An investigation of the potato eIF4E isoforms as targets for non-transgenic CRISPR/Cas9 genome editing for viral resistance

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dc.contributor.advisorLloyd, James Richarden_ZA
dc.contributor.advisorBurger, Johanen_ZA
dc.contributor.advisorCampa, Manuelaen_ZA
dc.contributor.authorHurst, Rebeccaen_ZA
dc.contributor.otherStellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.en_ZA
dc.date.accessioned2022-11-22T06:02:59Z
dc.date.accessioned2023-01-16T12:49:02Z
dc.date.available2022-11-22T06:02:59Z
dc.date.available2023-01-16T12:49:02Z
dc.date.issued2022-12
dc.descriptionThesis (MScAgric)--Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH ABSTRACT: Solanum tuberosum (potato) is an important food source in Southern Africa. Viral infection of potato plants leads to decreased tuber size and number, and symptomatic tubers are often unfit for consumption. Increased prevalence of the viruses Potato Virus Y (PVY) and Potato Leaf Roll Virus (PLRV) in South Africa threatens the country’s food security. The eukaryotic translation initiation factor 4E (eIF4E) gene family encode proteins that are involved in native and viral RNA translation. This mechanism is essential for viral survival and the eIF4E family are promising susceptibility factors that can be manipulated to confer viral resistance in plants. Expression of the potato eIF4E isoforms – eIF4E-1, eIF4E-2, eIF(iso)4E – and the related gene new RNA cap binding protein (nCBP), was investigated in tubers of PLRV-infected plants and compared to that of tubers from healthy plants. No significant difference was observed between the samples which may indicate that expression differences are tissue-specific, rather than stress-induced. A Bayesian maximum clade credibility tree was created to elucidate the emergence of eIF4E isoforms across plant evolution. Division of the plant RNA cap-binding proteins into two distinct groups - eIF4E and nCBP –occurred in the common ancestor of all land plants. The eIF4E-ancestor divided into eIF4E and eIF(iso)4E in at least the angiosperms and possibly as far back as the vascular plants. The further division of eIF4E into eIF4E-1 and eIF4E-2 only occurred recently, in an ancestor of the Solanaceous family. During infection, the essential interaction between the viral genome-linked protein (VPg) and the host translation machinery is facilitated by three amino acid residues in the eIF4E cap-binding pocket. Amino acid alignments of the VPg binding region of the potato eIF4E proteins indicate that all have the potential for interaction with the viral protein. This implies that the VPg binding capacity of all these genes would need to be disrupted to engineer complete resistance to PVY and PLRV. To begin the process of creating eIF4E-1, eIF4E-2 and eIF(iso)4E knockout mutants in potato, single guide RNAs were designed for all three genes. Additionally, a single guide RNA that is capable of simultaneously knocking out eIF4E-1 and eIF4E-2 was designed. Each of these guides were transcribed in vitro and complexed with Cas9. In vitro efficacy assays demonstrated that all ribonucleoproteins could induce double stranded breaks to the target genes. Transformation of the CRISPR/Cas9 ribonucleoproteins into protoplasts provides a transgene-free method of eIF4E gene editing. The isolation of viable potato protoplasts was established, and tissue culture of these cells yielded micro-calli.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Solanum tuberosum (aartappel) is ‘n belangrike voedselbron in suiderlike Afrika. Virus infeksie in aartappelplante lei tot ‘n afname in knol grootte en hoeveelheid en simptomatiese knolle is dikwels ongeskik vir verbruik. ‘n Verhoogde voorkoms in Suid-Afrika van die virusse “Potato Virus Y (PVY)” en “Potato Leaf Roll Virus (PLRV)” dreig die land se voedselsekuriteit. Die eIF4E-geenfamilie kodeer vir proteïene wat betrokke is by inheemse asook virale RNA-translasie. Hierdie meganisme is noodsaaklik vir virale oorlewing, dus is die manipulasie van die eIF4E-familie belowende vatbaarheidsfaktore om virale weerstand aan plante te verleen. Die uitdrukking van die aartappel eIF4E isovorme – eIF4E-1, eIF4E-2, eIF(iso)4E – en die verwante geen nCBP, is in knolle van PLRV-geïnfekteerde plante ondersoek en vergelyk met dié uitdrukking in knolle van gesonde plante. Geen beduidende verskil is tussen die monsters waargeneem nie, wat kan aandui dat die uitdrukkingsverskille weefselspesifiek is, eerder as stresgeïnduseerd. ‘n “Bayesian maximum clade credibility tree” filogenetiese boom is geskep om die opkoms van die eIF4E isovorme oor plant evolusie toe te lig. Die verdeling van die plantkap-bindende proteïene in drie afsonderlike groepe - eIF4E, eIF(iso)4E en nCBP – het in die gemeenskaplike voorouer van alle landplante plaasgevind. Die verdere verdeling van eIF4E in eIF4E-1 en eIF4E-2 het meer onlangs plaasgevind in ‘n voorouer van die Solanaceous-familie. Tydens infeksie word die noodsaaklike interaksie tussen die viral genome-linked protein (VPg) en die gasheer se translasiemasjienerie deur drie aminosuurreste in die eIF4E kap-bindende sak fasiliteer. Alignments van die aminosure van die VPg-bindingsgebied van twee afsonderlike klades dui aan dat al die gene in beide groepe potensiaal het vir interaksie met die virale proteïen. Hierdie impliseer dat die VPg-bindingskapasiteit van al hierdie gene ontwrig sal moet word om volledige weerstand teen PVY en PLRV te verkry. Om eIF4E-1, eIF4E- 2 en eIF(iso)4E mutante in aartappels te skep, is enkelgids-RNAs vir elke geen ontwerp. Verder is ‘n enkelgids- RNA ontwerp om gelyktydig eIF4E-1 en eIF4E-2 te muteer. Elkeen van hierdie gidse is in vitro getranskribeer en gekompleks met Cas9. In vitro doeltreffendheidstoetse het getoon dat al hierde ribonukleoproteïene dubbelstringbreuke aan die teikengene kan veroorsaak. Transformasie van die CRISPR/Cas9 ribonukleoproteïene in protoplaste bied ‘n transgeenvrye metode van eIF4E geenredigering. Die isolasie van lewensvatbare aartappelprotoplaste is vasgestel en weefselkultuur van hierdie selle het mikro-kallus opgelwer.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxiii, 83 pages : illustrations (some color)en_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/126079
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subjectPotatoes (Solanum tuberosum L.) -- South Africaen_ZA
dc.subjectVirus diseases of plants -- Genetic aspectsen_ZA
dc.subjectCRISPR-associated protein 9en_ZA
dc.subjectGene editingen_ZA
dc.subjectPotato leafroll virus -- South Africaen_ZA
dc.subjectEukaryotic translation initiation factor 4E (eIF4E)en_ZA
dc.subjectPotatoes -- Effect of stress onen_ZA
dc.subjectViral resistance in plantsen_ZA
dc.subjectUCTDen_ZA
dc.titleAn investigation of the potato eIF4E isoforms as targets for non-transgenic CRISPR/Cas9 genome editing for viral resistanceen_ZA
dc.typeThesisen_ZA
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