Fig. 3: E. adhaerens T4 nodules are prematurely stopped in their development and undergo a senescence process.

a-f Histological analysis of WSM419 (a–c) and T4 (d–f) nodule development. I, meristematic zone; II, infection zone; III, fixation zone and IV, premature senescence zone (IV). g–j Histological analysis of WSM419 (g, h) and T4 (i, j) nodule infected cells. v, vacuole; cv, collapsed vacuole; uc, uninfected cell and ic, infected cell. (Scale bars a–f, 200 μm; g–j, 40 μm). For a-j, similar results were observed using three independent kinetics. k RT-qPCR gene expression profiles of senescence marker genes MtCP2, MtCP3, MtCP5 and MtNAC969, and of symbiotic marker genes MtSymCRK, MtDNF2, MtRSD and MtLEGH1 along WSM419 (blue curves) and T4 (red curves) nodulation kinetics. Dots represent three independent biological replicates (n = 3). Gene accessions are provided (Supplementary Data 4). l–u Live/dead staining showing the viability of WSM419 and T4 bacteroids during nodulation. SYTO9 green signals indicate alive bacteroids. PI violet signals indicate dead bacteroids, cell walls, nuclei and nodule meristem. (Scale bars l–u, 100 µm). For l–u, the kinetics experiment was done once. v and w Live/dead staining showing WSM419 and T4 bacteroid viability and morphological differentiation within infected cells of 21-dpi nodules. v WSM419 nodule-infected cells were densely filled with morphologically differentiated and alive bacteroids. w T4 nodule-infected cells were occupied by dead and undifferentiated bacteroids. Some T4 bacteria were alive (Arrowhead). (Scale bars v and w, 20 µm). x-ab Morphological differentiation and cell size of WSM419 and T4 under free-living and bacteroid states. x–aa Fluorescence microscopy showing DAPI-stained undifferentiated free-living WSM419 (x), differentiated WSM419 bacteroids (y), undifferentiated free-living T4 (z) and undifferentiated T4 bacteroids (aa). (Scale bars x–aa, 10 µm). For x–aa, pictures were acquired from a single experiment. ab Cell length of WSM419 and T4 under free-living and bacteroid states. For each box-and-whisker plot, the box contains 50% of the data, the bottom and the top of the box represent Q1 and Q3, respectively, the center line indicates the median, the center cross indicates the mean, the whiskers indicate the data that range within 1.5 times the interquartile range and if they exist, outliers are shown (n = 100 cells). Asterisks indicate a significant difference compared to free-living WSM419 (***P value ≤ 0.001; P value in ab = 2.10^-84; Two-sided Student’s t-test). ac–aj Live/dead staining showing the viability of WSM419 and T4 bacteroids in A17 and Mtdnf1-1 at 8 and 12 dpi. In Mtdnf1-1, the death of T4 bacteroids is delayed compared to the wild type. (Scale bars ac–aj, 200 µm). The experiment was performed twice. Source data for Fig. 3k, ab are provided in Source Data file.