Genome-wide DNA methylation and transcriptomic profiles in the lifestyle strategies and asexual development of the forest fungal pathogen Heterobasidion parviporum

Abstract

Heterobasidion parviporum is the most devastating fungal pathogen of conifer forests in Northern Europe. The fungus has dual life strategies, necrotrophy on living trees and saprotrophy on dead woods. DNA cytosine methylation is an important epigenetic modification in eukaryotic organisms. Our presumption is that the lifestyle transition and asexual development in H. parviporum could be driven by epigenetic effects. Involvements of DNA methylation in the regulation of aforementioned processes have never been studied thus far. RNA-seq identified lists of highly induced genes enriched in carbohydrate-active enzymes during necrotrophic interaction with host trees and saprotrophic sawdust growth. It also highlighted signaling-and transcription factor-related genes potentially associated with the transition of saprotrophic to necrotrophic lifestyle and groups of primary cellular activities throughout asexual development. Whole-genome bisulfite sequencing revealed that DNA methylation displayed pronounced preference in CpG dinucleotide context across the genome and mostly targeted transposable element (TE)-rich regions. TE methylation level demonstrated a strong negative correlation with TE expression, reinforcing the protective function of DNA methylation in fungal genome stability. Small groups of genes putatively subject to methylation transcriptional regulation in response to saprotrophic and necrotrophic growth in comparison with free-living mycelia were also explored. Our study reported on the first methylome map of a forest pathogen. Analysis of transcriptome and methylome variations associated with asexual development and different lifestyle strategies provided further understanding of basic biological processes in H. parviporum. More importantly, our work raised additional potential roles of DNA methylation in fungi apart from controlling the proliferation of TEs.

The Pattern and Function of DNA Methylation in Fungal Plant Pathogens

“…According to the methylation data from more than 40 fungal species, most methylated cytosine bases exist in transcriptionally silent and repetitive loci, and they are absent in transcriptionally activated genes, indicating that fungal DNA methylation preferentially contributes to TE suppression for maintaining the genome integrity [58]. Analysis of genomic DNA methylation patterns confirmed that TEs are heavily methylated in both CpG (>90%) and nonCpG (>20%) sites in different developmental stages, and only a few TEs are expressed in H. parviporum [71]. Analogously, 5mC sites in mycelia are not evenly distributed but clustered across chromosomes, forming densely methylated domains around TE-rich and gene-poor regions [6,44].…”

“…In contrast, CG-enriched genes in fungi do not exhibit the same normal-like distribution of CG methylation across the gene body as plants and some insect species [58,74]. The methylation levels of transcribed genes in the CpG context are lower than in their flanking regions in H. parviporum [71]. In addition to methylation in TEs, approximately 20% of nonTE genes are also methylated in the mycelia of M. oryzae [44].…”

“…Moreover, 5mC levels are on average higher in Basidiomycota than in other phyla regardless of genomic location and sequence context [58]. Methylated cytosines are also preferentially found in CpG dinucleotides of repetitive DNA sequences and TEs across fungal genomes [44,71]. Global methylation levels in the range of 3.3-5.2% were observed in Heterobasidion parviporum with a pronounced preference for CpG dinucleotides (6.7-9.3%) over nonCpG nucleotide contexts (2.0-3.7%), which is a phenomenon also shared by most species for which methylation patterns have been studied [37,71].…”

“…Methylated cytosines are also preferentially found in CpG dinucleotides of repetitive DNA sequences and TEs across fungal genomes [44,71]. Global methylation levels in the range of 3.3-5.2% were observed in Heterobasidion parviporum with a pronounced preference for CpG dinucleotides (6.7-9.3%) over nonCpG nucleotide contexts (2.0-3.7%), which is a phenomenon also shared by most species for which methylation patterns have been studied [37,71]. In H. parviporum, the relative proportion of 5mC sites in the three nucleotide contexts is similar (47-52% in CHH, 30-36% in CpG, and 17-18% in CHG), indicating a methylation homogeneity in terms of the three types of nucleotide contexts [71].…”

“…Global methylation levels in the range of 3.3-5.2% were observed in Heterobasidion parviporum with a pronounced preference for CpG dinucleotides (6.7-9.3%) over nonCpG nucleotide contexts (2.0-3.7%), which is a phenomenon also shared by most species for which methylation patterns have been studied [37,71]. In H. parviporum, the relative proportion of 5mC sites in the three nucleotide contexts is similar (47-52% in CHH, 30-36% in CpG, and 17-18% in CHG), indicating a methylation homogeneity in terms of the three types of nucleotide contexts [71]. Importantly, DNA methylation in different genomic regions may differentially influence gene activity depending on the underlying sequence.…”