Editing of the urease gene by CRISPR-Cas in the diatom<i>Thalassiosira pseudonana</i>

Hopes, Amanda; Nekrasov, Vladimir; Kamoun, Sophien; Möck, Thomas

doi:10.1101/062026

Cited by 25 publications

(43 citation statements)

References 49 publications

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“…The genus Thalassiosira is comprised of centric bloomforming diatoms, widely distributed throughout the world's oceans (Malviya et al 2015). The Thalassiosira pseudonana is an important model system for studying diatom ecophysiology, cell biology, cell signaling, and metabolism due to the available genetic and genomic tools (Armbrust et al 2004, Poulsen et al 2006, Hennon et al 2015, Hopes et al 2016.…”

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confidence: 99%

Diurnal fluctuations in chloroplast GSH redox state regulate susceptibility to oxidative stress and cell fate in a bloom‐forming diatom

Volpert

Creveld

Rosenwasser

2018

Journal of Phycology

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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 (E ) in the chloroplast over diurnal cycles revealed distinct rhythmic patterns. Intriguingly, in the dark, cells exhibited reduced basal chloroplast E 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 E was observed in the light phase prior to the transition to darkness, suggesting an anticipatory phase. Rapid chloroplast E 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.

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confidence: 99%

Diurnal fluctuations in chloroplast GSH redox state regulate susceptibility to oxidative stress and cell fate in a bloom‐forming diatom

Volpert

Creveld

Rosenwasser

2018

Journal of Phycology

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“…Plasmid construction using Golden Gate cloning: Golden Gate cloning was carried out previously described 75 , in a design similar to Hopes at al. 72 (Supplementary Fig. 6a).…”

Section: Ptmc5 Expression and Purification: MC Expression Purificatimentioning

confidence: 99%

“…Protease inhibitors, in which the uncleavable fluoromethylketone (fmk) group is conjugated to the short peptide, z-VRPRfmk (25 µM, MC inhibitor) and z-VAD-fmk (100 µM, pan-caspase inhibitor) (both from abcam) were incubated for 30 min before the addition of the substrate. Importantly, the measurement of protease activity in cell extracts, in adequate activity buffer, does not represent the actual in vivo activity as MCs are often inactive zymogens, but the potential of MC typical activity upon activation signal.gRNA design for PtMC5 knockout: In order to inactivate PtMC5 we adapted for P. tricornutum the method established by Hopes et al for the diatom T. pseudonana72,73 .…”

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confidence: 99%

A redox-regulated type III metacaspase controls cell death in a marine diatom

Creveld¹,

Ben‐Dor

Mizrachi³

et al. 2018

Preprint

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Programmed cell death (PCD) in marine phytoplankton was suggested as one of the mechanisms that facilitates large scale bloom demise. Yet, the molecular basis for algal PCD machinery is rudimentary. Metacaspases are considered ancestral proteases that regulate cell death, but their activity and role in algae are still elusive. Here we biochemically characterized a recombinant metacaspase 5 from the model diatom Phaeodactylum tricornutum (PtMC5), revealing calcium-dependent protease activity. This activity includes auto-processing and cleavage following arginine. PtMC5 overexpressing cells exhibited higher metacaspase activity and were more sensitive to a diatom-specific infochemical compared to WT cells. Mutagenesis of potential disulfide-forming cysteines decreased PtMC5 activity, suggesting redox regulation. This cysteine pair is widespread in diatom type III metacaspases, but was not found in any other taxa. The characterization of a cell death associated protein in marine phytoplankton will enable deeper understanding of the ecological significance of PCD in bloom dynamics.

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“…Algae are key primary producers in aquatic environments and represent several emerging genetic model systems (Armbrust et al, 2004;Hopes et al, 2016;Nymark et al, 2016). They also play an increasingly important role in human nutrition (FAO, 2014).…”

Section: Introductionmentioning

confidence: 99%

Biotic interactions as drivers of algal origin and evolution

et al. 2017

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Contents 670 I. 671 II. 671 III. 676 IV. 678 678 References 678 SUMMARY: Biotic interactions underlie life's diversity and are the lynchpin to understanding its complexity and resilience within an ecological niche. Algal biologists have embraced this paradigm, and studies building on the explosive growth in omics and cell biology methods have facilitated the in-depth analysis of nonmodel organisms and communities from a variety of ecosystems. In turn, these advances have enabled a major revision of our understanding of the origin and evolution of photosynthesis in eukaryotes, bacterial-algal interactions, control of massive algal blooms in the ocean, and the maintenance and degradation of coral reefs. Here, we review some of the most exciting developments in the field of algal biotic interactions and identify challenges for scientists in the coming years. We foresee the development of an algal knowledgebase that integrates ecosystem-wide omics data and the development of molecular tools/resources to perform functional analyses of individuals in isolation and in populations. These assets will allow us to move beyond mechanistic studies of a single species towards understanding the interactions amongst algae and other organisms in both the laboratory and the field.

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Editing of the urease gene by CRISPR-Cas in the diatomThalassiosira pseudonana

Cited by 25 publications

References 49 publications

Diurnal fluctuations in chloroplast GSH redox state regulate susceptibility to oxidative stress and cell fate in a bloom‐forming diatom

Diurnal fluctuations in chloroplast GSH redox state regulate susceptibility to oxidative stress and cell fate in a bloom‐forming diatom

A redox-regulated type III metacaspase controls cell death in a marine diatom

Biotic interactions as drivers of algal origin and evolution

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