Development of a Pokeweed Mosaic Virus Vector as a Molecular Tool to Study Host Protein Function in Phytolacca Americana

American pokeweed (Phytolacca americana) is a plant known for both the synthesis of pokeweed antiviral protein (PAP) and the hyperaccumulation of heavy metals. Progress in understanding the factors behind viral resistance and hyperaccumulation has been slow due to lack of genetic tools to study pokeweed proteins in planta. Therefore, an infectious clone of Pokeweed mosaic virus (PkMV) was developed as the precursor of a viral vector that would allow the study of host protein function. PkMV is a potyvirus of the family Potyviridae and its 9.5 Kb single-stranded RNA genome shares the typical organization of potyviruses. PkMV was the ideal chassis to build an infectious clone to study pokeweed as it infects the plant in the wild despite pokeweed producing a potent antiviral protein. Here, the assembly of the first infectious clone of PkMV is described and its ability to both infect pokeweed and deliver a foreign protein into the plant is demonstrated. Instability of the PkMV clone was encountered during its generation, due to cryptic bacterial promoters within its genome. Manipulation in Agrobacterium was a limiting factor in modifying the PkMV clone, so a method to detect cryptic promoters throughout the viral genome was developed, which when silenced allowed recovery of an infectious clone that is readily manipulated in E. coli. This method relied on generation of a library of PkMV fragments that were integrated into an eGFP or chloramphenicol reporter construct. Application of the low-throughput method detected promoter activity by producing eGFP fluorescence. The high-throughput next generation sequencing variant sequenced colonies that survived high concentrations of chloramphenicol due to transcription of a chloramphenicol resistance gene. Both methods produced putative promoter regions (PPRs) containing a cryptic bacterial promoter. The +1 transcription start site (TSS) of each PPR was determined with template switching oligo (TSO)-5RACE, which allowed detection and silencing of the cryptic promoter. Silenced promoters in the PkMV genome restored stability of the viral clone in E. coli, permitting further manipulation. This work provides a straightforward method to detect and silence cryptic promoters in large viral genomes and begins to elucidate the mechanisms of plasmid toxicity in E. coli.