The elite grapevine cultivars often lack desirable traits, such as durable disease and pest resistance, which is specifically pronounced for resistance against phytoplasmas. Therefore, EU countries annually spend millions of euros to compensate for losses due to grapevine yellows. Since a specific control of phytoplasmas in plants is not possible in their host plants, grapevine growers rely mainly on indirect management of grapevine yellows by spraying against insect vectors, pruning of diseased parts of plants for eliminating infected tissues and thus prevent systemic infection and increase natural recovery. In the case of Bois noir disease, associated with ‘Candidatus Phytoplasma solani’, direct control of the vector is not effective. On the other hand, it works well in controlling FD associated with flavescence dorée phytoplasma but requires frequent use of pesticides. However, many chemicals that potentially control the vector have been banned due to increasing public awareness against such practices. Accordingly, there is no highly effective treatment against phytoplasma-infected grapevine available at the moment. Similarly, there are no potato cultivars resistant to phytoplasma ‘Ca. P. solani’.

For solving the problem, we urgently need new strategies of plant protection against phytoplasmas, which include identification of new potential S-genes, such as DMR6, which seems a promising candidate target for mutagenesis with a good perspective that its deactivation will increase the resistance to phytoplasmas and possibly to other pathogens, as well. The ability to obtain plants with new traits or mutations depends on the existence of a well-established in vitro plant regeneration protocol. Although this step is necessary for the analysis of gene function in whole plants, it is still very challenging in most agronomically important crops and has not been established yet for the regeneration of plants from protoplasts in the published CRISPR/Cas9 systems for grapevine. However, in our project team, the regeneration of plants from embryogenic callus-derived grapevine protoplasts is well established and we have published the entire protocol for regeneration of grapevine protoplasts in 2019.

Specific objectives of the proposed project

The main goal of this project is to engineer phytoplasma resistant grapevine/potato plants with the same level of changes as can occur in mutagenesis which is accepted at the global level. As an overall strategy, we suggest an adaptation of DNA-free genetically edited protoplasts of grapevine using direct delivery of purified CRISPR/Cas9 RNPs by targeting the DMR6 or other potential susceptible gene(s) S in order to achieve resistance or tolerance to ’Ca. P. solani’, which has not been reported yet.

If we confirm that the DMR6 or any other of targeted S genes is associated with increase in tolerance/resistance toward phytoplasmas as indicated by our transcriptomics analyses, the results will open a plethora of new possibilities in research of this important field of plant immunity.

To achieve the main aim of this project we have set the following specific objectives:

  • identification of candidate susceptible genes S to be targeted in order to obtain phytoplasma resistant grapevine/potato.
  • optimization of the protocols for CRISPR/Cas9 genome editing using S gene as a target and protoplast transformation for achieving maximum knockout efficiency in the targeted coding regions and optimization of protocol for detection of mutagenized plants;
  • establishing of an efficient system for regeneration of mutagenized grapevine plants from protoplasts;
  • testing the CRISPR/Cas9 mutagenized plants for the effects on phenotype and overall gene expression;
  • inoculation of the plants with phytoplasmas, and phenotyping for symptom development.