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

Breeds, Kelly (2017-12)

Thesis (MScAgric)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae – or Russian wheat aphid, RWA) is an economically important phloem-feeding pest of wheat and barley. The most effective method for controlling RWA infestation of wheat is the deployment of resistant cultivars. However, new biotypes – aphid populations expressing virulence towards these cultivars – continue to develop. Consequently, a dire need exists to understand the molecular mechanism underlying increases in aphid virulence. The epigenetic modification of methylation has been proposed as one such mechanism, yet its effect on virulence remains largely unexplored. The aim of the study was thus to determine if methylation plays a role in biotypification and the associated increase in aphid virulence. To this end, two methods, namely methylation-sensitive amplification polymorphism (MSAP or MS-AFLP) and restriction site-specific fluorescent labelling (RSSFL), were tested for their 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 22 polymorphic loci between the least and most virulent South African biotypes, SA1 and SAM, with 18 resulting from an increase in methylation during SAM‟s biotypification from SA1. Restriction site-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 the isoschizomers HpaII and MspI. Although unable to detect or quantify methylation, RSSFL was able to detect trends in methylation. Various aspects of the DNA methyltransferases (DNMTs), which catalyse methylation, were also investigated. A homology search identified four putative RWA DNMT genes, namely DNMT1, DNMT2, DNMT3A and DNMT3B. Sequencing of these genes detected only one single nucleotide polymorphism between biotypes SA1 and SAM. Baseline DNMT expression, quantified using RT-qPCR, revealed significant differences in DNMT3A expression, which could be explained by the virulence of the respective biotypes. An antibody specific to 5-methylcytosine (5mC) was used to quantify both the DNMT protein activity (by detecting 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. The 5mC 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 to 5-hydroxymethylcytosine (5hmC, a demethylation intermediate). Biotype SAM‟s 5hmC level was significantly higher than that of biotypes SA1, SA2 and SA3. Based on the results obtained, it is recommended that future studies of RWA methylation first perform RSSFL, followed by either MSAP or antibody-mediated methylation quantification (or both), depending on the needs of the specific study. The results also made clear the fact that methylation, and the removal thereof is related to differences in RWA virulence. Although many aspects of methylation were similar between the biotypes, local increases in methylation proved beneficial to the development of the highly virulent biotype SAM. During biotypification SAM also attained an increased ability to demethylate its genome, which affords this biotype greater flexibility to adapt to changing environments, by means of alterations in gene regulation. An increased demethylation capacity might therefore be a key contributory factor to increases in aphid virulence and hence biotypification.

AFRIKAANSE OPSOMMING: Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae – of Russiese koringluis, RKL) is ‟n ekonomies belangrike floeëmvoedende plaag van koring en gars. Die doeltreffendste beheermaatreël vir RKL-infestering in koring is die gebruik van weerstandige kultivars. Nuwe biotipes – koringluispopulasies wat virulensie teenoor hierdie kultivars toon – ontwikkel egter voortdurend. Gevolglik bestaan ‟n dringende behoefte om te verstaan watter molekulêre meganisme onderliggend aan toenemende koringluisvirulensie is. Die epigenetiese-modifikasie deur metilering is voorgestel as so ‟n meganisme, maar die effek daarvan op virulensie is nog nie goed ondersoek nie. Die doel van hierdie studie is dus om te bepaal of metilering ‟n rol in die vorming van biotipes en die gevolglike toename in koringluisvirulensie speel. Twee metodes, naamlik metileringsensitiewe amplifikasiepolimorfisme (MSAP of MS-AFLP) en beperkingsarea-spesifieke fluoresserende etikettering (RSSFL), is getoets vir hul vermoë om RKL-metilering uit te wys en te kwantifiseer. Eersgenoemde metode het beide gedoen, spesifiek in die konteks van die CG en CC dinukleotiedpare. Die gebruik van hierdie metode het ook 22 polimorfiese lokusse tussen die minste en mees virulente Suid-Afrikaanse biotipes, SA1 en SAM, uitgewys. Verder is bevind dat 18 lokusse die resultaat van ‟n toename in metilering tydens SAM se biotipevorming vanuit SA1 is. Beperkingsarea-spesifieke fluoresserende etikettering is ‟n nuwe tegniek wat gebruik maak van ‟n fluoresserend-gemerkde verbinder wat bind aan die beperkingsensiemoorhange wat ontstaan na beperkingsnyding van DNS deur isoskisomere HpaII en MspI te gebruik. Hoewel RSSFL nie metilering kon kwantifiseer nie, kon dit wel tendense in metilering uitwys. Verskeie aspekte van die DNS-metieltransferases (DNMTs) wat metilering kataliseer, is ook ondersoek. ‟n Homologiesoektog het vier vermeende DNMT-gene in die RKL-genoom geïdentifiseer, naamlik DNMT1, DNMT2, DNMT3A en DNMT3B. Volgordebepaling van hierdie gene het slegs een enkelnukleotied-polimorfisme tussen biotipes SA1 en SAM uitgewys. Basislyn-DNMT-uitdrukking, wat deur middel van RT-qPCR gekwantifiseer is, het betekenisvolle verskille in die uitdrukking van DNMT3A uitgewys, wat deur die virulensie van die onderskeie biotipes verklaar kan word. ‟n Teenliggaam, spesifiek aan 5-metielsitosien (5mC), is gebruik om sowel die DNMTproteïenaktiwiteit (deur vasstelling van die relatiewe aantal metielgroepe wat deur die DNMTs na ‟n universele substraat oorgedra is) as die globale 5mC-vlakke te kwantifiseer; beide het nie betekenisvolle verskille tussen die biotipes getoon nie. Die 5mC-vlakke het tussen 0.1% tot 0.16% gewissel en was in lyn met vlakke wat in verskeie ander insekte gemeet is. Globale vlakke van hidroksiemetilering is met ‟n teenliggaam wat spesifiek teen 5-hidroksiemetielsitosien (5hmC, ‟n demetileringstussenganger) is, gekwantifiseer. Biotipe SAM se 5hmC-vlak was betekenisvol hoër as dié van biotipes SA1, SA2 en SA3. Op grond van hierdie resultate word voorgestel dat toekomstige ondersoeke na RKL-metilering eerstens RSSFL uitvoer, gevolg deur óf MSAP óf teenliggaam-bemiddelde metileringskwantifisering (of beide), afhangende van die behoeftes van die betrokke ondersoek. Die resultate maak dit ook duidelik dat metilering en die verwydering daarvan verband hou met verskille in RKL-virulensie. Hoewel baie aspekte van metilering tussen die betrokke biotipes ooreenstem, het plaaslike toenames in metilering voordelig geblyk vir die ontwikkeling van die hoogs virulente biotipe SAM. Gedurende biotipevorming het SAM ook ‟n verhoogde vermoë om sy genoom te demetileer verkry, wat hierdie biotipe van groter buigsaamheid voorsien om by veranderende omgewings aan te pas. Laasgenoemde is waarskynlik deur middel van wysigings in geenregulering. ‟n Verhoogde vermoë om te demetileer is derhalwe moontlik ‟n sleutelfaktor wat tot toenames in plantluisvirulensie en gevolglik biotipevorming, bydra.

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