Tapping into the metabolic crosstalk between a host and its virus can reveal unique strategies employed during infection. Viral infection is a dynamic process that generates an evolving metabolic landscape. Gaining a continuous view into the infection process is highly challenging and is limited by current metabolomics approaches, which typically measure the average of the entire population at various stages of infection. Here, we took an innovative approach to study the metabolic basis of host–virus interactions between the bloom-forming alga Emiliania huxleyi and its specific virus. We combined a classical method in virology, the plaque assay, with advanced mass spectrometry imaging (MSI), an approach we termed ‘in plaque-MSI’. Taking advantage of the spatial characteristics of the plaque, we mapped the metabolic landscape induced during infection in a high spatiotemporal resolution, unfolding the infection process in a continuous manner. Further unsupervised spatially aware clustering, combined with known lipid biomarkers, revealed a systematic metabolic shift during infection towards lipids containing the odd-chain fatty acid pentadecanoic acid (C15:0). Applying ‘in plaque-MSI’ may facilitate the discovery of bioactive compounds that mediate the chemical arms race of host–virus interactions in diverse model systems. © 2019, The Author(s), under exclusive licence to Springer Nature Limited.

Diatoms are photosynthetic microorganisms of great ecological and biogeochemical importance, forming vast blooms in aquatic ecosystems. However, we are still lacking fundamental understanding of individual cells sense and respond to diverse stress conditions, and what acclimation strategies employed during bloom dynamics. We investigated cellular responses to environmental stress at single-cell level using the roGFP sensor targeted to various organelles in the diatom Phaeodactylum tricornutum. We detected cell-to-cell variability using flow cytometry cell sorting and a microfluidics system for live imaging of roGFP oxidation dynamics. Chloroplast-targeted roGFP exhibited a light dependent, bi-stable oxidation pattern in response to H2O2 and high light, revealing distinct subpopulations of sensitive oxidized cells and resilient reduced cells. Early oxidation in the chloroplast preceded commitment to cell death, and can be used for sensing stress cues and regulating cell fate. propose that light-dependent metabolic heterogeneity regulates diatoms’ sensitivity to environmental stressors in the ocean. © 2019, eLife Sciences Publications Ltd. All Rights Reserved.

Infection by large dsDNA viruses can lead to a profound alteration of host transcriptome and metabolome in order to provide essential building blocks to support the high metabolic demand for viral assembly and egress. Host response to viral infection can typically lead to diverse phenotypic outcome that include shift in host life cycle and activation of anti-viral defense response. Nevertheless, there is a major bottleneck to discern between viral hijacking strategies and host defense responses when averaging bulk population response. Here we study the interaction between Emiliania huxleyi, a bloom-forming alga, and its specific virus (EhV), an ecologically important host-virus model system in the ocean. We quantified host and virus gene expression on a single-cell resolution during the course of infection, using automatic microfluidic setup that captures individual algal cells and multiplex quantitate PCR. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and allowed detection specific host response in a subpopulation of infected cell which otherwise masked by the majority of the infected population. Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and susceptible cells within heterogeneous populations in the marine environment. We propose that resolving host-virus arms race at a single-cell level will provide important mechanistic insights into viral life cycles and will uncover host defense strategies. © 2019 Rosenwasser et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The cosmopolitan coccolithophore Emiliania huxleyi is a unicellular eukaryotic alga that forms vast blooms in the oceans impacting large biogeochemical cycles. These blooms are often terminated due to infection by the large dsDNA virus, E. huxleyi virus (EhV). It was recently established that EhV-induced modulation of E. huxleyi metabolism is a key factor for optimal viral infection cycle. Despite the huge ecological importance of this host–virus interaction, the ability to assess its spatial and temporal dynamics and its possible impact on nutrient fluxes is limited by current approaches that focus on quantification of viral abundance and biodiversity. Here, we applied a host and virus gene expression analysis as a sensitive tool to quantify the dynamics of this interaction during a natural E. huxleyi bloom in the North Atlantic. We used viral gene expression profiling as an index for the level of active infection and showed that the latter correlated with water column depth. Intriguingly, this suggests a possible sinking mechanism for removing infected cells as aggregates from the E. huxleyi population in the surface layer into deeper waters. Viral infection was also highly correlated with induction of host metabolic genes involved in host life cycle, sphingolipid, and antioxidant metabolism, providing evidence for modulation of host metabolism under natural conditions. The ability to track and quantify defined phases of infection by monitoring co-expression of viral and host genes, coupled with advance omics approaches, will enable a deeper understanding of the impact that viruses have on the environment.

Diatoms are one of the key phytoplankton groups in the ocean, forming vast oceanic blooms and playing a significant part in global primary production. To shed light on the role of redox metabolism in diatom's acclimation to light–dark transition and its interplay with cell fate regulation, we generated transgenic lines of the diatom Thalassiosira pseudonana that express the redox-sensitive green fluorescent protein targeted to various subcellular organelles. We detected organelle-specific redox patterns in response to oxidative stress, indicating compartmentalized antioxidant capacities. Monitoring the GSH redox potential (EGSH) in the chloroplast over diurnal cycles revealed distinct rhythmic patterns. Intriguingly, in the dark, cells exhibited reduced basal chloroplast EGSH but higher sensitivity to oxidative stress than cells in the light. This dark-dependent sensitivity to oxidative stress was a result of a depleted pool of reduced glutathione which accumulated during the light period. Interestingly, reduction in the chloroplast EGSH was observed in the light phase prior to the transition to darkness, suggesting an anticipatory phase. Rapid chloroplast EGSH re-oxidation was observed upon re-illumination, signifying an induction of an oxidative signaling during transition to light that may regulate downstream metabolic processes. Since light–dark transitions can dictate metabolic capabilities and susceptibility to a range of environmental stress conditions, deepening our understanding of the molecular components mediating the light-dependent redox signals may provide novel insights into cell fate regulation and its impact on oceanic bloom successions.

Author summary This study assesses the interplay between the globally distributed microalga Emiliania huxleyi and its specific lytic viruses, EhV, which drive the termination of vast oceanic blooms. E. huxleyi is characterized by a biphasic life cycle that alternates between morphologically dissimilar diploid and haploid cells. Here, we show that during viral infection, the bloom-forming diploid cells that are sensitive to EhV can produce virus-resistant cells. These latter cells are morphologically similar to the haploid phase but have diploid or aneuploid genomes. Therefore, a mechanism that mediates morphological remodeling appears to be activated during viral infection, enabling E. huxleyi to escape EhV. These results provide novel insights into morphological plasticity and viral resistance in marine phytoplankton, while highlighting the complexity of host–virus interactions in the oceanic microbial realm.

Communication between microorganisms in the marine environment has immense ecological impact by mediating trophic-level interactions and thus determining community structure1. Extracellular vesicles (EVs) are produced by bacteria2,3, archaea4, protists5and metazoans, and can mediate pathogenicity6or act as vectors for intercellular communication. However, little is known about the involvement of EVs in microbial interactions in the marine environment7. Here we investigated the signalling role of EVs produced during interactions between the cosmopolitan alga Emiliania huxleyi and its specific virus (EhV, Phycodnaviridae)8, which leads to the demise of these large-scale oceanic blooms9,10. We found that EVs are highly produced during viral infection or when bystander cells are exposed to infochemicals derived from infected cells. These vesicles have a unique lipid composition that differs from that of viruses and their infected host cells, and their cargo is composed of specific small RNAs that are predicted to target sphingolipid metabolism and cell-cycle pathways. EVs can be internalized by E. huxleyi cells, which consequently leads to a faster viral infection dynamic. EVs can also prolong EhV half-life in the extracellular milieu. We propose that EVs are exploited by viruses to sustain efficient infectivity and propagation across E. huxleyi blooms. As these algal blooms have an immense impact on the cycling of carbon and other nutrients11,12, this mode of cell–cell communication may influence the fate of the blooms and, consequently, the composition and flow of nutrients in marine microbial food webs.

The redox-sensitive proteome (RSP) consists of protein thiols that undergo redox reactions, playing an important role in coordinating cellular processes. Here, we applied a large-scale phylogenomic reconstruction approach in the model diatom Phaeodactylum tricornutum to map the evolutionary origins of the eukaryotic RSP. The majority of P. tricornutum redox-sensitive cysteines (76%) is specific to eukaryotes, yet these are encoded in genes that are mostly of a prokaryotic origin (57%). Furthermore, we find a threefold enrichment in redox-sensitive cysteines in genes that were gained by endosymbiotic gene transfer during the primary plastid acquisition. The secondary endosymbiosis event coincides with frequent introduction of reactive cysteines into existing proteins. While the plastid acquisition imposed an increase in the production of reactive oxygen species, our results suggest that it was accompanied by significant expansion of the RSP, providing redox regulatory networks the ability to cope with fluctuating environmental conditions.

Summary Viruses that infect marine photosynthetic microorganisms are major ecological and evolutionary drivers of microbial food webs, estimated to turn over more than a quarter of the total photosynthetically fixed carbon. Viral infection of the bloom-forming microalga Emiliania huxleyi induces the rapid remodeling of host primary metabolism, targeted towards fatty acid metabolism. We applied a liquid chromatography-mass spectrometry (LC-MS)-based lipidomics approach combined with imaging flow cytometry and gene expression profiling to explore the impact of viral-induced metabolic reprogramming on lipid composition. Lytic viral infection led to remodeling of the cellular lipidome, by predominantly inducing the biosynthesis of highly saturated triacylglycerols (TAGs), coupled with a significant accumulation of neutral lipids within lipid droplets. Furthermore, TAGs were found to be a major component (77%) of the lipidome of isolated virions. Interestingly, viral-induced TAGs were significantly more saturated than TAGs produced under nitrogen starvation. This study highlights TAGs as major products of the viral-induced metabolic reprogramming during the host?virus interaction and indicates a selective mode of membrane recruitment during viral assembly, possibly by budding of the virus from specialized subcellular compartments. These findings provide novel insights into the role of viruses infecting microalgae in regulating metabolism and energy transfer in the marine environment and suggest their possible biotechnological application in biofuel production.

The cosmopolitan coccolithophore Emiliania huxleyi is a unicellular eukaryotic alga responsible for vast blooms in the ocean. These blooms have immense impact on large biogeochemical cycles and are terminated by a specific large double-stranded DNA E. huxleyi virus (EhV, Phycodnaviridae). EhV infection is accompanied by induction of hallmarks of programmed cell death and production of reactive oxygen species (ROS). Here we characterized alterations in ROS metabolism and explored its role during infection. Transcriptomic analysis of ROS-related genes predicted an increase in glutathione (GSH) and H2O2 production during infection. In accordance, using biochemical assays and specific fluorescent probes we demonstrated the overproduction of GSH during lytic infection. We also showed that H2O2 production, rather than superoxide, is the predominant ROS during the onset of the lytic phase of infection. Using flow cytometry, confocal microscopy and multispectral imaging flow cytometry, we showed that the profound co-production of H2O2 and GSH occurred in the same subpopulation of cells but at different subcellular localization. Positively stained cells for GSH and H2O2 were highly infected compared with negatively stained cells. Inhibition of ROS production by application of a peroxidase inhibitor or an H2O2 scavenger inhibited host cell death and reduced viral production. We conclude that viral infection induced remodeling of the host antioxidant network that is essential for a successful viral replication cycle. This study provides insight into viral replication strategy and suggests the use of specific cellular markers to identify and quantify the extent of active viral infection during E. huxleyi blooms in the ocean.

Abstract Oxidative stress is generated in plants because of inequalities in the rate of reactive oxygen species (ROS) generation and scavenging. The subcellular redox state under various stress conditions was assessed using the redox reporter roGFP2 targeted to chloroplastic, mitochondrial, peroxisomal and cytosolic compartments. In parallel, the vitality of the plant was measured by ion leakage. Our results revealed that during certain physiological stress conditions the changes in roGFP2 oxidation are comparable to application of high concentrations of exogenous H2O2. Under each stress, particular organelles were affected. Conditions of extended dark stress, or application of elicitor, impacted chiefly on the status of peroxisomal redox state. In contrast, conditions of drought or high light altered the status of mitochondrial or chloroplast redox state, respectively. Amalgamation of the results from diverse environmental stresses shows cases of organelle autonomy as well as multi-organelle oxidative change. Importantly, organelle-specific oxidation under several stresses proceeded cell death as measured by ion leakage, suggesting early roGFP oxidation as predictive of cell death. The measurement of redox state in multiple compartments enables one to look at redox state connectivity between organelles in relation to oxidative stress as well as assign a redox fingerprint to various types of stress conditions.

Marine viruses are considered to be major ecological, evolutionary, and biogeochemical drivers of the marine environment, responsible for nutrient recycling and determining species composition. Viruses can re-shape their host's metabolic network during infection, generating the virocell–a unique metabolic state that supports their specific requirement. Here we discuss the concept of ‘virocell metabolism’ and its formation by rewiring of host-encoded metabolic networks, or by introducing virus-encoded auxiliary metabolic genes which provide the virocell with novel metabolic capabilities. The ecological role of marine viruses is commonly assessed by their relative abundance and phylogenetic diversity, lacking the ability to assess the dynamics of active viral infection. The new ability to define a unique metabolic state of the virocell will expand the current virion-centric approaches in order to quantify the impact of marine viruses on microbial food webs.