Application Of A Viral Vector For R Gene Interrogation In Nicotiana Tabacum
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abstract
Gene editing allows for precise targeted changes in genomic DNA to a single nucleotide resolution typically carried out through the creation of DNA double stranded breaks (DSBs). DSBs are then repaired by host molecular machinery causing insertion and deletions (indels) of nucleotide bases that, depending on where they are located within the genome, can inactivate genes. The RNA programmable endonuclease system, CRISPR/Cas9, has emerged as the premier gene editing tool due to the relative simplicity of its parts, consisting of a variable synthetic guide RNA (sgRNA) and Cas9 protein, for targeted genomic DSBs. On the other hand, one fundamental area of plant pathology research, and more specifically plant virology, is the discovery and study of host Resistance (R) genes. As the name suggests, R genes establish host plant resistance to specific pathogens by triggering a localized hypersensitive response in the presence of pathogen proteins (effectors) causing a small necrotic lesions on the leaves and subsequently purveying resistance to the pathogen. The R gene N, which conveys resistance to Tobacco mosaic virus (TMV), was one of the first R genes to be incorporated into commercial Nicotiana tabacum (tobacco) varieties to establish TMV resistance and has been important in understanding molecular R gene mechanisms. While much is known about the N gene there is still little information about how it is regulated during TMV infection such as how gene expression and transcript splice variants help facilitate host resistance or susceptibility. This hindrance is primarily due to the current availability of tools and resources, which is limited for dissecting these specific interactions.In this study we aim at using a plant virus to deliver CRISPR/Cas9 gene editing technology for targeted N gene mutagenesis in N. tabacum. We have previously established the TMV coat protein deletion mutant viral vector, TRBO, as an efficient method for delivering target specific synthetic guide RNAs (sgRNAs) capable of creating indels with the co-delivery of Cas9 to terminate a genes function. By using the virus as both a delivery tool for gene editing events and as a reporter of resistance or susceptibility of the virus, we can better understand the specifics TMV-N gene interactions as they occur within a plant. This goal will be accomplished through three research objectives. 1) Examine the ability of TRBO to deliver sgRNAs targeting the N gene in N. tabacum and thus eliminating or reducing the presence of necrotic lesions (resistance). 2) Establishing the TRBO-sgRNA delivery system as a biosensing tool that "reports" relative fitness of the virus during N gene targeting. 3) Study how alternate splice forms of N gene products affect virus replication and movement within the plant through transcript specific targeting. Through these objective we will be able to carefully dissect N gene regulation and better understand R gene function. The completion of this project will develop a standard for R gene interrogation in other R gene-pathogen interactions which could aid in R gene discovery or current R gene resistance improvement. Additionally, these results could lead to the production of commercially relevant plant species that are resistant to TMV or other plant pathogens.
