DNA Methylation In The Sexual Stage Of Cordyceps Militaris And Functions Of DNA Methyltransferases In Metarhizium Robertsii

DNA methylation is a basic epigenetic mechanism found in eukaryotes,but its patterns and roles vary significantly among diverse taxa.In fungi,DNA methylation has various effects on diverse biological processes,however,its function underlying the sexual development of fungi remains unclear.Cordyceps militairs,readily performing sexual reproduction,provides a remarkably rich model for understanding epigenetic processes in the sexual development.Metarhizium robertsii,a model system for studying insect-fungus interactions,has been used as an environmentally friendly alternative to chemical insecticide.Little,however,is known concerning the molecular basis for DNA methylation.Firstly,two genes encoding proteins(CmDIM-2 and CmDMTA)were confirmed as two “really” DNA methyltransferases by sequencing and enzyme activity assay.Secondly,we investigated the nature of DNA methylation in sexual and asexual development of C.militaris by generating a DNA methylation map at single-base resolution across the genome with the deep bisulfite sequencing,and roles of DNA methylation in sexual development were analyzed with RNA-Seq.Furthermore,we obtained MrRID and MrDIM-2 mutants by homologous recombination and the function of both DNMTases were explored.The results are as follows:(1)Analysis of DNA methyltransferases in C.militarisCmDIM-2 and CmDMTA were found in C.militaris through a BLAST search.Completed splicing and annotation of both gene full-length was performed with RACE sequencing and phylogenetic analysis.Subsequently,two prokaryotic expression vectors were constructed and cloned into E.coli BL21.SDS-PAGE showed that both recombinant proteins were induced expression successfully and specifically recognized by anti-His tag antibody.DNA methyltransferase activity assays of purified proteins showed both proteins have DNA methyltransferase activity.(2)Effect of DNA methylation on sexual development of C.militaris(1)Transcriptional profiling of two DMTase-encoding genes at different developmental stages: Both of genes were increasing expression in asexual development with the production of spores and got up to maximum at the stages of NF(sexual development).(2)Assays of DNA Methyltransferase activity and DNA methylation level of C.militaris: Based on results of Real-time PCR,the 3-day-old mycelium(asexualdevelopment)and nascent fruiting body(sexual development)were chose for further analysis.The results HPLC and enzyme assays showed that DNA Methyltransferase activity in NF is higher than it in 3M,and the levels of DNA methylation in NF and 3M are0.42% and 0.38%,respectively.(3)The characteristics of DNA methylation in 3M and NF: Initially we obtained overall genome-wide methylation patterns,0.41% including 0.39% at CG,0.38% at CHG and0.43% at CHH sites in NF while 0.39% including 0.37% at CG,0.39% at CHG and 0.40%at CHH sites in 3M.All the mC sites across the C.militaris genome evenly.There were no significant differences of % mC between two stages,however,obvious difference was found for m C sites of two samples,suggested that DNA methylation in the development of C.militaris is dynamic process.225 DMRs,of which 141 were located in intergenic regions while 84 DMRs are concentrated in 2-kb regions upstream of genes or gene bodies,were identified.136 DMR-associated genes were identified,GO and KEGG pathway analysis showed some genes among of involved in energy metabolisms and signal transmission.(4)The relationship between DNA methylation and sexual development: To reveal the functional consequences of DNA methylation,DMR-associated genes were further analyzed with RNA-Seq.In NF,5 down-regulated genes were found among 48 hypermethylated DMR-associated genes and 7 ones were found among 61 hypermethylated DMR-associated genes in 3M.Less than 12%(12/109)of genes correspond to the negative correlation between DNA methylation and gene expression.Therefore,our results showed that DNA methylation,contrast with previous study in animals and plants,has no direct effect on gene expression.Through combined analysis of genome and BS-Seq data,we found that C.militaris may undergone the RIP which showed strong consistence with DNA methylation.(3)Function analysis of DNA methyltransferases in Metarhizium robertsii(1)Bioinformatics of DNMTases in M.robertsii: two genes encoding proteins(MrRID and MrDIM-2)containing the DNMTase domain were found through a BLAST search.Our phylogenetic analysis showed that MrRID and MrDIM-2 are closely related to RID and DIM-2 in C.militaris and Neurospora crassa.(2)Roles of DNMTases for DNA methylation of M.robertsii: We obtained gene deletion mutants(?MrRID,?MrDIM-2 and ?RID/?DIM-2)by Agrobacterium tumefaciens-mediated homologous recombination method.~71 %,~10 % and ~8 %of the mC sites remained in ?MrRID,?MrDIM-2 and ?RID/?DIM-2,respectively,compared with the WT strains.Further analysis showed that MrRID in M.robertsii plays a role in regulating methylation specificity and some cytosine sites is accomplished by both MrRID and MrDIM-2.(3)Biological character analyses of ?MrRID,?MrDIM-2 and ?RID/?DIM-2 strains:We observed that the radial growth and conidial production of ?MrDIM-2 and?RID/?DIM-2 were more defective from that of wild type than ?MrRID.When comparing the mutant strains to wild type under UV irradiation or heat,spore viability was noticeably decreased,especially for ?MrDIM-2 and ?RID/?DIM-2.However,all of the DNMTase mutants showed a similar growth capability against stressful chemicals compared with control strains.Insect bioassays using a topical application of fungal conidia revealed severe attenuation in ?MrDIM-2 and ?RID/?DIM-2 with a mean lethal time to death(LT50)of 6.5 ± 0.9 and 7.3 ± 1.2 days while 4.6 ± 0.8 and 4.4 ± 0.5 days for ?MrRID and wild type,respectively,along with subsequent poor sporulation in the infected larvae.