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POSTERS: Biotechnology and genetic engineering

Editing of plant eIF4E genes for potyviral resistance
Zhongguo Xiong - University of Arizona. Zhixin Liu- Chinese Academy of Tropical Agricultural Sciences, Chunwei Zhang- Chinese Academy of Tropical Agricultural Sciences, Naitong Yu- Chinese Academy of Tropical Agricultural Sciences, Yadan Wu- Chinese Academy of Tropical Agricultural Sciences, Yu

Many positive-sense, single-stranded RNA viruses carry a viral protein (VPg) covalently linked to the 5’-terminus of their genomic RNA. VPg is functionally analogous to the cap of mRNA and serves as the binding site for the eukaryotic translation initiation factor 4E (eIF4Es, eIF(iso)4Es, and nCBPs). This interaction is a prerequisite for translation of viral genomic RNA, and therefore viral infection in host cells. To demonstrate the feasibility of engineering viral resistance by disrupting VPg-eIF4E interactions, we employed RNAi and CRISPR technologies to systematically knockdown and knockout the eIF4E family of genes in Nicotiana benthamiana, a ubiquitous host for many plant viruses. Seven eIF4E genes were cloned and the corresponding proteins were identified from N. benthamiana. Potential interactions of the eIF4E proteins with VPgs of selected potyviruses were screened by the yeast two-hybrid assay. RNAi and CRISPR constructs targeting the eF4E genes were designed and transformed to N. benthamiana to create knockdown and knockout lines of specific eIF4E genes, respectively. Significant reductions in the transcript level of the targeted eIF4E gene were observed in independent RNAi lines. In the CRSPR lines, random insertion and deletions were found around the eIF4E-coding sequences targeted by the CRSPR gRNAs. The RNAi knockdown lines and the CRSPR knockout lines are currently being characterized and evaluated for their resistance to potyviruses.