Biochemical Characterization of a Novel Redox-Regulated Metacaspase in a Marine Diatom

Programmed cell death (PCD) in marine microalgae was suggested to be one of the mechanisms that facilitates bloom demise, yet its molecular components in phytoplankton are unknown. Phytoplankton are completely lacking any of the canonical components of PCD, such as caspases, but possess metacaspases. Metacaspases were shown to regulate PCD in plants and some protists, but their roles in algae and other organisms are still elusive. Here, we identified and biochemically characterized a type III metacaspase from the model diatom Phaeodactylum tricornutum, termed PtMCA-IIIc. Through expression of recombinant PtMCA-IIIc in E. coli, we revealed that PtMCA-IIIc exhibits a calcium-dependent protease activity, including auto-processing and cleavage after arginine. Similar metacaspase activity was detected in P. tricornutum cell extracts. PtMCA-IIIc overexpressing cells exhibited higher metacaspase activity, while CRISPR/Cas9-mediated knockout cells had decreased metacaspase activity compared to WT cells. Site-directed mutagenesis of cysteines that were predicted to form a disulfide bond decreased recombinant PtMCA-IIIc activity, suggesting its enhancement under oxidizing conditions. One of those cysteines was oxidized, detected in redox proteomics, specifically in response to lethal concentrations of hydrogen peroxide and a diatom derived aldehyde. Phylogenetic analysis revealed that this cysteine-pair is unique and widespread among diatom type III metacaspases. The characterization of a cell death associated protein in diatoms provides insights into the evolutionary origins of PCD and its ecological significance in algal bloom dynamics

Rewiring Host Lipid Metabolism by Large Viruses Determines the Fate of Emiliania huxleyi

Marine viruses are major ecological and evolutionary drivers of microbial food webs regulating the fate of carbon in the ocean. We combined transcriptomic and metabolomic analyses to explore the cellular pathways mediating the interaction between the bloom-forming coccolithophore Emiliania huxleyi and its specific coccolithoviruses (E. huxleyi virus [EhV]). We show that EhV induces profound transcriptome remodeling targeted toward fatty acid synthesis to support viral assembly. A metabolic shift toward production of viral-derived sphingolipids was detected during infection and coincided with downregulation of host de novo sphingolipid genes and induction of the viral-encoded homologous pathway. The depletion of host-specific sterols during lytic infection and their detection in purified virions revealed their novel role in viral life cycle. We identify an essential function of the mevalonate-isoprenoid branch of sterol biosynthesis during infection and propose its downregulation as an antiviral mechanism. We demonstrate how viral replication depends on the hijacking of host lipid metabolism during the chemical “arms race” in the ocean

Unmasking cellular response of a bloom-forming alga to viral infection by resolving expression profiles at a single-cell level.

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

A single-cell view on alga-virus interactions reveals sequential transcriptional programs and infection states

The discovery of giant viruses infecting eukaryotes from diverse ecosystems has revolutionized our understanding of the evolution of viruses and their impact on protist biology, yet knowledge on their replication strategies and transcriptome regulation remains limited. Here, we profile single-cell transcriptomes of the globally distributed microalga Emiliania huxleyi and its specific giant virus during infection. We detected profound heterogeneity in viral transcript levels among individual cells. Clustering single cells based on viral expression profiles enabled reconstruction of the viral transcriptional trajectory. Reordering cells along this path unfolded highly resolved viral genetic programs composed of genes with distinct promoter elements that orchestrate sequential expression. Exploring host transcriptome dynamics across the viral infection states revealed rapid and selective shutdown of protein-encoding nuclear transcripts, while the plastid and mitochondrial transcriptomes persisted into later stages. Single-cell RNA-seq opens a new avenue to unravel the life cycle of giant viruses and their unique hijacking strategies.This work was supported by the European Research Council Consolidator Grant (CoG) (VIROCELLSPHERE consolidator grant no. 681715; A.V.) and the European Molecular Biology Organization Long-Term Fellowship (ALTF 1172-2016; C.K.

Organelles Contribute Differentially to Reactive Oxygen Species-Related Events during Extended Darkness1[C][W][OA]

Treatment of Arabidopsis (Arabidopsis thaliana) leaves by extended darkness generates a genetically activated senescence program that culminates in cell death. The transcriptome of leaves subjected to extended darkness was found to contain a variety of reactive oxygen species (ROS)-specific signatures. The levels of transcripts constituting the transcriptome footprints of chloroplasts and cytoplasm ROS stresses decreased in leaves, as early as the second day of darkness. In contrast, an increase was detected in transcripts associated with mitochondrial and peroxisomal ROS stresses. The sequential changes in the redox state of the organelles during darkness were examined by redox-sensitive green fluorescent protein probes (roGFP) that were targeted to specific organelles. In plastids, roGFP showed a decreased level of oxidation as early as the first day of darkness, followed by a gradual increase to starting levels. However, in mitochondria, the level of oxidation of roGFP rapidly increased as early as the first day of darkness, followed by an increase in the peroxisomal level of oxidation of roGFP on the second day. No changes in the probe oxidation were observed in the cytoplasm until the third day. The increase in mitochondrial roGFP degree of oxidation was abolished by sucrose treatment, implying that oxidation is caused by energy deprivation. The dynamic redox state visualized by roGFP probes and the analysis of microarray results are consistent with a scenario in which ROS stresses emanating from the mitochondria and peroxisomes occur early during darkness at a presymptomatic stage and jointly contribute to the senescence program

Morphological switch to a resistant subpopulation in response to viral infection in the bloom-forming coccolithophore <i>Emiliania huxleyi</i>

<div><p>Recognizing the life cycle of an organism is key to understanding its biology and ecological impact. <i>Emiliania huxleyi</i> is a cosmopolitan marine microalga, which displays a poorly understood biphasic sexual life cycle comprised of a calcified diploid phase and a morphologically distinct biflagellate haploid phase. Diploid cells (2N) form large-scale blooms in the oceans, which are routinely terminated by specific lytic viruses (EhV). In contrast, haploid cells (1N) are resistant to EhV. Further evidence indicates that 1N cells may be produced during viral infection. A shift in morphology, driven by meiosis, could therefore constitute a mechanism for <i>E</i>. <i>huxleyi</i> cells to escape from EhV during blooms. This process has been metaphorically coined the ‘Cheshire Cat’ (CC) strategy. We tested this model in two <i>E</i>. <i>huxleyi</i> strains using a detailed assessment of morphological and ploidy-level variations as well as expression of gene markers for meiosis and the flagellate phenotype. We showed that following the CC model, production of resistant cells was triggered during infection. This led to the rise of a new subpopulation of cells in the two strains that morphologically resembled haploid cells and were resistant to EhV. However, ploidy-level analyses indicated that the new resistant cells were diploid or aneuploid. Thus, the CC strategy in <i>E</i>. <i>huxleyi</i> appears to be a life-phase switch mechanism involving morphological remodeling that is decoupled from meiosis. Our results highlight the adaptive significance of morphological plasticity mediating complex host–virus interactions in marine phytoplankton.</p></div

Gene-expression dynamics of meiosis- and life-phase-specific genes during EhV infection.

<p>Detailed expression profiles of profiles of meiosis- and S-cell-specific for <i>E</i>. <i>huxleyi</i> RCC 1216 and CCMP 2090 were performed by qPCR over the course of infection and are presented as heatmaps. The results are presented as Log₁₀ fold-change (2^-ΔΔCt) relative to control, uninfected cultures over time to enable of a clear visualization of the expression data encompassing a large range of variability. Blank heatmap cells represent time points with undetected gene expression levels. Gene expression was undetected for days 6 and 7 and therefore this time period was removed from the heatmap (gene expression data is available in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006775#ppat.1006775.s008" target="_blank">S2 Table</a>). The mean values of duplicate cultures are shown.</p

Host–virus dynamics and ultrastructural features of <i>E</i>. <i>huxleyi</i> cells.

<p>(A–I) Dynamic of growth infection and ultrastructural features of <i>E</i>. <i>huxleyi</i> RCC 1216, a 2N calcified strain. (A) Temporal dynamics of <i>E</i>. <i>huxleyi</i> during infection by EhV-201 compared to uninfected, control conditions. The arrow denotes the time (7 dpi) at which virus-resistant, biflagellate cells are first detected by light microscopy. (B–D) Light microscopy (LM) and transmission electron microscope (TEM) imagery of RCC 1216 cells. The close-up (D) highlights the presence of remnants of the organic matrix of coccoliths as indicate by an asterisk, but not organic scales. (E) TEM micrograph of an EhV-infected cell with multiple virions detected as dark electron-dense particles in the cytoplasm. (F) LM image of a haploid cell (RCC 1217), shown for comparison (see text for details). (G–I) LM and TEM images of a biflagellate cell isolated postinfection. The close-up (I) denotes the presence of organic scales (arrow) with patterns of fibrils bound to the cell membrane. (J–R) Dynamic of growth infection and ultrastructural features of <i>E</i>. <i>huxleyi</i> CCMP 2090, a 2N noncalcified strain. (J) Temporal dynamics of <i>E</i>. <i>huxleyi</i> during infection by EhV-201 as compared to uninfected, control conditions. The arrow denotes the time (35 dpi) at which virus-resistant nonmotile scale-bearing cells (nonmotile-S cell) were first detected by LM. (K–M) LM and TEM images of CCMP 2090 cells. (M) Arrow denotes the absence of organic scales bound to the cell membrane. (N) Cell infected by EhV, as described above in (E). (O) Close-up of EhV virions upon cell lysis. (P–R) LM and TEM images of nonmotile-S cells postinfection. The close-up (R) denotes the presence of organic body scales (arrow), as described in (I). The mean ± standard deviation of duplicate cultures is shown. The scale bars are 1 μm for all TEM images, except for images D, I, M, O and R in which the scale bar = 0.2 μm.</p