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

Abstract

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.

Plant phenotyping relevance

植物のウイルス感染拡大と細胞死領域を共焦点顕微鏡で時空間的に追跡する再利用可能なプロトコルが中心であり、植物の病害状態・細胞死を画像から評価する方法に該当する。

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