Methods

We approach potyviral infection biology from different angles to gain insights into plant-virus interactions and their relevance for the infection. On the Viikki campus we have access to specialized instruments and in-house expert services in for example proteomics, microscopy, next-generation sequencing and particle purification. Our plants are maintained in environmentally controlled greenhouses on campus.

Methods in a nutshell

  • Pathosystems:
     
    • potato virus A-Nicotiana benthamiana
    • turnip mosaic virus-Arabidopsis thaliana
  • Agroinfiltration-based gene transfer to host plants. We also initiate infections by agrobacterium-mediated transfer of infectious viral cDNA to the plants. Check out our agroinfiltration video to see how it is done.
  • Quantitation of viral gene expression with a dual luciferase assay based on Renilla and firefly luciferase reporters
  • Quantitative real time PCR and other methods to study mRNA levels
  • Transient overexpression and RNA-silencing of host genes to study their role in potyviral infections
  • Affinity purification and characterization of viral ribonucleoprotein complexes and host plant interaction partners in the infection context
  • Recombinant protein expression and in vitro biochemical and structural analyses for studies on viral protein structures, functions and posttranslational modifications
  • Polysome purification and ribosome profiling for studying host translation in the infection context.
  • Microscopy: Epifluorescence and confocal microscopy to visualize in vivo localization and association of fluorescently tagged viral and host proteins. Electron microscopy for detailed imaging of infected cells and virus particles.
  • Purification and enrichment of virus particles, infection-linked structures and organelles by ultracentrifugation.