Transcriptomic and genetic analysis reveals a Zn2Cys6 transcription factor specifically required for conidiation in submerged cultures of Thermothelomyces thermophilus

Abstract

Filamentous fungi are important producers of enzymes and secondary metabolites. The industrial thermophilic species, Thermothelomyces thermophilus, is closely related to the model fungus, Neurospora crassa. A critical aspect of the filamentous fungal life cycle is the production of asexual spores (conidia), which are regulated by various stimuli, including nutrient availability. Several species of fungi, including T. thermophilus, produce conidia under submerged fermentation conditions, which can be detrimental to product yields. In this study, transcriptional profiling of T. thermophilus was used to map changes during asexual development in submerged cultures, which revealed commonalities of regulation between T. thermophilus and N. crassa. We further identified a transcription factor, res1, whose deletion resulted in a complete loss of conidia production under fermentation conditions, but which did not affect conidiation on plates. Under fermentation conditions, the ∆res1 deletion strain showed increased biomass production relative to the wild-type strain, indicating that the manipulation of res1 in T. thermophilus has the potential to increase productivity in industrial settings. Overexpression of res1 caused a severe growth defect and early conidia production on both plates and in submerged cultures, indicating res1 overexpression can bypass regulatory aspects associated with conidiation on plates. Using chromatin-immunoprecipitation sequencing, we identified 35 target genes of Res1, including known conidiation regulators identified in N. crassa, revealing common and divergent aspects of asexual reproduction in these two species.IMPORTANCEFilamentous fungi, such as Thermothelomyces thermophilus, are important industrial species and have been harnessed in the Biotechnology industry for the production of industrially relevant chemicals and proteins. However, under fermentation conditions, some filamentous fungi will undergo a switch from mycelial growth to asexual development. In this study, we use transcriptional profiling of asexual development in T. thermophilus and identify a transcription factor that specifically regulates the developmental switch to the production of unwanted asexual propagules under fermentation conditions, thus altering secreted protein production. Mutations in this transcription factor Res1 result in the loss of asexual development in submerged cultures but do not affect asexual sporulation when exposed to air. The identification of stage-specific developmental regulation of asexual spore production and comparative analyses of conidiation in filamentous ascomycete species have the potential to further manipulate these species for industrial advantage.

Keywords: Thermothelomyces; asexual development; conidiation; fungal biotechnology; transcriptional regulation.

Fig 1

Changes in gene expression patterns during T. thermophilus conidiation in submerged culture. (A) Time-course of hyphal and conidial development in submerged cultures in PCM (30). At 11.5 h, only vegetative hyphae were present. At 14 h, hyphal tips showed ampulliform swelling and hyphae showed lateral budding. At 16 h, conidiation had begun at hyphal tips and at lateral buds. At 20 h, conidia formation was more apparent, and at 48 h, conidia were mature with few vegetative hyphae. Black arrows indicate conidiophores and conidia. For a cartoon of conidiation steps, see Fig. S1. (B) Hierarchical clustering of differentially expressed genes during the 48 h time-course. Six distinct clusters of genes with differential expression at one point during the time course were revealed (see Data set S2). The heatmap shows the expression of each triplicate at the different time points normalized as z-score from log counts per million (logCPM). (C) UpSet plot of intersecting upregulated (top) and downregulated (bottom) genes between the different timepoints of the 48 h time-course with the number of differentially regulated genes over the bars. The total number of up- or downregulated genes at the time point is shown as a horizontal bar to the left of the time point.

Fig 2

Expression patterns of conserved conidiation regulators showing that the T. thermophilus fl homolog regulates conidiation in submerged culture in an environmental condition-dependent fashion. (A) Heatmap of hierarchically clustered, differentially expressed T. thermophilus homologs of conidiation-related genes during the 48-h time course. Heatmap shows z-score from log10 counts per million (logCPM). (B) Representative agar plates of T. thermophilus WT, ∆fluG, and ∆fl mutants after 5 days of growth on plates and the corresponding submerged culture phenotype in Vogel’s minimal medium (VMM) (39) and pre-culture medium (PCM) (30) after 20 and 48 h. Conidial quantification from submerged cultures of WT and the mutants from three biological replicates are shown below. Scale bar 10 µm. Conidia/conidiophores are indicated by black arrows. One-way ANOVA with Tukey’s post-test, ns = non-significant, **P < 0.01.

Fig 3

The conserved TF RES1 is required for conidia formation in submerged cultures of N. crassa and T. thermophilus. (A) VMM agar slant tubes of N. crassa WT (FGSC2489, FGSC4200) and ∆res-1 mutant (FGSC11130) and the corresponding conidia quantification. ns = non-significant, using a one-way ANOVA. (B) Representative microscopy pictures of three biological replicates N. crassa WT (FGSC2489) and the ∆res-1 mutant (FGSC11130) after 14 h and 20 h submerged growth in PCM medium. Cell walls were stained with calcofluor white (CFW) (1 µg/mL). (C) T. thermophilus WT and mutants ∆res1 (1), ∆res1 (5), and ∆res1 P_res1::res1-gfp were grown on VMM (39) agar plates and submerged in PCM (30) with conidia quantification under both conditions. ns = non-significant, ****P ≤ 0.0001 using a one-way ANOVA with a Tukey’s post hoc test. (D): T. thermophilus WT, ∆res1 (1), and ∆res1 P_res1::res1-gfp strains were grown in a main-culture medium and the total biomass was determined on day 1, day 2, and day 3. ****P ≤ 0.0001 using two-way ANOVA with a Tukey’s post hoc test. (E) Cellulase activity of T. thermophilus WT (ATCC42464), ∆res1 (1), and ∆res1 P_res1::res1-gfp strains. Strains were directly inoculated and grown in VMM 2% Avicel for 48 h and cellulase activity was measured in the supernatant. Normalization for biomass in all three cultures was performed by quantifying the total extracted protein from the collected mycelium. ns = non-significant, ****P ≤ 0.0001 using a one-way ANOVA with a Tukey’s post hoc test.

Fig 4

T. thermophilus Res1 regulates many genes during hyphal growth and conidial formation in submerged cultures. (A) The UpSet plot of intersecting upregulated (top) and downregulated (bottom) genes between T. thermophilus WT and the ∆res1 mutant at the indicated time points. Bars on the left indicate the total number of upregulated and downregulated genes at the time point and bars on top show the number of shared upregulated and downregulated genes at the indicated timepoints. (B) Enrichment analysis of GO terms at the timepoints 11.5 h, 14 h, and 16 h comparing WT vs the ∆res1 mutant (Data set S6). Displayed are the fold enrichment, number of genes, and FDR of the 10 highest enriched categories using ShinyGO (v0.8; http://bioinformatics.sdstate.edu/go/). Displayed genes had an FDR ≤ 0.05.

Fig 5

Overexpression of res1 causes premature conidiation in T. thermophilus. (A) Representative microscopic images of T. thermophilus P_H2A::res1-gfp and WT strains after 12 h of submerged growth in PCM (30). No conidiation is visible in the WT strain while res1 overexpression strains show conidia (black arrows). Scale bar: 10 µm. For plate phenotypes, see Fig. S7A. (B) RT-qPCR of res1 mRNA expression in P_H2A::res1-gfp strains relative to res1 expression in the WT strain after 12 h of growth in submerged cultures. ns = non-significant, *P ≤ 0.05, **≤ 0.001,****P ≤ 0.0001 using a one-way ANOVA with a Tukey’s post hoc test. (C) Fluorescence microscopic images of WT, control strains, ∆res1 Pres1::res1-gfp, and ∆res1 P_H2A::gfp, and ∆res1 P_H2A::res1-gfp #2 and #7 grown in PCM. Nuclei indicated by white arrows, scale bar 10 µm.

Fig 6

In T. thermophilus Res1 directly regulates genes with predicted roles in conidiation and nutrient sensing. (A) UpSet plots of upregulated and downregulated genes between the ∆res1 P_H2A::res1-V5 strain and either the ∆res1 mutant or WT strain at 6 h, 7.5 h, and 9 h in submerged cultures grown in PCM. (B) Functional category enrichment of shared genes between ∆res1 P_H2A::res1-V5, the ∆res1, or WT strains at all time points. Displayed are the fold enrichment, number of genes, and FDR in the highest enriched categories with FDR ≤ 0.05 using ShinyGO (v0.8) (33).

Fig 7

Res1 directly regulates genes with predicted roles in conidiation and nutrient sensing. (A) Heatmap of 28 genes that have a ChIPseq peak upstream of the translational start site and that were differentially regulated in the RNAseq data set comparing WT vs ∆res1. Heatmap shows z-score from log10 counts per million (logCPM). (B) Heatmap of 18 genes that have a ChIPseq peak upstream of the translational start site and that were differentially regulated in the RNAseq data set comparing P_H2A::res1-V5 vs ∆res1 and WT. Heatmap shows z-score from log10 counts per million (logCPM). (C) Res1-binding motif obtained from MEME suite v5.5.5 using ChIPseq binding peak sequences 4 kbp or less from the TSS. Shown are the motifs predicted by MEME (E-value 8.4 × 10⁻⁶, 18 bp) and by STREME (E-value 2.6 × 10⁻¹⁰, 19 bp) with a SEA rank of 2 and 4, respectively.