Biology subjects

Hopes, A.

Publications and source records attributed to Hopes, A..

4 recordsLinked to original sources

bioRxiv2022

Disentangling the genomic complexity of the Fragilariopsis cylindrus (CCMP1102) genome

Fragilariopsis cylindrus CCMP1102 is characterised by a complex genome with significant levels of heterozygosity between haplotypes, > 35% repeats, and an unknown karyotype. This complexity hindered prior assemblies, which show coverage discrepancies indicative of incompleteness. Here, we use a k-mer spectra analysis to reveal the coverage signature for a third haplotype. We applied a novel haplotype-specific assembly method to reconstruct the F. cylindrus CCMP1102 genome, producing 10 fully assembled chromosomes capped by telomeres, and a putative chromosome with a single breakpoint. Our analysis shows triploidy, two cases of aneuploidy, and several truncations. We also present evidence that F. cylindrus reproduces sexually. Taken together, our analytical approach is capable of haplotype-resolved assemblies from structurally complex, poly-ploid genomes, making it suitable for complex genomes of non-model organisms, including those with unknown karyotype.

genomics↗

bioRxiv2022

Cold-induced cyt elevations function to support osmoregulation in marine diatoms

Diatoms are a group of microalgae that are important primary producers in a range of open ocean, freshwater and intertidal environments. The latter can experience significant long- and short-term variability in temperature, from seasonal variations to rapid temperature shifts caused by tidal immersion and emersion. As temperature is a major determinant in the distribution of diatom species, their temperature sensory and response mechanisms likely have important roles in their ecological success. We have examined the mechanisms diatoms use to sense rapid changes in temperature, such as those experienced in the intertidal zone. We find that the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana exhibit a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in response to rapid cooling, similar to those observed in plant and animal cells. However, [Ca2+]cyt elevations were not observed in response to rapid warming. The kinetics and magnitude of cold-induced [Ca2+]cyt elevations correlate with the rate of temperature decrease. We do not find a role for the [Ca2+]cyt elevations in enhancing cold tolerance, but show that cold shock induces a Ca2+-dependent K+ efflux and reduces mortality of P. tricornutum during a simultaneous hypo-osmotic shock. As inter-tidal diatom species may routinely encounter simultaneous cold and hypo-osmotic shocks during tidal cycles, we propose that cold-induced Ca2+ signalling interacts with osmotic signalling pathways to aid in the regulation of cell volume. Our findings provide insight into the nature of temperature perception in diatoms and highlight that cross-talk between signalling pathways may play an important role in their cellular responses to multiple simultaneous stressors.

plant biology↗

bioRxiv2018

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

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.

microbiology↗

bioRxiv2017

Efficient CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana

CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase and there are no examples of efficient nuclease-meditated HR in any photosynthetic organism. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR for the first time in a diploid photosynthetic organism. HR was induced in T. pseudonana by means of sequence specific CRISPR/Cas, paired with a donor matrix, generating substitution of the silacidin gene by a resistance cassette (FCP:NAT). Approximately 85% of NAT resistant T. pseudonana colonies screened positive for HR using a nested PCR approach and confirmed by sequencing of the PCR products. The knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and any biotechnological application targeted on harnessing the metabolic potential of diatoms.

molecular biology↗