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Davies Group: Environmental sensing in a marine diatom

GFC / OTHER

Supervisor: Julia Davies (Plant Sciences
Co-Supervisor: Katherine Helliwell (Marine Biological Association)

Importance of the area of research:

Diatoms are a diverse group of unicellular algae characterised by their ability to produce a silicified cell wall (frustule). They are abundant primary producers in marine and freshwater ecosystems, supporting life at higher trophic levels in these environments. Diatoms are typified by their ability to divide rapidly when they encounter favourable conditions, and sophisticated signalling mechanisms most likely contribute to their ecological success. Recent evidence from Helliwell has indicated that marine diatoms have evolved novel mechanisms for environmental perception, which are distinct from those of plants and animals [1]. This work demonstrates that unique processes for environmental perception may have evolved in the oceans. Nevertheless, many aspects of how diatoms sense and respond to key environmental variables are poorly understood. A particularly important factor controlling diatom growth is the availability of nutrients, which can vary in abundance in space and in time. A growing body of evidence from plant research has led to the recognition of the importance of Ca2+ signalling in sensing nutrients (e.g., nitrate and zinc) in eukaryotes [2]. We have recently discovered a novel calcium-signalling pathway in diatoms for sensing the important macronutrient phosphate, which is in limiting supply in many marine ecosystems [3]. Our evidence suggests that this novel P-Ca2+ signalling pathway plays a vital role in regulating early acclimation responses of phosphate deplete cells to newly available phosphate. The aim of this project is to employ cutting-edge gene-editing approaches to characterise some of the molecular machinery that underpins this novel pathway. This work will provide important insight of the molecular underpinnings of a novel signalling pathway not previously described before in eukaryotes. Moreover, this work is vital to gaining a broader understanding of the diversity and evolution of signalling mechanisms across the eukaryote tree of life.

What the project will involve:

Functional characterisation of candidate genes of the novel phosphate Ca2+ signalling pathway

  • Pharmacological studies have identified inhibitors that block the P-Ca2+ signalling pathway and provide clues into the identity of the channel/s underpinning the phosphate induced Ca2+ signal. A key aim of the project will be to characterise these genes in Phaeodactylum via CRISPR-Cas9 gene knockout, localisation studies, and electrophysiology approaches with heterologous expression systems.
  • Other candidate genes identified via proteomics/literature review that we hypothesise to be involved in upstream/downstream processes will also be examined.

Develop genetically-encoded phosphate biosensor to measure phosphate homeostasis in single diatom cells

  • We have established transgenic strains of Phaeodactylum expressing the phosphate biosensor cpFLIPPi [4]. This provides a platform to examine phosphate dynamics in single cells and to better understand how phosphate homeostasis in the cell may be controlled by the P-Ca2+ signalling mechanisms. This will involve the study of cells exposed to different phosphate regimes, including resupply of phosphate following phosphate starvation. Development of mutant lines in the biosensor strain could moreover enable direct examination of the impacts of gene disruption on recovery of intracellular phosphate dynamics.

What the student will be doing:

The studentship will couple cell and molecular biology approaches using CRISPR-Cas9 genome-editing, algal physiology, microbiology and state-of-the-art live cell imaging, as outlined further below:

  • Molecular and cell biology: A key component of the project will be characterizing candidate genes (calcium channels, and other pathway components) in the model diatom Phaeodactylum. This will be done by employing CRISPR-Cas9 approaches that we have already established in diatoms. Techniques such as cloning, PCR, plasmid, guide RNA design, and algal transformation using biolistic bombardment will be employed for this purpose.
  • Algal physiology: mutants will be characterised via a range of approaches to determine gene function. Mutant and wild type lines will be grown in different phosphate regimes, and growth, photosynthetic efficiency, elemental stoichiometry, and cell morphology examined. Examination of mutants under other abiotic/biotic stress conditions will enable the broader impacts of the mutations to be examined.
  • Live-cell imaging: the project will employ cutting-edge genetically-encoded fluorescent biosensors to measure intracellular Ca2+ levels, and phosphate in live diatom cells. In addition, localization studies of candidate genes fused to fluorescent tags will determine their subcellular localization. A combination of epifluorescence and confocal microscopy will be employed for this work.
  • Bioinformatics and phylogenetics: Mining of genome and transcriptome databases will enable the abundance and distribution of candidate genes to be examined across algal lineages more broadly. This approach combined with phylogenetic trees will enable the evolutionary diversity of the channels to be assessed.

Training that will be provided:

  • Training will be provided in a range of algal physiology approaches (cell culture, measurement of growth (cell counting) and cell health parameters (photosynthetic efficiency). Part of this study will be based at the MBA with opportunities to gain field experience on the MBA research vessel at the Western Channel Observatory.
  • Supervision in molecular biology techniques (PCR, E. coli transformation, plasmid design, making and screening mutants via CRISPR-Cas9) will also be given.
  • Microscopy training in live-cell imaging and confocal/epifluorescence visualization of algal cells will also be provided. A state of the art microscopy suite is available at the MBA. Data analysis approaches using Image J and other programs will also be a key component of the project.
  • Bioinformatics training in performing sequence similarity searches, and domains analysis, and tree-building will also be given.
  • Professional development, including training in core verbal (preparing and giving effective conference talks and poster presentations) and written communication (paper writing, report and thesis writing) skills will be integral.

References:

  1. Helliwell K et al.. Alternative mechanisms for fast Na+/Ca2+ signaling in eukaryotes via a novel class of single-domain voltage-gated channels. Current Biology (2019) 29: 1503.
  2. Mattthus E., Wilken K., Swarbreck S., Doddrell N., Doccula G., Costa A., Davies J., Phosphate Starvation Alters Abiotic-Stress-Induced Cytosolic Free Calcium Increases in Roots. Plant Physiology (2019) 179: 1754.
  3. de Carvalho et al.. Noncoding and coding transcriptome responses of a marine diatom to phosphate fluctuations. New Phytologist (2016) 210: 497.
  4. Mukherjee et al., Live Imaging of Inorganic Phosphate in Plants with Cellular and Subcellular Resolution. Plant Physiology (2015) 167:628.

 

 

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