Browsing by Author "Hurst, Rebecca"

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    An investigation of the potato eIF4E isoforms as targets for non-transgenic CRISPR/Cas9 genome editing for viral resistance
    (Stellenbosch : Stellenbosch University, 2022-12) Hurst, Rebecca; Lloyd, James Richard; Burger, Johan; Campa, Manuela; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
    ENGLISH 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.