ZfpA-Dependent Quorum Sensing Shifts in Morphology and Secondary Metabolism in Aspergillus flavus

Abstract

Development of the fungal pathogen Aspergillus flavus involves the balance of asexual spores (conidia) and overwintering hardened hyphal masses (sclerotia). This balance is achieved by an oxylipin-based density-dependent mechanism regulating the switch from sclerotia to conidia as population density increases in A. flavus. Here, we show the transcription factor ZfpA, required for normal oxylipin synthesis, regulates the morphology switch. ZfpA overexpression (OE::zfpA) accelerates the shift leading to increased conidial production and reduced sclerotial production under conditions normally supporting sclerotia formation. In contrast, zfpA deletion (ΔzfpA) produces more sclerotia than wild-type control. These morphology changes are coupled with changes in tissue-specific secondary metabolites. Specifically, the production of four sclerotial metabolites (oxyasparasone A, hydroxyaflatrem, aflavinine, and kotanin) decreases in OE::zfpA whereas the hyphal metabolite aspergillic acid is upregulated in this mutant. Chemical profiling of OE::zfpA compared to a double mutant where the aspergillic acid non-ribosomal synthetase was deleted in the OE::zfpA background confirmed synthesis of known aspergillic acid pathway products as well as putative Val-derived pyrazinones involved in metal chelation. These findings offer valuable insights into the quorum sensing networks connecting fungal development and tissue-specific secondary metabolite production.

Keywords: aflatoxin; aspergillic acid; oxylipin; quorum sensing; sclerotia.

FIGURE 1

ppoA expression, 5,8‐diHODE production and lateral branching are regulated by ZfpA in A. flavus . (A) Quantification of 5,8‐diHODE from liquid shake glucose minimal medium (GMM) in A. flavus . p‐values were calculated using one‐way ANOVA with Tukey's multiple comparisons test (B) Concentration dependent increase in branching in A. flavus when treated with 5,8‐diHODE. p‐values were calculated using one‐way ANOVA with Tukey's multiple comparisons test. (C) Northern blot result shows the downregulation of ppoA expression in ΔzfpA compared to WT and OE::zfpA mutants. OE::zfpA mutant shows an upregulation of ppoA transcripts compared to the WT control. Transcript signals of ppoA and gpdA were quantified using ImageJ and signal ratios were normalised by the mean of the WT samples. (D) The bar graph shows 5,8‐diHODE production after 7 days of growth at 10⁴ spore density. The intensity of 5,8‐diHODE increased in the OE::zfpA mutant and decreased in the ΔzfpA mutant, comparable to the WT control. p‐values were calculated using one‐way ANOVA with Tukey's multiple comparisons test (E) Examination of branching in WT, ΔzfpA, and OE::zfpA strains grown in a 96‐well plate containing liquid GMM 21‐h post‐incubation at 30°C. p‐values were calculated using one‐way ANOVA with Tukey's multiple comparisons test. (F) The graph illustrates zfpA expression is needed for 5,8‐diHODE response. OE::zfpA mutant and WT control significantly increased branching in response to 0.1 μg/mL 5,8‐diHODE treatment. p‐values were calculated using two‐way ANOVA with Tukey's multiple comparisons test.

FIGURE 2

ZfpA impacts conidiation and sclerotia production at low and intermediate spore densities. (A) The images illustrate density‐dependent conidiation of ZfpA mutants after 7 days of growth. An overlay of low, intermediate, and high spore densities of mutants was cultivated on GMM containing 2% sorbitol, then incubated at 30°C. Four replicates of each mutant were grown for reproducibility. (B) The graph displays quantitative spore counts for ZfpA mutants. Plugs were taken from four replicates plates of each mutant after 7 days for conidia counts. p‐values were calculated using two‐way ANOVA with Tukey's multiple comparisons test. (C) The images show density‐dependent sclerotia production of ZfpA mutants after 7 days of growth. The black structures observed are sclerotia. (D) Graph illustrates gravimetric measurement of sclerotia collected from four replicates plates of each mutant after 7 days of growth. p‐values were calculated using two‐way ANOVA with Tukey's multiple comparisons test. (E) The images show sclerotia formation for WT plate. Large round black structures represent sclerotia. Cultures were grown in the dark using GMM plates supplemented with sorbitol to induce sclerotia. (F) The images show clustered sclerotia formation for OE::zfpA mutant plate. Cultures were grown in the dark using GMM plates supplemented with sorbitol to induce sclerotia.

FIGURE 3

Interactive principal component analysis and metabolomic analyses of A. flavus WT and ZfpA mutants across three spore densities. (A) Scores plot of the WT, ΔzfpA, and OE::zfpA strains of A. flavus at three different spore densities generated by XCMS v. 3. 7. 1. (B) Volcano plot shows upregulated and downregulated chemical features in the OE::zfpA mutant compared to WT control of 10⁶ spore density cultures. (C) Volcano plot shows upregulated and downregulated chemical features in the ΔzfpA mutant compared to WT control of 10⁶ spore density cultures. The plots were constructed based on Log₂ (fold‐change) of intensity with p‐values > 0.05 generated by XCMS at minimum intensity: 5 × 10⁵. The green and red colours indicate statistically significant features with Log₂(fold‐change) > 1 and < −1 on x–axis and –Log₁₀(p‐value) > 1.30103 on y–axis.

FIGURE 4

Production of secondary metabolites in A. flavus mediated by density dependence and ZfpA. (A) Scatter plot of production of six SMs mediated by population density dependence in WT in Log₁₀ height intensity. p‐values were calculated using two‐way ANOVA with Tukey's multiple comparisons test. (B) Bar charts of production of six SMs and their molecular structures in WT, ΔzfpA, and OE::zfpA strains presented in Log₁₀ height intensity at three spore densities. p‐values were calculated using two‐way ANOVA with Tukey's multiple comparisons test.

FIGURE 5

Metabolomic analyses of aspergillic acid related products and metal chelation assessment in WT and A. flavus mutants. (A) Comparison of total ion chromatograms in positive ion mode between WT and mutants at 10⁶ spore density. The OE::zfpA strain shows 11 upregulated features (B) Extracted ion chromatograms (m/z 225.1607) of the OE::zfpA strain at three spore densities. (C) Biosynthetic gene cluster (BGC) and pathway of asa producing aspergillic acid adapted from Lebar et al. (2018). (D) Molecular structures of analogues or derivatives of aspergillic acid. (E) Colony formation of WT and mutant strains grown on GMM with and without Fe supplementation. The OE::zfpA strain produced an orange colony due to ferriaspergillin production. (F) Chrome azurol S (CAS) assay assessing the metal chelation (distance of clearance) in WT and mutant strains. p‐values were calculated using ordinary one‐way ANOVA with Tukey's multiple comparisons test.