CRISPR-Mediated Susceptibility Gene Editing and Viral Interference for Plant Disease Resistance

Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Disruption of these host genes responsible for Potyviridae susceptibility confers disease resistance. CRISPR/Cas9 has emerged as a robust and specific gene editing technology. As a proof of concept for CRISPR-mediated susceptibility gene targeting, Arabidopsis thaliana was chosen as a model plant system for editing resistance to Turnip mosaic potyvirus (TuMV). CRISPR/Cas9 was employed to generate eif(iso)4e mutants in A. thaliana. eif(iso)4e mutants exhibited resistance to TuMV systemic spread and symptom development. Precise gene editing of A. thaliana eIF(iso)4E was also achieved in somatic cells via the CRISPR/Cpf1 tool./nCassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cassava encodes five eIF4E isoforms: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Yeast two-hybrid analysis detected interactions between both CBSV and UCBSV VPg proteins and cassava nCBP-1 and nCBP-2. CRISPR/Cas9-mediated genome editing was employed to generate eif4e, ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titer in storage roots relative to wild-type controls. However, full resistance to CBSD was not achieved, suggesting that remaining functional eIF4E isoforms may be compensating for the targeted mutagenesis of nCBP-1 and nCBP-2./nFurthermore, viruses of the family Geminiviridae threaten to global food security through severe crop loss. Tomato yellow leaf curl virus species (TYLCV; genus Begomovirus) cause widespread destruction of the tomato crop. The high levels of genetic variability and persistent insect vector distribution pose significant challenges for conventional and genetic control strategies. In addition to precise genome editing, CRISPR/Cas9 may be employed as a form of molecular immunity against plant DNA viruses. In this study, sgRNA were designed to target a broad range of the TYLCV species, mutagenize a stable region of the TYLCV genome, and disrupt expression of the virus replication machinery. Functionality of multiple TYLCV-targeting CRISPR/Cas9 expression systems was demonstrated in Nicotiana benthamiana. The utility of this TYLCV control strategy was then extended to tomato. CRISPR/Cas9 transgenic tomato lines exhibited reduced TYLCV symptom severity and viral DNA presence. CRISPR-mediated viral interference and viral susceptibility gene mutagenesis reflect the vast potential of the CRISPR technology as a solution to global food challenges.

Advisor: Staskawicz, Brian J