Chloroplast redox state changes indicate cell-to-cell signalling during the hypersensitive response

We performed detailed spatiotemporal analysis of chloroplast redox response to potato virus Y (PVY) infection in resistant Ny‐1-gene-bearing potato and its transgenic counterpart with impaired SA accumulation and compromised resistance. We found that the chloroplasts are highly oxidized in the cells adjacent to the cell death zone at different stages after virus inoculation in both genotypes. This hypothesis is further supported by highly induced formation of stroma filled tubules that extend from chloroplasts (stromules) in the cells adjacent to signalling cells. This dataset s a deposit of all the raw microscopy images of the study, plus the relevant metadata in ISA-tab compliant folder structure. 

Additional information about the microscopy images in this data deposit

Images of which name ends with ch00/ch01/ch02 are maximum projections from Z-stacks for each ROI for each of three channels: chlorophyll fluorescence (ch00), GFP fluorescence after excitation with 405 nm laser line (ch01) and GFP fluorescence after excitation with 488 laser line (ch02), exported from Leica LAS X software.

Only mesophyll cells are included in z-stack, except for the experiments with GFP-tagged PVY where additional z-stacks including  both mesophyll and epidermal cells were produced ( »_2« added in the image name, see images with the comment »epidermal cells included in z-stack« in S_PhenodataRedox).

Images of which name ends with ch00.tif and ch00.tif_ratio are analysed images, obtained using in-house Matlab script. These images were obtained by the analysis of ch00/ch01/ch02 images with the following steps: conversion to grayscale, filtering out pixels of low intensity, conversion to binary format using spatial adaptive thresholding, a round or erosion and dilation to remove single pixel noise around the chloroplasts, followed by size-based segmentation of individual chloroplasts. The ratios of fluorescence intensities 405/488 were then calculated for each pixel belonging to the chloroplast masks obtained in the previous step. Results were calculated per image (normalized to the fraction of pixels belonging to chloroplasts) and per individual chloroplast in each image.

In the experiments with GFP-tagged PVY, due to high background signal in the cell death zone as a result of virus derived GFP fluorescence, signal in the 488 channel and 405/488 ratio (images named »scaled 488« and »405/488 in chloroplasts« in ch00.tif_ratio) in the cell death zone are not accurate.

Figures of which name ends with ch00.tif_spatial_ROI1/ROI2 show the 405/488 ratios in ROI1 or ROI2, determined for each pixel inside chloroplast masks for each Bin. See Image analysis in Methods for details.

Experiment names and image names correspond to the names in S_PhenodataRedox. Details regarding experimental set-up and transgenic lines used are specified in S_PhenodataRedox.