Browsing by Author "Holm, Clara Cornelia"

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    Investigation of the DMR6 susceptibility genes in grapevine for improving phytoplasma resistance through CRISPR/Cas9 technology
    (Stellenbosch : Stellenbosch University, 2024-03) Holm, Clara Cornelia; Burger, Johan; Campa, Manuela; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Vi6culture is considered one of the most important crop industries worldwide but is con6nually placed under pressure from a vast array of pathogens. Phytoplasma diseases are a cri6cal challenge for the grapevine industry and cause considerable yield losses. Several breeding strategies aim to improve grapevine disease resistance through the introgression of resistance genes, or through the inac6va6on of suscep6bility (S) genes, using genome edi6ng. Recent advancements of genome edi6ng tools such as CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) allows for the targeted inac6va6on of important host S factors that play a role during pathogen infec6on. The Downy Mildew Resistant 6 (DMR6) genes were ini6ally described in Arabidopsis thaliana as a suscep6bility factor for bacterial and oomycete pathogens. Subsequent inac6va6on of these genes resulted in broad range pathogen resistance in various crops such as tomato, leBuce, cucumber, pepper, and citrus. Furthermore, there is evidence that the VviDMR6 genes play a similar role in grapevine. In fact, most recently the inac6va6on of the VviDMR6 genes lead to enhanced downy mildew resistance in grapevine. However, it’s precise role during pathogenesis remains unclear. To this end, this study aimed to func6onally characterize the grapevine DMR6 genes, with par6cular focus on their involvement in plant defense. Gene co- expression networks highlighted the involvement of VviDMR6.1 in defense, while VviDMR6.2 was associated with developmental pathways. Gene expression analyses across different grapevine 6ssues revealed that VviDMR6.1 was predominantly expressed in the roots. Furthermore, to beBer understand the role of VviDMR6 during pathogenesis, in vitro grown grapevine was treated with salicylic acid (SA) to examine its response in different 6ssue types. Both VviDMR6 genes were induced upon SA treatment across different 6ssues. Addi6onally, GUS-reporter assays were used to further inves6gate the 6ssue specificity of the VviDMR6 genes. This entailed the iden6fica6on and isola6on of the puta6ve promoter regions of VviDMR6.1 and VviDMR6.2 from Chardonnay. In silico analyses unveiled core promoter elements and puta6ve regulatory mo6fs associated with stress responsiveness within both VviDMR6 promoters. Subsequently, two promoterDMR6::GUS vectors were constructed consis6ng of the respec6ve promoters fused to GUS, and were subsequently introduced into A. thaliana. The proDMR6.1::GUS transgenic lines showed dis6nct promoter ac6vity in the roots of fully grown plants and demonstrated clear induc6on upon SA exposure. Finally, two previously designed CRISPR/Cas9 constructs containing two targets for VviDMR6.1 and a single target for VviDMR6.2 were introduced to rootstock cul6var Richter 110 embryogenic callus through Agrobacterium-mediated transforma6on. Successful edi6ng of VviDMR6.1 was achieved, with a muta6on frequency of 43%. Inference of CRISPR Edits (ICE) analysis revealed small indels, primarily 2-bp dele6ons, in the VviDMR6.1 edited lines. Notably, a single transgenic line demonstrated complete edi6ng, achieving 100% edi6ng efficiency. This contributes to the advancement of genome edi6ng tools for enhancing disease resistance in grapevine and yielded the first CRISPR/Cas9-edited grapevine on the African con6nent.