A targeted investigation of Diuraphis noxia (Hemiptera: Aphididae) methylation

[摘要] ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae – or Russian wheat aphid, RWA) is aneconomically important phloem-feeding pest of wheat and barley. The most effective method forcontrolling RWA infestation of wheat is the deployment of resistant cultivars. However, newbiotypes – aphid populations expressing virulence towards these cultivars – continue to develop.Consequently, a dire need exists to understand the molecular mechanism underlying increases inaphid virulence. The epigenetic modification of methylation has been proposed as one suchmechanism, yet its effect on virulence remains largely unexplored. The aim of the study was thus todetermine if methylation plays a role in biotypification and the associated increase in aphidvirulence. To this end, two methods, namely methylation-sensitive amplification polymorphism(MSAP or MS-AFLP) and restriction site-specific fluorescent labelling (RSSFL), were tested fortheir ability to detect and quantify RWA methylation. The former was successful on both counts,specifically in the CG and CC dinucleotide contexts. Use of this methodology also revealed 22polymorphic loci between the least and most virulent South African biotypes, SA1 and SAM, with18 resulting from an increase in methylation during SAM‟s biotypification from SA1. Restrictionsite-specific fluorescent labelling is a novel technique that makes use of a fluorescently labelled adaptor, which binds to the sticky ends produced after the restriction of DNA using theisoschizomers HpaII and MspI. Although unable to detect or quantify methylation, RSSFL was ableto detect trends in methylation. Various aspects of the DNA methyltransferases (DNMTs), whichcatalyse methylation, were also investigated. A homology search identified four putative RWADNMT genes, namely DNMT1, DNMT2, DNMT3A and DNMT3B. Sequencing of these genesdetected only one single nucleotide polymorphism between biotypes SA1 and SAM. BaselineDNMT expression, quantified using RT-qPCR, revealed significant differences in DNMT3Aexpression, which could be explained by the virulence of the respective biotypes. An antibodyspecific to 5-methylcytosine (5mC) was used to quantify both the DNMT protein activity (bydetecting the relative number of methyl groups transferred by the DNMTs to a universal substrate) and the global 5mC levels, both of which did not differ significantly between the biotypes. The5mC levels ranged from 0.1% to 0.16% and were in line with levels reported for numerous insects.Global hydroxymethylation levels were quantified using an antibody specific to5-hydroxymethylcytosine (5hmC, a demethylation intermediate). Biotype SAM‟s 5hmC level wassignificantly higher than that of biotypes SA1, SA2 and SA3. Based on the results obtained, it isrecommended that future studies of RWA methylation first perform RSSFL, followed by eitherMSAP or antibody-mediated methylation quantification (or both), depending on the needs of thespecific study. The results also made clear the fact that methylation, and the removal thereof isrelated to differences in RWA virulence. Although many aspects of methylation were similarbetween the biotypes, local increases in methylation proved beneficial to the development of thehighly virulent biotype SAM. During biotypification SAM also attained an increased ability todemethylate its genome, which affords this biotype greater flexibility to adapt to changingenvironments, by means of alterations in gene regulation. An increased demethylation capacitymight therefore be a key contributory factor to increases in aphid virulence and hencebiotypification.

[发布机构] Stellenbosch University