Projects / Programmes
Insights into the protein interactions involved in the Potato virus Y - potato relation
| Code |
Science |
Field |
Subfield |
| 7.00.00 |
Interdisciplinary research |
|
|
| Code |
Science |
Field |
| B230 |
Biomedical sciences |
Microbiology, bacteriology, virology, mycology |
| Code |
Science |
Field |
| 1.06 |
Natural Sciences |
Biological sciences |
Potato virus Y, PVY-potato interaction, protein - protein interaction, Surface plasmon resonance
Researchers (14)
Organisations (3)
Abstract
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 PVY-host 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 PVY-host 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.
Significance for science
The outcomes and tools derived from this project will have an important impact for the field of PVY-plant interactions and by extrapolation for the field of virus-host interactions in general. The SPR strategy that will be tested in this proposal is novel in the field and has the potential not only to complement but also to improve existing approaches such as yeast two hybrid, co-immuno precipitation or bimolecular complementation. SPR will offer the opportunity of searching for new interactions, confirming suspected ones and obtaining kinetics parameters of such interactions. The discovery of new candidate protein interactions within the PVY-potato system will contribute to an improved understanding of the virus biological cycle within the plant, including entry, movement and response to the plant defence mechanisms. In addition, the establishment of a PVY-VLP production system will allow for fast screening the effect of mutations in the CP on the interaction of PVY particles with selected ligand proteins. Moreover, nucleic acid-free filamentous PVY-VLPs, represent also an interesting scaffold which, upon proper modifications, could be used as nano biotechnological tools such as nanotubes or nanoparticles for carrier purposes. There is no 3D structure available for any PVY protein. Resolving the 3D structure of the coat protein, VLPs, or any other protein involved in PVY infection will have a big impact on understanding the function of this important protein for the virus infection. It will also allow discovering or confirming, motives or domains involved in such functions (i.e. protein interaction motives). All the produced data will also serve to enhance an already expanding model of plant defence signalling to extract novel knowledge on potato in interaction with PVY
Significance for the country
The results of the proposed project are included in the fields of biotechnology and systems biology for health and safe food, and therefore match the highest priorities of Slovenia and the European Union. New knowledge on the biology of the most important potato infecting virus (PVY) has a direct impact on agriculture manufacturing and therefore in the economy of potato production. A better understanding of the virus-plant interaction can ultimately result in the possibility of adopting new strategies for virus control, which will be beneficial for farmers, breeders and agriculture in general. During the project we will adapt a method based on CIM monolithic chromatography to purify PVY-VLP. CIM monoliths are manufactured by the Slovenian company BIA separations with which NIB partner maintains a long lasting fruitful collaboration. A new application for its products will impulse even more their already successful expansion into the international market, as well as their work with national high-tech industry. Moreover, all three partners NIB, IJS and KI have proven and fluent collaborations with pharmaceutical companies such as Lek - Sandoz or biotechnological companies such as the above mentioned BIA Separations, Oxford nanopore and others. All partners have various international patents granted or in application stage. They have also given birth to spin off companies such as Biosistemika (NIB partner). All these, ensures a rapid and fluent transfer of the basic-knowledge generated in this project to the industry in case a proper opportunity will be found. The Department of biotechnology and systems biology collaborates intensively for more than 15 years with representatives from the Ministry of agriculture and forestry, from the phytosanitary inspection services and with other experts from the field of plant protection. The knowledge obtained from the project will also be shared to such representatives.
Most important scientific results
Interim report,
final report
Most important socioeconomically and culturally relevant results
Interim report,
final report
