Over the summer I took part in a project at the James Hutton Institute attempting to illuminate the process of cell-to-cell transport of filamentous plant viruses through plasmodesmata. Such viruses are major crop pathogens with a global impact on the agro-economy and food security, yet the molecular mechanism by which they move from initially infected cells into surrounding tissues, which is crucial for infection of the host, remains unclear.
Live cell imaging studies in the host lab have shown that in Potato virus X (PVX) infections three movement proteins called Triple Gene Block (TGB) 1, 2 and 3 are responsible for the recruitment and insertion of the virus’ RNA-associated capsid proteins (CP) into the channel of the plasmodesmata for delivery into neighbouring cells. The lab now investigates the hypothesis that an interaction between TGB1 and CP is required for this. Within this context, my task was to analyse the previously described phenomenon of movement complementation of PVX CP mutants by the capsid protein of potyviruses. Potyviruses are an important class of filamentous plant viruses with virions structurally similar to those of PVX. Potyvirus CPs can rescue movement-deficient PVX mutants, which lack the C-terminal 10 amino acids of their CP (CPΔC10), the putative binding-site for TGB1.
My project investigated the hypothesis that complementation is due to formation of mixed-CP complexes, where both PVX and potyviral CP bind to the PVX RNA, and the potyviral CP restores interaction with the TGB1 protein. The experimental plan was to infect Nicotiana benthamiana plants with a transport-deficient PVX, bearing a GFP-fused CPΔC10, and co-express a Potato potyvirus Y (PVY) CP, followed by imaging of the plant tissue using confocal laser scanning microscopy to test if insertion of PVX CPΔC10 into plasmodesmata is restored. In parallel, a yeast-two-hybrid (Y2H) assay would be used to test for interaction between the PVY CP and PVX TGB1.
The first part of the project centred on the creation of the molecular constructs to be used for expressing PVY CP in plants, and for the Y2H assay. Plasmids were created using PCR and the Gateway system. Unfortunately, after infecting plants with a cDNA clone of the transport-deficient PVX mutant with a GFP-tagged CPΔC10 that was delivered either by agroinfiltration or biolistics, no expression of the PVY CP could achieved due to problems with the agrobacteria. When these were eventually resolved, there was insufficient time left to conduct the complementation assays. Likewise, the Y2H experiments could not be completed because the construction of the required plasmids took longer than anticipated.
Despite these setbacks I thoroughly enjoyed my time at the James Hutton Institute and would like to thank the BSPP for funding my project. I would also like to thank Dr Jens Tilsner for taking the time to help design the project with me, directly supervising me during it and teaching me all that I learned. Although results were not achieved in the timeframe of this project I still benefited greatly. I received valuable experience in laboratory techniques which as a 2nd year student I had so far only read about, as well as more general lab skills such as experimental design, lab book-keeping, and working with plant pathogens. The project allowed me to peek into what pursuing a career in academia feels like and really put plant pathology on the map as a possible professional direction.
Yordan Dolaptchiev
University of St Andrews
