Fig. 3: BID1 is required for proper ER-symbiosome proximal association.

a-e In qRT-PCR assay a group of ER stress-related marker genes were induced in 21 dpi bid1 nodules inoculated with ABS7 hemA::LacZ. Error bars represent standard deviation from three biological replicates. “**” and “***”, P < 0.01 and P < 0.001 in Student’s t-test respectively. Data were represented as means ± SEM of three independent amplifications. f Confocal microscopy analysis of ER structures in WT and bid1 nodule cells. 14 dpi Rm1021 pHC60-GFP inoculated mCherry-HDEL-expressing WT and bid1 nodules were analyzed. For both panels, bar=10 μm. Roi, region of interest. g and h Fluorescent intensity of symbiosome GFP and ER mCherry signals of indicated regions in WT and bid1 respectively. Fluorescent intensity (grey value of pixels) was measured by ImageJ. i TEM assay results of ER and symbiosome structures in 14 dpi ABS7 hemA::LacZ-inoculated WT and bid1 nodule cells. For both panels, bar=1 μm. j Reconstructed 3D structures of symbiosomes and ER in WT and bid1 cells varied significantly. Overview of several ER and symbiosomes and zoomed-in structures of individual symbiosome and ER were shown. Symbiosomes were labelled in blue, ER in orange red. Structures were obtained through 3D reconstruction of AutoCUTS-SEM tomography data using Imaris software. Scal bar, 1 μm. k and l Piano charts showing differences in ER width and area between WT and bid1, respectively. ER width and area in i were measured using ImageJ. “***”, P < 0.001 in One-way ANOVA assay. m Measurement of ER volumes in WT and bid1 by Imaris. “*”, P < 0.05 in one-way ANOVA assay. Numbers of ER analyzed were indicated. n Average distance between ER membrane and symbiosome was much larger in bid1. ER-symbiosome distances were measured using Imaris. y-axis, logarithmic scores of ER-symbiosome distance to the base 2. “***”, P < 0.001 in one-way ANOVA assay. For k, l and n, numbers of ER analyzed were indicated. For m, 3D ER structures were evenly divided into 20 segments, volumes of each segment were determined. Experiments were repeated 3 times with similar results.