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Transcription in fungal conidia before dormancy produces phenotypically variable conidia that maximize survival in different environments

Nature Microbiology volume 6, pages 1066–1081 (2021)Cite this article

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Abstract

Fungi produce millions of clonal asexual conidia (spores) that remain dormant until favourable conditions occur. Conidia contain abundant stable messenger RNAs but the mechanisms underlying the production of these transcripts and their composition and functions are unknown. Here, we report that the conidia of three filamentous fungal species (Aspergillus nidulans, Aspergillus fumigatus, Talaromyces marneffei) are transcriptionally active and can synthesize mRNAs. We find that transcription in fully developed conidia is modulated in response to changes in the environment until conidia leave the developmental structure. Environment-specific transcriptional responses can alter conidial content (mRNAs, proteins and secondary metabolites) and change gene expression when dormancy is broken. Conidial transcription affects the fitness and capabilities of fungal cells after germination, including stress and antifungal drug (azole) resistance, mycotoxin and secondary metabolite production and virulence. The transcriptional variation that we characterize in fungal conidia explains how genetically identical conidia mature into phenotypically variable conidia. We find that fungal conidia prepare for the future by synthesizing and storing transcripts according to environmental conditions present before dormancy.

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Fig. 1: Filamentous fungal conidia have robust transcription activities by all three RNA polymerases.
Fig. 2: A. nidulans conidia on the conidiophores have distinct transcriptional profiles under different growth conditions.
Fig. 3: A. nidulans conidia remain active until release from the developmental structure (conidiophore).
Fig. 4: A. nidulans and A. fumigatus conidial transcription activity affects gliotoxin content in conidia and accelerates protein expression and molecular response on germination.
Fig. 5: A. nidulans and A. fumigatus conidial transcription activity before dormancy affects germination, stress and drug resistance and secondary metabolite production capacity after germination.
Fig. 6: Proposed mechanism for how conidial transcription activity before dormancy drives phenotypic variation in conidia and in hyphae after germination.

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Data availability

Next-generation sequencing data are available from the National Center for Biotechnology Information Sequence Read Archive database under accession no. PRJNA602418 for the RNAP II ChIP–seq data, PRJNA602550 for the TBP and TFIIB ChIP–seq data, PRJNA602580 for the RNAP I and RNAP III ChIP–seq data, PRJNA602549 for the histone modifications ChIP–seq data and PRJNA607649 for the RNA-seq data. A list of figures that have associated raw data is given in Supplementary Table 10. Gene annotation and GO information were obtained from AspGD (http://www.aspgd.org) and FungiDB (https://fungidb.org). Source data are provided with this paper.

Code availability

All scripts reported in the Methods are available at Github (https://github.com/zqmiao-mzq/closest_gene_calling/blob/master/zqWinSGR-v4.pl; https://github.com/zqmiao-mzq/closest_gene_calling/blob/master/closest_gene_calling_v10.pl; and https://github.com/sethiyap/FungalSporeAnalysis) or at the bioinformatics analysis platform FungiExpresZ (https://cparsania.shinyapps.io/FungiExpresZ/) (Chirag Parsania, unpublished).

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Acknowledgements

We thank members of the Wong laboratory for comments and discussions throughout the study and M. J. Hynes, A. Andrianopoulos, R. B. Todd, M. Momany and K. Struhl for insightful discussions. We acknowledge the services and technical supports from the Genomics and Single Cell Analysis Core and the Drug and Development Core of the Faculty of Health Sciences at the University of Macau. This work was performed in part at the High-Performance Computing Cluster (HPC), which is supported by the Information and Communication Technology Office (ICTO) of the University of Macau. We thank L. Pardeshi and N. Shirgaonkar from the Genomics, Bioinformatics and Single Cell Analysis Core and Z. Miao and C. Parsania for Bioinformatics support, and J. Chan from ICTO for technical support on the HPC. We also acknowledge the support from the Science and Technology Development Fund, Macao S.A.R (FDCT) (project no. 0106/2020/A), the Research Services and Knowledge Transfer Office (project nos. MYRG2018-00017-FHS and MYRG2019-00099-FHS) and Faculty of Health Sciences of the University of Macau to K.H.W.

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Authors and Affiliations

  1. Faculty of Health Sciences, University of Macau, Macau SAR, China

    Fang Wang, Pooja Sethiya, Xiaohui Hu, Shuhui Guo, Yingying Chen, Ang Li, Kaeling Tan & Koon Ho Wong

  2. Drug Development Core, Faculty of Health Sciences, University of Macau, Macau SAR, China

    Xiaohui Hu

  3. Genomics, Bioinformatics and Single Cell Analysis Core, Faculty of Health Sciences, University of Macau, Macau SAR, China

    Kaeling Tan

  4. Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China

    Koon Ho Wong

  5. MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, China

    Koon Ho Wong

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  1. Fang Wang
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Contributions

F.W. and K.H.W. conceived the study, designed the experiments and interpreted the data. F.W. performed the experiments. F.W. performed the bioinformatics analysis with help from P.S. K.T. performed the Illumina sequencing and provided technical support. F.W. and X.H. performed the LC–MS analysis. F.W., S.G., Y.C. and A.L. engineered the strains. K.H.W. provided the funding. F.W. and K.H.W. wrote the manuscript.

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Correspondence to Koon Ho Wong.

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The authors declare no competing interests.

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Peer review information Nature Microbiology thanks Jan Dijksterhuis, Jean-Paul Latge and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Conidia on conidiophore have robust transcription activities.

a, Genome browser screenshots showing RNAP II ChIPseq and input DNA signals at selected genes with differential RNAP II occupancies for conidia and hyphae of A. nidulans, A. fumigatus and T. marneffei. b, Heatmap plots of RNAP II ChIPseq and input DNA signals at all annotated genes for conidia of the three species. The number of RNAP II occupied genes for each species is indicated on the colour lines at the right. c, A schematic diagram showing statistically significant (p-value < 0.01) GO terms enriched among transcribed genes for the three species. Colour intensity and size of diamonds indicate p-value and percentage of background, respectively. The p-value was calculated by Fisher’s Exact test and was corrected for multiple testing using Benjamini-Hochberg false discovery rate and the Bonferroni method. d-f, Heatmap plots showing RNAP II ChIPseq signal at (d) conidia-enriched-transcripts (CET), conidial maturation and trehalose biosynthesis genes, (e) conidiation initiation genes, and (f) septin and β-tubulin genes for A. nidulans conidia and hyphae. Input DNA signals for conidia were included as a negative control. g, A representative microscopy image example for a conidial preparation of A. nidulans for ChIPseq and other experiments. The experiment was repeated three independent times with similar results, and representative image was presented. h-j, Boxplots showing (h) RNAP III (TFIIIBHA and Rpo31HA), (i) RNAP I (Rpc40HA and Rpa43HA) and (j) RNAP II binding signals at (h) tRNAs (n = 180) and 5 S rDNAs (n = 52), (i) rDNAs (n = 16) and (j) protein encoding genes (n = 1,030) in A. nidulans conidia and hyphae. Lower and upper hinges of boxes represent 25th and 75th percentiles, respectively, and the centre of boxes represents the median. The p-values shown were calculated using two samples t-test by ggplot2. The asterisks *, **, *** and **** depict p-values of <0.05, <0.005, <0.001 and <0.0001, respectively, while ns represents a p-value of >0.05. k, A scatter plot showing the comparable mRNA levels of ribosomal protein genes detected by RNAseq in conidia and hyphae.

Extended Data Fig. 2 A. nidulans conidia transcriptionally respond to the environment, activating condition-specific physiological pathways.

a-b, Heatbox plots showing (a) RNAP II binding and (b) mRNA levels of heat-shock response genes in conidia subjected to heat (42 °C) for 0.5 or 4 h as measured by (a) ChIP-qPCR and (b) RT-PCR, respectively. c, A heatbox plot showing RNAP II binding level as measured by ChIP-qPCR of heat shock response genes in hyphae after heat-shock treatment (4 h). d, Schematic diagrams showing GO analysis of differentially transcribed genes for A. nidulans conidia subjected to temperature shocks (4 and 42 °C) and control conidia (37 °C) or between A. nidulans conidia grown in the presence of sodium chloride (NaCl) or potassium chloride (KCl) or in the absence of zinc (Zinc-starved) comparing to control conidia from ANM media. Colour intensity and size of diamonds represent p-value (expressed as –log10p-value) and percentage of background. The p-value was calculated by Fisher’s Exact test and was corrected for multiple testing using Benjamini-Hochberg false discovery rate and the Bonferroni method. e, Heatbox plots showing the RNAP II binding level of glycerol transport and oxidative stress response genes in NaCl and KCl conidia compared to Control conidia.

Extended Data Fig. 3 A. fumigatus conidia transcriptionally respond to the environment, activating condition-specific physiological pathways.

a, A heatmap plot showing differential expressed genes (presented as z-scores) among A. fumigatus conidia formed from ANM media in the presence of sodium chloride (NaCl) and potassium chloride (KCl) or in the absence of zinc (Zinc-starved). b, Schematic diagrams showing gene ontology analysis of differentially transcribed genes for A. fumigatus conidia grown in the presence of sodium chloride (NaCl) or potassium chloride (KCl) or in the absence of zinc (Zinc-starved), as compared to control conidia from ANM media. Colour intensity and size of diamonds represent p-value (expressed as –log10p-value) and percentage of background. The p-value was calculated by Fisher’s Exact test and was corrected for multiple testing using Benjamini-Hochberg false discovery rate and the Bonferroni method.

Extended Data Fig. 4 A. fumigatus conidia have transcription and ATP-consuming activities even after conidial development has completed.

a, Genome browser screenshots showing RNAP II ChIPseq and input DNA signals at selected genes with differential RNAP II occupancies for 3 and 17 day-old conidia. b, Heatmap plots showing RNAP II ChIPseq signals at genes occupied by RNAP II in either 3 or 17 day-old conidia. c, Heatmap plot showing differential expressed genes between 3 or 17 day-old conidia. d-f, Bar plots showing (d) the number of conidia, (e) viability and (f) intracellular ATP levels for conidia of different developmental ages (3, 6 or 17 day-old). d-f Data are presented as mean values + SD. Error bars represent the SD of three independent experiments (n = 3).

Source data

Extended Data Fig. 5 Conidial ATP level is linked to conidial viability in A. nidulans.

a-b, Bar graphs showing the mean (a) conidia numbers and (b) intracellular conidial ATP levels after the indicated time (that is, developmental age of conidia; 3, 6, 10, 20, 30, 40, 50, 60 and 70 day after inoculation) from two independent experiments. The same trend was observed in the biological repeats and the raw data are presented in Source Data Extended Data Fig. 5. c, A bar graph showing the viability of conidia same as (a-b). Data are presented as mean values + SD. Error bars represent the standard deviation of three independent experiments (n = 3). d, A gel electrophoresis result showing the quality and yield of chromatin extracted from conidia of different developmental ages collected in parallel with those conidia analyzed for a-c. e, A scatter plot showing the Pearson Correlation analysis of mRNA levels measured by RNAseq in attached and separated conidia as in Fig. 3h.

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Extended Data Fig. 6 Overlaps between RNAP II bound genes and targets of AtfA and WetA in A. nidulans conidia.

a, A Venn diagram showing the overlap of genes occupied by RNAP II in conidia and binding targets (by ChIPseq) of transcription factor AtfAMYC and WetAMYC in conidia. b, A scatter plot showing Pearson Correlation of RNAP II occupancies and mRNA levels for AtfA and WetA common ChIPseq binding targets (n = 536) in conidia. c, Heatmap plots showing ChIPseq signals of RNAP II, WetAMYC and AtfAMYC for selected conidium-specific genes in A. nidulans.

Extended Data Fig. 7 A. nidulans and A. fumigatus conidia formed from different conditions have dissimilar transcriptional response and protein expression patterns.

a-d, Heatbox plots showing RNAP II ChIPseq signals for genes encoding (a) conidia surface and intracellular proteins, (b) cell wall proteins, (c) copper and (d) sugar transporters in A. nidulans conidia formed under zinc deficiency (Zinc-starved conidia) or in the presence or absence (Control conidia) of NaCl and KCl (NaCl- and KCl-conidia, respectively). e, Western blot analysis showing the protein expression level of sugar transporters (for example XtrE and MstB) in control and NaCl conidia of A. nidulans. Histone H3 was used as a loading control. The Western blot experiment was repeated three independent times with similar result. f, Heatbox plots showing RNAP II ChIPseq signals for genes encoding conidia surface and intracellular proteins, dehydrin-like proteins, allergens and transporters in A. fumigatus conidia formed under zinc deficiency (Zinc-starved conidia) or in the presence or absence (Control conidia) of NaCl and KCl (NaCl- and KCl-conidia, respectively). Results of A. fumigatus are marked by green-shaded background.

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Extended Data Fig. 8 Disparities between transcription, mRNA and protein levels in A. nidulans conidia.

a, Western Blotting results showing protein levels of representative genes in A. nidulans conidia and hyphae. The Western blot experiment was repeated three independent times with similar result. b, Heatbox plots showing RNAP II ChIPseq and mRNA levels for the genes encoding those proteins analysed in (a) in conidia and hyphae.

Source data

Extended Data Fig. 9 Sporulation conditions and conidial age affect the transcription levels of transcription factor-encoding genes, protein expression efficiency following germination and germination behaviours of A. nidulans and A. fumigatus conidia.

a-b, Line plots showing the quantification of protein bands in the Western Blotting time-course analysis presented in Fig. 4f and g for (a) Hsp70 protein in heat-shocked and cold-shocked conidia during germination at high temperature (for example 42 °C) and (b) ZapA protein in control and zinc-starved conidia before and during germination in the absence of zinc. Histone H3 was used as a loading control. c, A heatbox plot showing the transcription levels for 48 transcription factor (TF)-encoding genes, which have detectable RNAP II ChIPseq signals, in Zinc-starved, NaCl-, KCl- and Control conidia of A. fumigatus. d-e, Line plots showing the germination kinetics of A. nidulans (d) conidia of different developmental ages (3 and 17 day-old) and (e) conidia formed under ANM media (Control) in the presence of sodium chloride (NaCl) or in the absence of zinc (Zinc-starved). Error bars represent the standard deviation of three independent experiments. The p-values shown were calculated using (d) unpaired t-test in two-tailed or (e) one way ANOVA with multiple comparisons test. f-g, Bar plots showing percentage of germination for A. nidulans conidia formed under the indicated conditions as in (f) and (g) at various germination times (for example 4 h, 4.5 h, 5 h, 6 h and 7 h). d-g, Data are presented as mean values + SD. Error bars represent the SD of three independent experiments (n = 3). The asterisks *, **, *** and **** depict p-values of <0.05, <0.005, <0.001 and <0.0001, respectively, while ns represents a p-value of >0.05. Results of A. fumigatus are marked by green-shaded background.

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Extended Data Fig. 10 Conidia formed under different sporulation conditions have different transcription levels for genes of various physiological pathways.

a-b, Heatbox plots showing RNAP II ChIPseq signals and expression changes of (a) oxidative stress response genes and (b) ergosterol biosynthesis genes for A. nidulans conidia formed under ANM media (Control) and ANM media with sodium chloride (NaCl). c-d, Growth tests of A. nidulans wildtype strains on solid ANM media containing different concentrations of the azole-related anti-fungal drugs (c) itraconazole (0, 0.05 and 0.1 µg/ml) and voriconazole (0, 0.05 and 0.1 µg/ml), and the non-azole-related anti-fungal drugs (d) caspofungin (0.2, 0.8 and 2.4 µg/ml) and flucytosine (10, 30 and 90 µg/ml) using conidia formed under ANM media containing sodium chloride (NaCl conidia) or not (Control conidia). e, Heatbox plots showing RNAP II ChIPseq signals and expression changes of gliotoxin protection and control genes for A. fumigatus conidia formed on ANM media with (Control) and without zinc (Zinc-starved). f, Heatbox plots showing expression difference of annotated secondary metabolite biosynthesis clusters genes in A. fumigatus between conidia formed under ANM media with (Control) and without zinc (Zinc-starved). g, Microscopy images showing the conidiation phenotype of the xylP(p)aflR strain with different levels of xylose induction (0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1%). Results of A. fumigatus are marked by green-shaded background. The experiment was performed three independent times with similar results and representative images are presented.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary

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Supplementary Tables 1–12

Supplementary Table 1: Genes with positive elongating RNAP II ChIP–seq signals in conidia and hyphae for Aspergillus nidulans, Aspergillus fumigatus and Talaromyces marneffei. Supplementary Table 2: GO analysis outputs for genes with positive elongating RNAP II ChIP–seq signals in conidia for A. nidulans, A. fumigatus and T. marneffei. Supplementary Table 3: Differentially expressed genes for conidia exposed to temperature shocks (for example, 4 or 42 °C) or conidia formed under different media (for example, ANM media with sodium chloride (NaCl) or potassium chloride (KCl) or without zinc) compared to control conidia (37 °C on ANM media). Supplementary Table 4: GO analysis outputs for genes transcribed in conidia exposed to temperature shocks (for example, 4 or 42 °C) or conidia formed under different media (for example, ANM media with sodium chloride (NaCl) or potassium chloride (KCl) or without zinc (zinc-starved)). Supplementary Table 5: Summary of transcription and mRNA levels for genes presented in Extended Data Fig. 8b. Supplementary Table 6: Strains used in this project. Supplementary Table 7: Summary of conidia treatments used in this study. Supplementary Table 8: Antibodies used in this project. Supplementary Table 9: Primers used in this project. Supplementary Table 10: List of figures that have associated raw sequencing data. Supplementary Table 11: Summary of the number of mapped reads and read length for each next-generation sequencing data sample. Supplementary Table 12: Raw data used in Supplementary Fig. 3a–c.

Source data

Source Data Fig. 3

Unprocessed immunoblots for Fig. 3i,j.

Source Data Fig. 4

Unprocessed immunoblots for Fig. 4b,f,g.

Source Data Extended Data Fig. 5

Unprocessed gel images for Extended Data Fig. 5d.

Source Data Extended Data Fig. 7

Unprocessed immunoblots for Extended Data Fig.7e.

Source Data Extended Data Fig. 8

Unprocessed immunoblots for Extended Data Fig. 8a.

Source Data Fig. 3

Raw data used in Fig. 3c,d,g,h,k.

Source Data Fig. 4

Raw data used in Fig. 4a,d,h,i.

Source Data Fig. 5

Raw data used in Fig. 5h,i,k.

Source Data Extended Data Fig. 4

Raw data used in Extended Data Fig. 4d–f.

Source Data Extended Data Fig. 5

Raw data used in Extended Data Fig. 5a–c.

Source Data Extended Data Fig. 9

Raw data used in Extended Data Fig. 9f,g.

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Wang, F., Sethiya, P., Hu, X. et al. Transcription in fungal conidia before dormancy produces phenotypically variable conidia that maximize survival in different environments. Nat Microbiol 6, 1066–1081 (2021). https://doi.org/10.1038/s41564-021-00922-y

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