Analysis of Virus Spread Around the Cell Death Zone at Spatiotemporal Resolution Using Confocal Microscopy.

The role of programmed cell death (PCD) in hypersensitive response (HR)-conferred resistance depends on the type of host-pathogen interaction and therefore has to be studied for each individual pathosystem. Here we present and explain the protocol for studying the role of PCD in HR-conferred resistance in potato plants in the interaction with the viral pathogen. As an experimental system, we use genotype Rywal, where the virus spread is restricted and HR PCD develops 3 days post potato virus Y (PVY) inoculation. As a control of virus multiplication and spread, we include its transgenic counterpart impaired in salicylic acid (SA) accumulation (NahG-Rywal), in which the HR-PCD occurs but the spread of the virus is not restricted. To follow the occurrence of virus-infected cells and/or virus multiplication outside the cell death zone, we use GFP-tagged PVY (PVY-N605(123)-GFP) which can be monitored by confocal microscopy. Any other plant-pathogen system which results in PCD development could be studied using a modified version of this protocol.