A cellular and molecular atlas reveals the basis of chytrid development
- Marine Biological Association, The Laboratory, Citadel Hill, United Kingdom
- School of Environmental Sciences, University of East Anglia, United Kingdom
- School of Biological and Marine Sciences, University of Plymouth, United Kingdom
Figures
Figure 1 with 1 supplement
Chytrids are a major fungal phylum with a dimorphic life cycle.
(A) Chytrids (phylum Chytridiomycota) are a major fungal lineage, many members of which exhibit cellular characteristics retained from the last common ancestor of branching (rhizoidal and hyphal) …
Figure 1—figure supplement 1
Representative images from confocal surveys conducted to assess the synchronicity of cell cultures for serial block face scanning electron microscopy (SBF-SEM) and RNA-Seq harvesting.
Cells diluted 1:1000, fixed in 0.2% formaldehyde, and stained with FM 1–43 FX to visualise cell membranes. Asterisks mark mature zoosporangia in mixed populations.
Figure 2 with 5 supplements
Serial block face scanning electron microscopy (SBF-SEM) reconstructions of the Rhizoclosmatium globosum life cycle.
(A) Representative SBF-SEM reconstructions of the first three life stages of the R. globosum life cycle. Bottom row shows the stages to scale. Organelle colours as in (B–D) and conserved throughout. …
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Figure 2—source data 1
Data associated with Figure 2.
- https://cdn.elifesciences.org/articles/73933/elife-73933-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Workflow of the image analysis protocol used to generate and visualise 3D reconstructions of chytrid cells from serial block face scanning electron microscopy (SBF-SEM) stacks.
Figure 2—figure supplement 2
Examples of subcellular components identified in this study taken from single serial block face scanning electron microscopy (SBF-SEM) slices.
(A) Whole cell slices of each chytrid life stage showing the localisation and orientation of subcellular structures in context. (B) High magnification images of individual subcellular structures …
Figure 2—figure supplement 3
Individual 3D serial block face scanning electron microscopy (SBF-SEM) reconstructions of Rhizoclosmatium globosum cells (not to scale) across life stages labelled with replicate IDs.
Organelle colours as in Figure 2A–D. Top row shows all replicates to scale.
Figure 2—figure supplement 4
Individual volumetric compositions of assigned organelles from Rhizoclosmatium globosum serial block face scanning electron microscopy (SBF-SEM) reconstructions across life stages labelled with replicate IDs.
Organelle colours as in Figure 2A–D.
Figure 2—figure supplement 5
Comparisons of volumetric proportions of subcellular structures across chytrid life stages (n = 5).
n.s p > 0.05 (not significant), *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3 with 5 supplements
Transcriptome analysis of the Rhizoclosmatium globosum life cycle.
(A) Shared and unique gene mRNA counts between life stages. Inset shows total gene mRNA counts per life stage. (B) Pairwise comparison of differentially expressed genes (DEGs) between germlings and …
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Figure 3—source data 1
Data associated with Figure 3.
- https://cdn.elifesciences.org/articles/73933/elife-73933-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Heatmap clustering of all differentially expressed genes (DEGs) between zoospore, germling, and immature thallus replicates.
Figure 3—figure supplement 2
Gene Ontology (GO) categories significantly downregulated (p < 0.05) in germlings relative to zoospores.
GeneRatio is number of genes significantly upregulated/total number of genes. Circle size represents numbers of genes.
Figure 3—figure supplement 3
Gene Ontology (GO) categories significantly upregulated (p < 0.05) in germlings relative to zoospores.
GeneRatio is number of genes significantly upregulated/total number of genes. Circle size represents numbers of genes.
Figure 3—figure supplement 4
Gene Ontology (GO) categories significantly downregulated (p < 0.05) in immature thalli relative to germlings.
GeneRatio is number of genes significantly upregulated/total number of genes. Circle size represents numbers of genes.
Figure 3—figure supplement 5
Gene Ontology (GO) categories significantly upregulated (p < 0.05) in immature thalli relative to germlings.
GeneRatio is number of genes significantly upregulated/total number of genes. Circle size represents numbers of genes.
Figure 4 with 1 supplement
Changes in lipid and lipid-associated cell structures occur with transitions between life stages.
(A) Fluorescent labelling of Rhizoclosmatium globosum shows distinct shifts in lipid structures across the chytrid life cycle and cell wall. Dashed line demarks cell boundary where not labelled in …
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Figure 4—source data 1
Data associated with Figure 4.
- https://cdn.elifesciences.org/articles/73933/elife-73933-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Comparison of sporulation times (as a proxy for normal cell development) for dye-labelled chytrid populations (n = 5) imaged by live-cell microscopy, relative to no-dye controls.
n.s. p > 0.05 (not significant).
Figure 5 with 1 supplement
The apophysis is a distinct subcellular structure characterised by increased endomembrane trafficking.
(A) Live-cell imaging (n = 5) of Rhizoclosmatium globosum population-level FM1–43-stained endomembrane dynamics. Purple = mean endomembrane fluorescence (±min/max), black = mean total cell area …
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Figure 5—source data 1
Data associated with Figure 5.
- https://cdn.elifesciences.org/articles/73933/elife-73933-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Comparisons of volumetric proportions of subcellular structures between immature thalli cell bodies and their corresponding apophyses (n = 5).
n.s. p > 0.05 (not significant), *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6 with 1 supplement
Developing zoospores in the zoosporangium displayed an amoeboid morphology with putative endocytotic activity.
(A) Fluorescent labelling of lipids, cell wall, and endomembrane in a Rhizoclosmatium globosum mature zoosporangium. Scale bar = 5 µm. (B–C) Serial block face scanning electron microscopy (SBF-SEM) …
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Figure 6—source data 1
Data associated with Figure 6.
- https://cdn.elifesciences.org/articles/73933/elife-73933-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Comparisons of volumetric proportions of subcellular structures between developing and mature zoospores (n = 5).
n.s. p > 0.05 (not significant), *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7
Summary of key components of the chytrid cell plan and biological processes associated with the transition between stages in the Rhizoclosmatium globosum life cycle.
Inner life cycle shows life stages to scale. Grey dashed lines indicate the beginning of the rhizoid system.
Videos
Video 1
Serial block face scanning electron microscopy (SBF-SEM) reconstructions allowed the structural comparison of life stages in Rhizoclosmatium globosum.
Representative SBF-SEM reconstructions of the zoospore, germling, and immature thallus life stages for comparison. Zoospore and germling cells shown to scale at the beginning of the video, and later …
Video 2
Structural shifts in lipid globules were observed across Rhizoclosmatium globosum life stages, associated with the change from catabolism/conversion to anabolism.
Representative serial block face scanning electron microscopy (SBF-SEM) reconstructions of the zoospore, germling, and immature thallus lipid structures for comparison.
Video 3
The zoospore lipid globule remained as an intact structure across the Rhizoclosmatium globosum life cycle.
Automated particle tracking of lipid globules (red) across the chytrid life cycle. Magenta circles mark individual lipid globules. Yellow track shows particle tracking of the initial lipid globule …
Video 4
The Rhizoclosmatium globosum apophysis is structurally dominated by endomembrane structures.
Representative serial block face scanning electron microscopy (SBF-SEM) reconstruction of a chytrid apophysis from an immature thallus.
Video 5
The Rhizoclosmatium globosum apophysis links intracellular trafficking between the rhizoids and cell body.
Live-cell imaging of endomembrane dynamics in the chytrid apophysis. The apophysis links endomembrane dynamics between the rhizoid system and thallus. Shown are DIC (left), endomembrane (centre), …
Video 6
Serial block face scanning electron microscopy (SBF-SEM) reconstruction of a Rhizoclosmatium globosum mature zoosporangium.
Video 7
Developingidual 3D serial block face scanning electron micr zoospores were more amoeboid than mature zoospores in Rhizoclosmatium globosum, putatively due to elevated endocytosis and trafficking.
Representative serial block face scanning electron microscopy (SBF-SEM) reconstructions of the ‘mature’ zoospore and developing zoospore life stages for comparison.
Additional files
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Supplementary file 1
Mean volumetric quantities of cellular structures recorded across chytrid life stages.
- https://cdn.elifesciences.org/articles/73933/elife-73933-supp1-v1.docx
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Supplementary file 2
Mean numerical quantities of cellular structures recorded across chytrid life stages.
- https://cdn.elifesciences.org/articles/73933/elife-73933-supp2-v1.docx
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Supplementary file 3
Mean volumetric percentages and statistical comparisons of cellular structures recorded across chytrid life stages.
- https://cdn.elifesciences.org/articles/73933/elife-73933-supp3-v1.docx
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Supplementary file 4
Mean volumetric percentages and statistical comparisons of cell bodies and their corresponding apophyses in immature thalli.
- https://cdn.elifesciences.org/articles/73933/elife-73933-supp4-v1.docx
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Supplementary file 5
Mean volumetric percentages and statistical comparisons of free-swimming and developing zoospores.
- https://cdn.elifesciences.org/articles/73933/elife-73933-supp5-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/73933/elife-73933-transrepform1-v1.docx
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Source code 1
Python script used to quantify population-level fluorescence of developing chytrid cells (Figures 4 and 5).
- https://cdn.elifesciences.org/articles/73933/elife-73933-code1-v1.zip
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Source code 2
Python script used to quantify single-cell Nile Red fluorescence of settled chytrid zoospores (Figure 4).
- https://cdn.elifesciences.org/articles/73933/elife-73933-code2-v1.zip
