CRISPR/Cas9 functional characterization of grapevine glutathione S-transferases VvGST40 and VvGST30 reveals a role in drought resilience

Abstract

Increasing climate variability and the rising frequency of drought events are intensifying the demand for grapevine (Vitis vinifera L.) genotypes with improved performance under water-limited conditions. In this context, New Plant Breeding Techniques (NPBTs), and particularly CRISPR/Cas9-mediated genome editing, enable targeted manipulation of candidate genes involved in abiotic stress adaptation, aligned with EU sustainability objectives for agriculture.

We describe a genome editing strategy to functionally characterize two grapevine Glutathione S-transferase genes, VvGST40 and VvGST30, selected based on homology to an Arabidopsis thaliana GST previously associated with enhanced drought tolerance. Candidate gene prioritization was further supported by transient VvGST40 downregulation using spray-induced gene silencing (SIGS) via dsRNAdelivery, which was associated with improved performance under water deficit.

Sixteen CRISPR-Cas9-positive lines were generated by Agrobacterium-mediated transformation. Targeted mutagenesis was validated by molecular analyses, and four independently edited lines carrying stable mutations were selected for downstream characterization. Apreliminary in vitro screening assessed root system architecture traits potentially relevant to water uptake. Subsequently, drought stress assays were performed to quantify plant performance under water deficit, integrating morphological and physiological parameters. Edited lines displayed improved drought-responsive phenotypes relative to controls, supporting a functional contribution of VvGST40 and VvGST30 to grapevine water-stress adaptation.