POSTERS: Plant defense response
A high throughput method for determining protease target sequences
Tyler Frailie - Indiana University. Roger Innes- Indiana University, Leina Joseph- Indiana University
Feeding the human population while reducing environmental costs requires crops resistant to a variety of pathogens. The PBS1/RPS5 decoy system can be used to this end. The Innes lab has shown that Arabidopsis detects the presence of the effector AvrPphB via its cleavage of PBS1 and resulting activation of the disease resistance protein RPS5, triggering a hypersensitive response. The AvrPphB cleavage site can be altered to be cleaved by other pathogen proteases that can then activate RPS5, as shown with the turnip mosaic virus protease NIa. For this to work, a protease’s target amino acid (AA) sequence must be determined. On this poster, I will describe a high throughput system for determining a protease’s target AA sequence. Specifically, we integrated into the E. coli chromosome an antibiotic resistance cassette repressible by a synthetic repressor. A randomized AA linker joins the repressor’s two halves. If a protease cleaves this sequence, then antibiotic resistance will be derepressed and selectable. This system enables rapid screening of random 7mer AA sequences for cleavage by proteases of interest. Components of this system are currently being assembled, and results are forthcoming. Because PBS1 cleavage activates resistance in many crop species, this assay will be instrumental in creating an array of PBS1-based decoy systems, allowing plants to resist a wider variety of pathogens. This would reduce pesticide use, lessening agriculture’s environmental impact.