The epigenetic reader SntB regulates secondary metabolism, development and global histone modifications in Aspergillus flavus

Abstract

Due to the role, both beneficial and harmful, that fungal secondary metabolites play in society, the study of their regulation is of great importance. Genes for any one secondary metabolite are contiguously arranged in a biosynthetic gene cluster (BGC) and subject to regulation through the remodeling of chromatin. Histone modifying enzymes can place or remove post translational modifications (PTM) on histone tails which influences how tight or relaxed the chromatin is, impacting transcription of BGCs. In a recent forward genetic screen, the epigenetic reader SntB was identified as a transcriptional regulator of the sterigmatocystin BGC in A. nidulans, and regulated the related metabolite aflatoxin in A. flavus. In this study we investigate the role of SntB in the plant pathogen A. flavus by analyzing both ΔsntB and overexpression sntB genetic mutants. Deletion of sntB increased global levels of H3K9K14 acetylation and impaired several developmental processes including sclerotia formation, heterokaryon compatibility, secondary metabolite synthesis, and ability to colonize host seeds; in contrast the overexpression strain displayed fewer phenotypes. ΔsntB developmental phenotypes were linked with SntB regulation of NosA, a transcription factor regulating the A. flavus cell fusion cascade.

Keywords: Acetylation; Aflatoxin; Hyphal fusion; Kojic acid; Sclerotia.

Figure 1:

Growth phenotypes of sntB mutants. A) Stains were point inoculated on GMM and grown at 30 °C for 7 days under constant light. B) Radial growth of plates was measured, all plates were grown in triplicate. C) Conidia were enumerated from a core taken from two day old overlay cultures on GMM. D) Sclerotia dry weight measured from overlay cultures grown on GMM+2% sorbitol grown in the dark at 30 °C for six days. E) Images of plates analyzed in D. Plates were washed with 70% EtOH to remove conidia and allow for visualization of sclerotia. P-value **p < 0.01.

Figure 2:

Loss of sclerotia in the deletion mutant is through transcriptional regulation of nosA. A) Semi-qPCR analysis of gene expression from three time points, vegetative growth at 24 hours, and then RNA from mycelia that was transferred to small GMM+2% sorbitol plates after one and three days. ubiD is used as a loading control. B) The number of heterokaryotic conidia was enumerated for three heterokaryotic crosses. Positive control was TJES19.1 (pyrG-) and TJES20.1, negative control ΔlaeA crossed with TJES20.1, and ΔsntB strain crossed with TJES20.1. P-value **p < 0.01.

Figure 3:

SntB is required for pathogenicity of corn. A) corn kernels were inoculated with 2×10⁵ spores and kept under a 12 hour light and dark cycle for five days. Each strain was inoculated with 5 reps, including a mock control which was kernels inoculated with 0.01% Tween 20. B) Spores were removed from surface of kernels and enumerated using a hemocytometer. Spore counts were corrected by weight of corn. C) Relative aflatoxin production of mutants compared to wild type. P-value, **p < 0.01, ***p < 0.001.

Figure 4:

Deletion of sntB leads to a greater change in secondary metabolite profile than overexpression. Metabolites were extracted from twelve-day-old cultures on PDA, run on a UHPLC-HRMS, and analyzed via XCMS. Experiment was completed in triplicate. A) Comparison of metabolite extracts from sntB deletion and wild type. Each dot represents a peak called by XCMS. The (–)log10 of the pvalue is plotted on the y-axis, with a gray dashed line indicating where the pvalue is equal to 0.05, values higher on the y-axis indicating higher statistical significance. Log2 of the fold change is on the x-axis, with values in the right half more abundant in the deletion strain, and values on the left half more abundant in the wild type. Red dots indicate known final products that were detected by the program including aflavarin, aflatoxin, asparasone A, ditryptophenaline, and leporin B. B) Same analysis comparing the overexpression of sntB to wild type.

Figure 5:

SntB is a global regulator of secondary metabolism in A. flavus. A) Individual graphs of known secondary metabolites produced by A. flavus detected via LCHRMS. Average peak area and standard error of mean was calculated from three biological repetitions. B) Wild type and sntB mutant strains grown on KAM, where kojic acid production is lost in the deletion strain. C) Structure of secondary metabolite analyzed. P-value **p < 0.01, n.d.-not detected.

Figure 6:

SntB regulates global levels of histone modifications. A) Levels of histone modifications were assessed by western blot from cultures grown for 72 hours in YES media. B) The relative intensity of the bands corresponding to histone H4 hyper acetylation (H4Ac) were calculated in Adobe PhotoshopTM, and standardized to the loading control, histone H3. Values were normalized to wild type. The same analysis was done for C (H3K4me3) and D (H3K9K14Ac). The deletion of sntB showed an increase in histone H3 acetylation, and a minimal increase in H3K4me3. Histone H3 is used as a loading control.

Figure 7:

Histone acetylation levels are not changed in sntB mutants at aflatoxin gene cluster promoters. Histone H4, H4Ac, H3, and H3K9K14Ac occupancy levels in wild type, deletion, and overexpression sntB mutants at the aflatoxin BGC after 36 hours of growth in YES media. Promoter regions of aflM and aflR were tested to represent the aflatoxin BGC. ubiD was chosen as an out of cluster control. Error bars represent standard error of mean, which was calculated from biological duplicates.