- Genome-wide DNA methylation analysis of Metarhizium anisopliae during tick. - Among the different epigenetic modifications, DNA methylation of cytosine bases is an important mechanism to control gene expression in several organisms. - Results: Using 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. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - 3 Rede Avançada em Biologia Computacional, RABICÓ, Petrópolis, RJ, Brazil Full list of author information is available at the end of the article. - 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. - Pest activities are one of the major problems associated with farming. - In Brazil, where agriculture is the main source of income, insect-pests cause an average an- nual loss of 7.7% in crop production (US$ 17.7 billion), resulting in the reduction of approximately 25 million tons of food, fiber, and biofuels [2]. - Chemical pesticides are still the usual method for arthropod-pests control causing great concern, in view of the known negative side effects to humans, animals and the environment.. - In recent years, genome sequencing, RNA-seq, and com- parative genomic analyses have been used for an explora- tory view of the genomes and for the discovery of new virulence determinants in Metarhizium spp. - How- ever, the still limited knowledge about Metarhizium-host interactions is one of the factors that limit in-depth ento- mopathogenic application for control of economic import- ant arthropods species. - DNA methylation of cytosine bases. - Striking, different isoforms of DNA methyltransferases (DNMTs) are enrolled in the process. - In view of DNA methylation importance in several organ- isms, including species in the Metarhizium genus, it is rea- sonable to expect that this epigenetic mark can regulate major steps, as well as virulence determinants, during ento- mopathogenic infection. - Additionally, we compared the Bisulfite sequencing (BS-Seq) results with previous RNA-seq data obtained in the same experimental conditions and the re- sults were further confirmed employing quantitative reverse transcription PCR (RT-qPCR) in the presence or absence of DNMT inhibitor. - Global mapping of DNA methylation in rich medium (saprophytic-like condition) and tick cuticles (mimicked infection condition). - In order to understand the impact of DNA methylation in M. - The experiments herein analyzed followed the recommendations of the Standards and Guidelines for Whole Genome Shotgun Bisulfite Se- quencing of the NIH Roadmap Epigenomics Mapping Consortium, which suggests the use of at least two bio- logical replicates with an average coverage of at least 30 times [13]. - Notably, a higher proportion of the identified methylated sites was found in the 48hTC condition (0.89% of total cytosines detected) compared to the 48hRM condition (0.60% of total cytosines detected). - It consisted in the identification of 5mCs in the open reading frames (ORFs) of each gene and their re- spective 500 bp flanking regions. - e., 390 mRNA genes were uniquely methylated in the 48hTC condition) when compared with 48hRM (i. - e., 135 mRNA genes were uniquely methylated in the 48hRM) with 145 mRNA genes methylated in both conditions (Fig. - However, no differences could be found in the content of methylation in these 145 putatively methylated genes when the two conditions were compared.. - 5.1%) of the putatively methylated protein coding genes did not presented an associated predicted domain (Additional file 2).. - Notably, 55 GO terms were uniquely found in the 48hTC condition, 9 GO terms were uniquely found in the 48hRM condition and 9 GO terms were found in both con- ditions (Fig. - DNA methylation and secondary metabolite backbone genes. - g., polyketide synthases [PKS], non-ribosomal peptide synthetases [NRPS], hybrids Table 1 Patterns of putative 5mCs sites distribution in the. - Thus, the methylation pattern of the backbone genes from 73 BGCs found in M. - b Seventy-three GO terms were over-represented, with 55 GO terms in the 48hTC condition, 9 GO terms in the 48hRM condition and 9 GO terms in both conditions. - 2 The impact of DNA methylation on secondary metabolite backbone genes. - b Expression and differential expression profile of the 44 putatively methylated backbone genes on the comparison 48hRM x 48hTC performed by [6]. - led to decreased compound synthesis [16], the explor- ation of the methylated pattern of the backbone gene, as well as transcription activity of the backbone gene can be a indicator of BGC active/inactive state. - In the 48hTC condition, 14 backbone genes were putative methylated, while 9 backbone genes were putative meth- ylated in 48hRM condition and 21 backbone gene were putative methylated in both conditions, which corres- pond to near 60% of the total of BGCs found in M. - e., 48hRM and 48hTC, each in biological duplicates) [6], the expression of the putative methylated backbone genes was inferred from the RNA-seq data, looking for possible correlations be- tween methylated state and expression profile. - Ten out of 44 BGCs ( 22.7%) displayed detectable expression in the RNA-seq data (RPKM ≥2), but there were no statis- tical differences (ND) between conditions (Fig. - Three out of putative meth- ylated BGCs were down-regulated (Down) in the RNA- seq data (Fig. - Six out of puta- tive methylated BGCs were among those up-regulated in the RNA-seq data (Fig. - Note- worthy, 25 out of 44 BGCs (56.8%) did not have detect- able expression (NE) in the RNA-seq data in both conditions (RPKM <. - However, it is important to notice that nearly half of the BGCs (among the 73 identified) were silent under the conditions evaluated in the RNA-seq [7]. - Pattern of expression of the putative methylated mRNA genes inferred from the RNA-seq data. - We extended the evaluation of the patterns of expres- sion using the RNA-seq data for all putative methylated protein coding genes found. - 70%) putative methylated protein coding genes displayed detectable expression in the RNA-seq data (RPKM ≥2), but there were no statistical differences be- tween conditions (48hRM x 48hTC) (Additional file 4).. - This contrasts with the subset of putative methylated BCGs backbone genes, which did not display detectable expression in the RNA-seq data (Fig. - methylated genes that fall in the ND category are abun- dant in all conditions. - 73% for protein coding genes only methylated in the 48hTC condition. - 66% for pro- tein coding genes only methylated in the 48hRM condi- tion and ~ 67% for protein coding genes methylated in both conditions) (Additional file 4). - 12%) putative methylated protein coding genes did not have detectable expression in the RNA-seq data (RPKM <. - 6%) putative methylated mRNA genes were down-regulated in the RNA-seq data and 77 out of 670. - 11%) putative methylated mRNA genes were up-regulated in the RNA- seq data (Additional file 4). - Although, the vast majority of putative methylated mRNA genes were expressed, a clear pattern of down-regulation or up-regulation linked to DNA methylation could not be observed. - 024437 is the backbone gene for the destruxin BGC (MaNRPS1) [7], which was strongly up-regulated in the comparison 48hRM x 48hTC (Fig. - MANI_023437 is another back- bone gene, which codes for a protein putatively enrolled in the biosynthesis of a xenolozoyenone-like metabolite [MaNRPS-PKS3]) [7]. - MANI_023437 was down-regulated in the comparison 48hRM x 48hTC (Fig. - Additionally, while MANI_026638 was putative methylated in 48hRM and 48hTC, MANI_111160 was only putative methylated in the 48hTC condition (Additional file 1). - codes a putative exo-beta-1,3-glucanase from family 17 of glycoside hydrolases, was included in the analysis. - The gene expression patterns of the seven chosen genes were explored using three incubation periods (24, 48, and 72 h), which spans the period between the early interaction between fungal cells and tick cuticles to the establishment of the infection. - identified methylated genes, 5-AZA treatment led to increased expression in, at least, one of the incubation times analyzed (Fig. - The impact of the epigenetic machinery, specifically DNA methylation, in the lifecycle of fungal species from Metarhi- zium genus have started to be addressed in M. - Exploring the changes in the methylation pattern between the conidia and mycelia stages, Li and coworkers (2017) showed that approximately 0.38% of the total number of cy- tosines were putatively methylated in conidia, while 0.42% of the total number of cytosines were putatively methylated in mycelia [9]. - However, the impact of DNA methylation in the infection process was not evaluated in M. - In the experiments conducted here, we started to address this problem, employing a mimicked infection condition that has been used before and a saprophytic-like condition as a. - Noteworthy, previous results have shown that virulence determinants were up-regulated in the mim- icked infection condition [6, 7]. - anisopliae, the proportion of putative methylated sites in the CHH, CpG and CHG residues were similar between the Metarhi- zium spp., with ~ 57, 23 and 20% of the methylation sites in CHH, CpG and CHG residues, respectively, in M. - Furthermore, the BS-Seq results support the impact of DNA methylation modification on the modulation of M.. - It is assumed that, in the presence of glucose, other catabolic pathways and virulence deter- minants should be repressed in M. - Whereas, in the infection condition, these pathways would be available, in view of host’s/nutrient’s complex- ity. - However, what we found was the contrary of that hy- pothesis, with more genes putatively methylated in the infection condition. - Moreover, the pro- duction of destruxin, evaluated by the expression of the destruxin BGC (MaNRPS1), have previously displayed a temporal pattern of regulation during the mimicked in- fection condition [7]. - As several backbone genes enrolled in the biosynthesis of SM are putatively methylated, it is possible that M.. - anisopliae employs DNA methylation as an additional mechanism to control SM-associated gene expression. - Moreover, the importance of the DNA methylation on cell morphogenesis is further supported by the GO analysis, reiteratively demonstrating the im- portance of this epigenetic modification in the lifecycle and infection of M. - in the infection process, the expression of virulence determinants can be crucial even when the virulence determinants are potentially. - In the absence of host’s stimuli, the energetic expenditure should be real- located. - In this scenario, the DNA methylation may be an important mechanism. - Each of the two biological replicates consisted of mycelium grown over approxi- mately 5 g (wet weight) of tick cuticles, which are dis- posed onto 5 Petri dishes. - The sequencing of the librar- ies was performed with TrueSeq DNA Methylation kit (Illumina) using as input the four bisulfite-treated single-stranded DNA samples. - All the steps of the proto- col were carried out following manufacturer’s instruc- tions. - Quality control of the distinct libraries was performed using the 2100 Bioanalyzer System with the Agilent High Sensitivity DNA Kit (Agilent). - recover of the 10 best hits. - and the 2 DNMTs found in the genome of M. - A melting curve analysis was performed at the end of the reaction to confirm the presence of single PCR products. - Expression and differential expression profile of the 670 putatively methylated mRNA genes extracted from previously published RNA-seq data.. - The datasets generated during the current study are available in the NCBI ’ s repository, BioSample accessions SAMN11941230, SAMN11941231, SAMN11941232, SAMN11941233.. - The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.. - Secondary metabolite gene clusters in the entomopathogen fungus Metarhizium anisopliae: genome identification and patterns of expression in a cuticle infection model. - DNA methylation: superior or subordinate in the epigenetic hierarchy? 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