Investigation of the DMR6 susceptibility genes in grapevine for improving phytoplasma resistance through CRISPR/Cas9 technology

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dc.contributor.advisorBurger, Johanen_ZA
dc.contributor.advisorCampa, Manuelaen_ZA
dc.contributor.authorHolm, Clara Corneliaen_ZA
dc.contributor.otherStellenbosch University. Faculty of Agrisciences. Dept. of Genetics.en_ZA
dc.date.accessioned2024-02-23T13:36:38Z
dc.date.accessioned2024-04-26T12:22:23Z
dc.date.available2024-02-23T13:36:38Z
dc.date.available2024-04-26T12:22:23Z
dc.date.issued2024-03
dc.descriptionThesis (MScAgric)--Stellenbosch University, 2024. en_ZA
dc.description.abstractENGLISH 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.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Wingerdbou word beskou as een van die belangrikste gewasbedrywe wêreldwyd, maar dit ervaar voortdurend druk van 'n wye verskeidenheid patogene. Fitoplasmas is 'n kri6eke uitdaging vir die wingerdboubedryf en veroorsaak aansienlike opbrengsverliese. Verskeie strategieë is gemik op die verbetering van wingerd siektebestandheid deur die introgressie van weerstandbiedende gene, of deur die uitskakeling van vatbaarheid (S) gene, deur genoomredigering. Onlangse vordering in genoomredigeringstegnologieë soos CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) maak dit moontlik om die doelgerigte uitskakeling van belangrike S-faktore wat 'n rol speel tydens patogeen-infeksie, uit te voer. Die Downy Mildew Resistant 6 (DMR6) gene is aanvanklik in Arabidopsis thaliana beskryf as 'n S geen vir bakteriese en oomisete patogene. Daaropvolgende uitskakeling van hierdie gene het gelei tot breë-spektrum patogeenweerstandigheid in verskeie gewasse soos tama6e, slaai, komkommer, rissie en sitrus. Daar is verdere bewyse dat die VviDMR6-gene 'n soortgelyke rol in wingerd vervul. Die onlangse inak6vering van die VviDMR6-gene het gelei tot verhoogde weerstand teen downy mildew infeksie in wingerd. Die presiese rol daarvan tydens patogeen infeksie bly egter onduidelik. Met hierdie doelwit in gedagte, het hierdie studie gepoog om VviDMR6-gene funksioneel te karakteriseer, met spesifieke fokus op hul betrokkenheid by plant verdedigingsmeganismes. Geen-interaksie netwerke het VviDMR6.1 se betrokkenheid by verdediging beklemtoon, terwyl VviDMR6.2 verband gehou het met ontwikkelingspaaie. Geenuitdruking-analises het beklemtoon dat VviDMR6.1 hoofsaaklik in die wortels uitgedruk word in wingerd. Verder is in vitro gekweekte wingerd met salisielsuur (SA) behandel om die rol van VviDMR6 tydens patogenese infeksie beter te verstaan. Beide VviDMR6- gene is geïnduseer tydens SA-behandeling regdeur verskeie weefsels. Daarbenewens is GUS- raporteerde-toetse opgestel om die weefselspesifisiteit van die VviDMR6-gene verder te ondersoek. Dit het die iden6fikasie en isolasie van die vermeende promotor-areas van VviDMR6.1 en VviDMR6.2 vanaf Chardonnay ingesluit. In siliko-analises het kern-promotor elemente en vermoedelike regulatoriese mo6ewe wat verband hou met stres-reak6ewiteit binne beide VviDMR6-promotors onthul. Daarna is twee promotorDMR6::GUS-vektore ontwerp wat bestaan uit die onderskeie promotors, gekoppel aan GUS, en is daarna in A. thaliana getransformeer. Die proDMR6.1::GUS transgeniese lyne het duidelike promotorak6witeit in die wortels van volwasse plante getoon en duidelike induksie tydens SA-blootstelling aangetoon. Laastens is twee voorheen-ontwerpte CRISPR/Cas9 konstrukte wat twee teikens vir VviDMR6.1 en een teiken vir VviDMR6.2 bevat, deur middel van Agrobacterium-gemedieerde transformasie na Richter 110 embryogeniese kallus oorgedra. Suksesvolle redigering van VviDMR6.1 is behaal, met 'n mutasie-frekwensie van 43%. Inferensie van CRISPR Redigering (ICE) analise het klein indels, hoofsaaklik 2-bp delesies, binne die VviDMR6.1 lyne onthul. Merkwaardig genoeg het 'n enkele transgeniese lyn volledige redigering gedemonstreer, wat 100% redigeringsdoeltreffendheid aandui. Hierdie resultate dra by tot die vooruitgang van genoomredigeringstegnologieë vir die verbetering van siektebestandheid in wingerd en het die eerste CRISPR/Cas9-redigeerde wingerd op die Afrika-kon6nent opgelewer.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxviii, 107 pages : illustrationsen_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/130294
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshDowny mildew diseases -- Genetic aspectsen_ZA
dc.subject.lcshGene mapping -- Techniqueen_ZA
dc.subject.lcshGrape powdery mildew disease -- Controlen_ZA
dc.subject.lcshGrapes -- Effect of pesticides onen_ZA
dc.subject.lcshPlants -- Disease and pest resistance -- Genetic aspectsen_ZA
dc.subject.lcshPhytoplasma diseasesen_ZA
dc.subject.nameUCTDen_ZA
dc.titleInvestigation of the DMR6 susceptibility genes in grapevine for improving phytoplasma resistance through CRISPR/Cas9 technologyen_ZA
dc.typeThesisen_ZA
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