BioProject

Facultative heterochromatin marked by histone H3 lysine 27 trimethylation (H3K27me3) is an important regulatory layer for secondary metabolite (SM) gene silencing and important for fungal development in the genus Fusarium. While this histone mark is essential in some (e.g., the rice pathogen Fusarium fujikuroi), it appears dispensable in other fusaria. Here, we show that deletion of FpKMT6 is detrimental but not lethal in the plant pathogen Fusarium proliferatum, a member of the Fusarium fujikuroi species complex (FFSC). Loss of FpKmt6 results in aberrant growth, and expression of a large set of previously H3K27me3-silenced genes is accompanied by increased H3K27 acetylation (H3K27ac) and an altered H3K36me3 pattern. Next, H3K9me3 patterns are affected in ∆fpkmt6, indicating a crosstalk between both heterochromatic marks that became even more obvious in a strain deleted for FpKMT1 encoding the H3K9-specific histone methyltransferase. In ∆fpkmt1, all H3K9me3 marks present in the wild-type strain are replaced by H3K27me3, a finding that likely explains the subtle phenotype of ∆fpkmt1 strains which stands in marked contrast to other filamentous fungi. A large proportion of SM-encoding genes is allocated with H3K27me3 in the wild-type strain, and loss of H3K27me3 result in elevated expression of 49% of them. Interestingly, genes involved in the biosynthesis of the phytohormones gibberellins (GA) are among the most upregulated genes in ∆fpkmt6. Although several FFSC members harbor GA biosynthetic genes, its production in planta is largely restricted to F. fujikuroi with few exceptions. We show that GA gene silencing is mediated by H3K27me3 in F.proliferatum and in at least one additional FFSC member, possibly outlining the distinct lifestyles of these notorious plant pathogens. Overall design: The F. proliferatum strain NRRL62905, FpWT, was used as parental strain and compared to the deletion strain of fpkmt6: FpKMT6. For fungal liquid cultivations, mycelia were pre-cultured in 100 mL Darken medium in a 300 mL Erlenmeyer flask for 3 days in the dark (30°C, 180 rpm). Then, 0.5 mL of the pre-culture was transferred to ICI media supplemented with either 6 mM glutamine sole nitrogen source . Fungal cultures were incubated on an orbital shaker at 180 rpm, 30°C for 3 dpi . Total RNA for subsequent sequencing was extracted using TRIzol Reagent as described above, and sent to the Vienna BioCenter (Vienna, Austria) for quality control, library preparation and sequencing. Experiments were performed in duplicates. Library prep and sequencing was performed using poly-A enrichment kit (NEB) and Nextera Library prep kit. 75 bp single end sequencing was performed using a NextSeq550 Illumina sequencer. Obtained sequences were de-multiplexed, quality controlled, filtered using trimmomatic 0.36 and mapped on the new genome assembly (needs also to be submitted). Mapping was performed using BWA and reverse transcripts were counted using python script HTSeq . Normalization and statistics were done using R/Bioconductor and the limma and edgeR packages, using mean-variance weighting (voom) and TMM normalisation . A significance cut-off of p < 0.01 and differential expression of +/-1 (2-fold) was applied for analysis. Transcription levels are log2 read counts per kilobase of exon per million library reads (RPKM).