Transgene-free CRISPR-Cas and cisgenesis approaches for resistance to powdery and downy mildew in grapevine

The development of disease-resistant tree crops is essential to sustainable agriculture, particularly under increasing biotic stresses driven by climate change. This project explores a dual strategy to confer resistance to powdery mildew (PM) and downy mildew (DM) in grapevine employing (i) a transgene-free CRISPR-Cas genome editing platform based on ribonucleoprotein (RNP) delivery, and (ii) a cisgenic transformation approach utilizing native resistance (R) genes. Both approaches rely on efficient production of embryogernic callus and regeneration of modified grapevine plants. Our CRISPR-Cas RNP system enables precise, transient gene editing without the integration of foreign DNA, targeting key susceptibility (S) genes known to be involved in PM and DM pathogenesis–specifically, MLO and DMR6 genes, respectively. In parallel, we are implementing a cisgenic strategy to introduce naturally occurring R genes from sexually compatible and disease-resistant donor genotypes. This strategy uses a binary vector system carrying a heat-inducible recombination cassette. Following selection, regenerated plants containing the cisgenic construct and the desired R gene are subjected to controlled heat shocks to activate the recombinase, leading to the excision of unwanted transgenic sequences. The resulting plants retain only the native R gene sequence. We present initial results demonstrating successful simultaneous editing of one MLO and two DMR6 genes. In cisgenic lines, we report stable expression of the Resistance to Plasmopara viticola1 (RPV1) gene. Both approaches are designed to align with emerging regulatory frameworks and enable the preservation of the genetic backrounds of elite grapevine cultivars such as Chardonnay and Merlot, which are disrupted through conventional breeding. All resulting lines from both cisgenic and gene editing strategies will undergo sequencing to assess and ensure the absence of unwanted genomic modifications.