PMC

M. pudica were co-inoculated with Fix⁺ and Fix^- C. taiwanensis at a 1/1 ratio and sections of nodules collected at 14 dpi (ABFG), 16 dpi (C) or 35 dpi (DEHI) were observed under bright field (panels 1) or fluorescent microscopy (panels 2 and 3), and after PI staining (panels with an *). Panels with the same letters represent the same nodule section. (F3), magnification of (F2) visualized by confocal microscopy. (A) and (D), sections of nodules infected with a GFP-labeled Fix⁺ strain. (B) (C) and (E), sections of nodule infected with a GFP-labeled Fix^- strain. (F), nodule co-infected with a GFP-labeled Fix⁺ and a mCherry-labeled Fix^- strain. (G) and (H), nodules co-infected with a GFP-labeled Fix^- and an unlabeled Fix⁺ strain. (I), nodules co-infected with a GFP-labeled Fix⁺ and an unlabeled Fix^- strain. The white and yellow dotted lines in (GHI) delimit the areas occupied by the Fix^- and Fix⁺ strains in a co-infected nodule, respectively. Note that neither the Fix⁺ (D3) nor the Fix^- bacteroids (B3G3) are red-labeled by PI staining at 14 dpi whereas a few cells are PI-stained in the Fix^--occupied nodule at 16 dpi ([C3], arrows), and Fix^- are mostly PI-labeled (dead) at 35 dpi (E3H3I3). Note that bacteria of the infection zone are still alive at 35 dpi (arrow, E2E3). Note that nodule cells filled with Fix^- are browner than nodule cells filled with Fix⁺ (G1H1I1). Scale bars correspond to 100 µm except for F3 (30 µm).

(A) Fix⁺ (CBM382) or Fix^- (CBM2568) C. taiwanensis were inoculated on M. pudica. Box plots represent the distribution of the number of bacteria recovered per nodule on plates. Box plots were constructed as described in . R, ratios of the median number of Fix⁺ bacteria per nodule on the median number of Fix^- bacteria per nodule. The number of nodules analyzed at each time point is indicated in brackets. The number of plants analyzed at each time point is indicated in red. Results are from two independent experiments (). *Significantly different from the number of Fix⁺ bacteria per nodule (p<0.05 multiple comparison test after Kruskal-Wallis). (B, C) Theoretical reproductive fitness of Fix⁺ (B) and Fix^- bacteria (C) following single-inoculation of M. pudica as compared to experimental data. Dotted lines represent bacterial populations per plant averaged over 200 replicate simulations (). Box plots represent the distribution of the number of bacteria experimentally recovered per plant. Experimental data are from (A).
10.7554/eLife.28683.008Figure 2—source data 1.Reproductive fitness of nodule bacteria following single-inoculations with either Fix⁺ (CBM382) or Fix^- (CBM2568) C. taiwanensis.
10.7554/eLife.28683.009Figure 2—source data 2.Simulation data for the reproductive fitness of Fix⁺ and Fix^- bacteria following single inoculations of M. pudica.

Segregation of Fix⁺, Fix⁻, and Fix^+/− phenotypes in the progeny of RHL-NFS1. (A) Plants forming Fix⁺, Fix⁻, and Fix^+/− nodules were visually distinguishable by their growth vigor. (B–D) Confocal microscopy of Fix⁺ (B), Fix^+/− (C), and Fix⁻ (D) nodules infected by GFP-tagged Rm41. Samples were contrasted with propidium iodide (red). The Fix⁺ and Fix⁻ nodules were structurally similar to those formed on the DZA315 and A17 roots, respectively. The Fix^+/− nodules, however, were smaller in size, contained smaller numbers of symbiotic cells, and had an extended zone of senescence compared with the Fix⁺ nodules. (B and C) Prepared with multiple images. (Scale bars: B–D, 100 µm.)

Segregation of the Fix⁺, Fix⁻, and Fix^+/− phenotypes in the progeny of RHL-NFS2. (A–C) Plants forming the Fix⁺, Fix⁻, and Fix^+/− nodules are visually distinguishable by their growth vigor and nodule size. (Scale bars, 1 cm.) (D–F) Confocal microscopy of the Fix⁺ (D), Fix^+/− (E), and Fix⁻ (F) nodules infected by GFP-tagged Rm41. Panels D and E were prepared with multiple images. (Scale bars, 100 µm.) (G–I) Plant dry weight (G), nodule dry weight (H), and nodule numbers (I) differ significantly between plants forming Fix⁺, Fix^+/−, and Fix⁻ nodules.