Genome-wide DNA methylation analysis of Metarhizium anisopliae during tick mimicked infection condition

Abstract

BackgroundThe Metarhizium genus harbors important entomopathogenic fungi. These species have been widely explored as biological control agents, and strategies to improve the fungal virulence are under investigation. Thus, the interaction between Metarhizium species and susceptible hosts have been explored employing different methods in order to characterize putative virulence determinants. However, the impact of epigenetic modulation on the infection cycle of Metarhizium is still an open topic. Among the different epigenetic modifications, DNA methylation of cytosine bases is an important mechanism to control gene expression in several organisms. To better understand if DNA methylation can govern Metarhizium-host interactions, the genome-wide DNA methylation profile of Metarhizium anisopliae was explored in two conditions: tick mimicked infection and a saprophytic-like control.ResultsUsing a genome wide DNA methylation profile based on bisulfite sequencing (BS-Seq), approximately 0.60% of the total cytosines were methylated in saprophytic-like condition, which was lower than the DNA methylation level (0.89%) in tick mimicked infection condition. A total of 670 mRNA genes were found to be putatively methylated, with 390 mRNA genes uniquely methylated in the tick mimicked infection condition. GO terms linked to response to stimuli, cell wall morphogenesis, cytoskeleton morphogenesis and secondary metabolism biosynthesis were over-represented in the tick mimicked infection condition, suggesting that energy metabolism is directed towards the regulation of genes associated with infection. However, recognized virulence determinants known to be expressed at distinct infection steps, such as the destruxin backbone gene and the collagen-like protein gene Mcl1, were found methylated, suggesting that a dynamic pattern of methylation could be found during the infectious process. These results were further endorsed employing RT-qPCR from cultures treated or not with the DNA methyltransferase inhibitor 5-Azacytidine.ConclusionsThe set of genes here analyzed focused on secondary metabolites associated genes, known to be involved in several processes, including virulence. The BS-Seq pipeline and RT-qPCR analysis employing 5-Azacytidine led to identification of methylated virulence genes in M. anisopliae. The results provided evidences that DNA methylation in M. anisopliae comprises another layer of gene expression regulation, suggesting a main role of DNA methylation regulating putative virulence determinants during M. anisopliae infection cycle.

An efficient Agrobacterium tumefaciens-mediated transformation method for Simplicillium subtropicum (Hypocreales: Cordycipitaceae)

“…This strain is deposited in the fungal collection “Coleção de Culturas de Entomopatógenos Oldemar Cardim Abreu” located in São Paulo, Brazil. Before subsequent experiments, this strain was grown at 28 °C in solid Cove’s Complete Medium (MCc), as previously described ( Sbaraini et al ., 2019 ). Maintenance of transformants were in solid Cove’s Medium (MC), as described by Sbaraini and coworkers ( 2019 ), and 10 μg/mL of Chlorimuron Ethyl (CE) was employed.…”

“…Before subsequent experiments, this strain was grown at 28 °C in solid Cove’s Complete Medium (MCc), as previously described ( Sbaraini et al ., 2019 ). Maintenance of transformants were in solid Cove’s Medium (MC), as described by Sbaraini and coworkers ( 2019 ), and 10 μg/mL of Chlorimuron Ethyl (CE) was employed. Escherichia coli TG2 was employed in routine cloning, and Agrobacterium tumefaciens strains EHA105 and LBA1100 were employed to perform the ATMT of S. subtropicum strain IBCB 79.…”

The mechanisms and factors that induce trained immunity in arthropods and mollusks

“…DNA methylation is a stable covalent cytosine modification in a cytosine-guanine dinucleotide (CpG) context, which in the genomes of vertebrates, are highly methylated but sparsely methylated in invertebrates ( 156 ). This genomic methylation is regulated by DNA methyltransferases (DNMTs), a key mechanism for controlling gene expression in various organisms ( 157 , 158 ) ( Figure 3 ). The oxidation of 5-methylcytosine (5mC) at CpG dinucleotides to form 5-hydroxymethylcytosine (5-hmC) serves as an epigenetic marker ( 159 ) because 5-hmC is enriched at the enhancers of most highly transcribed genes ( 160 ).…”

Frequent horizontal chromosome transfer between asexual fungal insect pathogens

“…Here we have the unique opportunity to study how epigenetic marks were affected by such a horizontal transfer, because we have access to both ancestral strains as well as the recipient strain that now harbors chrA. We focused on DNA-methylation, which is known to vary between different Metarhizium strains (34)(35)(36). We hypothesized that chrA may possess a distinct DNA methylation pattern compared to the rest of the genome in the donor strain and that its pattern might be affected by the horizontal transfer to R3.…”