Fig 1.
WetA is required for proper conidial maturation and contains both a transcription activation domain and a nuclear localization signal in a variety of fungi.
(A) A model for the roles of the central regulators in Aspergillus conidiogenesis. WetA is activated by AbaA and is responsible for conidia wall maturation. The black square illustrates the graphic view of the wall structure of the mature conidium, including the crenulated electron-dense outer layer C1, the carbohydrate-condensed layer C3, the electron-thin layer C2, and the innermost layer C4. Note: some Aspergillus species lack metulae (ex. A. parasiticus), and some species can have both metulae-phialides or phialides-only conidiophores (ex. some A. flavus variants) [11]. (B) Unrooted phylogeny of WetA amino acid sequences of A. flavus NRRL3357 XP_002383329.1 (AFL), A. fumigatus Af293 XP_751508.1 (AFU), A. nidulans FGSC4 XP_659541.1 (ANI), A. oryzae RIP40 XP_001816745.1 (AOR), Penicillium chrysogenum Wisconsin 54–1255 XP_002564365.1 (PCH), P. digitatum Pd1 XP_014534725.1 (PDI), Fusarium graminearum PH-1 I1S0E2.2 (FGR), and Beauveria bassiana ARSEF 2860 XP_008599445.1 (BBA) [12–18]. The sequences were aligned using MAFFT, version 7.1.5 [19]. The WetA protein phylogeny was calculated using the maximum likelihood optimality criterion, as implemented in PAUP [20], version 4.0a152; we used the WAG model of amino acid evolution [21], with empirical amino acid frequencies and allowing for rate heterogeneity among sites. Values near internal branches correspond to bootstrap support values (only values above 70% are shown). Branch lengths correspond to the estimated number of amino acid substitutions per site–the internal branch leading to the FGR and BBA sequences has been truncated for optimal visualization. (C) The predicted WetA protein architecture. The red circle and the red box represent the transcription activation domain (TAD) which was predicted by 9aaTAD using the “Less stringent Pattern” setting [22]. The blue diamond and the blue box represent the nuclear localization signal (NLS) predicted by NLStradamus using the 4 state HMM static model [23]. The orange rectangle and the orange box represent the ESC1/WetA-related domain (PTHR22934) predicted by the PANTHER classification system [24]. The consensus sequence and the consensus histogram are shown under the amino acid sequence multiple sequence alignment.
Fig 2.
WetA is necessary for the proper formation of conidia in Aspergillus flavus.
(A) Phenotypes of WT (NRRL3357), ΔwetA, and C’wetA grown on solid MM at 30°C for 3, 4, 16 days after asexual induction. The white triangles indicate the liquid droplets formed on the autolyzing conidiophores of ΔwetA strain. (B) TEM images of 2-day-old conidiophores/conidia of WT and ΔwetA strains. Note: the remnant of lysed conidia formed a wet-vesicle-like structure on the top of the conidia chain, and most of the conidiophore/conidia contents were lost in the ΔwetA strain. The bottom panels show the conidia wall structures of WT and ΔwetA strains. Arrows indicate the locations of the C1 and C2 layers while the arrowheads indicate the C2 layer thickness. (C, D) The average diameter of conidia and thickness of the C2 layer of WT and ΔwetA conidia. At least seven WT and ΔwetA intact conidia from different sample slices were measured. (E, F) Quantification of conidia content (β-(1,3)-glucan (E) and trehalose (F)) of WT, ΔwetA, and C’wetA 2-day-old conidia The error bars indicate one standard deviation from the mean and the asterisks the level of significance (*, p < 0.05; **, p < 0.01). (G) The relative viability of WT, ΔwetA, and C’wetA conidia grown on solid MM at 30°C for 4, 7, 18 days after inoculation. The conidial viability at day 4 of each strain was set as 100%. ** (p < 0.01). The error bars indicate one standard deviation from the mean viability of triplicates. (H) Tolerance of WT, ΔwetA, and C’wetA 2-day-old conidia to heat (50°C), oxidative (H2O2), osmotic (KCl), and UV stresses. The control indicates untreated conidia. The viability of the untreated conidia of each strain was set as 100%. ** (p < 0.01). The error bars indicate one standard deviation from the mean viability of triplicates.
Fig 3.
(A-C) WetA affects vegetative growth. (A) The colony image of WT, ΔwetA, and C’wetA strains on solid MM at 5 days after point inoculation under light and dark conditions. (B) Colony edge image of WT and ΔwetA strains under light and dark conditions. Vs: single-layer vegetative hyphae region. Vm: multi-layer vegetative region. As: sparse aerial hyphae region. Ad: dense aerial hyphae region. (C) Colony growth rates of WT, ΔwetA, and C’wetA strains after point inoculation on solid MM. The error bars indicate one standard deviation. * (p < 0.05) and ** (p < 0.01). (D, E) Hyphal branching rates of WT and ΔwetA strains. (D) Microscopy images show WetA regulates hyphal branching. Loss of wetA leads to reduced hyphal branching rate in both solid and submerged cultures. (E) Average PGU values of A8. ** (p < 0.01). The error bars indicate one standard deviation. (F, G) Aflatoxin quantification by HPLC of WT and ΔwetA submerged culture after 5-days cultivation. (F) AFB1 amount (per g dry weight) in WT and ΔwetA vegetative cells. ** (p < 0.01). The error bars indicate one standard deviation. (G) The HPLC chromatograms of AFB1 and AFB2 in the culture medium of WT and ΔwetA strains. (H) WT and ΔwetA strains were induced for asexual development and observed after 8 h incubation at 30°C on solid MM plate. The white arrows indicate conidiophores. Note: the abundant conidiophore formation in ΔwetA culture. (I) Northern blot analysis of brlA, abaA, wetA, and vosA mRNA levels in WT and ΔwetA strains at 6, 12, 24, 48 h after conidiation induction.
Fig 4.
(A) The numbers of genes whose mRNA levels were similar (Unaffected, grey), or different between WT and ΔwetA conidia (DEG, green), with down (blue) and up (yellow) in the ΔwetA conidia compared to WT. A DEG is defined by having a > 2-fold change of mRNA levels between WT and ΔwetA conidia and an adjusted p-value of less than 0.05. (B) Functional categories of DEGs in conidia. The yellow bars represent genes whose mRNA levels increased in the ΔwetA conidia, whereas the blue bars represent those genes whose mRNA levels decreased in the ΔwetA conidia. The pink shaded box represents the biological process GO categories; the yellow shaded box represents the molecular function GO categories; the blue shaded box represents the cellular component GO categories. “Number of Genes”: the total number of DEGs assigned to the specific GO category. “% of Gene Number in GO”: the number of DEGs divided by the total number of genes assigned to the specific GO category in the genome as a whole. (C) The schematic diagram and mRNA expression profile of the G-protein regulatory pathways controlling development, stress response, and aflatoxin biosynthesis.
Table 1.
DEGs of interest.
Fig 5.
Summary of WetA functions and a model for WetA-mediated developmental regulation in A. flavus.
(A) Schematic diagram of the WetA-mediated regulatory model of conidia architecture. The question mark indicates that the WetA-mediated activation/repression function needs to be verified by further experiments. (B) A comprehensive model for WetA-mediated regulation of asexual development based on transcriptomic, genetic, and biochemical data. In this model, those genes with increased and decreased mRNA levels in the ΔwetA conidia are labeled in yellow (WetA-inhibited) and blue (WetA-activated), respectively. The functions and references of each gene in this model are listed in S9 Table.
Table 2.
DEGs involved in spore maturation.
Table 3.
DEGs within the secondary metabolite biosynthesis clusters.