CRISPR/Cas9 Targeted Editing of Genes Associated With Fungal Susceptibility in Vitis vinifera L. cv. Thompson Seedless Using Geminivirus-Derived Replicons

Abstract

The woody nature of grapevine (Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system in vivo and evaluate their editing capability in individuals derived from Agrobacterium-mediated gene transfer experiments of ‘Thompson Seedless’ somatic embryos. Preliminary assays using a BeYDV-derived vector for green fluorescent protein reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including AUXIN INDUCED IN ROOT CULTURE 12 (VviAIR12), SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4 (VviSWEET4), LESION INITIATION 2 (VviLIN2), and DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.

Powdery Mildew Resistance Genes in Vines: An Opportunity to Achieve a More Sustainable Viticulture

“…This, together with new rapidly developing Vitis models, such as Microvine or Picovine, have helped to accelerate the discovery of new target genes to decipher the resistance of Vitis to powdery mildew, such as the PATHOGENESIS-RELATED 4b (VvPR4b) gene, whose loss of function decreases Vitis resistance to downy mildew [39]. As expected, the overexpression of VvPR4b is related to enhanced resistance to E. necator [40], while the DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1) double-cut transgenic Vitis has 90% fewer symptoms of powdery mildew infection than the control plants [40].…”

The new directions in genetics, breeding and biotechnology of ornamental and berry crops in the N.I. Vavilov Institute of Plant Genetic Resources (VIR)

“…В последние годы успешно разрабатываются подходы к модификации генов винограда с помощью системы геномного редактирования CRISPR/ Cas9. Проводятся исследования по оптимизации компонентов системы редактирования (Olivares et al, 2021;Ren et al, 2021); получены первые практические результаты. С помощью универсального вектора на основе репликона гермивируса (pGMV-U) получены крупные делеции в генах винограда, вовлеченных в контроль устойчивости к грибным патогенам.…”

Transgene-free genome editing and RNAi ectopic application in fruit trees: Potential and limitations

“…Besides eliminating transgenes after generating transgenic gene-edited plants through conventional stable transformation methods, direct transgene-free editing can be performed through either transient expression of plasmids or directly using CRISPR elements as ribonucleoproteins. Transient expression of CRISPR elements without integration into the plant genome has been reported in the grapevine using a Geminiviral replicon system ( Olivares et al., 2021 ). But direct gene editing using CRISPR elements as ribonucleoproteins (RNP), without the use of DNA, first demonstrated by ( Woo et al., 2015 ), is the most promising and desirable option to generate transgene-free plants because it avoids DNA insertions and accomplishes gene editing in one generation without unwanted crossings in most clonal crops.…”