The project will consist of 6 work packages (WP), five designed to address the set objectives and one WP addressing the dissemination of results and data management. The overview of activities is shown in Fig. 1.

Fig 1: Schematic overview of planned activities

WP1. Structure-function studies of CP involved in viral movement (M1-18; NIB, KI)

In this work package, we will study the contribution of different CP regions to viral movement within the infected plant. The design strategy will be based on the results published in Kežar et al. (Kežar et al., 2019). We have there shown that the C-terminal region of CP is essential for the establishment of efficient viral replication. In contrast, deletion of 50 N-terminal amino acids of CP reduces viral ability for cell-to-cell movement. This data combined with structural information, open the possibility of a rational and detailed functional study of the multitasking protein CP. It will enable us to identify critical amino acids for the cell-to-cell movement of PVY and examine the potential critical role of CP assemblage in this step of the viral cycle. The WP will involve the participation of the National Institute of Biology (NIB) and National Institute of Chemistry (KI).

WP2. Structural studies of the viral-host protein complexes (M1-36; All partners)

This WP will involve the participation of Biotechnical Faculty (BF), KI and NIB and the collaboration with Prof Dr Kristiina Makinen’s lab (UH). The WP2 will focus the efforts on purification of the protein complexes that we observed by negative-stain TEM within different steps during purification of the virus and obtaining structural data describing the complex.

WP3. Localisation of viral proteins during the infective cycle (M3-30, NIB)

Within this WP, we will prepare PVY clones with different fluorescently-tagged viral proteins involved in viral complexes and we will follow their localisation during the infection cycle using confocal microscopy.

WP4. Analysis of the protein-protein interaction network (M12-33, NIB)

In this WP, we will study the virus-host protein interaction network. We will search for the interacting partners of one selected PVY protein based on the outcomes of WP3 at different stages of the infection.

WP5. Modelling of PVY-plant interactome (M27-36, NIB)

All the resulting data including novel interactions as well as additional kinetic and structural data will be fed into the model of plant defence signalling (Miljkovic et al., 2012; Ramsak et al., 2018) that was developed within the ARRS project N40026. Data gathered in previous WPs will be used to assert the existence of connections or add new connections in the potato/PVY resulting network. Methods of network topological analysis will be performed, using both distance and connectivity measures in program Pajek (Batagelj and Mrvar, 1998), to search for shortest paths, walks and trails or extract highly connected modules. The results will be either further analysed or visualised using Cytoscape and its corresponding add-ons (Saito et al., 2012). Thus, apart from improving the potato-PVY network topology, we will also be able to develop new hypotheses about the mechanistic of PVY infection.

WP6. Dissemination, exploitation and data management (M1-36, all partners)

Data will be managed according to FAIR principles. All partners will throughout the project ensure dissemination of activities and results both in the scientific community as well as broader.