Browsing by Author "Spencer, Katarina Paula"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    CRISPR-based genome editing tools for virus resistance in grapevine
    (Stellenbosch : Stellenbosch University, 2022-12) Spencer, Katarina Paula; Burger, Johan; Campa, Manuela; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Grapevine (Vitis vinifera) is an important fruit crop which contributes significantly to the South African agricultural sector, and is a major produce crop worldwide. Grapevine viruses are widespread and cause serious diseases which impact the quality and quantity of crop yields. More than 80 viruses plague grapevine, with RNA viruses constituting the largest of virus pathogens. Clustered regularly interspaced, short palindromic repeat (CRISPR), along with its CRISPR- associated (Cas) proteins, is a system which has been harnessed from the prokaryotic immune system and adapted for genome editing technologies. The first CRISPR system to be adapted for genome editing was CRISPR/Cas9, which is characterised by its ability to target double-strand DNA. A recent extension to the CRISPR armoury is the Cas13 effector, which exclusively targets single-strand RNA. CRISPR/Cas has been implemented as a defence mechanism in plants, against both DNA and RNA viruses, by being programmed to directly target and cleave the viral genomes. The efficacy of the CRISPR/Cas tool in plants is dependent on efficient delivery of its components into plant cells. Geminiviruses, a group of small DNA viruses with a useful replication mechanism, have been reconstructed into efficient expression vectors and used for the delivery of genome editing components. By harnessing the CRISPR/Cas tool, and implementing the use of a viral vector for the expression thereof, a robust approach to induce virus resistance in plants can be achieved. To this end, the first aim of this study was to use CRISPR/CasRx to target an infectious clone of the RNA virus, grapevine virus A (GVA). GVA naturally infects V. vinifera, but can infect the model plant Nicotiana benthamiana, making it a helpful model to study virus infection in grapevine. The second aim of this study sought to use a geminivirus vector based on the bean yellow dwarf virus (BeYDV) to deliver and express CRISPR/Cas9 components in N. benthamiana. Firstly, constructs harbouring CasRx and a guide RNA (gRNA) targeting the replicase gene of GVA were assembled, and used for Agrobacterium-mediated transformation of N. benthamiana. Transgenic lines were infiltrated with the GVA infectious clone, but no consistent GVA interference was observed. To improve virus targeting, gRNAs were designed against the coat protein (CP) gene of GVA. N. benthamiana plants expressing CasRx were co-infiltrated with the infectious clone, and with a tobacco rattle virus (TRV)-gRNA expression vector, harbouring a CP gRNA. Results indicated more consistent GVA reductions, specifically CP gRNA 2, which demonstrated a significant negative correlation with GVA accumulation, as well as multiple gRNA co-infiltrations which similarly showed reduced GVA titre. When the pRIC BeYDV vector was used for gene targeting with CRISPR/Cas9, exogenously-delivered enhanced green fluorescence protein (eGFP), as well as endogenous N. benthamiana genes phytoene desaturase (PDS), and green fluorescence protein (GFP), from the transgenic N. benthamiana 16c line, were successfully cleaved and edited. By establishing a virus-targeting defence system in plants, and utilising a high- expressing geminivirus vector for the delivery of genome editing components, efficient virus interference mechanisms can be established and applied to major crops, such as grapevine.