genética , Olfato/fisiología , Tioglicolatos/farmacología , Triazoles/farmacologíaRESUMEN
Napier grass stunt (NGS) phytoplasma, a phloem-limited bacterium, infects Napier grass leading to severe yield losses in East Africa. The infected plants are strongly inhibited in growth and biomass production. In this study, phytoplasma-induced morphological changes of the vascular system and physiological changes were analyzed and compared with uninfected plants. The study showed that the phytoplasmas are more abundant in source leaves and range from 103 bacteria/µg total DNA in infected roots to 106 in mature Napier grass leaves. Using microscopical, biochemical, and physiological tools, we demonstrated that the ultrastructure of the phloem and sieve elements is severely altered in the infected plants, which results in the reduction of both the mass flow and the translocation of photoassimilates in the infected leaves. The reduced transport rate inhibits the photochemistry of photosystem II in the infected plants, which is accompanied by loss of chloroplastic pigments in response to the phytoplasma infection stress eventually resulting in yellowing of diseased plants. The phytoplasma infection stress also causes imbalances in the levels of defense-related antioxidants, glutathione, ascorbic acid, reactive oxygen species (ROS), and-in particular-hydrogen peroxide. This study shows that the infection of NGS phytoplasma in the phloem of Napier grass has an impact on the primary metabolism and activates a ROS-dependent defense response.
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Phytoplasma , Floema , Enfermedad por Fitoplasma , Enfermedades de las Plantas , Hojas de la PlantaRESUMEN
Flavonoids may mediate UV protection in plants either by screening of harmful radiation or by minimizing the resulting oxidative stress. To help distinguish between these alternatives, more precise knowledge of flavonoid distribution is needed. We used confocal laser scanning microscopy (cLSM) with the "emission fingerprinting" feature to study the cellular and subcellular distribution of flavonoid glucosides in the giant duckweed (Spirodela polyrhiza), and investigated the fitness effects of these compounds under natural UV radiation and copper sulphate addition (oxidative stress) using common garden experiments indoors and outdoors. cLSM "emission fingerprinting" allowed us to individually visualize the major dihydroxylated B-ring-substituted flavonoids, luteolin 7-O-glucoside and luteolin 8-C-glucoside, in cross-sections of the photosynthetic organs. While luteolin 8-C-glucoside accumulated mostly in the vacuoles and chloroplasts of mesophyll cells, luteolin 7-O-glucoside was predominantly found in the vacuoles of epidermal cells. In congruence with its cellular distribution, the mesophyll-associated luteolin 8-C-glucoside increased plant fitness under copper sulphate addition but not under natural UV light treatment, whereas the epidermis-associated luteolin 7-O-glucoside tended to increase fitness under both stresses across chemically diverse genotypes. Taken together, we demonstrate that individual flavonoid glucosides have distinct cellular and subcellular locations and promote duckweed fitness under different abiotic stresses.
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Organismos Acuáticos/metabolismo , Araceae/metabolismo , Flavonoides/metabolismo , Glucósidos/metabolismo , Organismos Acuáticos/fisiología , Araceae/fisiología , Flavonoides/fisiología , Fluorescencia , Microscopía Confocal , Estrés Oxidativo , Estrés Fisiológico , Rayos UltravioletaRESUMEN
The examination of phylogenetic and phenotypic characteristics of the nervous system, such as behavior and neuroanatomy, can be utilized as a means to assess speciation. Recent studies have proposed a fundamental tradeoff between two sensory organs, the eye and the antenna. However, the identification of ecological mechanisms for this observed tradeoff have not been firmly established. Our current study examines several monophyletic species within the obscura group, and asserts that despite their close relatedness and overlapping ecology, they deviate strongly in both visual and olfactory investment. We contend that both courtship and microhabitat preferences support the observed inverse variation in these sensory traits. Here, this variation in visual and olfactory investment seems to provide relaxed competition, a process by which similar species can use a shared environment differently and in ways that help them coexist. Moreover, that behavioral separation according to light gradients occurs first, and subsequently, courtship deviations arise.
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Antenas de Artrópodos/fisiología , Ojo Compuesto de los Artrópodos/fisiología , Drosophila/genética , Drosophila/fisiología , Especiación Genética , Conducta Sexual Animal , Animales , Cortejo , Ecología , Ecosistema , Femenino , Procesamiento de Imagen Asistido por Computador , Masculino , Fenotipo , Filogenia , Olfato , Especificidad de la Especie , Visión Ocular , Alas de Animales/fisiologíaRESUMEN
Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, we identify two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. Our results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner.
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Drosophila melanogaster , Atractivos Sexuales , Animales , Drosophila/metabolismo , Drosophila melanogaster/fisiología , Femenino , Masculino , Vías Olfatorias , Feromonas/genética , Feromonas/metabolismo , Atractivos Sexuales/fisiología , Conducta Sexual Animal/fisiologíaRESUMEN
Endoparasitic larval stages of Eoxenos laboulbenei were documented with different techniques, with a main focus on the male tertiary larva. Two discrete endoparasitic stages occur, the secondary and the tertiary larva. The presence of large compound eyes and externally visible wing buds in the tertiary larva is a unique feature within Holometabola. The brain with large optic lobes is followed by a single postcephalic ganglionic complex. The cephalic musculature is greatly reduced but pharyngeal dilators and muscles associated with the mouth field are present. Postcephalic sclerites are absent except for the pronotum. The segmented legs bear filiform pretarsal claws. The indirect flight muscles fill up a large part of the metathorax. The 10-segmented abdomen lacks appendages. Pleural folds are present on the thorax and abdomen. The digestive tract is characterized by a very short oesophagus. The large midgut and the narrow hindgut are disconnected. Six short Malpighian tubules are present. Large testes fill out almost the entire abdomen. In contrast to the tertiary larva, the muscles of the secondary larva are not fully differentiated. Cephalic appendages are present as bud-shaped anlagen. The legs lack a pretarsal claw. The developmental transformations are outlined and discussed, also with respect to phylogenetic implications.
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Holometabola/anatomía & histología , Animales , Croacia , Femenino , Holometabola/crecimiento & desarrollo , Larva/anatomía & histología , Larva/crecimiento & desarrollo , MasculinoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Optical imaging of intracellular Ca2+ influx as a correlate of neuronal excitation represents a standard technique for visualizing spatiotemporal activity of neuronal networks. However, the information-processing properties of single neurons and neuronal circuits likewise involve inhibition of neuronal membrane potential. Here, we report spatially resolved optical imaging of odor-evoked inhibitory patterns in the olfactory circuitry of Drosophila using a genetically encoded fluorescent Cl- sensor. In combination with the excitatory component reflected by intracellular Ca2+ dynamics, we present a comprehensive functional map of both odor-evoked neuronal activation and inhibition at different levels of olfactory processing. We demonstrate that odor-evoked inhibition carried by Cl- influx is present both in sensory neurons and second-order projection neurons (PNs), and is characterized by stereotypic, odor-specific patterns. Cl--mediated inhibition features distinct dynamics in different neuronal populations. Our data support a dual role of inhibitory neurons in the olfactory system: global gain control across the neuronal circuitry and glomerulus-specific inhibition to enhance neuronal information processing.
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Proteínas de Drosophila , Odorantes , Neuronas Receptoras Olfatorias , Receptores Odorantes , Animales , Drosophila , Proteínas de Drosophila/metabolismo , Femenino , Potenciales de la Membrana , Vías Olfatorias/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Receptores Odorantes/metabolismo , OlfatoRESUMEN
Flagellated oomycetes frequently infect unicellular algae, thus limiting their proliferation. Here we show that the marine oomycete Lagenisma coscinodisci rewires the metabolome of the bloom-forming diatom Coscinodiscus granii, thereby promoting infection success. The algal alkaloids ß-carboline and 4-carboxy-2,3,4,9-tetrahydro-1H-ß-carboline are induced during infection. Single-cell profiling with AP-MALDI-MS and confocal laser scanning microscopy reveals that algal carbolines accumulate in the reproductive form of the parasite. The compounds arrest the algal cell division, increase the infection rate and induce plasmolysis in the host. Our results indicate that the oomycete manipulates the host metabolome to support its own multiplication.