The KdmB-EcoA-RpdA-SntB (KERS) chromatin regulatory complex controls development, secondary metabolism and pathogenicity in Aspergillus flavus

Abstract

The filamentous fungus Aspergillus flavus is a plant and human pathogen predominantly found in the soil as spores or sclerotia and is capable of producing various secondary metabolites (SM) such as the carcinogenic mycotoxin aflatoxin. Recently, we have discovered a novel nuclear chromatin binding complex (KERS) that contains the JARID1-type histone demethylase KdmB, a putative cohesion acetyl transferase EcoA, a class I type histone deacetylase RpdA and the PHD ring finger reader protein SntB in the model filamentous fungus Aspergillus nidulans. Here, we show the presence of the KERS complex in A. flavus by immunoprecipitation-coupled mass spectrometry and constructed kdmBΔ and rpdAΔ strains to study their roles in fungal development, SM production and histone post-translational modifications (HPTMs). We found that KdmB and RpdA couple the regulation of SM gene clusters with fungal light-responses and HPTMs. KdmB and RpdA have opposing roles in light-induced asexual conidiation, while both factors are positive regulators of sclerotia development through the nsdC and nsdD pathway. KdmB and RpdA are essential for the productions of aflatoxin (similar to findings for SntB) as well as cyclopiazonic acid, ditryptophenaline and leporin B through controlling the respective SM biosynthetic gene clusters. We further show that both KdmB and RpdA regulate H3K4me3 and H3K9me3 levels, while RpdA also acts on H3K14ac levels in nuclear extracts. Therefore, the chromatin modifiers KdmB and RpdA of the KERS complex are key regulators for fungal development and SM metabolism in A. flavus.

Fig. 1.. Identification of the KERS complex in Aspergillus flavus.

(A) A. flavus KdmB domain architecture and alignment of Jumonji C (JmjC) domain with S. cerevisiae Jhd2, Neurospora crassa NCU01238, A. nidulans KdmB, and A. fumigatus AFUA_5G03430. (B) Protein expression of KdmB::GFP fusion protein assayed by a Western blot analysis. KdmB::GFP fusion immunoblot was prepared from crude extract of vegetative growth at 30 °C for 24 h. As a loading control, a subunit of SCF complex, SkpA was used. (C) Subcellular localization of KdmB expressed under native promoter. Nuclei staining was performed at the end of 16 h submerged growth by treating samples with 1:10,000 DRAQ5 dye for 30 min at room temperature. (D) LC-MS² identification of KERS complex. Table shows the identified complex components with unique peptide numbers and total coverage. Demethylase KdmB interacts with putative acetyltransferase EcoA, histone deacetylase RpdA and Ring finger protein SntB to form the tetrameric KERS complex in vivo. Two biological replicates of KdmB::sGFP and KdmB::3xHA fusion strains and WT as negative control were immunoprecipitated and run in LC-MS². The protein list obtained from the WT control was subtracted from KdmB::sGFP (Table S1) and KdmB::3xHA (Table S2) purifications to eliminate non-specific contaminants. (E) Domain architectures of multidomain KERS complex proteins. Like KdmB, SntB contains the histone binding plant homeodomain (PHD) which is uniquely involved in chromatin regulation.

Fig. 2.. KdmB and RpdA are essential for sclerotia development.

(A) For conidiation analysis, ~5x10³ spores of WT, kdmB and rpdA mutants were spot inoculated onto PDA plates and grown for 4 days at 30 °C under illumination (upper lane). For sclerotia induction, PDA (5 days) and WKM plates (21 days) were incubated in dark conditions at 30 °C. Lower section shows the stereomicroscopic images of sclerotia formation in WT which was totally abolished in kdmB and rpdA mutants. (B) Percentage of conidiation and sclerotia production in WT and deletion strains. Final values were normalised with respect to WT representing 100% production. (C) RT-qPCR expression analysis of conidia regulatory genes abaA, brlA, flbA, flbB and (D) sclerotia regulatory genes nsdC and nsdD. Fungal mycelia were shifted onto agar plates from submerged cultures grown for 24 h at 30 °C. For the analysis of conidia regulatory genes, total mRNA was obtained by harvesting fungal mat grown on PDA plates for 3 days at 30 °C under illumination. For the analysis of the mRNA expression profiles of sclerotia regulatory genes, WKM agar plates were cultured for 5 days at 30 °C in dark conditions. WT mRNA levels were adjusted to 1.0. RT-qPCR experiments were carried out in two independent biological replicates and six technical replicates. (E, F, G) The effects of kdmB and rpdA on various stress agents. Glucose minimal media (GMM) agar plates were supplemented with various stress inducing agents (oxidative stress, menadione and H2O2, cell wall stress, SDS, congo red and calcofluor, cytoskeleton stress, Nocodazole and Benomyl, DNA damage, Camptothecin (CPT)) at the concentrations indicated and were incubated for 3 days under light conditions. All phenotypic tests were carried out in three independent biological replicates.

Fig. 3.. KdmB and RpdA are essential for aflatoxin production and play vital roles in peanut seed contamination.

(A) Aflatoxin B₁ analysis by reversed-phase HPLC in WT, kdmB and rpdA mutant strains grown on YES agar media. WT represents 100 % production. Lower panel shows the chromatogram of aflatoxin B1 peaks obtained from standard (Sigma), WT and kdmB, rpdA mutants. (B) Relative expression analysis of aflatoxin regulatory gene clusters aflC, aflD, aflM and aflR. Total mRNA was obtained by harvesting fungal mat grown on PDA plates for 3 days at 30 °C under dark conditions. (C) Peanut infection assay of WT and mutant strains. Peanut seeds were infected with ~ 5x10³ spores of WT, kdmBΔ and rpdAΔ strains and incubated for 5 days at 30 °C in a dark environment. Mock control peanut seeds without any fungal treatment are not included in the figure. (D) The number of sclerotia produced on seeds infected with WT and mutant strains. Sclerotia number grown on each peanut were manually counted and adjusted relative to WT set at 100. (E) RP-HPLC analysis of aflatoxin from infected peanut seeds. All values are the average of three independent biological replicates and error bars represent standard errors.

Fig. 4.. Secondary metabolite analysis of deletion strains via LC-MS.

(A) Base peak chromatograms generated from ES⁻ mode of WT and kdmBΔ and rpdAΔ mutant strains. Peaks correspond to; 2 = CPA (Cyclopiazonic Acid), 4 = Leporin B. (B) Individual graphs of known secondary metabolites produced by A. flavus. Average peak area and standard deviation were calculated from four biological replicates. Statistical significance was calculated using one way ANOVA. P-value **p < 0.01, ****p < 0.0001.

Fig. 5.. KdmB and RpdA are global regulators of secondary metabolite production in A. flavus.

(A) Four representatives of RT-qPCR expression analysis showing either upregulation, downregulation or no change compared to WT. Grey box represents no change, red box represents down-regulation by 30 % or more and green box represents 30% upregulation or more. cDNA was obtained from total mRNA extracted from fungal samples grown on PDA plates for 72 h at 30 °C in dark conditions. (B) mRNA profiles of 52 secondary metabolite cluster backbone genes corresponding to predicted NRPS, PKS, dimethylallyl tryptophan synthase, 3-oxoacyl carrier protein synthase and a hypothetical protein (HP). All values are the average of two independent biological replicates and 6 technical replicates. The error bars represent standard errors. See Supplementary S3 Table for full description of secondary metabolite gene clusters.

Fig. 6.. KERS complex has in vivo HDM and HDAC activities.

(A) Histone PTM analysis using various antibodies against methylated and acetylated histone lysine residues. For nuclear enrichment, approximately 2x10⁶ spores were inoculated into GMM with required supplements and grown for 24 h at 30 °C submerged culture. Quantification of band intensities was performed from two independent biological replicates using imageJ software. H3 was used as loading control and as a reference signal intensity for quantifications (B) Schematic model representing the role of the KERS complex on the fungal development and SM production. The KERS complex negatively affects global H3K4me3, H3K9me3, H3K36me3 methylation levels. RpdA remarkably represses H3K14 acetylation. KdmB and RpdA are positive regulators of sclerotia development and aflatoxin biosynthesis through regulation of nsdC, nsdD and AF gene cluster pathways respectively.