RNAi-based approaches to induce resistance against grey mould disease in strawberry

Strawberry (Fragaria×ananassa) is one of the most diffused soft fruit species, which counts about 372.360 cultivated hectares worldwide (FAOSTAT, 2018). This perennial herbaceous species is subjected to several fungal diseases. Among them, grey mould, caused by the ascomycete Botrytis cinerea, is considered one of the most damaging pathogens for this crop, causing significant economic losses for both cultivated strawberry plants and harvested fruits. Recently a new biotechnological approach, named Spray-induced gene silencing (SIGS), which is based on RNA interference (RNAi) mechanism, has been demonstrated to be effective in the containment of grey mould disease. In particular, Wang et al. (2016) induced resistance against B. cinerea on different plant organs from different plant species, by the exogenous application of double-strand RNA (dsRNA) molecules targeting both dicer like1 (DCL 1) and 2 (DCL2) genes of B. cinerea. We are validating the effectiveness of these RNAi molecules also through their stable expression in strawberry plants [Host-induced gene silencing (HIGS)]. Strawberry (Fragaria×ananassa) in vitro explants were genetically transformed by Agrobacterium tumefaciens to stably express two different hairpin-based gene constructs targeting both DCL1 and DCL2 genes of B. cinerea alone or combined with DCL1/2 of Verticillium dahliae (Wang et al., 2016). Putatively transgenic lines were obtained, and the effectiveness of these hairpin-based gene construct for the resistance against B. cinerea is under evaluation on HIGS-strawberry lines, in comparison with the untransformed control plants. Meanwhile, wild type potted strawberry plants were sprayed with dsRNAs (SIGS approach) targeting the same B. cinerea genes. These studies aim to confirm the efficiency of SIGS approach in inducing resistance against B. cinerea in strawberry in a different environment than the controlled environment of a laboratory, confirming RNAi systems as useful tools for plant pathogen protection.