Mulberry MITE Transposon And Dynamic Methylation In Response To Botrytis Cinerea

Transposable elements(TEs)are widely distributed in the prokaryotic and eukaryotic genomes.They can increase their copy number by moving from one part of the chromosome to another,a process known as transposable.Transposons play an important role in genome size,production of new genes,gene expression regulation,and production of small RNAs.Miniature inverted-repeat transposable elements(MITE)are a kind of truncated DNA transposon,which has terminal inverted repeat(TIR)sequence and target site duplication(TSD)sequence.MITE themselves do not contain ORF region.Their transposition relies on homologous autonomous DNA transposons provided transposases.MITE are widely distributed in the eukaryotic genomes.At present,the research on MITE mainly focuses on species identification,content analysis,and transpose mechanism.The research on the function of MITE is still relatively few,especially in woody plants.In addition to being widely planted for silkworm breeding,mulberry is a perennial deciduous woody plant with important ecological and medicinal values.Mulberry is the representative plant of Moraceae.The sequencing of its whole genome was completed in 2013,which provides an opportunity to comprehensively identify and study transposon function at the whole genome level.At present,the research on MITE of mulberry is only on the simple identification and classification,and the detailed systematic research has not been reported.In this paper,MITE-Hunter,MITE-Digger,and RSPB were used to identify the MITE in the genome of Morus notabilis C.K.Schneid.The systematic superfamily and family classification were carried out for the identified MITE.By means of bioinformatics,the pattern of MITE transposon amplification,insertion preference,small RNA generation,and the correlation with alternative splicing were analyzed.PCR and transgenic methods were used to verify the polymorphism of the genome caused by MITE transposons,the regulatory effect of MIETs transposons and the alternative splicing caused by MITE transposons.The mulberry leaves mock-treated or inoculated with Botrytis cinerea were analyzed by methylomes,transcriptome,and small RNA omics.The dynamic methylation of genes and transposons in response to gray mold was clarified and the important role of dynamic methylation was analyzed.Subsequently,virus-induced gene silencing(VIGS)was used for verification.The main results of this study are as follows: 1.The types and contents of translatons in the genome of Morus notabilisA total of 286122 MITE transposon related sequences were obtained from the M.notabilis genome.MITE transposons account for 13.83 % of the genome of mulberry,which is higher than that of rice(10%).According to TSD and TIR sequences,mulberry MITE transposons were divided into four superfamilies: PIF/Harbinger,Tc1/Mariner,Mutator,and h AT.Among the four known MITE transposon superfamilies,Tc1/Mariner accounted for the highest proportion of the genome(5.11%).2.Amplification mode and insertion preference of MITE in the mulberry genomeBy analyzing the nucleotide difference degree of each family of MITE,51 families presented unimodal distribution,44 families presented bimodal distribution,and the remaining families presented multi-peak distribution or non-peak smooth distribution.The phylogenetic tree was further constructed using the adjacency method.The phylogenetic tree of families with unimodal distribution showed obvious stellate shape without obvious branches,indicating that the members of these families were rapidly amplified from the same ancestor.The phylogenetic tree of families with bimodal or even multipeaked distributions had several distinct branches,suggesting that the families may have branched off from their ancestors during the evolution.In order to further explore MITE transposon amplification,we investigated the insertion polymorphisms of four MITE transposons in different ploidy mulberry resources.Two MITE transposons were detected in all mulberry resources,indicating that these two MITE transposons were fixed in different ploidy mulberry resources,while the other two MITE transposons were activated in the process of mulberry chromosome increase.In order to analyze the preference of MITE transposons inserted into the genome,the MITE transposons inserted into the gene sequence(GS)and the intergenic sequence(IS)were firstly counted.The results indicated that the MITE transposons were uniformly arranged on the genome in a linear manner.The distribution of each MITE transposon superfamily in gene structure regions,as well as the 2k sequences upstream of the start codon and downstream of the stop codon were then compared.It was found that except for the Tc1/Mariner superfamily,the other three superfamilies tended to be inserted near genes.In order to analyze the proportion of MITE transposon accompanying gene expression,the MITE transposon inserted into the gene exon was counted.Among the MITE transposon superfamily,the expression rate of PIF/Harbinger was the highest.The expression ratios of MITE transposon inserted genes in five tissues were flower(1.65%),bud(1.58%),leaf(1.58%),bark(1.53%),and root(1.40%).These results showed that the gene expression rates with MITE transposon insertion were relatively consistent in the five analyzed tissues.3.MITE regulation and its influence on alternative splicingMn M2 was inserted near the gene and there were polymorphisms in different mulberry resources.In order to further study whether it had regulatory effect,it was inserted near the Mn ANR gene and heterogeneously expressed.The expression level of Mn ANR in transgenic seedlings with Mn M2 was significantly higher than that in transgenic seedlings without Mn M2.The proportion of MITE transposon related alternative splicing in the five tissues were flower(5.00%),leaf(4.26%),root(4.02%),bark(3.31%),and bud(2.58%).The proportion of MITE transposon at splicing sites in five tissues was flower(0.51%),bark(0.50%),bud(0.48%),root(0.45%),and leaf(0.43%).To further verify the effect of MITE transposons on alternative splicing,we analyzed one Mn PR-4 gene from three mulberry resources,whose intron contains a MITE transposon(Mn P4),leading to the alternative splicing of the second exon.At the same time,Mn P4 polymorphism exists in different mulberry resources,and Mn PR-4 alternative splicing is also missing in mulberry resources with Mn P4 deletion.4.Gene and transposon methylation patterns and differential methylationAveraged across the genome,the percentages of m CG,m CHG,and m CHH out of total CG,CHG,and CHH sites were 35.5%,26.6%,and 37.9%,respectively,in the Mock samples and 33.8%,25.5%,and 40.7%,respectively,in the Inoculated samples.Compared with Mock treatment,Inoculated samples showed increased levels of C methylation in genes and transposon bodies and their flanking regions.The m CG level was higher in both gene bodies and their flanking regions,while the m CHG and m CHH levels were lower in gene bodies and higher in the gene flanking regions.In transposons,the levels of m CG,m CHG,and m CHH were much higher in the body regions than in the flanking regions.This finding suggested that DNA methylation plays an important role in transposon silencing in mulberry.The MITE methylation model was consistent with the transposon model,indicating that MITE transposons play a leading role in transposon methylation.Gene promoter differential methylation was analyzed,and m CG,m CHG and m CHH had 976(811 hypodifferential methylation),1,003(774 hypodifferential methylation),and 12,779(2,842 hypodifferential methylation),respectively.KEGG pathway analysis revealed that plant-pathogen interaction(87.50% hypodifferential methylation)is major gene categories in differential methylation of m CG,carbon metabolism(73.68% hypodifferential methylation)is major gene categories in differential methylation of m CHG,and metabolism(21.83% hypodifferential methylation)is major gene categories in differential methylation of m CHH.These results indicated that m CG and m CHG participate in resistance to B.cinerea by enhancing the expression of genes related to disease-resistance and carbon metabolism,and that m CHH contributes to resistance by inhibiting the expression of genes related to metabolic pathways.We analyzed the proportions of CG,CHG,and CHH differential methylation in genes and TEs.The proportion of DMRs,especially in CHG and CHH,was significantly higher in TEs than in genes,and 63.59% of CHH differential methylation were distributed in TEs.Further analyses showed that the differential methylation was mainly distributed in the MITE superfamily of TEs.Gene promoter regions affect transcriptional regulation and differences in promoter methylation affect transcriptional expression.The proportion of CG,CHG and CHH differential methylation in TEs were 40.60%,43.13% and 67.22%,respectively.Differential methylation was mainly distributed in the MITE superfamily,suggesting that MITE plays an important role in regulating gene expression through the Rd DM pathway.5.The silence of Mn MET1 enhances the resistance of mulberry leaves to B.cinereaVirus induced gene silencing(VIGS)was used to silence Mn MET1,and the plants that silenced Mn MET1 had obvious inhibition on the growth of B.cinerea.Meanwhile,it was identified that a resistance gene Morus002632 was enhanced in the plants that were silenced by Mn MET1.The results indicated that silence of Mn MET1 could enhance the expression of resistance gene of mulberry,thus enhancing the resistance of mulberry to B.cinerea.The present study systematically identified MITE and then comprehensively analyzed the functions of MITE transposons in mulberry trees.The data of we obtained is helpful to further understand the evolution of the mulberry genome.Combining the analyses of methylome,transcriptome and small RNA omics,the relationship between transposon-gene-methylation-Botrytis cinerea was addressed.The importance of MITE transposon regulating genes through Rd DM was clarified for the first time.