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Protein-protein interactions

Image showing reporter gene activiation
This depth of screening has proven highly successful and has enabled the targets of a range of effectors to be identified.

The principal method we use to discover novel interacting proteins is yeast two hybrid (Y2H) screening. Candidate interactors are then verified by cell biological approaches, such as split YFP, or affinity chromatography. The system we have adopted for Y2H screening relies on three reporter genes and low expression levels of the fusion proteins in order to reduce false positives. At present the bulk of the Y2H screening has made use of a Phytophthora infestans infected potato leaf cDNA library. This library has been screened with over forty P. infestans effectors along with a small number of P. capsici, aphid and nematode effectors.

We expect to extend the range of available libraries to include nematode infected root libraries and aphid infected leaf libraries. We are also developing related technologies to allow interactions between membrane proteins to be revealed e.g. split ubiquitin libraries. Typically a screen consists of generating between 2 and 10 million yeast co-transformants which are then assayed for reporter gene activation. This depth of screening has proven highly successful and has enabled the targets of a range of effectors to be identified.

Research

Areas of Interest


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The James Hutton Research Institute is the result of the merger in April 2011 of MLURI and SCRI. This merger formed a new powerhouse for research into food, land use, and climate change.