Deletion of gene clusters to identify novel secondary metabolites in Fusarium graminearum

CGRB Fall Conference 2015Previously we generated Fusarium graminearum mutants in which the gene for a histone methyltransferase (kmt6) was deleted [4]. In normal cells, this histone mark, trimethylated histone H3 lysine 27 (H3K27me3), is responsible for gene silencing or downregulation of ~25% of all genes. Many of these genes are in regions that are enriched for secondary metabolite gene clusters, compounds not required for primary growth but often associated with survival in hostile environments. Many secondary metabolites have been used as useful bioactive compounds or as antibiotics [2]. This is a promising approach as bacteria develop resistance to new compounds more quickly [1] In the kmt6 mutant many compounds were accumulating compared to wild type. As nearly 2,000 genes are upregulated or newly expressed, it is unclear which specific genes are associated with previously known or novel products. Our strategy is to delete key genes required for the production of metabolites in the kmt6 mutant to assign gene function to almost 650 overexpressed genes in 67 gene clusters. As proof‐of‐principle experiment, we focused on five clusters, replacing each cluster with a selectable maker that conveys antibiotic resistance. We validated transformants by PCR and Southern blots to show successful deletion of four of the five loci. The next step will be to analyze the metabolite profile of these mutants

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits

The <i>Fusarium graminearum</i> Histone H3 K27 Methyltransferase KMT6 Regulates Development and Expression of Secondary Metabolite Gene Clusters

<div><p>The cereal pathogen <i>Fusarium graminearum</i> produces secondary metabolites toxic to humans and animals, yet coordinated transcriptional regulation of gene clusters remains largely a mystery. By chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing. H3K27me3 was found predominantly in regions that lack synteny with other <i>Fusarium</i> species, generally subtelomeric regions. Di- or trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found in mutually exclusive regions of the genome. To find functions for H3K27me3, we deleted the gene for the putative H3K27 methyltransferase, KMT6, a homolog of <i>Drosophila</i> Enhancer of zeste, E(z). The <i>kmt6</i> mutant lacks H3K27me3, as shown by western blot and ChIP-seq, displays growth defects, is sterile, and constitutively expresses genes for mycotoxins, pigments and other secondary metabolites. Transcriptome analyses showed that 75% of 4,449 silent genes are enriched for H3K27me3. A subset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in <i>kmt6</i>. A largely overlapping set of genes showed increased expression in <i>kmt6</i>. Almost 95% of the remaining 2,720 annotated silent genes showed no enrichment for either H3K27me3 or H3K4me2/3 in <i>kmt6</i>. In these cases mere absence of H3K27me3 was insufficient for expression, which suggests that additional changes are required to activate genes. Taken together, we show that absence of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity factors. Results from this study provide the framework for novel targeted strategies to control the “cryptic genome”, specifically secondary metabolite expression.</p></div

Distribution of histone modifications across the complete set of <i>F. graminearum</i> genes.

<p>The left panel for each modification shows read counts (reads/gene) across all genes that were aligned to make the known or presumed transcriptional start site (TSS) position “0”. The middle panel shows read counts across all genes (reads/gene) normalized by gene length (x-axis indicates percent of gene length). The right panel shows the distribution of normalized read counts (NLCS; density) per gene. For most modifications two peaks are observed, background levels of reads (“B”) and enrichment (“E”). Results are for low nitrogen experiments, but indistinguishable results were obtained with samples grown on high nitrogen medium.</p

Heatmaps for individual genes in primary (A) and secondary (B) metabolism (left panels) and clusters of genes around eight centers (right panels).

<p>Few primary metabolic genes are affected by <i>kmt6</i> mutation compared to the majority of secondary metabolic genes that have increased expression in <i>kmt6</i>. Values for the log₂ of the ratio of RPKMs for <i>kmt6</i>/WT or high/low nitrogen conditions for each strain are color-coded and the scale shown on the right. For example, in the left image of (A) red represents log₂ (<i>kmt6</i>/WT) of 10, which means the <i>kmt6</i> RPKM is 1024-fold higher than the WT RPKM. Primary metabolic genes in clusters 2 and 5, upregulated in <i>kmt6</i>, and cluster 7, downregulated in high nitrogen, are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003916#pgen.1003916.s007" target="_blank">Table S2</a> with their putative functions. Secondary metabolic genes in clusters 1, 4, and 7, upregulated in <i>kmt6</i>, and cluster 8, downregulated in high nitrogen, are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003916#pgen.1003916.s007" target="_blank">Table S2</a> with their putative functions.</p

The <i>kmt6</i> mutant displays altered growth and development.

<p>A. WT and <i>kmt6</i> strains were grown on minimal (MIN) or YPD medium. The mutant shows sparse growth on MIN and dense, slow-growing and intensely orange mycelium on YPD. The lower panel shows YPD plates with a complemented strain (<i>kmt6</i>+<i>kmt6⁺</i>) and a plasmid control (<i>kmt6</i>+plasmid). B. WT and <i>kmt6</i> were grown through race tubes on MIN or YPD to measure linear growth for 25 to 33 days. Results are the average of three replicates, and bars indicate standard error. C. Sexual development was induced on carrot agar (CAR) plates. Black patches in the WT plate indicate numerous fully developed perithecia, while no perithecia formed in <i>kmt6</i> plates. The complemented strain (<i>kmt6</i>+<i>kmt6⁺</i>) is able to produce perithecia and ascospores at similar levels as WT. Protoplast fusions to complement <i>kmt6 neo⁺</i> strains with WT <i>hph⁺</i> nuclei do not restore formation of perithecia. These heterokaryons did not break down, as colonies resistant to both G418 and Hyg were isolated from different areas of the CAR plate and grew on YPD+G418+Hyg (center and edge).</p

Global transcriptional analysis and correlation with histone modifications.

<p>A. Biological replicates are highly correlated. The FPKM for biological replicate 1 is plotted against biological replicate 2 for each gene, for all four conditions, demonstrating strong correlation between replicate experiments (r is the correlation coefficient). B. Pairwise comparisons of RPKMs for each gene, WT <i>vs. kmt6</i> and low <i>vs.</i> high nitrogen for each strain shows global changes in gene expression. While there are some changes in expression based on nitrogen availability, more drastic changes are observed between WT and <i>kmt6</i> (orange and green area). C. Histone modifications are correlated to expression state. For each gene, expression (RPKM) is plotted <i>vs.</i> histone enrichment (NLCS) for H3K27me3 (orange), H3K4me2 (green), H3K4me3 (gray) and H3K36me3 (black) for WT grown in low nitrogen. For each modification, except for H3K36me3, two clusters of points can be seen on the y-axis, correlating to the background and enriched gene groups from the distribution curves (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003916#pgen-1003916-g005" target="_blank">Fig. 5</a>). D. Histone modifications associated with silent and expressed genes in low nitrogen conditions. Genes were classified as silent (≤3 RPKM) or expressed (>3 RPKM) and enrichment of H3K27me3, H3K4me2, and H3K4me3 determined by EpiChIP NLCS values. Venn diagrams were drawn to show histone enrichment for each silent or expressed gene in WT (A–B) or <i>kmt6</i> (C–D).</p

Summary of expression data for 45 secondary metabolite clusters of <i>F. graminearum</i>.

<p>The 19 non-ribosomal peptide synthase (NRPS), 15 polyketide synthase (PKS) and 11 terpenoid-producing clusters were arranged into classes according to recently published nomenclature <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003916#pgen.1003916-Wiemann1" target="_blank">[37]</a>. Products are known or predicted based on similarity of gene clusters with other <i>Fusarium</i> species. The last four columns summarize our results, showing enrichment with H3K27me3, repression by high nitrogen levels, and expression patterns in <i>kmt6</i> and WT for key cluster genes.</p><p>doi:10.1371/journal.pgen.1003916.t002</p