Rhizobium-legume symbiosis in the absence of Nod factors: two possible scenarios with or without the T3SS

Abstract

The occurrence of alternative Nod factor (NF)-independent symbiosis between legumes and rhizobia was first demonstrated in some Aeschynomene species that are nodulated by photosynthetic bradyrhizobia lacking the canonical nodABC genes. In this study, we revealed that a large diversity of non-photosynthetic bradyrhizobia, including B. elkanii, was also able to induce nodules on the NF-independent Aeschynomene species, A. indica. Using cytological analysis of the nodules and the nitrogenase enzyme activity as markers, a gradient in the symbiotic interaction between bradyrhizobial strains and A. indica could be distinguished. This ranged from strains that induced nodules that were only infected intercellularly to rhizobial strains that formed nodules in which the host cells were invaded intracellularly and that displayed a weak nitrogenase activity. In all non-photosynthetic bradyrhizobia, the type III secretion system (T3SS) appears required to trigger nodule organogenesis. In contrast, genome sequence analysis revealed that apart from a few exceptions, like the Bradyrhizobium ORS285 strain, photosynthetic bradyrhizobia strains lack a T3SS. Furthermore, analysis of the symbiotic properties of an ORS285 T3SS mutant revealed that the T3SS could have a positive or negative role for the interaction with NF-dependent Aeschynomene species, but that it is dispensable for the interaction with all NF-independent Aeschynomene species tested. Taken together, these data indicate that two NF-independent symbiotic processes are possible between legumes and rhizobia: one dependent on a T3SS and one using a so far unknown mechanism.

Figure 1

Bradyrhizobium elkanii is able to induce nodules on Aeschynomene indica in a T3SS-dependent manner. (a) Number of nodules per plant elicited by B. elkanii USDA61, BEnodC, BErhcJ, BEttsnod and Bradyrhizobium ORS278 strain on A. indica plants at 14 days post inoculation (d.p.i.). Error bars represent s.d. (n=5). (b) Comparison of the growth of the plants (aerial part), non-inoculated (NI) or inoculated with B. elkanii USDA61 and Bradyrhizobium ORS278 (at 14 d.p.i.). (c–f) Nodules elicited by B. elkanii USDA61 (c), BEnodC (d), BErhcJ (e), ORS278 (f). Bars, 1 mm. (g) Transversal section of a nodule elicited by B. elkanii tagged with gusA and stained with X-Gluc. Bar, 200 μm. Black arrows indicate the presence of necrotic area. (h and i) Confocal microscopy observation of nodules elicited by B. elkanii tagged with GFP. Bars, 200 μm (h) and 10 μm (i). A full colour version of this figure is available at the ISME journal online.

Figure 2

The ability to induce nodules on NF-independent Aeschynomene species is widespread among Bradyrhizobium species. Neighbor joining phylogenetic relationship among the Bradyrhizobium strains tested for their ability to form nodules on A. indica based upon aligned sequences of 16S-23S rDNA IGS regions. Percent of bootstrap replicates (1000 replicates) are indicated at each tree node (only if >50%). In red, negative strain; in green, positive strains; in blue, photosynthetic Bradyrhizobium strains that are able to induce nitrogen-fixing nodules on A. indica; in bold, strains for which an intracellular infection is observed. A full colour version of this figure is available at the ISME journal online.

Figure 3

Symbiotic phenotypes of Bradyrhizobium STM3560 and STM6978 on A. indica. (a and e) Nodules elicited by Bradyrhizobium STM3560 (a) and STM6978 (e). Bars, 1 mm. (b, f and h–j) Transversal section of nodules elicited by Bradyrhizobium STM3560 (b and h), STM6978 (f and i) and ORS278 (j). Bars, 200 μm. (d) Comparison of the growth of the plants (aerial part), non-inoculated (NI) or inoculated with STM6978, and Bradyrhizobium ORS278 (at 14 d.p.i.). (c, g and n) Confocal microscopy observation of nodules elicited by Bradyrhizobium STM3560 (c), STM6978 (g) and ORS278 (n). (l, m and o) Confocal microscopy images showing the morphology of the intracellular bacteria. Note that STM6978 bacteroids showed mainly a rod shape (l) and rarely a spheric shape (m) whereas the ORS278 bacteroids were all spherical (o). Live bacteria are stained green, whereas dead bacteria stain red owing to uptake of propidium iodide. Bars, 200 μm (c, g and n) and 10 μm (l, m and o). (k) A. indica roots inoculated with Bradyrhizobium STM6978ΔT3SS. Bar, 1 mm. A full colour version of this figure is available at the ISME journal online.

Figure 4

Comparison of the symbiotic properties of Bradyrhizobium ORS285 and the ΔT3SS mutant on various NF-dependent and NF-independent Aeschynomene species. (a) Number of nodules per plant elicited by Bradyrhizobium ORS285 (blue bars) and ORS285ΔT3SS (red bars) on various Aeschynomene species. (b) Acetylene-reducing activity (ARA) in various Aeschynomene plants inoculated with Bradyrhizobium ORS285 (blue bars) and ORS285ΔT3SS (red bars). A.U., Arbitrary Unit. Error bars in a and b represent s.d. (n=5). (c–e, h–j). Aspect of the nodules elicited by ORS285 and 285ΔT3SS on A. nilotica (c and h), A. uniflora (d and i) and A. afraspera (e and j). Bars, 1 mm. (f–k, g–l) Cytological observations of the A. afraspera nodules elicited by ORS285 (f and g) and 285ΔT3SS (k and l). Bars, 500 μm (f and l) and 100 μm (g and l). A full colour version of this figure is available at the ISME journal online.