Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides

Abstract

In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongated-branched, depending on the host plant. In Medicago truncatula, this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.

Keywords: legume; nitrogen-fixing bacteroids; nodule; nodule-specific cysteine-rich peptides; symbiosis.

Fig. 1.

Numbers of NCR peptides in different IRLC legumes are correlated with bacteroid morphology. Numbers of NCR peptides predicted from nodule transcriptomes or genome sequences of 10 IRLC legumes are shown in relation to the morphotype of the bacteroids (A). There is a positive correlation between average bacteroid length and the size of the NCR family (B). Pearson correlation coefficient: 0.90 (P value > 0.001).

Fig. 2.

Isoelectric point profiles of NCR peptides and the relative expression of NCRs with different pIs in the IRLC legumes and nodule zones in M. truncatula. The frequency of nodule-expressed NCRs with given pI values is shown graphically; grouping of the NCRs from different legume species is based on the S, E, SP, and EB morphotypes of bacteroids (A–D, respectively). The contribution [% RPKM or DEseq normalized values (29)] of different isoelectric point categories to whole-nodule NCR expression for seven IRLC legumes is provided (E). The pattern of NCR expression [% DEseq normalized values (29)] in different zones of M. truncatula nodules is shown in F.

Fig. 3.

SEM and biofilm formation of S. meliloti treated with synthetic M. truncatula NCR peptides. SEM images of S. meliloti cells incubated for 3 h in phosphate buffer (A, control) supplemented with anionic (B, NCR095), neutral (C, NCR084), or cationic (D, NCR055) peptides at 8 µM. Biofilm formation was evaluated by crystal violet assays after incubation for 24 h in LSM medium with a range of NCRs with different isoelectric points (E). SEs were calculated with three biological replicates using averages based on three technical repeats. P values of <0.05, 0.01, and <0.001 are marked with one, two, or three asterisks, respectively (Student’s t test). (Scale bars, 1 µm.)

Fig. 4.

Morphology and viability of S. meliloti mutants challenged with NCR055 or NCR124. SEM and confocal microscopy images of S. meliloti mutants affected in the exoA, exoB, and lpsB genes after incubation in phosphate buffer only (untreated) or phosphate buffer supplemented with 8 µM NCR124 or NCR055. After incubation, membrane integrity was evaluated by staining with PI (red fluorescence) and SYTO9 (green fluorescence). T.L., transmitted light. (Scale bars, 1 µm.)