Image4_Whole-genome DNA methylome analysis of different developmental stages of the entomopathogenic fungus Beauveria bassiana NCHU-157 by nanopore sequencing.JPEG

The entomopathogenic fungus (EPF), Beauveria bassiana, is an important and commonly used EPF for microbial control. However, the role of DNA methylation has not been thoroughly studied. Therefore, the whole genomic DNA methylome of one promising EPF isolate, B. bassiana NCHU-157 (Bb-NCHU-157), was investigated by Oxford Nanopore Technologies (ONT). First, the whole genome of Bb-NCHU-157 was sequenced by next-generation sequencing (NGS) and ONT. The genome of Bb-NCHU-157 contains 16 contigs with 34.19 Mb and 50% GC content, which are composed of 10,848 putative protein-coding genes. Two putative DNA methyltransferases (DNMTs) were found, including Dim-2 and C-5 cytosine-specific DNA methylases. Both DNMTs showed higher expression levels in the mycelium stage than in the conidia stage, indicating that development of DNA methylation in Bb-NCHU-157 might occur in the mycelium stage. The global methylation level of the mycelium stage (5 mC = 4.56%, CG = 3.33%, CHG = 0.74%, CHH = 0.49%) was higher than that of the conidial stage (5 mC = 2.99%, CG = 1.99%, CHG = 0.63%, CHH = 0.37%) in both the gene and transposable element (TE) regions. Furthermore, the TE regions showed higher methylation frequencies than the gene regions, especially for CHH site methylation, suggesting regulation of genomic stabilization during mycelium development. In the gene regions, high methylation frequencies were found around the transcription start site (TSS) and transcription end site (TES). Moreover, CG and CHG methylation mainly occur in the promoter and intergenic regions, while CHH methylation occurs in the TE region. Among the methylated regions, 371, 661, and 756 differentially DNA methylated regions (DMRs) were hypermethylated in the mycelium in CG, CHG, and CHH, while only 13 and 7 DMRs were hypomethylated in the mycelium in CHG, and CHH, respectively. Genes located in the DMR shared the GO terms, DNA binding (GO: 0003677), and sequence-specific DNA binding (GO: 0043565) for hypermethylation in the mycelium, suggesting that methylation might regulate gene expression from the initial process. Evaluation of the DNA methylome in Bb-NCHU-157 by ONT provided new insight into this field. These data will be further validated, and epigenetic regulation during the development of B. bassiana will be explored.