Comparing different siRNA delivery systems to target Diuraphis noxia

Swiegers, Hendrik Willem (2018-12)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Diuraphis noxia, also known as the Russian wheat aphid, is a major pest of wheat. Breeding for resistance against D. noxia has been relatively successful in wheat as there has been many resistance genes incorporated into wheat in the past. However, this resistance has more often than not been counteracted by D. noxia through the development of a new biotype. The mechanism with which D. noxia is able to do this is not well understood. Previously, a highly virulent, laboratory generated biotype, known as SAM (South African Mutant), was compared to its avirulent progenitor, SA1, through proteome analysis of the salivary glands and complete genome sequence analysis. It was found that, among other differences, the cuticle protein, Dncprr1-8, containing a Rebers and Riddiford consensus was present in the salivary gland of SAM but not SA1. The gene also contained single nucleotide polymorphisms (SNPs) between the biotypes. In this study the function of Dncprr1-8 was investigated through RNA interference (RNAi). As RNAi has never been performed in D. noxia, several methods of siRNA delivery to this organism were compared. Injection of siRNA into the aphid haemolymph and ingestion of siRNA through artificial feeding medium was not successful. Allowing D. noxia to feed on wheat inoculated with a virus-induced gene silencing (VIGS) vector modified to contain D. noxia transcript sequence was partly effective, but overall had variable results. Finally, siRNA delivery through injection into wheat and allowing D. noxia to feed around the injection site, proved to be the most effective. Delivery of Dncprr1-8-siRNA using this method resulted in reduced survival and fecundity of biotype SAM while feeding on resistant wheat. The phenotypic responses were then compared to that of another aphid species, Myzus persicae, feeding on Arabidopsis thaliana injected siRNA targeting the same gene. M. persicae did not display reduced survival, but did produce fewer nymphs. Collectively, the results were then used to draw conclusions on the putative function of Dncprr1-8 in the plant-aphid interaction.

AFRIKAANSE OPSOMMING: Diuraphis noxia, ook bekend as die Russiese koringluis, is ‘n belangrike plaag van koring. Koring wat weerstandig is teen D. noxia is met relatiewe sukses geteel omdat vele weerstandbiedende gene al voorheen in koring geïnkorporeer is. Hierdie weerstand word dikwels afgebreek deur D. noxia deur die ontwikkeling van ‘n nuwe biotipe. Die meganisme waardeur D. noxia nuwe biotipes vorm word nog nie goed verstaan nie. ‘n Hoogs virulente laboratorium-gegenereerde biotipe, bekend as SAM (Suid-Afrikaanse Mutant), was voorheen vergelyk met sy stamvader, SA1, deur middel van proteïoomanalise van die speekselkliere asook deur volledige genoomanalise. Onder andere was daar gevind dat die kutikula-proteïen, Dncprr1-8 (wat ‘n Rebers en Riddiford konsensusvolgorde bevat), teenwoordig was in die speekselklier van SAM, maar nie in SA1 nie. ‘n Enkelnukleotiedpolimorfisme was ook tussen die twee biotipes opgemerk. Die funksie van Dncprr1-8 was deur middel van RNS-inmenging (RNSi) in hierdie studie ondersoek. Verskeie klein inmengende-RNS (kiRNS)-toediendingsmetodes was met mekaar vergelyk, aangesien RNSi nog nie van tevore in D. noxia uitgevoer is nie. Toediening via die inspuit van kiRNS direk in die hemolimf van die plantluis en inname van kiRNS deur kunsmatige voeding was nie suksesvol nie. D. noxia wat voed op koring wat geïnokuleer is met ʼn virusgeïnduseerde geen onderdrukkingsvektor wat gemodifiseer is om ‘n D. noxiatranskripvolgorde te bevat was gedeeltelik suksesvol, maar die resultate was inkonsekwent. Laastens was kiRNS in koringblare ingespuit en D. noxia toegelaat om rondom die inspuitingsarea te voed – hierdie metode was die effektiefste. Toediening van Dncprr1-8- siRNS deur middel van hierdie metode het tot ‘n verminderde oorlewing en vrugbaarheid van biotipe SAM gelei terwyl dit op weerstandige koring gevoed het. Hierdie fenotipiese reaksies was met ‘n ander plantluisspesie, Myzus persicae, vergelyk. Dit het op Arabidopsis thaliana gevoed, wat ingespuit is met kiRNS wat dieselfde geen, cprr1-8, teiken. M. persicae het nie verminderde oorlewing getoon nie, maar het wel minder nimfe produseer. Gesamentlik was die resultate gebruik om gevolgtrekkings oor die vermeende funksie van Dncprr1-8 in plant-plantluis interaksies te formuleer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/104892
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