TOWARD EDITING OF ZUCCHINI GENOME FOR POWDERY MILDEW RESISTANCE

Powdery mildew caused by the fungal species Golovinomyces cichoracearum and Podosphaera xanthii is considered to be one of the main biotic threat for the cultivation for zucchini (Cucurbita pepo L.). The Mildew Locus O (MLO) gene family encodes for plant-specific proteins harbouring seven transmembrane domains. More recently, knocking-out or knocking-down of specific MLO genes was shown to lead to powdery mildew resistance in several plant species, such as Arabidopsis, tomato, pea, pepper and wheat. Several approaches of reverse genetics are available to breeders interested in MLO gene knock-out or knock down, such as those based on RNA interference (RNAi), targeted induced local lesions in genomes (TILLING), transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeat (CRISPR) technology. Noteworthy, TALEN and CRISPR/Cas9 technology have been recently successfully used to introduce mlo resistance in bread wheat. For this reason, we used CRISPR/Cas9 technologies in Cucurbita Pepo to introduce targeted mutations in two genes that encode MILDEW-RESISTANCE LOCUS (MLO) proteins. We designed The CRISPR/Cas9 constructs to target two MLO genes contained two single guide RNAs for creating large specific deletions. We tested it by Agrobacterium mediated transient assay. We are going further to use CRISPR-Cas9 technology to generate transgenic zucchini plants that carry mutations in MLO alleles. Thus, with our study we provide information on targets of future activities addressed to the inactivation of susceptibility genes and, thus, the selection of resistant genotypes.