Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum

Abstract

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin.

Fig. 1

Multiple sequence alignment of F. graminearum HEP1 with other putative or characterized fungal Heterochromatin protein 1 homologs. The alignment was performed using ClustlalW and similarities are visualized with Boxshade. f_oxg, Fusarium oxysporum FOXG_03019.2; f_ver, Fusarium verticillioides FVEG_01876.3; n_crassa, Neurospora crassa hpo (HP1); m_gri, Magnaporthe grisea AAR19295.1; a_fla, Aspergillus flavus AFL2G_02774; a_nid, Aspergillus nidulans HepA. The open horizontal bar is positioned above the predicted chromodomain and the filled horizontal bar is positioned above the predicted chromoshadow domain. Asterisks indicate residues that form a putative hydrophobic pocket presumably binding the N-methyl group in H3K9 (Nielsen et al., 2002).

Fig. 2

Deletion of Hep1 causes overproduction of aurofusarin and reduction in DON levels. (A) The red pigment aurofusarin strongly accumulates in the Hep1 deletion mutants (shown here for the transformant T-V7) in liquid FMM media after 72 h (upper panel) and on solid FMM after 5 days (lower panel) of incubation. Plates are shown from the bottom and from the top. (B) Quantification of aurofusarin and DON metabolite levels in FMM culture supernatants of PH-1 (open bars) and two different Hep1Δ deletion mutants (filled bars). Metabolite levels for PH-1 have been arbitrarily set to 100%. Error bars indicate the standard deviation of two biological and two technical repetitions in two different Hep1Δ strains (T-V7 and T-X1).

Fig. 3

The aurofusarin biosynthetic cluster is upregulated in the Hep1 deletion mutant. (A) Schematic representation of the AUR gene cluster. Only the relative position of genes analyzed in this study is indicated. Dotted vertical lines indicate the proposed 3′ and 5′ limits of the cluster. FGSG_2319.3 is the first predicted gene that is not proposed to form part of the cluster and is therefore drawn here as the first gene positioned immediately outside the AUR cluster. B. RT-qPCR transcriptional analysis of the AUR-cluster regulator Aur1, the biosynthetic gene Pks12 and the predicted non-cluster gene FGSG_2319.3 in the wild type strain PH-1 (open bars) and in the Hep1Δ strain (filled bars). Relative amount of the specific transcripts were obtained by normalization against the amount of beta-tubulin transcript. Average values and standard deviations are derived from two biological and two technical repetitions.

Fig. 4

The cluster for DON biosynthesis is repressed in the Hep1 deletion mutant. (A) Schematic representation of the DON gene cluster. Only the relative position of genes analyzed in this study is indicated. Dotted vertical lines represent the proposed 3′ and 5′ limits of the cluster. FGSG_3531.3 is the first predicted non-cluster gene. (B) RT-qPCR transcriptional analysis of the DON-cluster regulator Tri6, the biosynthetic gene Tri5 and the predicted non-cluster gene FGSG_3531.3 in the wild type strain PH-1 (open bars) and in the Hep1Δ strain (filled bars). Relative amounts of the specific transcripts were calculated by normalization against the amount of beta-tubulin transcript. Expression of the non-cluster gene is shown in a separate graph due to the roughly 10-fold stronger overall values obtained in qPCR. Average values and standard deviations are derived from two biological and two technical repetitions.

Fig. 5

Heterochromatic marks are lost in the Hep1Δ strain. Chromatin immunoprecipitation (ChIP) analysis of the heterochromatic mark K9H3me3 analyzed in the promoter region of two AUR cluster (panel A) and two DON cluster (panel B) genes. Promoters of the predicted genes FGSG_2319.3 and FGSG_3531.3 flanking the AUR and DON clusters, respectively, were used as non-cluster controls. Relative amounts of DNA precipitated with the H3K9me3-specific antibody were calculated relative to the amount of DNA precipitated with the antibody recognizing the histone H3 C-terminus. Wild type H3K9 trimethylation levels (filled bars) were arbitrarily set to 1 and mutant levels (open bars) are shown relative to the wild type. Average values and standard deviations are derived from two biological and two technical repetitions.