We have developed a technology for somatic cell genetics that leads rapidly to genetic agents that modulate cellular phenotypes. The technology utilizes genetic assays in combination with perturbagens, peptides that inhibit specific biochemical processes in cells, causing a phenotypic shift. Our perturbagen technology is potentially able to endow plants with novel phenotypes that would be very difficult to obtain by conventional plant breeding. The primary objective of the proposed research is to explore the use of perturbagen methods to identify peptide aptamers that confer resistance to the model viral pathogen tobacco etch virus (TEV), a member of the economically important potyviruses. Phase I research involves: (1)The identification of peptides that bind to TEV NIa and HC-Pro proteins and TEV RNA genome using the technology developed at Arcaris (specifically, perturbagen libraries) via modified yeast two-and three-hybrid systems. (2)The expression of these peptides in cultured tobacco cells to identify peptides that inhibit TEV replication. Protoplasts derived from these tobacco cells will be infected with transcripts of TEV that has been modified to include the b-glucuronidase (GUS) gene in its genome. The impact of these peptides on TEV replication will be monitored by GUS activity.
In contrast to viral sequence-derived resistance, perturbagen technology circumvents any potential risks associated with the expression of virus-derived transgenes. This approach has the potential to engineer highly durable virus resistance in plants by expression of peptides that interfere with the function several essential viral proteins. In addition, this technology can be used to rapidly identify perturbagens with fungicidal, bacteriocidal, and insecticidal properties that cna be expressed in plants.
