Imaging Potato Virus in Leaves by HCR FISH

• Rebekah Tuminaro, Applied Molecular Biology & Biotechnology, University of Delaware

• Mona Batish, , University of Delaware

Potato virus Y (PVY) is the largest constraint on potato production, reducing crop yields by up to 80%. There are three different strains of potato virus Y, with over 30 substrains identified as it can rapidly mutate and adapt to different environments through genetic recombination and selective pressures. Recently, the occurrence of strain PVYNTN has been increasing, while PVYO has been decreasing, likely as a result of antagonistic interactions between the two strains. Multiple strains of potato virus routinely coinfect cells, which can lead to recombination and the emergence of  new, more harmful, strains, making it important to understand this superinfection and the resulting creation of new strains. 

The potato virus helper component proteinase (HcPro) has been documented to function as a host antiviral RNA silencing suppressor. This prevents the replication of a related secondary virus, known as superinfection exclusion (SIE), preventing multiple strains from coinfecting a cell and recombining to create a new variant, and plants preinfected with a mild isolate of a virus can also acquire protection from secondary infections of the same virus. 

For our experiments, HcPro cistrons were amplified from PVYNTN and PVYO strains and then introduced into an entry vector. The plasmids were then transformed into A. tumefaciens (a strain of Agrobacterium). This Agrobacterium was then used to inoculate the plants. Samples from these leaves were then collected at different time points. We have optimized an HCR FISH (hybridization chain reaction fluorescence in situ hybridization) protocol for use in these samples. Samples are fixed, permeabilized, and then incubated with fluorescent probes that attach to the virus, allowing them to be visualized and quantified. Now that this protocol has been developed, we will be able to visualize a timeline of this virus and how it spreads, and better understand the different strains and their interactions.