J4-2544

The main goal of this project is to engineer an improved phytoplasma resistance in grapevine and potato after knockout of the DMR6 gene by the CRISPR/Cas9 system. In parallel, transcriptomic data on phytoplasma infected grapevine, collected in previous studies in Slovenia, Austria and South Africa, will be combined by computing and modelling in order to identify some new suitable candidate susceptibility genes S, other than DMR6, to be targeted for increased resistance of grapevine to phytoplasmas.

We suggest an adaptation of DNA-free genetically edited protoplasts, using a direct delivery of purified CRISPR/Cas9 ribonucleoproteins targeting the chosen plant S gene. The regenerated plants will be tested for the effects on phenotype and overall gene expression and compared with the wild type plants. The proposed approach of mutagenesis has better chances that the plants engineered by it will be accepted at the global level. As an
alternative procedures we will introduce a nanoparticle-based delivery of CRISPR/Cas9 genetic elements into plant tissues and/or Agrobacterium-mediated transformed S gene knockout plants with the application of the in-house protocols.

All regenerated grapevine and potato plants will be inoculated with studied phytoplasmas to test for possible acquired resistance/tolerance against them. Confirmation of an association of the target S gene with an increase in resistance/tolerance toward phytoplasmas will open a plethora of new possibilities in research in the important field of plant immunity, with the potential to lead to novel improved potato and grapevine cultivars.