DNA Methylation and Demethylation Are Regulated by Functional DNA Methyltransferases and DnTET Enzymes in Diuraphis noxia

Abstract

Aphids are economically important insect pests of crops worldwide. Despite resistant varieties being available, resistance is continuously challenged and eventually broken down, posing a threat to food security. In the current study, the epigenome of two related Russian wheat aphid (Diuraphis noxia, Kurdjumov) biotypes (i.e., SA1 and SAM) that differ in virulence was investigated to elucidate its role in virulence in this species. Whole genome bisulfite sequencing covered a total of 6,846,597,083 cytosine bases for SA1 and 7,397,965,699 cytosine bases for SAM, respectively, of which a total of 70,861,462 bases (SA1) and 74, 073,939 bases (SAM) were methylated, representing 1.126 ± 0.321% (SA1) and 1.105 ± 0.295% (SAM) methylation in their genomes. The sequence reads were analyzed for contexts of DNA methylation and the results revealed that RWA has methylation in all contexts (CpG, CHG and CHH), with the majority of methylation within the CpG context (± 5.19%), while the other contexts show much lower levels of methylation (CHG − ± 0.27%; CHH − ± 0.34%). The top strand was slightly (0.02%) more methylated than the bottom strand. Of the 35,493 genes that mapped, we also analyzed the contexts of methylation of each of these and found that the CpG methylation was much higher in genic regions than in intergenic regions. The CHG and CHH levels did not differ between genic and intergenic regions. The exonic regions of genes were more methylated (±0.56%) than the intronic regions. We also measured the 5mC and 5hmC levels between the aphid biotypes, and found little difference in 5mC levels between the biotypes, but much higher levels of 5hmC in the virulent SAM. RWA had two homologs of each of the DNA methyltransferases 1 (DNMT1a and DNMT1b) and DNMT3s (DNMT3a and DNMT3b), but only a single DNMT2, with only the expression of DNMT3 that differed significantly between the two RWA biotypes. RWA has a single ortholog of Ten eleven translocase (DnTET) in the genome. Feeding studies show that the more virulent RWA biotype SAM upregulate DnDNMT3 and DnTET in response to wheat expressing antibiosis and antixenosis.

Phenotypic Plasticity: What Has DNA Methylation Got to Do with It?

“…DNA methylation is reversible and the ten-eleven-translocase (TET) family of enzymes have a role in this process by converting 5-methylcytosine to 5-hydroxymethylcytosine [40,41], which can, in mammals, be subsequently oxidised to 5-formyl-and 5-carboxyl-cytosine [42]. 5-hydroxymethylcytosine is present in insect genomes at appreciable levels, e.g., [43][44][45], but the presence of 5-formyland 5-carboxyl-cytosine in insects and the function of these modifications remain unknown. In addition, it is proposed that DNA replication in the absence of maintenance of DNA methylation could result in passive demethylation [42].…”

The PWWP domain and the evolution of unique DNA methylation toolkits in Hymenoptera

“…Besides Hymenoptera, there are other insect in families such as Coleoptera and Orthoptera that have a copy of DNMT3 with the consensus domain architecture, although some claims that aphids (Hemiptera) have a conserved DNMT3 57 , 58 need to be re-evaluated because the available NCBI data suggest that the DNMT3 hallmark domains, PWWP and ADDz are encoded by separate genes. In Acyrthosiphon pisum , the PWWP-ADDZ domains are encoded by GenBank: XP_029348651.1 , and the MTase domain by GenBank: XP_016662566.1 , possibly exemplifying yet another highly diverse DNA methylation toolkit.…”

Whole-body transcriptome mining for candidate effectors from Diuraphis noxia

“…Primers were used in a primerBLAST analysis against the assembled RWA SAM biotype reference genome (GCA_001465515.1) to ensure specificity. The relative expression of the transcripts of interest in aphid heads, salivary glands and aphid bodies was quantified as previously described [86]. A five point, two times serial dilution of a body tissue sample was used to generate quantification standards.…”

“…All samples and standards were quantified with three technical repeats across three biological repeats along with a no template control for all genes of interest (Additional file 6: Table S5). The ribosomal genes L27 and L32 were used as reference genes as in previous studies [86,87]. A CFX96 Real-Time System (Bio-Rad) was used to perform the PCR analysis.…”