Fig. 1: Symbiosome differentiation is impaired in nodule cells of bid1.

a bid1 mutant only had small and white nodules when inoculated with S. meliloti ABS7 hemA::LacZ. Representative pictures of nodules were taken at 21 dpi (days post inoculation). Bar=1 mm. b Symbiosomes were not differentiated in bid1 nodule cells from a in nodule sectioning assay. Note that infected bid1 nodule cells were also smaller than WT. Nodules from a were sectioned into 5 μm slides and stained with toluidine blue. Bar=20 μm. c Symbiosomes in bid1 nodule cells were undifferentiated. 21 dpi nodules inoculated with S. meliloti RM021 pHC60-GFP were stained with PI (Propidium iodide, red) and analyzed under confocal microscopy. Bar=10 μm. d Symbiosomes were undifferentiated in bid1 nodule cells in TEM assay. 21 dpi nodules inoculated with ABS7 hemA::LacZ were used for analysis. Letter “b” indicated bacteroids. Bar=2 μm. e Representative pictures showing differences between isolated symbiosomes from WT and bid1. Symbiosomes were isolated from nodules in c. Bar=10 μm. f bid1 symbiosomes were much shorter in length in the quantification assay of symbiosomes from e. Piano plot to show the minimum, 25th percentile, median, 75th percentile and maximum symbiosome lengths. Numbers of symbiosome analyzed were indicated respectively. “***”, P < 0.0001 in one-way ANOVA assay. g nifH::GUS reporter was not activated in bid1 nodule cells. GUS staining was performed on WT and bid1 nodules 21 days after inoculation with S. meliloti RM1021 nifH::GUS at 21 dpi. Bar=750 μm. h, Expression of NCR001 was totally blocked in bid1 nodule cells. qRT-PCR assay was performed using samples from nodules 21 dpi with ABS7 hemA::LacZ. ND, not detected. i and j, Rhizobium ExoY and CtrA genes were highly expressed in bid1 nodule cells compared with WT. qRT-PCR assays were done using the same samples as in h. For h, i and j, “**” and “***” meant P < 0.01 and P < 0.001 in Student’s t-test, respectively. Data were represented as means ± SEM of three independent amplifications. Experiments were repeated at least 3 times with similar results.