Deliverables
The domain composition of these R-proteins is crucial in their functioning. Where at the N-terminus of these proteins several domain types are found, the central NB domain makes that R-proteins likely function as a molecular switch.
A mechanism that is also found in other apoptosis proteins such as Apaf and CED and proteins that play a role in innate immunity of animals such as NOD proteins. Specific recognition of a pathogen effector, either directly or indirectly, via the LRR then triggers the release of the intramolecular autoinhibition, and allowing, among others, a change in nucleotide-binding status and the binding of further signaling components or oligomerisation.
Eventually, the specific pathogen recognition leads to a complex resistance response, including the production of anti-pathogenic compounds, the induction of a reactive oxygen burst and often a local programmed cell death or so called hypersensitive response (HR). R proteins are translocated to the nucleus where they interact directly with transcription factors.
One way to accelerate the throughput rate of screening for protein-protein interactions is to implement library-versus-library screening. Recent developments in protein-array technologies provide the possibility to combine this technique with phage display, thereby screening in parallel for interactors. Fluorescent phages provide a possibility to automate and determine the interactions in real time.
The fluorescent T7 phage is based on a translational frameshift producing both the wild type capsid protein, necessary for the integrity of the capsid, and the a few copies of the fusion between YFP and the capsid protein. Microspheres coated with the antibody Y-5 were visible by fluorescence microscopy after incubation with fluorescent T7 phages that also expressed the antigen recognized by the antibody.
Microspheres coated with non-specific antibodies did not show any sign of fluorescence, meaning that no aspecific binding phage could be detected. These results point out that the fluorescent phages can be applied to assess the specificity of the binding between a bait and the proteins displayed on the fluorescent T7 phages.
Production of fluorescent phages is fully compatible with the available T7 phage display system and a rational next step would be to test value of the system in a parallel bait setup.
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