Figure 5. Viability of Fix⁺ and Fix^- bacteroids.

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).