Gene editing based modulation of stomatal activity and drought resistance in grapevine

Grapevine (Vitis vinifera) is one of the most important fruit crops in the world, but the cultivars typically planted are not well adapted to abiotic and biotic stresses. As a consequence of climate change, drought is now one the biggest concerns for agriculture. Indeed, global climate models predict a significant increase in aridity in the near future, and water deficit may become a limiting factor in wine production and quality. As an example, the grape growing region of South Africa recently experienced a severe drought, which had significant negative impacts on agricultural productivity. It is therefore important to investigate how grapevine tolerance to water stress can be improved. In this regard we have focussed our attention on the MYB60 transcription factor. In Arabidopis, AtMYB60 has been linked to stomatal regulation in response to ABA and, interestingly Arabidopsis myb60 knockouts proved to be more tolerant to drought. The expression of the gene encoding the grapevine ortholog, VvMYB60, appears very specific to the guard cells and is able to revert the phenotype of the Arabidopsis myb60 mutant. The development of genome editing via CRISPR/Cas9 in the last few years has paved the way to new opportunities in the field of crop genetic improvement. The technology is precise, efficient and flexible, and can be used for biotechnological projects, as well as for studies on gene function. Here, we have implemented a genome editing approach to better understand the role of VvMYB60 in grapevine in relation to water stress. CRISPR-P and CRISPR RGEN online software tools were used to select four gRNAs targeting VvMYB60. gRNAs were then cloned into the pDIRECT_22C binary vector that generate multigene knockouts (Voytas Lab Plant Genome Engineering Toolkit). The vector was electroporated in Agrobacterium EHA105 and grapevine (cv ‘Chardonnay’ and ‘Sugraone’) transformation was performed. Embryogenic calli are currently in germination medium and molecular analysis of regenerated plants will be performed to detect the presence of Cas9 and mutations in VvMYB60. Expression analysis, phenotypical and physiological characterisation, including field trials, will then follow