Small RNA mobility through RNAi grafted grapevine plants and disease protection effects on grey mould

Worldwide, with an average of 77 million tons per year produced (FAOSTAT, 2020), the production of table and wine grapes is a leading sector, with continued growth in Europe in the area devoted to vine cultivation. During the growing season, most plant organs are challenged by pathogenic organisms, such as fungal and oomycete, leading to important economic losses and causing detrimental effects on fruit quality. The increasingly scarce availability of fungicidal products, often due to their negative impact on the environment, coupled with the emergence of pathogen resistance to these products, make defence increasingly challenging. In this perspective, RNAi-interference (RNAi) an evolutionarily conserved mechanism activated by double-stranded RNA (dsRNA) molecules, can be used to modulate the expression of infection-related genes. The constitutive in planta expression of dsRNAs targeting genes of grapevine pathogens is a new strategy to enhance disease tolerance in specific cultivars. For plants clonally propagated by grafting, the trans-grafting mobility of small RNA (sRNA) could offer the possibility to use RNAi-expressing rootstocks to confer stable resistance to the grafted cultivars. Following this approach, we have modified grapevine by stably expressing a hairpin construct designed to silence the Dicer-like genes 1 and 2 of Botrytis cinerea. Several independent BcDCL1/2-dsRNA- expressing lines were used either as rootstock or scion and sRNA accumulation and mobility were studied in different graft combinations. RNA-Seq analysis revealed the presence of specific siRNAs in leaves collected from unmodified scion grafted on dsBcDCL1/2-expressing rootstocks in the absence of B.cinerea, an indication of sRNA mobility through the grafting junction. The different BcDCL1/2-dsRNA- expressing lines used in the rootstock/scion combinations induced a different accumulation level of sRNAs and resistance response to on the leaf infection with B. cinerea. This result highlights the possibility of using dsRNA rootstocks to induce resistance in grafted grape varieties, with a strong impact on the reduction of environmental impact due to the limitation of pesticide use and greater safety for the consumer. Furthermore, the obtained plant material, besides an applied interest in crop protection, can contribute to increasing the knowledge, beyond the state of the art, on the mobility of the silencing signal in grafted plants.