Phytogene therapy development with RNAi mediated gene silencing in crops for disease control without GM

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AimThe aim of this project is to use small antisense (as) oligodeoxynucleotides (ODNs) or as phosphorothioates (PTOs) to specifically silence plant and fungal genes that are necessary for the establishment of fungal disease in cereals. The predicted outcome is the development of epigenetic strategies to protect barley and wheat against major cereal pathogens. BackgroundDelivery of small asRNA for RNAi-based gene silencing is extensively used in animal science, but only emerging in plant sciences. Small synthetic specific asODNs or their chemically modified phophorothioates (PTOs) are being used successfully for targeted gene silencing in plants, delivering "naked" 18-19 mers ODNs/PTOs via xylem or by direct infiltration through stomata. Virulence factors (effectors) of barley powdery mildew (Blumeria graminis f.sp. hordei; Bgh) were silenced in planta by Host Induced Gene Silencing (HIGS). HIGS, involves cloning the target gene in a sense/as hairpin construct for biolistic transformation and in planta expression. In HIGS, only a fraction of the cells are transformed, thus it was not possible to link infection phenotype to decreased expression of the targeted gene. Moreover HIGS might also silence "off-target" genes and does not allow practical applications. Preliminary data-We demonstrated successful uptake of fluorescent ODNs by cells in barley leaves (cut/ infiltrated). -asPTOs were more effective than asODNs to decrease infection success rate.-asPTOs targeting specific fungal or plant genes reduced barley infection by Bgh. Successful targets included Blumeria effectors BEC1011 (RNAse like), the universal metalloprotease like BEC1019 and the plant susceptibility gene Blufensin-1 (BLN-1).-Preliminary qRT-PCR data indicated that the reduced infection phenotypes correlated with reduced gene expression.-Recent patents suggest successful uptake from spray application for disease control.Workplan and objectives -Optimising PTOs and other ODNs chemistries, and delivery methods from excised leaves or tillers/ ears to entire plants. -Transferring the PTO-gene silencing technology to control wheat pathogens such as B. graminis f.sp tritici (Bgt), Fusarium graminearum (Fg) and Zymoseptoria tritici (Zt). -Silencing and querying BEC1019 universality in Bgt, Fg and Zt. This will be confirmed by using knock out constructs to delete the BEC1019 homolog in Fg and Zt.-Designing and testing of new asPTOs for silencing other fungal genes effectors or general virulence factors, plant susceptibility genes facilitating fungal infection. Successful asPTOs will then be tested in agro-industry fungicide screening facilities at Syngenta, Stein Crop Protection, CH.