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Editor's Pick Research Article | Molecular Biology and Physiology

Pathways of Pathogenicity: Transcriptional Stages of Germination in the Fatal Fungal Pathogen Rhizopus delemar

Poppy C. S. Sephton-Clark, Jose F. Muñoz, Elizabeth R. Ballou, Christina A. Cuomo, Kerstin Voelz
Aaron P. Mitchell, Editor
Poppy C. S. Sephton-Clark
aInstitute for Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom
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  • ORCID record for Poppy C. S. Sephton-Clark
Jose F. Muñoz
bInfectious Disease and Microbiome Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA
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Elizabeth R. Ballou
aInstitute for Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom
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Christina A. Cuomo
bInfectious Disease and Microbiome Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA
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  • ORCID record for Christina A. Cuomo
Kerstin Voelz
aInstitute for Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom
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Aaron P. Mitchell
Carnegie Mellon University
Roles: Editor
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DOI: 10.1128/mSphere.00403-18
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  • FIG 1
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    FIG 1

    Phenotypic characterization of germinating spores. (A) Spores germinated in SAB were imaged at hours (indicated by white numbers) postgermination. Scale bar = 50 µm for all images. Micrographs representative of >3 replicate experiments are shown. (B) Diameter of ungerminated spore bodies (n = 3; time [T] = 0 h) compared to spore body size measured immediately prior to germ tube emergence for each spore (n = 3; T = 4 to 6 h). (C) Spore germination as a percentage over time, determined by live-cell imaging (n = 3). (D) Fungal mass over time, determined by optical density at 600 nm.

  • FIG 2
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    FIG 2

    Principal-component analysis of 7,942 genes differentially expressed across all time points (n = 3 for each time point; T = 0, 1, 2, 3, 4, 5, 6, 12, 16, or 24 h postgermination). Each time point is color coded.

  • FIG 3
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    FIG 3

    Resting-spore-specific expression. (Top) Heat map displaying the absence (blue) or presence (red) of 10 or more transcripts for a given gene over time. The average percentage of the transcriptome expressed at any given time point is given below. (Bottom) Coexpression diagram, where each node represents a gene only expressed in ungerminated spores. Nodes linked to 10 or more others are highlighted in yellow, with their functions shown adjacent.

  • FIG 4
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    FIG 4

    Clustering of expression over time. (A) Heat map displaying differentially expressed genes. Expression levels are plotted in log2, space and mean centered (FDR of <0.001) across the entire time course. k-means clustering has partitioned genes into 7 clusters, as indicated by colored bars and numbered graphs below the heat map. (B) Graphs displaying cluster expression over time (0 to 24 h). (C) Table displaying categories enriched (hypergeometric test, corrected P value of <0.05), indicated in red, for clusters 1 to 7.

  • FIG 5
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    FIG 5

    Differential gene expression over time. (A) The number of genes significantly differentially expressed (multiply corrected P value of <0.05) between time points, shown over time. Green bars indicate genes with an increase in expression (log fold change [FC] of >2), while red bars indicate genes with a decrease in expression (log FC of <−2). (B) Enriched categories for the up- or downregulated genes over time. Green boxes indicate an overall upregulation of this category, red indicates an overall downregulation and red-green hatching indicates mixed regulation of this category. (C) Expression profiles of transcripts in specific categories over time, with the number of transcripts represented by each trend shown in parentheses.

  • FIG 6
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    FIG 6

    Cell wall dynamics and inhibition of germination. (A) Spores germinated for 0, 3, 6, 12, and 24 h, stained with calcofluor white (CFW), fluorescein (FITC), and ROS stain carboxy-H2DCFDA (ROS). (B) Germination is inhibited by 5 mM hydrogen peroxide and over 1.5 nM antimycin A, as determined by live-cell imaging, after 5 h of germination in SAB. The hydrogen peroxide control consists of an equivalent volume of H2O, and the antimycin A control consists of an equivalent volume of 100% ethanol.

  • FIG 7
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    FIG 7

    Number of homologous genes significantly differentially expressed (multiply corrected P value of <0.05) between time points, shown over time. Green represents the number of A. niger genes, red represents the number of R. delemar genes, and dark red represents the number of R. delemar genes found in high-synteny regions of the R. delemar genome.

Supplemental Material

  • Figures
  • FIG S1

    Table displaying genome annotation statistics, comparing the original R. delemar annotation (column 1) and the annotation updated with our RNA-Seq data (column 2). Download FIG S1, TIF file, 0.1 MB.

    Copyright © 2018 Sephton-Clark et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S2

    Sum of squares cluster analysis of PCA data. Download FIG S2, TIF file, 0.4 MB.

    Copyright © 2018 Sephton-Clark et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S1

    Listing of enriched terms. Download Table S1, TXT file, 0.03 MB.

    Copyright © 2018 Sephton-Clark et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S3

    Upregulation (red) of the Fe-S cluster biosynthetic pathway, determined with PathwayTools. Download FIG S3, TIF file, 16 MB.

    Copyright © 2018 Sephton-Clark et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Pathways of Pathogenicity: Transcriptional Stages of Germination in the Fatal Fungal Pathogen Rhizopus delemar
Poppy C. S. Sephton-Clark, Jose F. Muñoz, Elizabeth R. Ballou, Christina A. Cuomo, Kerstin Voelz
mSphere Sep 2018, 3 (5) e00403-18; DOI: 10.1128/mSphere.00403-18

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Pathways of Pathogenicity: Transcriptional Stages of Germination in the Fatal Fungal Pathogen Rhizopus delemar
Poppy C. S. Sephton-Clark, Jose F. Muñoz, Elizabeth R. Ballou, Christina A. Cuomo, Kerstin Voelz
mSphere Sep 2018, 3 (5) e00403-18; DOI: 10.1128/mSphere.00403-18
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    • ABSTRACT
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KEYWORDS

RNA-seq
Rhizopus delemar
fungi
germination
mucormycosis
pathogens
spores
time course
transcription

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