Potato virus Y (PVY), member of the genus Potyvirus, infects potato and other Solanaceae and was recently classified within the top 10 most important viruses from economic and scientific point of view. During its infection cycle, the coat protein (CP) and other viral proteins interact with each other and with the host plant proteins to ensure optimal virus replication, encapsidation and movement. The plant also relies on these interactions as part of its defence. There are studies that describe protein interactions in the Potyvirus host system, however, many of the host proteins that interact with the virus remain unknown. A method that could help to search for new PVYhost protein interactions and at the same time study already known ones would be an important contribution to the field. The main objective of this proposal is to gain better understanding of the protein interactions in the PVYhost plant system. To do this we will select both viral proteins and proteins of interest from the host plants and we will clone and express them using a high throughput strategy. Expressed proteins will be purified and challenged to protein protein and virus particle protein interactions using Surface Plasmon Resonance (SPR). We will use mutagenesis to explore the importance of particular amino acid residues and/or protein motives in the interactions. In parallel we will use SPR to search in plant extracts of different complexity for proteins that interact with immobilized PVY particles. Proteins that bind to the PVY particles will be eluted from the SPR chip and identified using mass spectrometry. In addition, the PVY CP will be expressed and its assembly into virus like particles (VLP) will be optimized. The VLPs will be purified and used in SPR studies, where they will allow the evaluation of different CP mutations in the interaction with different proteins with. We will also attempt to obtain the 3D structure of the PVY CP using X-ray diffraction and electron microscopy. The generated data including novel interactions as well as additional kinetic and structural data will be fed into a model of plant defence signalling. In summary, the proposed project will strongly contribute to a better understanding of the protein interactions that occur during the PVY infection and will shed light into the mechanism of action of this important virus and enable new strategies of virus control. In addition the project will contribute to the field with new valuable research tools, such as SPR for measuring virus host interactions, and PVY-VLP which represent also promising tools to be used in other biotechnological applications such as epitope carrier for immunization purposes. The extensive expertise that the involved partners, National Institute of Biology, National Institute of Chemistry and Josef Stefan Institute, have in PVY plant interaction, molecular interactions, and protein structure, will ensure a successful outcome of the proposed work.