Fig 1.
(A) Schematic representation of the RNA-seq analysis experimental design, including macrophage phagocytosis and saprophytic conditions. The assays included wild-type (WT) spores with and without macrophages (WT-M and WT-C), and r3b2Δ spores with and without macrophages (r3b2Δ-M and r3b2Δ-C). (B) Fluorescence microscopy of macrophages stained with LysoTracker Green DND-26 (green) and Hoechst 3342 (blue) during early interaction with Mucor spores from WT and r3b2Δ strains (scale bar = 15 µm). (C) Light microscopy images showing the interaction between Mucor spores and macrophages at early (1 h) and late (5 h) time points (scale bar = 50 µm). (D) Quantification of internalized versus free spores during early and late stages of phagocytosis. Data are represented as mean ± SD. Statistical significance: **** p < 0.0001 (Welch’s t-test). (E) Comparative transcriptomic analysis of the WT Mucor strain during early and late phagocytosis. (F) Differentially expressed genes (DEGs) shared between early and late phagocytosis, classified by expression patterns: red indicates genes upregulated at both time points; blue, downregulated at both; orange, upregulated early and downregulated late; and green, downregulated early and upregulated late.
Fig 2.
Early transcriptomic changes of the fungal spores during their phagocytosis by macrophages.
(A) Volcano plots of the WT strain fungal spores with and without macrophages (WT-M/WT-C), the r3b2∆ versus the WT interacting with macrophages (r3b2∆-M/ WT-M), the r3b2∆ with and without macrophages (r3b2∆-M/ r3b2∆-C), and the r3b2∆ versus the WT in saprophytic conditions (r3b2∆-C/ WT-C). Red and blue dots indicate the upregulated and downregulated genes, respectively, while black dots show not differentially expressed genes. (B) Venn diagrams illustrating the overlap of DEGs among the different comparisons. The upper left panel shows common DEGs between WT and r3b2∆ strains during macrophage interaction. The upper right panel shows shared upregulated genes between the macrophage-interacting and saprophytic conditions within each strain. The lower panel (three-set Venn diagram) depicts the intersection of DEGs identified in WT-M versus WT-C, r3b2∆-M versus r3b2∆, and r3b2∆ versus WT-M, highlighting genes consistently regulated across conditions. (C) Enrichment analysis of the DEG of the WT during its interaction with macrophages versus its saprophytic growth, the enrichment analysis of those DEGs during the interaction of the mutant with macrophages versus its growth under non-stressful conditions, and the enrichment of the DEGs when comparing the mutant versus the WT during their phagocytosis.
Fig 3.
The degradome analysis based on the correspondence between the sRNA production and mRNA expression.
(A) Volcano plots showing differential sRNA expression in the following comparisons: WT interacting with macrophages versus saprophytic growth (WT-M/WT-C), r3b2Δ interacting with macrophages versus WT interacting with macrophages (r3b2Δ-M/WT-M), r3b2Δ interacting with and without macrophages (r3b2Δ-M/r3b2Δ-C), and r3b2Δ versus WT under saprophytic conditions (r3b2Δ-C/WT-C). (B) Analysis of differentially expressed genes (DEGs) from the mRNA dataset based on their corresponding sRNA levels across the following comparisons: WT-M/ WT–C, r3b2∆-M/ WT-M, r3b2∆-M/ r3b2∆–C, and r3b2∆–C/ WT–C. The number of upregulated and downregulated sRNAs was determined for each condition. (C) Scatter plots illustrating the relationship between the expression of the sRNA and mRNA in the WT-M/ WT-C, r3b2∆-M/ WT-M, r3b2∆-M/ r3b2∆-C, and r3b2∆-C/ WT-C. Blue dots indicate genes downregulated at both RNA levels with, black dots are genes upregulated at the sRNA level and downregulation at the mRNA level, the red dots are genes with upregulation in both RNAs, and the green dots are genes with downregulated sRNA and upregulated mRNAs. (D) Validation of RNA-seq data by RT-qPCR. Gene expression was evaluated in WT spores interacting with macrophages versus saprophytic growth (left panel), as well as in the r3b2∆ mutant under the same conditions (right panel). Target genes included a permease, three transcription factors (prd, box, and brca1), and an LTR-transposable element. Data represent mean ± SD of three biological replicates, with individual replicates shown as circles. Statistical significance was determined using an unpaired t-test with Welch’s correction, with significance levels indicated above comparisons: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 4.
(A) Domain architecture of candidate genes encoding the transcriptional regulator Brca1, and the chromatin-associated proteins Hist1 and Hda10. (B) Temporal expression profile of brca1, box, hist1, and hda10 in the WT strain (M. lusitanicus) during macrophage interaction (30 min-5 h post-infection) relative to saprophytic conditions. Data represent mean ± SD of 3 biological replicates; individual replicates are shown as circles. Statistical significance was determined using an unpaired t-test with Welch’s correction. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Sporulation of the mutants with disruptions in transcriptional regulators and chromatin-related genes in R. microsporus. (D) Radial growth of the same mutants monitored every 24 hours over 5 days. (E) Spore production per cm² in the WT strain and each mutant after 2 days of growth. Data represent mean ± SD of three biological replicates; individual replicates are shown as circles. Statistical significance was assessed using one-way ANOVA followed by post hoc pairwise comparisons with Welch’s t-test. Significant differences are indicated by asterisks (* p < 0.05). (F) Survival plots of G. mellonella larvae infected with spores from WT and box mutant strains (upper panel, M. lusitanicus; lower panel, R. microsporus). Data represent the average of three independent experiments. (G) Survival plot of the murine model infected with spores of R. microsporus mutants disrupted in brca1, box, hist1, or hda10, along with virulent and avirulent control strains. Survival was analyzed using the log-rank (Mantel–Cox) test (p ≤ 0.05). DPI, days post-injection. (H) Quantification of fungal DNA in mouse lungs and brains after four days post-infection. Data mean ± SD of three biological replicates, with individual replicates shown as circles. Statistical significance was determined using one-way ANOVA. Significance levels: **p < 0.01, ****p < 0.0001.
Fig 5.
Schematic representation of the regulation of genes induced during the interaction with macrophages and targeted by RNAi.
Below, a schematic depiction of the brca1 gene disruption in R. microsporus and its effects on pathogenic capacity is shown.