Fig 1.
Construction and verification of the 500 bp C-terminal of TE deletion and complemented strain of F. monophora.
(A) Schematic diagram of gene deletion. The resistance gene (hph) replacement strategy was used to destruct the 500 bp C-terminal of TE in F. monophora. Through homologous recombination, overlapping DNA fragments of the hygromycin resistance gene cassette (hph) were used for gene replacement. PCR identification analysis of the wild-type and deletion mutant strains. (B) Schematic diagram of gene complementation. Another resistance gene (neo) replacement strategy was used to complement the 500 bp C-terminal of TE in F. monophora. Through homologous integration, overlapping DNA fragments of the neomycin resistance gene cassette (neo) were used for gene replacement. PCR identification analysis of the wild-type and complementation strain. (C) Colony morphology and microscopic morphology examination of the wild-type and mutant strain. (D) The mRNA expression of conserved functional domains of AYO21_03016 protein in F. monophora were detected, including starter ACP transacylase (SAT), β-ketoacyl synthase (KS), acetyl transferase (AT), dehydratase (DH), acyl carrier protein (ACP) and thioesterase (TE). TE gene expression was negative in Δpks1-TE-C500 and other 5 genes (SAT, KS, AT, DH, ACP) were significantly less expressed than WT. All statistical analysis were performed using two-tailed t-test (**, P<0.01; ***, P<0.001; ND means not detected, relative mRNA expression = 0). All the assays were performed in triplicate.
Table 1.
Strains and plasmids used in this study.
Fig 2.
Effects of the 500 bp C-terminal of TE on the radial growth, biomass and conidiation of F. monophora.
(A) The conidia of each strain were inoculated on PDA and incubated at 25°C and 37°C for 14 days. The radial growth rate of the Δpks1-TE-C500 strain was significantly than that of the wild-type and Complemented strain. (B) After the growth of each strain on PDA for 14 days at 25°C and 37°C, the cells were collected, dried, and weighed. The Δpks1-TE-C500 had a significantly lower biomass. (C) After the growth of each strain on PDA for 14 days, samples of fungi (1 cm2) were collected; the spores were separated, and the number of spores was counted. The Δpks1-TE-C500 had fewer spore. All statistical analysis were performed using two-tailed t-test (**, P<0.01; ***, P<0.001; ****, P<0.0001).
Fig 3.
The 500 bp C-terminal of TE is involved in the stress response in vitro.
In order to evaluate the resistance of Δpks1-TE-C500, all strains were inoculated on PDA with a 5 μl suspension containing 1×106/ml conidia under specific conditions. For salt stress, 0.2, 0.4, and 0.6 M potassium chloride were added. For oxidative stress, 0.5, 1, 2, and 2.5 mM H2O2, 0.225, 0.45 and 0.9 mM SNAP, or 15, 30, and 60 μM vitamin K were added. For osmotic stress, 0.25, 0.5, and 1 M sorbitol were added. Then, the plates were incubated for 14 days at 25°C (The straight line represents the basic level on the fifth day (0.6cm)). For UV stress, the strains were irradiated with UV light for 5, 10, and 15 min. Compared to the wild-type, the growth of the Δpks1-TE-C500 decreased significantly with the increase of stress factor concentration or UV irradiation time, especially oxidative stress and UV irradiation. Statistical significance was determined by ANOVA test (ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).
Fig 4.
Effects of the 500 bp C-terminal of TE on metabolites.
All strains were incubated at 25°C and collected after 14 days for the analysis of secondary metabolites. (A) PCA diagram of the identified metabolites of the WT and mutant strains. (B) Venn diagram of the WT and mutant strains. (C) KEGG pathway enrichment analysis of the WT and mutant strains. (D) The DOPA-melanin pathway. Glutathione (labeled by red circle) is implicated in the biogenesis of the pheomelanin. (E) The yields of L-glutathione (reduced), S-lactoylglutathione, and niacinamide were increased in the albino strain. All statistical analysis were performed using two-tailed t-test (*, P<0.05; **, P<0.01; ****, P<0.0001). There were three biological replicates in each experiment.
Fig 5.
The 500 bp C-terminal of TE is essential for survival in macrophages.
(A) The wild-type and Δpks1-TE-C500 strain were co-cultured with RAW264.7 macrophages for 24 and 48 h. In comparison with the wild-type strain, the conidia of the Δpks1-TE-C500 were more sensitive to cytotoxic activity and were more easily killed and cleared by macrophages. The wild-type hyphae penetrated the cells in 48 h. (B) TEM analysis after 24 h of co-culture. The wild-type and complemented strain maintained the integrity of the cell wall of the conidia after 24 h in cytoplasm, and macrophage mitochondria were swollen (black arrow). In contrast, the conidia of the Δpks1-TE-C500 were deformed in phagosome, and the mitochondria were normal (white arrow). (C) Conidial survival was detected by counting CFU on PDA after the infected macrophages lyses. The number of surviving colonies of the Δpks1-TE-C500 strain was much lower than that of the wild-type strain. The statistical analysis was performed using two-tailed t-test (**, P<0.01).
Fig 6.
Effect of 500 bp C-terminal of TE deletion on virulence in the F. monophora infection mice model.
The conidia (1×106) of all strains injected into the footpads of mice to infect mice. (A and B) The footpad thickness of mice were photographed and measured 3, 5, 7 and 14 days after infection. The footpad thickness of mice in Δpks1-TE-C500 group were significantly thinner than those of mice in the wild-type group and the complemented group in different stages. (C) The fungal burden of footpads was then detected. The number of CFU for the Δpks1-TE-C500 was significantly reduced compared with that of the wild-type and complemented strain at 14 days. (D) Histopathology of footpads injected with all strains at different time points (day 3, 7, and 14). In comparison with footpads injected with the wild type and complemented strain, footpads injected with the Δpks1-TE-C500 were less swollen, and the number of CFU in the tissues was significantly reduced. In addition, the number of inflammatory cells of Δpks1-TE-C500 group in the pathological tissues was decreased. All statistical analysis were performed using two-tailed t-test, and the results were statistically significant (*, P<0.05; ***, P<0.001; ****, P<0.0001).
Fig 7.
Association of 500 bp C-terminal of TE deletion with a lack of immune response.
(A-C) RNA-seq analysis of the whole-genome transcript profiles of mice infected with all strains and PBS. (A) PCA showed the Δpks1-TE-C500 group was relatively similar to the PBS group on the 3rd and 7th day. (B) Heatmap of the RNA-seq data for all groups. (C) Heatmap of gene expression in neutrophil chemotaxis in all groups. (D) Specific proinflammatory cytokines and neutrophil chemokines were significantly reduced in Δpks1-TE-C500 group on the 3rd, 7th and 14th day after injection, compared with wild-type group. Statistical significance was determined by two-tailed t-test (**P<0.01; ***P<0.001; ****P<0.0001).
Fig 8.
The 500 bp C-terminal of TE affects the recruitment of neutrophils in the footpads.
The infiltration of neutrophils was detected in the footpads on the different days after injection. (A) Immunohistochemistry was performed to assess the expression of MPO in the footpads of infected mice. The Δpks1-TE-C500 strain had a limited effect on neutrophil recruitment. (B) Western blotting was performed to detect MPO in footpad tissues. MPO was highly expressed in footpad tissues on the 3rd, 7th, and 14th day after injection with the wild-type and complemented strains. (C) After each strain was co-cultured with human blood-derived neutrophils for 1 h and 2 h, the phagocytosis rate was determined by flow cytometry, and the survival rate of conidia was measured by counting CFU on PDA. Statistical significance was determined by two-tailed t-test (****, P<0.0001). The Δpks1-TE-C500 had higher phagocytosis and survival rate.