Convergent Evolution of Endosymbiont Differentiation in Dalbergioid and Inverted Repeat-Lacking Clade Legumes Mediated by Nodule-Specific Cysteine-Rich Peptides

Abstract

Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.

Figure 1.

Properties of free-living cultured Bradyrhizobium sp. ORS285 bacteria and ORS285 bacteroids isolated from A. afraspera or A. indica nodules. A, Normaski bright-field (BF; top row) and fluorescence microcopy of bacteria and bacteroids stained with 4′,6-diamidino-2-phenylindole (DAPI; middle row) and PI (bottom row). Bars = 1 µm. B, DNA content of DAPI-stained bacteria and bacteroids measured by flow cytometry. Colors are as follows: blue, free-living bacteria; green, bacteroids isolated from A. afraspera; and red, bacteroids isolated from A. indica.

Figure 2.

Distribution of S- and E-morphotype bacteroids among Aeschynomene ssp. The species A. indica, A. evenia ssp. serrulata, A. evenia spp. evenia, A. virginica, A. scabra, A. sensitiva, A. afraspera, A. nilotica, and A. aspera were nodulated by the ORS285 GFP-tagged strain; the species A. americana, A. schimperi, and A. pfundii were nodulated by Bradyrhizobium spp. strains ORS301, ORS302, and ORS305, respectively, and bacteroids were stained by the SYTO 9 fluorescent probe of the live/dead stain. Bars = 10 μm.

Figure 3.

Alignment and Cys signature of Aeschynomene spp. NCR peptides. A, The deduced protein sequences from the up-regulated NCR genes from A. indica (AiNCRs) or A. afraspera (AaNCRs) were aligned using Multalin (http://multalin.toulouse.inra.fr/multalin/), and the alignment obtained was adjusted manually. Highly conserved amino acids are in red, and conservative substitutions are in blue. The putative location of the signal peptide is indicated below the alignment. B, Comparison of the Cys-rich motifs of Aeschynomene spp. NCR peptides with those of Medicago spp. and defensin-like peptides. Red and green Cys residues correspond to the NCR and defensin signatures, respectively. The peptides that do not contain any motif are gray shaded in A.

Figure 4.

NCR expression during the symbiotic process. NCR expression is shown during A. afraspera (A) and A. indica (B) nodule development. Plants were inoculated with the Bradyrhizobium sp. ORS285 strain, and the roots were sampled at time 0 (T0) and at different time points as indicated. The relative expression level of the NCR peptides was determined by RT-qPCR and normalized by the expression of Elongation Factor 1α (EF1α). As a control of nodule development, the expression of LegHb (LEG) was also measured.

Figure 5.

NCR genes are specifically expressed in the infected symbiotic cells of Aeschynomene spp. nodules. Sections of 14-dpi A. indica and A. afraspera nodules, infected with the Bradyrhizobium sp. ORS285 strain, were analyzed by in situ hybridization with antisense probes of LegHb (B and E) or NCR (C and F). Control hybridizations were done with the sense NCR probe (A and D). Bars = 100 μm.

Figure 6.

DNF1 silencing affects bacteroid differentiation in Aeschynomene spp. A, Alignment of the deduced protein sequence of the A. evenia ssp. serrulata DNF1 ortholog with the corresponding Medicago spp. protein (accession no. TC121074). The two protein sequences were aligned using Multalin (http://multalin.toulouse.inra.fr/multalin/). B to E, Roots of different lines of A. evenia ssp. serrulata transformed with an RNAi construct directed against AeDNF1 encoding the SPC22 subunit of the SPC were inoculated with a GFP-tagged Bradyrhizobium sp. ORS285 strain. At 21 dpi, the nodules were harvested, and the shape of the bacteroids was observed by confocal microscopy on nodule sections. As a control, a line with no RNAi effect (1% reduction) was used (B). The lines 3, 7, and 53, with expression levels reduced by 62%, 65%, and 66%, respectively, are presented in C to E. Bars = 10 µm.